See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/348568444

Integrating Biomaterials and Food Biopolymers for Cultured Meat Production

Article in Acta Biomaterialia · January 2021

DOI: 10.1016/j.actbio.2021.01.017

CITATIONS READS

64 3,446

2 authors, including:

Motoichi Kurisawa
Agency for Science, Technology and Research (A*STAR)
113 PUBLICATIONS 7,106 CITATIONS

SEE PROFILE

All content following this page was uploaded by Shengyong Ng on 15 August 2021.

The user has requested enhancement of the downloaded file.

JID: ACTBIO
ARTICLE IN PRESS [m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx

Contents lists available at ScienceDirect

Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actbio

Review article

Integrating biomaterials and food biopolymers for cultured meat

production
Shengyong Ng∗, Motoichi Kurisawa∗
Institute of Bioengineering and Nanotechnology (A∗ STAR), 31 Biopolis Way, The Nanos, 138669, Singapore

a r t i c l e i n f o a b s t r a c t

Article history: Cultured meat has recently achieved mainstream prominence due to the emergence of societal and indus-
Received 2 September 2020 trial interest. In contrast to animal-based production of traditional meat, the cultured meat approach en-
Revised 18 December 2020
tails laboratory cultivation of engineered muscle tissue. However, bioengineers have hitherto engineered
Accepted 11 January 2021
tissues to fulfil biomedical endpoints, and have had limited experience in engineering muscle tissue for
Available online xxx
its post-mortem traits, which broadly govern consumer definitions of meat quality. Furthermore, existing
Keywords: tissue engineering approaches face fundamental challenges in technical feasibility and industrial scala-
Cultured meat bility for cultured meat production. This review discusses how animal-based meat production variables
Food biopolymers influence meat properties at both the molecular and functional level, and whether current cultured meat
Muscle approaches recapitulate these properties. In addition, this review considers how conventional meat pro-
Adipose ducers employ exogenous biopolymer-based meat ingredients and processing techniques to mimic desir-
Tissue engineering
able meat properties in meat products. Finally, current biomaterial strategies for engineering muscle and
adipose tissue are surveyed in the context of emerging constraints that pertain to cultured meat produc-
tion, such as edibility, sustainability and scalability, and potential areas for integrating biomaterials and
food biopolymer approaches to address these constraints are discussed.

Statement of Significance

Laboratory-grown or cultured meat has gained increasing interest from industry and the public, but cur-
rently faces significant impediment to market feasibility. This is due to fundamental knowledge gaps in
producing realistic meat tissues via conventional tissue engineering approaches, as well as translational
challenges in scaling up these approaches in an efficient, sustainable and high-volume manner. By defin-
ing the molecular basis for desirable meat quality attributes, such as taste and texture, and introducing
the fundamental roles of food biopolymers in mimicking these properties in conventional meat products,
this review aims to bridge the historically disparate fields of meat science and biomaterials engineering
in order to inspire potentially synergistic strategies that address some of these challenges.
© 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction tured meat field to mature and demonstrate its commercial

Global trends in climate change and meat consumption high-
light an increasing need to complement animal-based meat pro-
duction with alternative dietary protein sources. The emergence of
cellular agriculture start-ups on the technological horizon, begin-
ning with a focus on plant-derived proteins, is punctuated with
a recent thrust towards cultured meat that comprises animal-
derived proteins [1]. This trend points to an increasing demand
for bioengineering solutions that would allow the nascent cul-
∗
Corresponding authors.
E-mail addresses: syng@ibn.a-star.edu.sg (S. Ng), mkurisawa@ibn.a-star.edu.sg
(M. Kurisawa).
feasibility.
Biomaterial-based scaffolds for engineering muscle or adipose
tissue are typically designed to fulfil biomedical endpoints, includ-
ing efficient cellular differentiation, functional robustness and post-
transplantation viability. While these design principles fulfil neces-
sary intermediary requirements for cultured meat, they may not
suffice to meet its ultimate functional endpoint, which is to sat-
isfy consumer definitions of meat quality more cost-efficiently and
scalably than current cultured meat manufacturing models. It may
therefore be instructive for bioengineers to understand how meat
quality attributes like appearance, texture, flavour and nutritional
composition fundamentally relate to complex molecular processes
that occur during meat production, ranging from in vivo muscle

https://doi.org/10.1016/j.actbio.2021.01.017
1742-7061/© 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article as: S. Ng and M. Kurisawa, Integrating biomaterials and food biopolymers for cultured meat production, Acta
Biomaterialia, https://doi.org/10.1016/j.actbio.2021.01.017
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
development to its post-mortem transformation into meat, and its
subsequent processing into meat products. A deeper insight into
how the meat production industry augments the quality, value and
range of conventional meat products, using food-grade ingredi-
ents and processing techniques in cost-effective and scalable ways,
could open up opportunities for combinatorial approaches that in-
tegrate food and biomedical materials to design practicable strate-
gies that address the above challenges.
In this review, we first discuss how animal-based meat pro-
duction variables influence molecular-level material properties
of meat, how these molecular properties relate functionally to
macroscopic meat properties that the consumer experiences, and
whether current cell-based meat production recapitulates these
properties. We then illustrate how biopolymer-based meat ingre-
dients contribute to desirable meat properties in semi-structured
and unstructured meat products, and explore how they may pro-
mote consumer acceptance of future cultured meat products. Next,
we survey conventional bioengineering strategies for promoting
expansion and differentiation of muscle and adipose precursor
cells and critically evaluate their feasibility for cultured meat pro-
duction. Finally, we consider novel biomaterial constraints, such
as the use of degradable, edible and animal-free scaffolds that
mitigate culture media costs and contribute to desirable meat
quality attributes, and explore the integration of biomaterials and
food engineering approaches for cultured meat production. Al-
though this review focuses on specific scientific and technolog-
ical challenges that the cultured meat field faces, we recog-
nize that its long-term market feasibility also depends on ad-
dressing challenges posed by the environmental, socio-political
and regulatory domains, as discussed in several recent reviews
[2–4].
2. How animal-based meat production influences meat
material properties and quality
A fundamental motivation of the cultured meat industry is
to complement traditional meat production to satisfy increas-
ing consumer demand for sustainable meat products. However,
this demand is tempered by consumer unwillingness to com-
promise on the organoleptic properties of meat [5]. Therefore,
to increase consumer acceptance and ensure commercial viabil-
ity, cultured meat should closely mimic conventional meat in
terms of appearance, structure, texture, flavour and nutritional
composition [6]. The wide variety of conventional meat prod-
ucts may facilitate earlier market entry of cultured meat prod-
ucts, as different products present different levels of technologi-
cal barriers to effective mimicry. For example, a full-cut steak re-
quires a much higher similarity to freshly slaughtered meat than
a fine-ground sausage does. The latter demonstrates the degree
to which conventional meat processing alters tissue and cellu-
lar structures in the final product [6,7], suggesting that com-
plete replication of original muscle structure may not always be
necessary.
A complex interplay of pre- and post-mortem variables influ-
ence animal-based meat properties. Inter- or intra-species genetic
variation due to natural or artificial selection, or individual vari-
ation due to gender or age, rearing environment and diet rep-
resent diverse pre-mortem factors that meat scientists study to
optimize meat properties [8–11]. Concomitantly, the post-mortem
transformation of muscle tissue into meat is a sophisticated sci-
ence, with post-mortem processing, storage conditions and pre-
slaughter animal conditioning designed to preserve desirable meat
quality [8,11–13]. Post-mortem transformation of muscle tissue be-
gins with the development of tissue hypoxia after blood perfusion
ceases, which prompts a metabolic switch to anaerobic respiration
and results in decreased intracellular pH [8,11–13]. The exhaus-
2
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
tion of intracellular glycogen triggers irreversible actomyosin com-
plex formation and permanent myofibre contraction (rigour mortis),
whereas a steady state acidic pH that matches the isoelectric point
of the actomyosin complex results in a decrease in water binding
capacity [8,11–13].
Current cultured meat technology entails the expansion and
subsequent differentiation of muscle precursor cells into fused
myotubes that comprise the fundamental building block of mus-
cle tissue, which is indispensable for structured meat produc-
tion. However, unstructured meat, which may not preserve tissue-
level structure, could require a simplified production process in-
volving only cell expansion or even industrial fermentation for
recombinant meat protein production [6]. The analogous ver-
sion of rigour mortis in cultured meat would theoretically oc-
cur with a comparable time delay after perfusion within the dif-
ferentiation bioreactor with culture medium is terminated, like
in animal-derived meat. In contrast to animal-based meat, post-
mortem metabolism of cultured meat is relatively uncharacter-
ized, and basic studies to assess this process vis-à-vis animal-
based meat are required given its significant contribution to meat
properties.
2.1. Appearance
The appearance of meat is a function of its colour and opac-
ity, and greatly influences the consumer decision to purchase it
[14]. Myoglobin, a major determinant of meat colour, exists in a
brownish, oxidized, non-oxygen-binding form (metmyoglobin), or a
purplish-red, reduced form (reduced myoglobin) that binds oxygen
to form the attractive bright red oxymyoglobin that characterizes
fresh meat [8,11]. Meat oxygenation levels, which determine rela-
tive concentrations of myoglobin isoforms, is a complex function
of pre-slaughter animal stress levels and post-slaughter conditions
[8,11]. Furthermore, intrinsic myoglobin concentration varies with
animal species and tissue location, with beef being darker than
poultry, and chicken thigh tissues being darker than chicken breast
tissue, for example [10,15].
In contrast to meat colour, the typical opaque appearance of
meat compared with live muscle tissue is largely a function of the
decreased myofibre water-binding capacity that results from post-
mortem tissue acidification [8,11,12,16]. Pre-slaughter animal stress
may cause premature depletion of glycogen stores, decreased post-
mortem anaerobic respiration, and a higher resultant pH than the
isoelectric point of actomyosin, leading to increased water binding,
less light-scattering and a darker appearance that appears overly
dry and less fresh to consumers, thus illustrating the role of opac-
ity in meat appearance [8,12].
Conventional myogenic differentiation protocols culture muscle
precursor cells at ambient oxygen conditions (20–21% O2 ), under
which myoglobin expression is suppressed and the cultured mus-
cle typically exhibits a pale appearance [6,17]. Possible approaches
to increasing myoglobin expression include introducing hypoxia
[17–19] or supplementing iron, lipids or even myoglobin during
culture [19,20]. The addition of myoglobin or iron-containing mim-
ics, such as soy leghaemoglobin used in Impossible©, the plant-
based meat analogue [21], or natural colorants such as beetroot
juice [22,23], offer alternative methods to replicate the reddish
colour of meat post-culture. To ensure that cultured meat appears
similar to animal-based meat, the colour of the former should be
quantified according to the three-dimensional colour space sys-
tem developed by the Commission Internationale d’Léclairage (CIE),
CIE L∗ a∗ b∗ , where L∗ indicates the brightness axis, a∗ indicates the
red-green axis and b∗ indicates the yellow-blue axis [8,24]. Both
instrumental methods (e.g. colorimeters, spectrophotometers) and
trained visual appraisal panels may be used to assess meat colour
[8,24].
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
2.2. Flavour
Although the cooking processing generates much of meat
flavour, various pre-cooking factors contribute significantly too.
Meat flavour intensity increases with animal age at slaughter, and
free-range animals raised on curated or foraged diets generate
terroir-specific flavours [8,25–27]. Fat is a major determinant of
species-specific flavours, which are associated with distinct fatty
acid profiles and the varied diet-derived aromatic compounds that
fat is a vehicle for [28,29]. Post-mortem ageing of meat further
generates umami-generating amino acids and aromatic compounds
via proteolysis and lipolysis respectively [8,11]. Cooking intensi-
fies meat flavour by generating volatile compounds from thermal
degradation of fat, and carbonyl compounds from the Maillard re-
action associated with the browning flavour of well-cooked meat
[8,30–32].
The specialization of several start-ups on cell-based fat produc-
tion highlights the potential importance of fat in cultured meat
flavour [33,34]. While differentiation of fat precursor cells into
adipocytes is technically feasible [35–39], certain limitations re-
main to be overcome, including the use of small molecules with
uncharacterized food safety profiles and the generation of imma-
ture adipocytes [40,41]. Furthermore, the fabrication of structured
meat containing both muscle and adipose cells requires complex
co-culture or post-culture assembly methods that are currently
lacking. In contrast, incorporating cultured fat into unstructured
meat products should be relatively facile and analogous to the mix-
ing of fat and lean meat from different sources in conventional
finely ground meat products [8].
Given the importance of post-mortem ageing and cooking in
generating flavour precursor molecules, it is critical to simulate
both processes when comparing the flavour of cultured meat pro-
totypes with animal-based meat [6]. The gold standard of meat
flavour analysis involves the assessment of cooked meat flavour
by trained consumer sensory panels, using quantitative descriptive
analysis of meat aroma, tastes and aftertastes [42], which requires
relatively large samples. In contrast, conventional electromagnetic
methods (e.g. ultrasound, light diffraction, optical impedance) mea-
sure meat composition in terms of fat and collagen content,
thereby providing an estimate of meat flavour, whereas emerging
molecular profiling methods (e.g. liquid chromatography – mass
spectrometry) directly quantify levels of meat flavour molecules
using smaller sample sizes that current cultured meat methods can
produce [11,31,43,44].
2.3. Texture
Meat texture, an interplay of tenderness, juiciness and lubric-
ity at the consumer level, is a complex function of molecular-
level properties such as firmness, and fat or water binding capacity
[45], which in turn are linked to the hierarchical organization of
myofibers, connective tissue and intramuscular fat, and the effect
of post-mortem ageing [6,8,12,46,47]. To ensure tender and juicy
meat, meat producers slaughter younger and fatter animals, hang
carcasses to minimize post-mortem muscle contraction prior to the
onset of rigour mortis, age meat to facilitate endogenous enzymatic
degradation, and control temperatures to avoid meat toughening
[6,8,11,46,48]. Pre-cooking strategies, such as adding enzymes or
acid to degrade or solubilize connective tissue, and slow cook-
ing methods for tougher cuts, further enhance meat tenderness
[8,11,48,49].
The pioneer cultured meat prototype exhibited a dry texture,
which was attributed to it comprising only myotubes, and high-
lights the importance of fat to the texture of future cultured
meat products [17]. Other limitations of current cultured meat ap-
proaches include low levels of actin and myosin, immature isoform
3
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
expression [50], a lack of overall myotube alignment, and the ab-
sence of an appropriate connective tissue mimetic, any of which
could affect the various properties that underlie meat texture
[47,51]. Appropriate scaffold design may enhance myofibre align-
ment and maturation, or promote cellular production of extracel-
lular matrix (ECM) molecules that comprise connective tissue, thus
recapitulating their textural contributions in cultured meat (see
Section 4.2.2). Additionally, gel-forming or texturized biopolymers
may independently substitute for meat texture, a common strat-
egy in unstructured meat and meat analogues (see Sections 3.2,
3.5) [52,53].
Given the major role of post-mortem processes on meat texture,
their time- and environment-dependant effects on cultured meat
after the termination of culture medium perfusion should be char-
acterized. Well-established instrumental methods in the meat in-
dustry that directly quantify meat texture include the texture pro-
file analysis (a two-bite simulation compression test), which mea-
sures firmness, springiness, cohesiveness, gumminess and chewi-
ness [54], and the Warner-Bratzler shear force test, which mea-
sures the perpendicular shear force needed to slice through mus-
cle fibres [42,55]. Other indirect quantitative methods measure wa-
ter holding capacity and myofibre anisotropy using various elec-
tromagnetic modalities to infer meat texture [11,44,55,56]. As it is
non-trivial to correlate instrumental readouts with consumer ac-
ceptance of meat texture, standardized “descriptive attribute” sen-
sory evaluations of meat juiciness, connective tissue amount and
tenderness, performed by trained consumer panellists, are ulti-
mately required to capture the complexity of the consumer ex-
perience and provide a more objective evaluation [42]. Due to an
increasing appreciation of the importance of texture on poten-
tial consumer acceptance of cultured meat, recent studies have
performed texture analysis or sensory panel studies on cultured
meat prototypes in comparison with animal-based meat, and high-
light the importance of scalable production methods for testing
[6,57,58].
2.4. Nutrition
Although consumers do not typically consider nutritional con-
tent when purchasing meat, meat is a nutrient-dense food that
provides important amino acids, fatty acids, vitamins and other
minerals not readily found in other food classes. To fundamen-
tally address the longer-term challenge of sustainably producing
alternative dietary protein, cultured meat producers should aim to
match the nutritional content of animal-based meat. Given that
current in vitro methods for generating cultured muscle and adi-
pose tissue result in incomplete maturation [50], increasing cell
differentiation efficiency and thereby cellular protein or lipid pro-
duction is a subject of ongoing research in the biomedical field.
The likely requirement for biomaterial-based scaffolds to
facilitate scalable cell expansion and cell differentiation (see
Sections 4.1, 4.2) introduces both challenges and opportunities
in designing the nutritional composition of cultured meat. One
technical challenge is the need to support high cell densities to
match those in native tissue (108 – 109 cells/mL) [59]. However,
most research into engineering muscle tissue has been performed
at relatively low cell densities (107 cells/mL) [58,60–63], and the
need for high encapsulated cell densities may require scaffold re-
formulation for ensuring fabrication feasibility and cellular mi-
croenvironmental optimization. Alternative approaches to maxi-
mize protein content in cultured meat prototypes include the use
of edible, proteinaceous scaffolds, or biodegradable scaffolds that
are replaced by cells and their secreted ECM proteins during cul-
ture [6].
Not all nutritional components of meat are produced by en-
dogenous cells [6] and may require supplementation. For example,
JID: ACTBIO
ARTICLE IN PRESS [m5G;February 21, 2021;17:50]

S. Ng and M. Kurisawa Acta Biomaterialia xxx (xxxx) xxx

Molecular process Gelling Water binder Thickening Fat

agent (G) (W) agent (T) emulsifier (F)
Function in meat
Texture modulation G W T F G W T F G W T F
Flavour modulation G W T F G W T F G W T F
Bulk structural integrity G W T F G W T F G W T F
Water, brine retention G W T F G W T F G W T F
Spoilage delay G W T F G W T F G W T F
Fat replacement G W T F G W T F G W T F
Protein replacement G W T F G W T F G W T F
Emulsified/ Comminuted/
Structured
Meat product type unstructured chopped/
(steak, whole-
(Examples) (fine-grain semi-structured
muscle ham)
sausage) (burger, meatloaf)

Fig. 1. Role of ingredients added to conventional meat products.

fatty acids such as conjugated linoleic acid and docosahexaenoic
acid are synthesized by ruminant gut-specific biohydrogenation or
algae respectively, and not by cultured adipocytes [29,64]. Other
micronutrients are either not produced by muscle or fat tissue,
such as microbial-derived vitamin B12 [65], or are packaged in a
more easily absorbed form in meat, such as haem-bound iron and
zinc [6]. Increasing consumer preference for healthier diets also
encourage meat producers to introduce healthier fatty acids into
their products [66].
3. Enhancing meat quality attributes with food-grade
biopolymers
Meat processing is integral to the production of various meat
products, ranging from structured whole-muscle hams, semi-
structured burgers, to unstructured meat emulsions such as
sausages [8,11,52], and offer bioengineers practical lessons in the
use of food-safe biopolymers and industrial processing methods
to maximize the organoleptic and economic value of slaughtered
meat without compromising consumer health. Ingredients added
to meat fulfil several broad functions that overlap, including tex-
ture and flavour modulation, product stability, fat or protein re-
placement, and delivery of bioactive compounds (Fig. 1). These
functions define meat quality and are underpinned by molecular-
level solubilisation of meat proteins, resulting in secondary molec-
ular processes such as water binding, thickening, fat emulsification
and gelation [8,11,52].
The use of phosphates and salt in the first step of traditional
meat processing illustrates several of these fundamental processes.
Alkaline phosphates ionically disrupt the actin-myosin complexes
formed during rigour mortis, thereby solubilizing this major source
of meat protein. Both phosphate and salt increase meat pH away
from the isoelectric point of actin-myosin, thus increasing water
binding capacity [8,11,12,16]. Protein solubilisation and subsequent
water binding lead to an overall increase in meat tenderness and
juiciness. Additionally, solubilized meat proteins intrinsically emul-
sify fat, which helps to retain its lubricity, and also undergo in-
termolecular binding and gel formation, which contribute to struc-
tural integrity in less structured meat products [8,11]. Although the
protein solubilisation activity of phosphate is irreplaceable, the wa-
ter binding capacity, fat emulsification and protein gelation that it
engenders and their desirable contributions to meat texture and
flavour can be replicated by various biopolymers (Table 1), which
were developed partly to mitigate the potentially negative health
effects of excessive dietary phosphates and salt and are detailed in
the following sections.
3.1. Product stability
Due to the loss of native tissue structure, restructured and un-
structured meat products often require the addition of biopoly-
mers that provide either thickening or gelling functions to enhance
product formability and retain structural integrity under storage
and cooking conditions. Hydrocolloids that swell and are typically
non-gelling at low temperatures, such as xanthan gum, guar gum,
locust bean gum and pre-swollen starches, act as thickeners that
improve the formability of uncooked minced meat products and
often suffice for maintaining product integrity (Table 1) [8,11,52]. In
addition, their water binding capacity contributes to reduced water
loss during storage and cooking.
Gel-forming materials are often employed to enhance product
formability where thickeners alone do not suffice, such as in re-
structured meat products comprising larger meat pieces (reformed
steaks, surimi) [8,11,52]. In addition to their structural roles, gels
applied as films or coatings also reduce water loss during storage
and act as an oxidation barrier, thereby improving storage stabil-
ity [66,67]. Typical examples of gelling biopolymers include hy-
drocolloids (e.g. alginate, κ -carrageenan, gellan gum, agar), pre-
swollen starches and proteins (e.g. gelatine, soy or pea isolates,
and whey) (Table 1) [8,11,52], the majority of which form phys-
ically crosslinked hydrogels in a thermo-responsive manner, with
the key exception of alginate which forms an ionically crosslinked,
cold-set gel (Box 1). A common gelation mechanism involves the
spontaneous aggregation of biopolymer chains upon cooling a
heated biopolymer solution below its sol-gel transition temper-
ature, which characterizes κ -carrageenan, gellan gum, agar, pre-
swollen starches and gelatine gels [8]. In contrast, physical gela-
tion of soy, pea and whey protein isolates involve thermal denatu-
ration of the globular proteins and subsequent hydrophobic aggre-
gation in a pH- and salt-dependant manner [68,69]. The require-
ment for high temperatures to solubilize gelling hydrocolloids or
denature gelling proteins limits the use of most physical crosslink-
ing methods to the production of cooked meat products. However,
the development of enzymatic methods using microbial transglu-

4
JID: ACTBIO
ARTICLE IN PRESS [m5G;February 21, 2021;17:50]

S. Ng and M. Kurisawa Acta Biomaterialia xxx (xxxx) xxx

Table 1
Biopolymer ingredients in conventional meat products.

Biopolymer Gelling agents Water binder Thickener, stabilizer Fat emulsifier Fat replacer Protein replacer
√ √ √ √
Polysaccharides Alginate
√ √ √ √
κ -carrageenan
√ √ √ √
Gellan gum
√
Agar
√ √ √
Konjac ∼ 1
√2 √ √
Pectin
√ √ √ √
Xanthan gum ∼ 3
√ √ √
Locust bean ∼ 4
gum
√ √
Guar gum
√ √
Cellulose
√ √ √ √ √
Modified starch
√ √ √ √ √ √
proteins gelatine
√ √ √ √ √ √
Soy isolate
√ √ √ √ √
Pea isolate
√ √
Whey
√
Wheat gluten
√
Lipids Lecithin
√

Monoglycerides
& diglycerides
1. Forms thermoreversible gel when mixed with xanthan, κ -carrageenan or starch; also forms thermostable gel at pH > 9.5 and above 85°C but would
denature meat proteins and is not used in meat processing.
2. Low-methoxyl pectin forms thermoreversible, calcium-dependant ionic gels at pH < 6, unlike the 55% sugar and pH < 3.5 required for high-methoxyl
pectin to gel.
3. Forms gel when mixed with konjac or locust bean gum.
4. Forms gel when mixed with xanthan or κ -carrageenan. References: [8,11,52].

taminase and recombinant fibrinogen/thrombin allows the use of
gelling proteins for uncooked meat products by supporting the for-
mation of covalently crosslinked proteins gels at low temperatures
[70].
3.2. Texture and flavour modulation
From a consumer perspective, desirable meat texture is per-
ceived as a complex interplay of tenderness, juiciness and lubricity
[45], and biopolymer ingredients added to meat generally aim to
replicate these properties by promoting water binding, gelation or
fat stabilization (Table 1) [8,11,52].
As fat is a major source of meat flavour, effective fat stabiliza-
tion contributes significantly to flavour retention. Effective emulsi-
fiers reduce the separation of fat and water in meat products un-
der both storage and cooking conditions, thus contributing to the
retention of fat-associated flavour, juiciness and lubricity [8,11]. Ex-
ogenous proteins from animal (e.g. caseinate, egg) and plant (e.g.
gluten, soy, pea) sources are introduced to supplement the emul-
sifying properties of solubilised meat proteins [8,52]. Non-protein
emulsifiers are commonly employed and include various lipids (e.g.
lecithin, monoglycerides and diglycerides of fatty acids), modified
starches and more recently, pectin [52,71,72].
Non-gelling gum- and starch-based thickeners stabilize emulsi-
fied fat primarily via their viscosity, whereas their high water bind-
ing capacities efficiently retain injected brines in raw meat prod-
ucts. These properties decrease fat and water loss during both stor-
age and cooking, therefore enhancing the lubricity, juiciness and
flavour of the cooked meat [8,52].
Gel-forming biopolymers contribute significantly to the replica-
tion of meat-like firmness in unstructured products that do not
preserve native tissue structure, such as sausages. Even restruc-
tured whole-muscle ham is injected with gelling agents like car-
rageenan to compensate for the partial loss of original meat tex-
ture due to the protein solubilisation required for optimal meat
binding and fat emulsification [8]. In addition to providing firm-
ness, gelation facilitates the retention of moisture and fat, and en-
ables the delivery of antioxidants in meat products, therefore con-
tributing to juiciness, lubricity and inhibiting the development of
rancid flavours [8,52,73]. However, the degree of gelation should
be judiciously optimized as it may negatively affect the appearance
(unnatural, glassy, stretch marks) and flavour intensity of meat
[8,74,75].
3.3. Fat and protein replacement
Due to increasing consumer demand for healthier products,
meat producers have attempted to incorporate either healthier fat
or less fat in their products [66,76]. The first approach is to sim-
ply replace fat with combinations of water-binding starch, hydro-
colloids, or proteins [52,77,78], which contribute fewer calories
and partially substitute fat-derived succulence with increased wa-
ter content. A second approach is to mimic fat textural properties
using certain starches that exhibit a soft and creamy mouthfeel
due to their rheological similarity to fat, such as oat-derived mal-
todextrin and rice starch [8,52]. A third approach is to replace fat
with healthier versions like plant-based oils, which tend to exhibit
low melting points and require oil bulking agents such as alginate,
konjac and cellulose or microencapsulation techniques to improve
their retention [66,79–81].
Meat proteins are often replaced with non-meat, non-protein
extenders like plant-derived fibre and hydrocolloids for economic
reasons [8,11,52], which merely substitute the mass, but not the
nutritional value, of the replaced protein. Non-meat proteins may
also replace meat proteins, and are used in meat analogue pro-
duction for vegetarians. In addition to being a nutritional re-
placement, these non-meat proteins should also fulfil the typical
molecular functions of meat proteins described above, including
fat emulsification, gelation, and water binding. Soy and pea pro-
tein are major plant-based protein replacers, but tend to exhibit
favourable properties only in highly purified protein isolate forms
[8,52]. For example, soy isolates containing 92% protein are in-
jected into whole muscle hams to add firmness via its gelling be-
haviour, but soy concentrates containing 71% protein cannot form
a hydrogel due to the presence of insoluble carbohydrates [8].
Novel alternative protein sources include algae, fungi and insects,

5
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
and currently exist as niche, standalone products that are not yet
widely used as protein replacers in conventional meat products
[82,83].
3.4. Relevance of food-grade biopolymers to cultured meat
Table 1 summarizes major examples of biopolymers employed
to replicate meat quality attributes during post-slaughter meat
processing or meat analogue production. In principle, similar
biopolymer ingredients and processes could be applied to cultured
meat during post-culture processing to endow them with compa-
rable properties to animal-based meat products.
The lower content of immature actin-myosin isoforms in my-
otubes and lack of connective tissue in current cultured meat pro-
totypes [50] could lead to a smaller effect of rigour mortis, which
may decrease the need for phosphate-dependant protein solubili-
sation and suggest a supplementary role for protein-based ingredi-
ents in order to ensure nutritional similarity to slaughtered meat.
In addition, the lack of tissue-level structure in current cultured
meat models may necessitate the use of gelling agents that provide
structural integrity and replicate meat-like texture. Gelling agents
may continue to play major roles in future cultured meat products
that are designed to comprise single cells, cell lysates or even re-
combinant meat proteins [6].
When future cultured meat development reaches the stage of
generating macroscale cultured meat units that resemble coarsely
chopped meat, gelling agents could play novel roles in their post-
culture modular assembly into even larger cultured meat con-
structs using additive manufacturing techniques [84], analogous to
their current role in restructured meat production (see Box 1). If
cultured meat technology attains the stage of one-step production
of cultured full-cut meat, water-binding agents like cold-swelling
gums or starches may become helpful to retain water and augment
product juiciness upon cooking. Biopolymer-based microencapsula-
tion or coating strategies for the delivery of bioactive compounds
can also be readily adapted to post-culture processing of cultured
meat at various stages of complexity.
While not a typical component of conventional meat process-
ing, the structuring processes employed to produce completely
animal-free meat analogues may be instructive for cultured meat
production. Such methods aim to mimic the fibrous texture of
structured meat and include both top-down (e.g. extrusion, shear
cell, or freeze structuring) and bottom-up (e.g. wet spinning, elec-
trospinning) methods [53,85–94]. Although developed primarily
for non-animal protein sources such as plants or fungi [95–97],
such structuring methods could allow whole-cell cultured my-
ocytes, cell lysates, or even recombinant animal protein, to be in-
troduced seamlessly to form hybrid meat analogues comprising
a portion of culture-derived animal proteins, thus potentially ex-
panding the range of cultured meat production methods.
Beyond post-culture processing of cultured meat, some food-
grade biopolymers may provide potential sources of scaffold mate-
rials for muscle and fat culture that are intrinsically cost-effective,
scalable, and sustainable, by virtue of having met these constraints
within the context of the meat industry (see Section 4.4).
Box 1: Alginate in animal-based meat products
Alginate is a seaweed-derived hydrocolloid that forms a phys-
ically crosslinked gel in the presence of bivalent cations such
as calcium. As it forms hydrogels at low but non-freezing
temperatures, alginate is particularly useful in the produc-
tion of restructured raw meat products, such as meatloaves,
meatballs, or seafood-based surimi, where a larger piece of
raw meat is formed from several smaller pieces. The first de-
6
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
scribed use of alginate in restructured meat utilized a com-
bination of alginate, calcium carbonate and an acid precur-
sor glucono-delta-lactone (GDL). GDL delays calcium ion re-
lease from calcium carbonate and enables proper mixing of
meat parts with alginate before ionic gelation [98]. The use
of alginate improves upon the use of phosphates and salt in
earlier versions of restructured meat, which solubilize meat
proteins and increase meat binding, but also introduce di-
etary concerns and require frozen storage to retain product
integrity. However, the use of calcium for alginate gelation
imparts some bitterness to the product, which inevitably re-
quires masking via sodium addition and partially negates the
envisioned role of alginate in reducing dietary sodium in pro-
cessed meats.
The calcium-induced gelation of alginate at low temper-
ature further enables its use as an oil bulking agent and
enables the replacement of animal fat in meat batters and
sausages with healthier plant-derived oils, which would oth-
erwise be poorly retained due to their relatively low melting
temperatures [79].
Although the gelation mechanism of alginate limits it to
batch-type production processes, this is beneficial in certain
seafood analogues that replace animal proteins with non-
animal soy protein isolates, where alginate layering tech-
niques are applied to mimic the flake-like structure of fish
fillets [67,85].
As a film coating, alginate helps to delay rancidity and
colour changes in frozen meat and fish [67,99]. Alginate
coated meat patties also exhibit improved water retention,
which reduces product shrinkage during storage, and in-
creases cooking yields, tenderness and flavour retention dur-
ing cooking [100].
4. Adapting biomaterial design strategies from biomedical
engineering to the cultured meat process
The original cultured meat prototype in 2013 comprised thou-
sands of self-assembled myotubes that were individually cultured
in manually fabricated chambers, and these myotubes represent
the most mature form of muscle cells currently achievable [101].
Although absent from this initial prototype, adipose tissue will be
considered an essential cell type in future cultured meat prod-
ucts. Broadly, the generation of mature myotubes and adipose cells
comprises two main stages: 1) expansion of progenitor cells, and
2) differentiation of expanded progenitor cells into myotubes and
subsequently myofibers, or adipocytes. Suitable progenitors include
lineage-committed cells, such as myoblasts and muscle satellite
cells from skeletal muscle, or preadipocytes from adipose tissue.
Alternatively, lineage-uncommitted cells such as bone marrow-
derived mesenchymal stem cells (BM-MSCs), adipose-derived stem
cells (ADSCs) or induced pluripotent stem cells (iPSCs) may be
considered. With the exception of immortal iPSCs, the other cell
sources exhibit limited and variable proliferation capacities, and
would require either regular isolation from animal biopsies or im-
mortalization.
Bioengineers have elucidated various design criteria that bio-
material scaffolds should fulfil for engineering tissue for biomed-
ical applications [102–107]. Nevertheless, both cell expansion and
differentiation stages face significant technological hurdles to in-
dustrial translation. We first contextualize and evaluate the feasi-
bility of current biomaterials strategies for promoting efficient ex-
pansion and differentiation of muscle and adipose precursor cells
within the setting of large-scale bioreactors, and identify possi-
ble avenues for improvement (Table 2A). In addition, we consider
novel constraints specific to cultured meat production, such as the
role for material degradability at different stages of cultured meat
JID: ACTBIO
ARTICLE IN PRESS [m5G;February 21, 2021;17:50]

S. Ng and M. Kurisawa Acta Biomaterialia xxx (xxxx) xxx

Table 2
Scaffold design principles for cultured meat production.

(A) Conventional design criteria for engineering muscle and adipose tissue

Cell expansion:
 Surface chemistry
 Cell adhesion
 Scaffold stiffness

Additional criteria for cell differentiation:

 Cell alignment
 Mechanical stimuli (passive & dynamic)
 Electrical stimuli

(B) Additional design criteria for adapting classical tissue engineering approaches for cultured meat production

Scaffold fabrication:
 Compatibility with bioreactors for cell expansion & differentiation
 Scalability of fabrication method for cultured meat production

Scaffold crosslinking: chemical (elasticity) versus physical (viscoelasticity)

 Mechanical stability during bioreactor culture
 Role of elasticity or viscoelasticity on cell expansion & differentiation
 Post-culture product stability & organoleptic properties (texture)

Scaffold degradability:
 Ease of harvesting expanded cells
 Responsiveness to cellular modelling during differentiation
 Organoleptic, stabilizing and nutritional functions post-differentiation

Scaffold edibility:
 Optional for cell expansion, necessary for cell differentiation
 Compatibility with food-safe cultured meat production

Scaffold sustainability:
 Animal-free, natural or recombinant sources

Cultured meat cost:

 Growth factor release, sequestration & mimicry to mitigate culture medium cost
Adoption of food biopolymers to mitigate scaffold costs

Product fulfils meat quality attributes: appearance, flavour, texture, nutrition

 Meat quality evaluation with conventional methods like textural profile analysis, human sensory testing

production, the potential for biomaterials to mitigate high growth
media costs, the need for edible, cost-effective, sustainable scaf-
fold materials and their potential sources (Table 2B), and conclude
by exploring how bioengineers may repurpose common biopoly-
mer meat ingredients as novel multifunctional biomaterial scaf-
folds that support engineered muscle and adipose tissue produc-
tion and contribute to desirable meat quality attributes.
4.1. Conventional biomaterial design strategies for cell expansion
4.1.1. Bioreactor considerations
Cell expansion currently appears to be less challenging to scale
up than differentiation, given earlier efforts by the cell therapy in-
dustry to expand adherent MSCs for allogeneic use on an indus-
trial scale using microcarriers in stirred tank bioreactors (STBs)
[102,108]. The possibility of bead-to-bead transfer as a mode of
cell passaging offers the attractive prospect of a semi-continuous
or even continuous seed train process that minimizes cell han-
dling and processing time, and therefore maximizes productivity
of the cell expansion stage (Fig. 2A) [109]. Nevertheless, the re-
quired scale of cell expansion required for cultured meat produc-
tion is orders of magnitude larger than that for biomedical ap-
plications [1,110,111], and the relatively low working cell densities
(105 – 106 cells/mL) and modest working volumes (50 L) of cur-
rently used bioreactors may pose significant scalability limitations
[108]. In contrast to an estimated batch size of 5 × 1010 cells pro-
duced by this method, 1 kg of muscle cells contains approximately
3 × 1011 cells [111], and average daily global meat consumption is
approximately 8 × 108 kg [112]. Microcarriers may be compatible
with other bioreactor types that support higher cell densities, such
as packed or fluidized bed bioreactors, or larger volumes, such as
aerated (bubble column, airlift) bioreactors that could support up
to 1 × 106 L (Fig. 2B) [110].
Alternatively, hollow fibre bioreactors (HFBs) (Fig. 2C) have ex-
hibited the potential for expanding bone marrow-derived MSCs
[113–115], embryonic stem cells [116], myoblasts [117,118] and
adipose-derived stem cells [114,119,120]. Such systems provide ma-
jor advantages of higher cell densities (108 – 109 cells/mL) and vol-
ume efficiencies [108,111,121], which could provide a space-saving
conduit in the earlier part of a seed train process [109]. However,
high microfibre and cell densities may pose significant limitations
to cell harvesting efficiencies in the intermediate rounds of cell ex-
pansion [122], which suggests greater practicality of HFBs in the
cell differentiation stage of cultured meat production, where the
scaffold is designed to be edible without requiring separation from
differentiated cells.
4.1.2. Scaffold design principles
Surface chemistry, particularly charge and hydrophilicity, pri-
marily promote initial cell attachment on microcarriers under dy-
namic seeding conditions [123]. Positively charged surfaces gen-
erally increase cell attachment efficiency due to the slightly neg-
atively charged cell surface [124–127], whereas moderately hy-
drophilic surfaces increase cell attachment indirectly by promot-
ing surface protein adsorption, which mediates cell attachment
[127–130]. However, microcarrier cell attachment is often a com-
plex, empirical function of inter-dependant factors including net
surface charge, charged group density, hydrophilicity, surface to-
pography, curvature and shear rates [123]. A salient demonstration
of this complexity is the observation that bovine myoblasts exhib-
ited similar cell attachment rates to net-positively charged Cytodex
1 microcarriers and net-negatively charged Synthemax microcar-

7
JID: ACTBIO
ARTICLE IN PRESS [m5G;February 21, 2021;17:50]

S. Ng and M. Kurisawa Acta Biomaterialia xxx (xxxx) xxx

Table 3
Common food biopolymers for tissue engineering.

Biopolymer Biocompatible scaffold fabrication strategies1 Applications2 Key review

Carrageenan Sol-gel transition + ionic gelation: composite hydrogel with alginate [307], hydrogel [314–316] Cartilage Yegappan et al.
Ionic gelation + polyelectrolyte complexation: composite microfibres with chitosan [313] [314,315] 2018 [311]
Acrylate-acrylate photopolymerization: methacrylated carrageenan hydrogels [308,310]
Sol-gel transition + freeze-drying: porous hydrogel [317]
Polyelectrolyte complexation + freeze-drying: composite porous hydrogel with chitosan [309]
Konjac Alkaline thermal gelation + freeze drying: porous hydrogel [355,357] Bone [357] Yang et al.
Electrospinning + Schiff-base formation: nanofibres [358] 2017 [384]
Water-in-oil emulsion: DEAE-modified microgel carriers [359]
Semi-interpenetrating network: composite hydrogel with polyphenol crosslinked fibrillar collagen
[356,360]
Pectin Ionic gelation: microgel carriers [318], interpenetrating networks with photopolymerized methacrylated Muscle [137] Noreen et al.
gelatine [321] Bone [318,322] 2017 [385]
Ionic gelation + freeze-drying: porous hydrogel [319] Skin [320]
Chemical gelation with (3-glycidyloxypropyl)trimethoxysilane (GPTMS) + freeze-drying: porous hydrogel Cartilage [325]
[323]
Electrospinning + Schiff-base formation: nanofibres [324]
Thiol-ene photopolymerization: norbornene-modified pectin hydrogel [320]
Disulphide bond formation: thiolated pectin hydrogel [137]
Acrylate-acrylate photopolymerization: methacrylated pectin hydrogels [322]
Schiff-base formation: composite hydrogel of aldehyde-modified HA and hydrazide-modified pectin
[325]
Polyelectrolyte complexation: composite hydrogel membranes with chitosan [326]
Gellan gum Sol-gel transition + ionic gelation: hydrogel [341,342,345], composite hydrogel with xanthan [346] Muscle [342] Stevens et al.
Sol-gel transition + ionic gelation + freeze-drying: porous hydrogel [347] Cartilage 2016 [339]
Acrylate-acrylate photopolymerization: methacrylated gellan gum microgels [340,348] [340,341,345]
Schiff-base formation: composite hydrogel of aldehyde-modified gellan gum and hydrazide-modified Neural [343]
gelatine [344] Cardiac [344]
Water-in-oil emulsion: microgel carriers [304] Bone [346]

Xanthan gum Ionic gelation: microcapsules of carboxymethylated xanthan [349] Bone [346] Kumar et al.
Sol-gel transition + ionic gelation: composite hydrogel with gellan [346] 2018 [351]
Borate-diol complexation: weak hydrogel or composite hydrogel with cellulose [350]
Hydrophobic-driven self-assembly: microcapsules of palmitoyl-modified xanthan [352] or
phospholipid-modified xanthan [353]
Thermal casting + chemical gelation with citric acid: hydrogel film [354]
Guar gum Acrylate-acrylate photopolymerization: methacrylated guar gum hydrogels [335] - George et al.
Borate-diol complexation + gas-foaming + freeze-drying: Porous composite hydrogel with polyvinyl 2019 [386]
alcohol [337]
Schiff-base formation: composite hydrogel of aldehyde-modified guar gum and collagen [336]
Chemical gelation with sodium trimetaphosphate + freeze-drying: porous hydrogel [338]
Cellulose Bacterial nanofibre self-assembly + freeze-drying: composite porous hydrogel with agarose/gelatine Muscle [178] Hickey et al.
[158,330,331] Skin [334] 2019 [159]
Decellularization: prevascularized plant scaffold [329,333] Cardiac [329]
Spin-coating of cellulose nanorods: nanofilms [178] Bone [330,331]
Electrospinning: nanofibres [328]
Rotary jet spinning: composite nanofibres with soy [334]
Chemical gelation + freeze-drying: ether-crosslinked composite porous hydrogels with soy isolate [332]
Chemical gelation + solvent casting: epoxide-crosslinked composite hydrogel films with soy [327]
Starch Melt spinning/fibre bonding: porous composite microfibre mesh with polycaprolactone (PCL) [374,375] Bone Hemamalini
Melt spinning/fibre bonding + electrospinning: composite microfibre mesh with PCL filled with [163,373,374,376] et al. 2018
nanofibres [373] Cartilage [377] [387]
Wet spinning: composite microfibres with polycaprolactone [163] Vascular [375]
Electrospinning: composite nanofibres with PCL [377] or with PEG [378]
Electrospinning + Schiff-base formation: nanofibres [358]
Thiol-ene + acrylate-acrylate photopolymerization: interpenetrating network of pentanoate-modified
starch with methacrylated gelatine [376]
Soy isolate Thermal gelation + freeze-drying: porous hydrogel [370] Muscle [58] Tansaz et al.
Chemical gelation + freeze-drying: porous hydrogel that is transglutaminase-crosslinked [367], Skin [334] 2016 [388]
glutaraldehyde-crosslinked [370], carbodiimide-crosslinked composites with gelatine/alginate/pectin Adipose [365]
[369] or ether-crosslinked composites with cellulose [332]
Electrospinning: nanofibres [365]
Rotary jet spinning: composite nanofibres with cellulose [334]
Wet spinning: microfibres [366]
Extrusion: texturized scaffold [58]
Chemical gelation + solvent casting: epoxide-crosslinked composite hydrogel films with cellulose [327]
Zein (corn) Solvent casting: nanoparticulate films [134], smooth hydrated films [389] Bone Paliwal et al.
Salt leaching + freeze-drying: Porous scaffold [362,364,383] [362,364,383] 2014 [390]
Electrospinning: nanofibres [361]
Thermal gelation + compression moudling; smooth composite film with pea [372]
1
Biocompatibility is defined as a quantitative or qualitative demonstration of cell viability by a conventional biochemical or microscopic method (e.g. redox assay or
calcein AM/propidium iodide labelling) over at least two time points (e.g. 1 and 3 days) after the seeding of any cell type in the fabricated biopolymer scaffold in either
two- or three-dimensions. As such, studies that perform indirect cytotoxicity tests involving the exposure of cells to culture medium supernatants from prior incubation
with the biopolymer scaffold are excluded.
2
A minimal tissue engineering application of a biopolymer scaffold involves the quantitative or qualitative demonstration of at least one relevant differentiated pheno-
type (e.g. biochemical assays, immunohistochemical labelling, or expression of genes or proteins associated with the differentiated phenotype) that typically characterizes
functional cells from the tissue of interest.

8
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
Fig. 2. Bioreactors for cultured meat production. (A) A generic seed train process for expansion of precursor muscle or adipose cells. (B, C) Types of suspension and
perfusion bioreactors that are potentially suitable for scalable cell expansion and cell differentiation.
riers [131], as the enhanced protein adsorption conferred by the
hydrophilicity of the negatively charged Synthemax microcarriers
outweighed the adverse effect of its net negative charge on cell at-
tachment [132].
Cell adhesion moieties presented at optimal densities signal
cell survival and proliferation after initial attachment, and are
typically introduced by physically coating or covalently modify-
ing the microcarrier surface or biomaterial scaffold with ECM pro-
teins (e.g. collagen, fibronectin) [126], or cell adhesion peptides
(e.g. fibronectin-derived RGD, laminin-derived YIGSR) [133]. If re-
combinant proteins or synthesized peptides prove too costly for
cultured meat production, empirical screening of natural products
could reveal cheaper alternative sources of cell-adhesive proteins
[134–136]. Whole proteins presenting cell adhesion domains may
also be incorporated into composite scaffolds [137].
Scaffold stiffness influences cell adhesion and proliferation in
multiple cell types [138–141], and these cell type-specific re-
sponses may correlate with the rigidity of the in vivo cellular envi-
ronment [142,143]. For example, primary murine myosatellite cells
9
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
proliferated faster on hydrogels with an elastic modulus (E) of
12 kPa, which mimic that of natural muscle, than on much stiffer
tissue-culture polystyrene [144]. Similarly, primary or immortal-
ized murine myoblasts exhibited proliferation rates that increased
with substrate stiffness and plateaued above E = 12 kPa [104,145],
whereas increased muscle stiffness in aged muscle impaired en-
dogenous myoblast proliferation [146]. In contrast to the well doc-
umented increase in proliferation of BM-MSCs on stiffer substrates
[147–149], the relationship between substrate stiffness and ADSC
proliferation is currently less firmly established [105,150,151].
4.2. Conventional biomaterial design strategies for cell
differentiation
4.2.1. Bioreactor considerations
Myogenic differentiation of myoblasts/satellite cells is presum-
ably more challenging to scale up than adipogenic differentiation
of preadipocytes/ADSCs, because of a greater need to accommodate
intra-scaffold cellular self-assembly and provide dynamic differen-
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
tiation stimuli in the former, whereas the latter may only require a
switch in culture medium and static scaffold properties. The type
of cultured meat product is an important consideration when de-
signing biomaterial scaffolds for the cell differentiation stage, as
the required degree of native muscle tissue mimicry depends on
the structural complexity of the final product [6].
For unstructured meat applications that do not preserve tissue-
level structure [6], replicating muscle tissue structure is unneces-
sary. Cell differentiation could occur on edible microscale scaffolds
(μm – mm scale), which remain compatible with any suspension
bioreactor type used for cell expansion (Fig. 2B). However, this ap-
proach requires post-culture assembly of the microscale cultured
meat units into a macroscale product, perhaps leveraging conven-
tional meat structuring processes (see Section 3) or newer additive
manufacturing technologies [84] and conventional food biopoly-
mers to substitute for meat texture and protein content [52,53].
For structured meat applications, differentiation should ideally
occur in edible scaffolds with final macroscale dimensions (cm –
dm scale) that match the desired product (e.g. steaks or slices) to
eliminate the need for secondary assembly of microscale cultured
meat units. Both fixed bed bioreactors and HFBs can conceivably
generate monolithic cultured tissue constructs with the requisite
macroscale dimensions and are scalable via bioreactor paralleliza-
tion (Fig. 2C) [1]. However, this necessitates the development of
non-trivial methods for scaffold incorporation into the bioreactor,
cell seeding, and harvesting of cultured tissues while minimizing
handling and retaining product integrity and sterility [1,152].
Fixed bed bioreactors are potentially feasible for scaffolds with
sufficiently large pores that balance the need to support high cell
densities, which requires sufficiently high perfusion rates for ef-
ficient intra-scaffold fluid transport, and maintain cell viability,
which requires minimizing fluid shear stresses [1,110,153]. This
may limit the application of nanoporous hydrogels previously de-
veloped for myogenesis or adipogenesis [154] due to the high
shear stresses needed to ensure adequate perfusion, which could
limit cell viability [110,153]. Current fixed bed bioreactors accom-
modate porous scaffolds with sizes ranging from millimetre to
decimetre dimensions, but generally utilize inedible or animal-
derived scaffold materials such as glass, collagen and synthetic
polymers (e.g. polyesters and polystyrene) [152,155,156]. While
millimetre-sized scaffolds are limited to unstructured meat appli-
cations, centimetre to decimetre-sized scaffolds may be suitable for
structured meat applications. The commercial availability of single-
use fixed bed bioreactors that are pre-loaded with centimetre-
sized polyester carriers, such as iCELLis R
[155], supports the fea-
sibility of developing similar bioreactors that accommodate edible,
porous macro-scaffolds with centimetre to decimetre dimensions
for structured meat applications. Well-defined co-cultures of mul-
tiple cell types offer a potential strategy to represent both mus-
cle and adipose cells in future structured meat products produced
by fixed bed bioreactors, as exemplified by the self-organization of
tri-cultures comprising myoblasts, fibroblasts and endothelial cells
into engineered vascularized muscle [157], and the recent gener-
ation of engineered bovine muscle for cultured meat applications
using a similar approach [58].
HFBs may overcome the limited perfusion rates of fixed bed
bioreactors by separating the media and cellular compartments,
thereby protecting cells from shear stress, and the fibres may con-
veniently provide topographical cues for myoblast alignment [121].
Existing HFBs are often designed for the production of secreted
proteins and viruses, and the entrapped cells are not the de-
sired end-product [121]. As such, current hollow fibres generally
comprise inedible synthetic polymers (e.g. polysulfones, polyethy-
lene, polyesters), with the exception of potentially edible cellulose
derivatives [158,159]. Wet spinning of hollow fibres, a common
fabrication method, could be adapted to edible materials such as
10
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
alginate, starch, recombinant gelatine or soy protein [93,94,160–
163], in conjunction with shear patterning to introduce micro-
striations that further guide cell alignment [164,165], and biofunc-
tionalisation strategies for cell adhesiveness. Given that the perfu-
sion mode in HFBs can be intracapillary (intra-fibre) or extracap-
illary (between fibres) [121], direct fabrication of solid or tubu-
lar cell-encapsulated microgel fibres via a single wet spinning step
prior to HFB incorporation is conceivable [166–170]. Such microfi-
bre cell encapsulation approaches could eliminate potential chal-
lenges in uniformly filling pre-fabricated hollow fibres with a cell-
encapsulated hydrogel, and offer a facile way to represent both
muscle and adipose cells in the same HFB construct by combining
microfibres encapsulating different precursor cell types. The extra-
capillary space could be filled with an edible hydrogel to obtain a
monolithic construct suitable for structured meat applications, and
could also encapsulate cells.
4.2.2. Scaffold design principles
Myoblast alignment is a crucial step that precedes myotube for-
mation [101]. The provision of cellular alignment cues could occur
via the use of aligned fibrous scaffolds [57,169,171–175], microto-
pographically patterned surfaces [164,165,173,176–181], anisotropic
or directional pore formation [182,183], biochemically or mechan-
ically patterned materials [142,184], naturally aligned, decellular-
ized plant-derived scaffolds [185] or dynamic mechanical stimula-
tion [186–190]. However, not all cell alignment strategies are in-
dustrially scalable, and those that are implementable in macrop-
orous or fibrous formats for fixed bed bioreactors or HFBs, respec-
tively, are likely to be advantageous.
The static mechanical environment, in particular substrate
stiffness, influences both myogenic and adipogenic differentia-
tion efficiency [104,142,145,191–197]. Mimicking the stiffness of
fat tissue (E ≈ 1 – 3 kPa) [76,198,199] consistently enhances ADSC
adipogenesis in vitro [194–197,200], highlighting the relevance
of modulating scaffold stiffness for optimizing adipogenesis of
bovine adipose precursors. In contrast, myotube formation oc-
curs over a broad range of substrate rigidity [104,142,145,193],
and late-stage myotube maturation in the form of actomyosin
striations exhibits a context-dependant relationship with stiff-
ness that may involve other biochemical, topographical and cel-
lular variables [104,142,193]. For example, when cultured on
polyacrylamide gels presenting anisotropic collagen micropat-
terns, C2C12 myoblasts exhibited maximal striation at moderate
gel stiffnesses matching native mouse muscle (E ≈ 12 kPa) [142].
In contrast, culturing C2C12 myoblasts on unpatterned poly-l-
lysine/hyaluronic acid polyelectrolyte films or primary myoblasts
on entactin/collagen/laminin-coated polyacrylamide gels monoton-
ically increased myotube striation with increasing stiffness, with
maximal striation on the stiffest control substrates tested [104,193].
Therefore, the feasibility of optimizing bovine myogenic differenti-
ation via scaffold stiffness modulation is currently unclear and re-
quires further investigation. Importantly, increasing scaffold stiff-
ness often requires increasing material concentration or crosslink-
ing densities, which concomitantly decreases pore size, and may
limit maximal perfusion rates and therefore engineered tissue
thickness in the context of fixed bed bioreactor-based production.
Encapsulating myoblasts in a hydrogel between two anchoring
posts and the subsequent passive development of static uniaxial
tension is a well-established method to generate relatively mature
myofibers of macroscopic dimensions in vitro [101,201–204], and
its limited industrial scalability may be circumvented by utilizing
appropriate scaffold rigidity as a source of cellular tension [205].
Increased protein production due to static tension [204] increases
levels of texture-contributing actin/myosin proteins, as well as
flavour- and colour-contributing myoglobin protein in meat [206].
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
In addition to static tension, dynamic mechanical stimuli
further increases myogenic maturation [187–190,207–209], but
scaling dynamic mechanical stimuli industrially is non-trivial
and would require substantial bioreactor development [210,211].
Emerging advanced methods that remotely actuate intra-scaffold
mechanical stimuli could offer potential pathways towards large-
scale mechanical stimulation of myogenesis, and include the use
of moderate fluid shear stress [212,213], light-responsive nanopar-
ticle actuators of cell adhesion receptors [214], or magnetically ac-
tuated biphasic ferrogels that undergo large, fatigue-resistant de-
formations [215].
Exogenous electrical stimuli also promote various facets of my-
otube maturation, including cross-striation, MHC isoform expres-
sion, myofibrillar protein level, and contractility [216–222], but
also require substantial bioreactor development to ensure indus-
trial scalability [210,211,223]. A unique approach to introduce scal-
able electrical stimuli is by using electrically conductive bioma-
terials such as polyaniline, polypyrrole and polythiophenes [224],
which increase myogenesis when incorporated into scaffolds, even
without exogenous electrical stimulation [225–228]. Such conduc-
tive materials may synergize with other differentiation cues, such
as cell alignment, to further promote myogenesis [229,230], but
their suitability for human consumption is uncertain.
4.3. Novel constraints on biomaterial design for cultured meat
production
4.3.1. Scaffold crosslinking and fabrication
Biomaterial scaffolds may be fabricated in various morpholo-
gies using physical or chemical crosslinking approaches. Natural
biopolymers often exhibit intrinsic gelation via physical crosslink-
ing, most commonly thermal (collagen, gelatine, agar) or ionic
(alginate), and may naturally undergo enzyme-mediated covalent
crosslinking (thrombin/fibrin) [231–233]. In order to improve their
gelation kinetics and mechanical properties, biopolymers are of-
ten conjugated with reactive side-groups that enable chemical
crosslinking, such as acrylates, thiols or phenols [231–233], some
of which undergo biocompatible crosslinking reactions that sup-
port viable cell encapsulation during scaffold fabrication. These
include small molecule-based reactions such as azide-alkyne cy-
cloaddition, Michael-type addition, Schiff-base formation, photo-
initiated free radical reactions, and enzymatic crosslinking reac-
tions such as phenol-phenol (peroxidase), phenol-amine (tyrosi-
nase) and amine-amine (lysyl oxidase) reactions [231,232].
For cultured meat production, precursor cells will likely be
seeded on pre-fabricated microcarriers for expansion and poten-
tially for differentiation (for unstructured meat). In principle, ei-
ther physical or chemical crosslinking approaches may be appli-
cable, as long as the microcarriers fulfil critical design elements
that support cell expansion or differentiation. Certain physically
crosslinked biomaterials that intrinsically lack mechanical stabil-
ity under dynamic culture conditions (e.g. collagen, fibrin) may
be unsuitable without secondary chemical crosslinking. Increasing
awareness that physically crosslinked hydrogels exhibit viscoelastic
properties (e.g. stress relaxation), whereas chemically crosslinked
hydrogels are purely elastic [234], coupled with knowledge of
the viscoelastic nature of native muscle and fat [234–236], has
helped to elucidate the independent effects of scaffold viscoelas-
ticity and elasticity (stiffness) on cell behaviour. For example, my-
oblasts exhibit increased cell spreading and proliferation on vis-
coelastic compared to elastic hydrogels of the same mechanical
stiffness [236,237], which suggests a potential role of scaffold vis-
coelasticity in cell expansion optimization.
As discussed earlier, cell differentiation would likely require
the use of porous scaffolds or microfibres that are loaded post-
fabrication into fixed bed or hollow fibre bioreactors respectively
11
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
[110,121,154]. Cells could conceivably be encapsulated either dur-
ing scaffold fabrication or after fabrication, given that biocompati-
ble crosslinking and fabrication strategies exist for both porous and
fibrous morphologies [238,239]. The choice of physical or cova-
lent crosslinking approaches should be informed by whether clas-
sical scaffold design criteria for cell differentiation are fulfilled,
and may require further studies on how their resultant viscoelas-
tic properties influence cell differentiation and post-culture proper-
ties. For example, osteogenic differentiation of MSCs and chondro-
cyte production of cartilage matrix were both accelerated in vis-
coelastic compared with elastic alginate hydrogels [240,241], but
the contribution of viscoelasticity to myogenesis and adipogene-
sis is currently unknown. The texture of cultured meat and its
stability during storage and cooking are two post-culture prop-
erties that could be affected in opposite ways by scaffold vis-
coelasticity. To illustrate this, gelling agents used in meat prod-
ucts that undergo cooling-induced sol-gel transitions may lose
structural integrity and exhibit substantial water loss during cook-
ing, which could be overcome by transglutaminase-mediated cova-
lent crosslinking between meat proteins. However, the viscoelas-
tic properties of animal-based meat [242,243] suggests that there
may be a threshold degree of covalent crosslinking in meat prod-
ucts beyond which the increased elasticity may noticeably affect
meat texture, as exemplified by increased hardness and chewiness
exhibited by transglutaminase-crosslinked restructured meat prod-
ucts [244–246]. Given the potentially complex effects of scaffold
viscoelasticity on cultured meat properties, holistic characteriza-
tion of its effects will be required.
4.3.2. Scaffold degradability
Expanded cells would likely require multiple rounds of passag-
ing before final re-seeding in a different type of scaffold and biore-
actor for cell differentiation [109]. In this scenario, expanded cells
have to be efficiently harvested from the scaffold, which could be
non-degradable or degradable.
Harvesting cells from non-degradable scaffolds would re-
quire efficient cellular dissociation and post-dissociation separa-
tion strategies. Dissociation methods could include recombinant
enzymes [247,248], chemical mimetics of enzymes [123], mechan-
ical agitation [249–251], and thermo-responsive polymer coatings
based on poly(N-isopropylacrylamide) (pNIPAAm), methylcellulose,
hydroxybutyl chitosan and xyloglucan [252–256]. Separation could
occur via filtration, centrifugal, inertial, or magnetic methods [257–
260], as long as the scaffold exhibits sufficiently different size, den-
sity or magnetism compared with the harvested cells. However,
the accumulated inefficiency of both dissociation and separation
steps, together with a desire for high cell purity to minimize the
re-seeding of the non-degradable material in the subsequent differ-
entiation bioreactor, could result in significant cell loss [123,257].
The use of degradable scaffolds could simplify cell harvesting by
eliminating the need for dissociation and separation, and such ma-
terials may degrade hydrolytically, chemically, enzymatically, pho-
tolytically, or in response to pH or temperature changes [123].
However, most biomaterials that undergo pH-dependant, thermal
or photo-degradation are unlikely to be cell-compatible, whereas
those that undergo hydrolysis, such as poly(lactic-co-glycolic acid)
(PLGA) and polycaprolactone (PCL), may release cells prematurely.
Therefore, only chemical, enzymatic and pH-dependant degrada-
tion remain viable options that are potentially fast, robust and
stimulus-specific. Chemically degradable biomaterials include ion-
ically crosslinked polygalacturonic acid (PGA) and alginate, which
degrade in response to sequestrants of bivalent cations such as
EDTA, citrate or phosphate. Enzymatically degradable biomateri-
als may include various polysaccharides, such as dextran, alginate,
pectin and cellulose, and any non-animal-derived protein for which
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
Fig. 3. Integrating biomaterials and food biopolymers for cultured meat production. This schematic provides a side-by-side comparison of conventional and cultured
meat production, and highlights the many steps other than the initial engineering of cultured muscle or adipose tissue in vitro that are involved in determining meat quality
attributes. Conventional food biopolymers may contribute to future cultured meat production by providing edible, sustainable and cost-efficient tissue engineering scaffold
materials, and supporting post-culture assembly and processing of cultured meat products.
recombinant enzymes that function under cell-compatible condi-
tions are available [123].
The requirement for degradability of the differentiation scaffold
depends on a balance between the need to accommodate cellu-
lar remodelling during differentiation and the potential organolep-
tic or nutritional roles played by the scaffold in the final meat
product. Differentiation scaffolds that comprise biopolymer-based
meat ingredients could conceivably fulfil different functions dur-
ing culture and post-culture, which could make degradability op-
tional. For example, wet-spun fibrous scaffolds comprising edible
hydrocolloids may be designed to provide alignment and mechani-
cal cues that support myogenesis during culture, and may also con-
tribute post-culture to structural and storage stability, and mod-
ulation of texture and flavour, in the final cultured meat prod-
uct via their gelation and water retention behaviour (see Table 1,
Sections 3.1, 3.2). From a nutritional perspective, scaffolds con-
taining indigestible polysaccharides such as cellulose may supple-
ment dietary fibre, which is often underrepresented in modern di-
ets [261], whereas those containing residual protein content post-
culture could supplement dietary protein.
Scaffolds could even be molecularly designed to degrade into
molecules with positive organoleptic or nutritional properties
[154], such as flavour-generating aromatic compounds or essential
vitamins [65]. One possibility is the degradation-dependant release
of specific fatty acids (e.g. omega-3) that act as maturation factors
during adipogenesis [262] and contribute flavour, mouthfeel and
nutritional benefits post-culture.
4.3.3. Culture medium cost mitigation
Culture medium is predicted to account for 55 – 95% of the
marginal cost of industrial cultured meat production [109,263] due
to the need for chemically defined, animal-free medium, which
requires the replacement of bovine sera with relatively expen-
sive recombinant growth factors. A recent analysis estimated the
marginal cost of culture medium alone for cultured meat produc-
tion to range between $2100 – $15,0 0 0/kg cells produced at cur-
rent medium component prices, in contrast to wholesale prices of
conventional animal-based meat ($3.3 – $4.4/kg) [263]. Through
reductions in the cost of both growth factors and basal medium
via incremental methods that do not rely on fundamental new
technologies or innovations, the same analysis predicted that it is
likely that cultivated meat can achieve price parity with conven-
tional meat once produced at industrial scale [263].
12
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
Possible strategies to mitigate this significant cost include: (1)
reducing growth factor requirements by cell line adaptation, en-
gineering growth factors with improved stability or binding affin-
ity, or substituting growth factors with small molecule mimetics
or plant/microbial hydrolysates, (2) reducing growth factor cost
by producing them at industrial scale (e.g. for industrial enzymes
like lipase and cellulase), (3) reducing basal medium cost by using
food-grade rather than pharmaceutical-grade ingredients, or using
cheaper antioxidants or buffer systems [109,263]. However, these
approaches do not address the fundamental inefficiency that arises
from the provision of soluble growth factors at supraphysiological
bulk concentrations in order to ensure sufficient concentrations at
the cellular level [264].
Engineered biomaterials may increase the efficiency of exoge-
nous growth factor utilization by localizing their sustained release
from the scaffold [264,265], or by controlling their spatial pre-
sentation with matrix-bound forms [264], which may exhibit pro-
longed biological activity [266] or enable multivalent interactions
that strengthen signal transduction [267]. Alternatively, biomate-
rials could mimic the non-covalent sequestration of endogenously
secreted growth factors by ECM via the incorporation of peptides
that mimic the binding domain of growth factor receptors or ap-
tamers that specifically bind to growth factors [264,268], thereby
amplifying myogenic or adipogenic autocrine signals [269–271].
Biomaterials could even directly incorporate growth factor mimet-
ics based on peptides, aptamers or small molecules as part of the
scaffold backbone [264]. While sustained local delivery approaches
could be conceivably adapted to various food-grade biopolymers
like lipids and hydrocolloids in various micro- or nano-scale forms
with existing encapsulation technologies [272,273], engineered ma-
terials that present new chemical entities such as molecular se-
questrants or growth factor mimetics would require more exten-
sive food safety evaluation.
4.3.4. Scaffold edibility, sustainability and cost
In principle, scaffolds for cell expansion could be either inedible
or edible. In the likely scenario where expanded cells are harvested
for re-seeding in a differentiation scaffold, the use of an edible
scaffold could increase the tolerance level for remaining quantities
of the scaffold in the harvested cells, thus allowing for increased
harvesting yields [123]. However, if a single scaffold is designed
to support both cell expansion and subsequent cell differentiation
into the final product, scaffold edibility may become indispensable
[123]. The scaffold used for cell differentiation must be edible as
JID: ACTBIO
ARTICLE IN PRESS [m5G;February 21, 2021;17:50]

S. Ng and M. Kurisawa Acta Biomaterialia xxx (xxxx) xxx

the final engineered muscle and fat tissues are likely to possess
intrinsically complex tissue-level structures that may be practically However, the marginal cost of microcarriers is indepen-
inseparable from the remaining portion of their original scaffold. dent of bioreactor type and size as long as microcarrier con-
Amongst current biomaterial scaffolds that support myo- centrations remain within reasonable limits compatible with
genic and adipogenic differentiation, including collagen or gela- the maintenance of cell viability, in contrast to the marginal
tine [202,203,274,275], hyaluronic acid (HA) [276–280], fibrin cost of culture medium, which varies with the maximum cell
density achievable in culture and is therefore dependant on
[281–283], alginate [278,284–287], chitosan [288], PEG [289,290],
bioreactor type.
PLGA [205,278,291,292], PCL [289,293] and decellularized tissues In the current analysis, we compare microporous Cytodex
[107,185,294–296], only alginate, gelatine and PEG are routinely 1/3 and macroporous Cytopore 2 microcarriers, and assumes
used in food applications. The use of decellularized tissues will a confluent cell density of 1 × 105 cells/cm2 [302] and a
probably be limited to plant or fungal sources and may face chal- generic cell mass of 3.4 × 10−12 kg/cell [109,111]. Microcarrier
lenges in both scalability and cost-efficiency. Although synthetic cost estimates are from the current retail prices of the largest
polymers like PEG, PCL and PLGA are approved for clinical appli- unit size of each microcarrier type at Cytiva Life Sciences
cations, such as drug delivery, sutures, and dermal fillers, and are (November 2020).
relatively cheaply produced, it is currently unclear if the larger
Cytodex 1 Cytodex 3 Cytopore 2
amounts of such polymers that may be regularly consumed as
part of cultured meat products will be considered food-safe. Al- Microcarrier cost ($/g 4.88 5.24 31.5
microcarrier)
though naturally derived biomaterials like HA, fibrin and chitosan
Microcarrier growth area 4400 2700 11,000
are likely also safe for consumption, they are conventionally de- (cm2 /g microcarrier)
rived from animal sources, which limits their use in cultured meat [302]
production to recombinant versions from transgenic plant, fun- Maximum cell productivity 4.4 × 108 2.7 × 108 1.1 × 109
(cells/g microcarrier)
gal or microbial sources [297–301]. Such recombinant sources are
Maximum cell mass 1.50 0.92 3.74
likely to remain prohibitively costly for cultured meat production productivity (kg cells/kg
until true economies of scale are realized with future increases in microcarrier)
demand. For example, microbial-derived HA is currently available Marginal microcarrier cost 3262 5708 8422
at a food-grade scale at $130/kg, in contrast to food-grade alginate, ($/kg cells produced)

which has a wholesale price of $10/kg. Assuming that every ex-

panded precursor cell is arbitrarily seeded at twice its own volume B) Scaffolds for differentiation:
in a 10 mg/mL (1% w/v) differentiation scaffold within a perfusion As scaffolds for differentiation may come in various forms
(macroporous hydrogels, microcarriers, fibres), this analysis
bioreactor, the marginal cost of raw materials for an alginate- or
assumes the use of a volume-averaged scaffold material con-
HA-based differentiation scaffold would be $0.23/kg or $2.95/kg of
centration of 10 mg/mL (1% w/v). We also arbitrarily assume
cells produced respectively (Box 2). Given that many food biopoly- that expanded precursor cells are re-seeded in a perfusion
mers used in conventional meat production (Table 1) are available bioreactor at twice the total volume of cells, to cater for the
at industrial scale and at prices that are competitive with alginate void space needed for the scaffold itself and its perfusion
(Box 2), the application of these biopolymers as potential differ- with culture medium [263]. Given an average meat density
entiation scaffold materials (see Section 4.4) could conceivably en- of 881 kg/m3 [263], which results in a volume of 1135 mL/kg
sure that the marginal cost of such scaffolds remain within a frac- of cells, the total mass of scaffold material required per kilo-
tion of the wholesale price of conventional meat. gram of cells seeded is:
In contrast to scaffolds for myogenic and adipogenic differentia- mL kg scaffold
tion, microcarriers for adherent cell expansion are widely available 1, 135 x 2 x 10−5
kg cells mL
commercially, and comprise a variety of materials such as dextran
= 0.023kg scaffold/kg cells
(Cytodex 1/3), gelatine (Cultispher), cellulose (Cytopore), polyethy-
lene (Cytoline), polystyrene (Synthemax II, SoloHill, Hillex), most Cost estimates for potential scaffold raw materials are de-
of which are either inedible or animal-derived. In addition to the rived from the median lower-bound prices of food-grade ma-
cell density at confluence, which is dependant on cell type, the terial, at the kilogram scale, from multiple suppliers at Al-
ibaba (November 2020). Importantly, this cost analysis con-
marginal cost of microcarriers for cell expansion also depends on
siders only the raw material cost and excludes processing
both the cost and the growth area per mass of microcarriers. As-
costs, such as those arising from chemical modification to in-
suming a cell density of 1 × 105 cells/cm2 at confluence [302], the troduce necessary biological functions (e.g. cell adhesiveness
marginal cost of microcarriers would be a prohibitively expensive or chemical crosslinking), or scaffold fabrication.
$320 0 – $840 0/kg of cells produced, at current retail prices (Box
2). The application of existing food biopolymers as materials for Food-grade material Marginal scaffold
cost ($/kg) cost ($/kg cells)
microcarrier fabrication, such as gellan gum and zein [303,304],
could potentially mitigate microcarrier costs and ensure microcar- Alginate 10 0.23
rier edibility. HA (recombinant, 130 2.95
microbial)
Carrageenan 5 0.11
Box 2: Marginal cost analysis of scaffolds for cultured meat produc- Pectin 11.5 0.26
tion Gellan gum 19 0.43
Xanthan gum 2 0.05
Cellulose 3 0.07
A) Microcarriers for cell expansion: Starch 0.5 0.01
The marginal cost of microcarriers for cell expansion is Soy isolate 2 0.05
dependant on: Zein (corn) 2 0.05
(1) Cell size, which is cell type-dependant and determines
the maximum achievable cell density at confluence,
(2) Microcarrier cost per mass, and
(3) Microcarrier growth area per mass

13
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
4.4. Repurposing food biopolymers for cultured meat production
As discussed in Section 3, a wide range of edible biopoly-
mers, from polysaccharides, proteins, lipids to synthetics have had
long-standing applications as added ingredients in meat prod-
ucts, but amongst these, only alginate and gelatine are widely
applied as tissue engineering scaffolds [305,306], and even so,
only recently have bioengineers started to apply them expressly
for cultured meat applications [57,284]. Due to an increasing in-
terest in more sustainable and low-cost sources of tissue engi-
neering scaffolds, biomaterials engineers have started to fabri-
cate ECM-mimetic scaffolds with bioreactor-compatible morpholo-
gies (porous hydrogels, fibres, microcarriers) using other edible
biopolymers and evaluating their biocompatibility, including car-
rageenan [307–317], pectin [318–326], cellulose [158,159,178,327–
334], guar gum [335–338], gellan gum [304,339–348], xanthan
gum [346,349–354], konjac [355–360], protein isolates from soy or
corn (zein) [58,134,332,334,361–372], and starches [163,358,373–
378] (Table 3).
Many of these edible biopolymers are useful as biomaterials
due to their high water binding capacities, which enable them
to recapitulate the highly hydrated state of native ECM, or their
gelation behaviour, which could supplement the mechanical prop-
erties of softer materials [307,310,344,351,379]. However, biopoly-
mer composites and chemical modifications that enable covalent
crosslinking are often employed to enable gelation of non-gelling
agents, overcome harsh physical gelation conditions that are not
cytocompatible, and improve the range and control of gel physico-
chemical properties [158,311,380,381]. For example, unmodified κ -
carrageenan undergoes potassium-dependant ionic gelation upon
cooling from its heat-solubilized form, but starts gelling at 50 °C
to form a brittle, thermoreversible hydrogel that exhibits a non-
linear variation of mechanical stiffness with cooling tempera-
ture, and dissociates in physiological buffers [382]. In contrast,
methacrylated κ -carrageenan forms photopolymerized, covalently
crosslinked gels that are thermally and mechanically stable in
physiological buffers, and retain the viability of encapsulated cells
[308]. Other gelling biopolymers that undergo similar cooling-
dependant gelation (agar, gellan gum and gelatine) or conversely
require high temperatures to induce thermal gelation driven by hy-
drophobic aggregation (konjac, methycellulose, soy and pea protein
isolates), are also unable to support cell encapsulation and offer
limited control over hydrogel properties unless modified to sup-
port more biocompatible gelation mechanisms.
The general lack of cell adhesiveness amongst edible biopoly-
mers, particularly polysaccharides, also requires direct chemical
modification with cell adhesion moieties or the development
of composites that incorporate a cell-adhesive protein [158]. An
example of industrial effort in this direction is the development
of pectin-thiopropionylamide conjugates by Modern Meadows,
which forms a covalently crosslinked hydrogel with thiol-modified
proteins that naturally contain the RGD adhesive peptide domain,
such as cardosin at a cytocompatible pH [137], in contrast with
the non-cytocompatible acidic and high sugar conditions required
to form native pectin physical gels [52]. amongst the above
biocompatible and edible biopolymers, carrageenan [314,315],
pectin [318,320,322,325], cellulose [178,329–331,334], gellan gum
[340–346], konjac [357], starch [163,373–377], soy protein isolate
[58,334,365] and corn-derived zein protein [362,364,383] have
demonstrated feasibility as non-composite or composite scaf-
folds for various engineered tissues [304,314,318,320,322,329–
331,334,340–343,345,346,357,364,371], although to date, only
pectin, cellulose, gellan gum and soy protein isolate have been
investigated for their suitability in generating engineered muscle
tissue [58,137,178,342] (Table 3).
14
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
The relative novelty of the cultured meat field, and the concen-
tration of cultured meat research in industrial start-ups as com-
pared with academia, are two reasons for the current dearth of
data on the meat quality attributes of cultured meat prototypes.
To date, there are only two studies of engineered bovine muscle
tissue and one of engineered bovine adipose tissue for cultured
meat applications [57,58,284]. Importantly, in addition to charac-
terizing the phenotype of bovine cells during culture, these stud-
ies utilized potentially scalable scaffold fabrication methods to fab-
ricate macroscale cultured meat prototypes. Moreover, with these
macroscale prototypes, they demonstrated the application of con-
ventional methods for meat quality evaluation, such as textural
profile analysis and human sensory panels, thereby paving the way
for future integrative studies at the interface of tissue engineering
and meat science.
5. Outlook and conclusion
Cultured meat production, while conceptually attractive, faces
fundamental challenges in technical feasibility and industrial scal-
ability. Due to additional novel constraints on scaffold edibility,
sustainability and cost-efficiency, it is unlikely that a piecemeal
merger of the meat science and tissue engineering fields, where
tissue engineers produce live muscle tissue and meat scientists
characterize the post-mortem tissue via their respective conven-
tional methods, will suffice to solve these non-trivial challenges.
Rather, bridging conceptual and knowledge divides between the
historically disparate fields of meat science and biomaterials engi-
neering may help to illuminate novel strategies to address some of
these challenges (Fig. 3). A holistic consideration of the constraints
discussed in this review with an integrated perspective into bio-
materials and meat science earlier on in the cultured meat de-
sign process could accelerate successful incorporation into existing
meat manufacturing pipelines and provide an advantage in even-
tual market adoption.
Declaration of Competing Interest
There is no conflict of interest and disclosure associated with
the manuscript.
Acknowledgements
This work was funded by the Institute of Bioengineering and
Nanotechnology, Biomedical Research Council, Agency for Science,
Technology and Research (A∗ STAR), Singapore.
References
[1] E.A. Specht, D.R. Welch, E.M. Rees Clayton, C.D. Lagally, Opportunities for
applying biomedical production and manufacturing methods to the devel-
opment of the clean meat industry, Biochem. Eng. J. 132 (2018) 161–168,
doi:10.1016/j.bej.2018.01.015.
[2] N. Stephens, L. Di Silvio, I. Dunsford, M. Ellis, A. Glencross, A. Sexton, Bringing
cultured meat to market: technical, socio-political, and regulatory challenges
in cellular agriculture, Trends Food Sci. Technol. (2018), doi:10.1016/j.tifs.2018.
04.010.
[3] N. Stephens, A.E. Sexton, C. Driessen, Making Sense of Making Meat: key Mo-
ments in the First 20 Years of Tissue Engineering Muscle to Make Food, Front.
Sustain. Food Syst. (2019), doi:10.3389/fsufs.2019.0 0 045.
[4] S. Chriki, J.F. Hocquette, The Myth of Cultured Meat: a Review, Front. Nutr. 7
(2020) 1–9, doi:10.3389/fnut.2020.0 0 0 07.
[5] W. Verbeke, A. Marcu, P. Rutsaert, R. Gaspar, B. Seibt, D. Fletcher, J. Barnett,
Would you eat cultured meat?”: consumers’ reactions and attitude formation
in Belgium, Portugal and the United Kingdom, Meat Sci (2015), doi:10.1016/j.
meatsci.2014.11.013.
[6] I. Fraeye, M. Kratka, H. Vandenburgh, L. Thorrez, Sensorial and Nutritional
Aspects of Cultured Meat in Comparison to Traditional Meat: much to Be In-
ferred, Front. Nutr. 7 (2020), doi:10.3389/fnut.2020.0 0 035.
[7] S. Glorieux, L. Steen, D. Van de Walle, K. Dewettinck, I. Foubert, I. Fraeye, Ef-
fect of Meat Type, Animal Fat Type, and Cooking Temperature on Microstruc-
tural and Macroscopic Properties of Cooked Sausages, Food Bioprocess Tech-
nol. (2019), doi:10.1007/s11947- 018- 2190- 6.
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
[8] G. Feiner, Meat Products Handbook, Practical Science and Technology, Wood-
head, 2006.
[9] M.V. Dodson, R.E. Allen, M. Du, W.G. Bergen, S.G. Velleman, S.P. Poulos,
M. Fernyhough-Culver, M.B. Wheeler, S.K. Duckett, M.R.I. Young, B.H. Voy,
Evolution of meat animal growth research during the past 50 years, J. Anim.
Sci. 93 (2015) 457–481, doi:10.2527/jas2014-8221.
[10] S. Chriki, B. Picard, Y. Faulconnier, D. Micol, J.P. Brun, M. Reichstadt, C. Jurie,
D. Durand, G. Renand, L. Journaux, J.F. Hocquette, A data warehouse of muscle
characteristics and beef quality in France and a demonstration of potential
applications, Ital. J. Anim. Sci. (2013), doi:10.4081/ijas.2013.e41.
[11] Y.H. Hui, J.L. Aalhus, L. Cocolin, I. Guerrero-Legarreta, L.M. Nollet, R.W. Pur-
chas, M.W. Schilling, P. Stanfield, Y.L. Xiong, Handbook of Meat and Meat Pro-
cessing, 2nd Edition, 2012.
[12] K.L. Pearce, K. Rosenvold, H.J. Andersen, D.L. Hopkins, Water distribution and
mobility in meat during the conversion of muscle to meat and ageing and the
impacts on fresh meat quality attributes - A review, Meat Sci (2011), doi:10.
1016/j.meatsci.2011.04.007.
[13] S.K. Matarneh, E.M. England, T.L. Scheffler, D.E. Gerrard, The Conver-
sion of Muscle to Meat, Lawrie’s Meat Sci., 8th Ed., 2017, doi:10.1016/
B978- 0- 08- 100694- 8.00005- 4.
[14] K.G. Grunert, L. Bredahl, K. Brunsø, Consumer perception of meat quality and
implications for product development in the meat sector - A review, Meat
Sci. (2004), doi:10.1016/S0309-1740(03)00130-X.
[15] M. Rødbotten, E. Kubberød, P. Lea, Ø. Ueland, A sensory map of the meat
universe. Sensory profile of meat from 15 species, Meat Sci. (2004), doi:10.
1016/j.meatsci.2004.02.016.
[16] G. Offer, P. Knight, R. Jeacocke, R. Almond, T. Cousins, J. Elsey, N. Parsons,
A. Sharp, R. Starr, P. Purslow, The structural basis of the water-holding,
appearance and toughness of meat and meat products, Food Microstruct.
(1989).
[17] M.J. Post, J.F. Hocquette, New Sources of Animal Proteins, Vitro Meat, Elsevier
Ltd„ 2017, doi:10.1016/B978- 0- 08- 100593- 4/00017- 5.
[18] S.B. Kanatous, P.P.A. Mammen, P.B. Rosenberg, C.M. Martin, M.D. White,
J.M. DiMaio, G. Huang, S. Muallem, D.J. Garry, Hypoxia reprograms calcium
signaling and regulates myoglobin expression, Am. J. Physiol. - Cell Physiol.
(2009), doi:10.1152/ajpcell.00428.2008.
[19] A.E. Schlater, M.A. De Miranda, M.A. Frye, S.J. Trumble, S.B. Kanatous, Chang-
ing the paradigm for myoglobin: a novel link between lipids and Myoglobin,
J. Appl. Physiol. (2014), doi:10.1152/japplphysiol.00973.2013.
[20] R. Simsa, J. Yuen, A. Stout, N. Rubio, P. Fogelstrand, D.L. Kaplan, Extracellular
heme proteins influence bovine myosatellite cell proliferation and the color
of cell-based meat, Foods. (2019), doi:10.3390/foods8100521.
[21] Y. Jin, X. He, K. Andoh-Kumi, R.Z. Fraser, M. Lu, R.E. Goodman, Evaluating Po-
tential Risks of Food Allergy and Toxicity of Soy Leghemoglobin Expressed in
Pichia pastoris, Mol. Nutr. Food Res. (2018), doi:10.10 02/mnfr.20170 0297.
[22] M.N. Hamilton, C.E. Ewing, Food coloring composition, CA2314727C, 2005.
[23] P. Eisner, R. Osen, Extruded vegetable protein product with coloring vegetable
ingredients and method of preparation, DE102015203653A1, 2016.
[24] A.M.S. AssociationAMSA Meat Color Measurement Guidelines, AMSA, 2012.
[25] M.I. Khan, C. Jo, M.R. Tariq, Meat flavor precursors and factors influencing
flavor precursors-A systematic review, Meat Sci (2015), doi:10.1016/j.meatsci.
2015.08.002.
[26] C.R. Calkins, J.M. Hodgen, A fresh look at meat flavor, Meat Sci (2007), doi:10.
1016/j.meatsci.2007.04.016.
[27] J.D. Wood, G.R. Nute, R.I. Richardson, F.M. Whittington, O. Southwood, G. Plas-
tow, R. Mansbridge, N. Da Costa, K.C. Chang, Effects of breed, diet and muscle
on fat deposition and eating quality in pigs, Meat Sci. (2004), doi:10.1016/j.
meatsci.20 04.01.0 07.
[28] M.S. Arshad, M. Sohaib, R.S. Ahmad, M.T. Nadeem, A. Imran, M.U. Arshad,
J.H. Kwon, Z. Amjad, Ruminant meat flavor influenced by different factors
with special reference to fatty acids, Lipids Health Dis. 17 (2018) 1–13,
doi:10.1186/s12944-018-0860-z.
[29] J.D. Wood, M. Enser, A.V. Fisher, G.R. Nute, P.R. Sheard, R.I. Richardson,
S.I. Hughes, F.M. Whittington, Fat deposition, fatty acid composition and meat
quality: a review, Meat Sci. (2008), doi:10.1016/j.meatsci.2007.07.019.
[30] D.S. Mottram, Flavour formation in meat and meat products: a review, Food
Chem. (1998), doi:10.1016/S0308-8146(98)0 0 076-4.
[31] F. Shahidi, L.J. Rubin, L.A. D’Souza, Meat Flavor Volatiles: a Review of the
Composition, Techniques of Analysis, and Sensory Evaluation, Crit. Rev. Food
Sci. Nutr. (1986), doi:10.1080/10408398609527435.
[32] M. Kosowska, M.A. Majcher, T. Fortuna, Volatile compounds in meat and meat
products, Food Sci. Technol. (2017), doi:10.1590/1678-457X.08416.
[33] C. Lamb, Barcelona’s Cubiq Foods Raises €5 M to Produce Better-for-
You Cultured Fat, (2020). https://thespoon.tech/barcelonas- cubiq- foods-
raises- e5m- to- produce- better- for- you- cultured- fat/ (accessed July 27, 2020).
[34] O. Morrison, Coronavirus will boost clean meat trend, says supplier,
(2020). https://www.foodnavigator.com/Article/2020/04/16/Coronavirus-will-
boost- clean- meat- trend- says- supplier (accessed July 27, 2020).
[35] P. Wu, K. Sato, F. Suzuta, Y. Hikasa, K. Kagota, Effects of lipid-related factors
on adipocyte differentiation of bovine stromal-vascular cells in primary cul-
ture, J. Vet. Med. Sci. 62 (20 0 0) 933–939.
[36] K. Sato, N. Nakanishi, M. Mitsumoto, Culture conditions supporting adipose
conversion of stromal-vascular cells from bovine intramuscular adipose
tissues, J. Vet. Med. Sci. 58 (1996) 1073–1078 http://www.mendeley.
com/research/geology- volcanic- history-eruptive-style-yakedake-volcano-
group- central- japan/.
15
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
[37] S.B. Smith, H. Kawachi, C.B. Choi, C.W. Choi, G. Wu, J.E. Sawyer, Cellular regu-
lation of bovine intramuscular adipose tissue development and composition,
J. Anim. Sci. 87 (2009) 72–82, doi:10.2527/jas.2008-1340.
[38] A.C. Grant, G. Ortiz-Colòn, M.E. Doumit, D.D. Buskirk, Optimization of in vitro
conditions for bovine subcutaneous and intramuscular preadipocyte differen-
tiation, J. Anim. Sci. 86 (2008) 73–82, doi:10.2527/jas.2007-0379.
[39] A.J. Lengi, B.A. Corl, Factors influencing the differentiation of bovine
preadipocytes in vitro, J. Anim. Sci. 88 (2010) 1999–2008, doi:10.2527/jas.
2009-2439.
[40] K.D. Fish, N.R. Rubio, A.J. Stout, J.S.K. Yuen, D.L. Kaplan, Prospects and chal-
lenges for cell-cultured fat as a novel food ingredient, Trends Food Sci. Tech-
nol. 98 (2020) 53–67, doi:10.1016/j.tifs.2020.02.005.
[41] E.D. Rosen, O.A. MacDougald, Adipocyte differentiation from the inside out,
Nat. Rev. Mol. Cell Biol. 7 (2006) 885–896, doi:10.1038/nrm2066.
[42] American Meat Science Association (AMSA), Research Guidelines for Cook-
ery, Sensory Evaluation, and Instrumental Tenderness Measurements of Meat,
2016. http://www.meatscience.org.
[43] U. Andjelković, D. Josić, Mass spectrometry based proteomics as foodomics
tool in research and assurance of food quality and safety, Trends Food Sci.
Technol. (2018), doi:10.1016/j.tifs.2018.04.008.
[44] J.L. Damez, S. Clerjon, Meat quality assessment using biophysical methods re-
lated to meat structure, Meat Sci. (2008), doi:10.1016/j.meatsci.2008.05.039.
[45] J.X. Guinard, R. Mazzucchelli, The sensory perception of texture and mouth-
feel, Trends Food Sci. Technol. (1996), doi:10.1016/0924-2244(96)10025-X.
[46] E. Huff Lonergan, W. Zhang, S.M. Lonergan, Biochemistry of postmortem
muscle - Lessons on mechanisms of meat tenderization, Meat Sci. (2010),
doi:10.1016/j.meatsci.2010.05.004.
[47] P. Ertbjerg, E. Puolanne, Muscle structure, sarcomere length and influences on
meat quality: a review, Meat Sci (2017), doi:10.1016/j.meatsci.2017.04.261.
[48] R.D. Warner, C.K. McDonnell, A.E.D. Bekhit, J. Claus, R. Vaskoska, A. Sikes,
F.R. Dunshea, M. Ha, Systematic review of emerging and innovative technolo-
gies for meat tenderisation, Meat Sci. (2017), doi:10.1016/j.meatsci.2017.04.
241.
[49] A.A. Bekhit, D.L. Hopkins, G. Geesink, A.A. Bekhit, P. Franks, Exogenous Pro-
teases for Meat Tenderization, Crit. Rev. Food Sci. Nutr. (2014), doi:10.1080/
10408398.2011.623247.
[50] L. Thorrez, H. Vandenburgh, Challenges in the quest for ‘clean meat, Nat.
Biotechnol. (2019), doi:10.1038/s41587- 019- 0043- 0.
[51] S.T. Joo, G.D. Kim, Y.H. Hwang, Y.C. Ryu, Control of fresh meat quality through
manipulation of muscle fiber characteristics, Meat Sci. (2013), doi:10.1016/j.
meatsci.2013.04.044.
[52] R. Tarté, Ingredients in meat products: properties, functionality and applica-
tions, 2009. 10.1007/978-0-387-71327-4.
[53] B.L. Dekkers, R.M. Boom, A.J. van der Goot, Structuring processes for meat
analogues, Trends Food Sci. Technol. (2018), doi:10.1016/j.tifs.2018.08.011.
[54] M.C. Bourne, Texture Profile Analysis, Food Technol. 32 (1978) 62–66.
[55] J.R. Claus, Methods for the Objective Measurement of Meat Product Texture,
in: Reciprocal Meat Conf. Proceedings, Am. Meat Sci. Assoc., 1995, pp. 96–101.
[56] K.O. Honikel, Reference methods for the assessment of physical character-
istics of meat, Meat Sci. 49 (1998) 447–457, doi:10.1016/S0309-1740(98)
0 0 034-5.
[57] L.A. MacQueen, C.G. Alver, C.O. Chantre, S. Ahn, L. Cera, G.M. Gonza-
lez, B.B. O’Connor, D.J. Drennan, M.M. Peters, S.E. Motta, J.F. Zimmerman,
K.K. Parker, Muscle tissue engineering in fibrous gelatin: implications for
meat analogs, Npj Sci. Food. (2019), doi:10.1038/s41538- 019- 0054- 8.
[58] T. Ben-Arye, Y. Shandalov, S. Ben-Shaul, S. Landau, Y. Zagury, I. Ianovici,
N. Lavon, S. Levenberg, Textured soy protein scaffolds enable the generation
of three-dimensional bovine skeletal muscle tissue for cell-based meat, Nat.
Food. (2020), doi:10.1038/s43016- 020- 0046- 5.
[59] L.B. Kleinert, J.B. Hoying, S.K. Williams, The neointima formed in endothelial
cell sodded ePTFE vascular grafts results from both cellular-hyperplasia and
extracellular-hypertrophy, Cell Transplant. (1996), doi:10.1016/0963-6897(96)
0 0 024-3.
[60] V. Mudera, A.S.T. Smith, M.A. Brady, M.P. Lewis, The effect of cell density on
the maturation and contractile ability of muscle derived cells in a 3D tissue-
engineered skeletal muscle model and determination of the cellular and me-
chanical stimuli required for the synthesis of a postural phenotype, J. Cell.
Physiol. (2010), doi:10.1002/jcp.22271.
[61] C.S. Cheng, B.N.J. Davis, L. Madden, N. Bursac, G.A. Truskey, Physiology and
metabolism of tissue-engineered skeletal muscle, Exp. Biol. Med. (2014),
doi:10.1177/1535370214538589.
[62] L. Madden, M. Juhas, W.E. Kraus, G.A. Truskey, N. Bursac, Bioengineered hu-
man myobundles mimic clinical responses of skeletal muscle to drugs, Elife.
(2015), doi:10.7554/eLife.04885.
[63] N.M. Wragg, D.J. Player, N.R.W. Martin, Y. Liu, M.P. Lewis, Development of
tissue-engineered skeletal muscle manufacturing variables, Biotechnol. Bio-
eng. (2019), doi:10.1002/bit.27074.
[64] J. Bradbury, Docosahexaenoic acid (DHA): an ancient nutrient for the modern
human brain, Nutrients (2011), doi:10.3390/nu3050529.
[65] R. Obeid, S.G. Heil, M.M.A. Verhoeven, E.G.H.M. van den Heuvel, L.C.P.G.M. de
Groot, S.J.P.M. Eussen, Vitamin B12 intake from animal foods, biomarkers, and
health aspects, Front. Nutr. (2019), doi:10.3389/fnut.2019.0 0 093.
[66] F. Jimenez-Colmenero, L. Salcedo-Sandoval, R. Bou, S. Cofrades, A.M. Herrero,
C. Ruiz-Capillas, Novel applications of oil-structuring methods as a strategy to
improve the fat content of meat products, Trends Food Sci. Technol. (2015),
doi:10.1016/j.tifs.2015.04.011.
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
[67] Y. Qin, J. Jiang, L. Zhao, J. Zhang, F. Wang, Chapter 13: Applications of
Alginate as a Functional Food Ingredient, Elsevier Inc., 2018, doi:10.1016/
B978- 0- 12- 811449- 0.0 0 013-X.
[68] X.D. Sun, S.D. Arntfield, Gelation properties of salt-extracted pea protein iso-
late induced by heat treatment: effect of heating and cooling rate, Food
Chem. (2011), doi:10.1016/j.foodchem.2010.07.063.
[69] S. Comfort, N.K. Howell, Gelation properties of soya and whey protein isolate
mixtures, Food Hydrocoll. (2002), doi:10.1016/S0268- 005X(02)00033- 4.
[70] D. Santhi, A. Kalaikannan, P. Malairaj, S. Arun Prabhu, Application of microbial
transglutaminase in meat foods: a review, Crit. Rev. Food Sci. Nutr. (2017),
doi:10.1080/10408398.2014.945990.
[71] M.M. Kasprzak, W. Macnaughtan, S. Harding, P. Wilde, B. Wolf, Stabilisation of
oil-in-water emulsions with non-chemical modified gelatinised starch, Food
Hydrocoll. (2018), doi:10.1016/j.foodhyd.2018.03.002.
[72] E.D. Ngouémazong, S. Christiaens, A. Shpigelman, A. Van Loey, M. Hendrickx,
The Emulsifying and Emulsion-Stabilizing Properties of Pectin: a Review,
Compr. Rev. Food Sci. Food Saf. (2015), doi:10.1111/1541-4337.12160.
[73] B. Gómez, F.J. Barba, R. Domínguez, P. Putnik, D. Bursać Kovačević, M. Pateiro,
F. Toldrá, J.M. Lorenzo, Microencapsulation of antioxidant compounds through
innovative technologies and its specific application in meat processing,
Trends Food Sci. Technol. (2018), doi:10.1016/j.tifs.2018.10.006.
[74] K.G.C. Weel, A.E.M. Boelrijk, A.C. Alting, P.J.J.M. Van Mil, J.J. Burger, H. Grup-
pen, A.G.J. Voragen, G. Smit, Flavor release and perception of flavored whey
protein gels: perception is determined by texture rather than by release, J.
Agric. Food Chem. (2002), doi:10.1021/jf0202786.
[75] D. Renard, F. Van De Velde, R.W. Visschers, The gap between food gel struc-
ture, texture and perception, Food Hydrocoll. (2006), doi:10.1016/j.foodhyd.
2005.10.014.
[76] A.R. Patel, R.A. Nicholson, A.G. Marangoni, Applications of fat mimetics for
the replacement of saturated and hydrogenated fat in food products, Curr.
Opin. Food Sci. 33 (2020) 61–68, doi:10.1016/j.cofs.2019.12.008.
[77] W.T. Kao, K.W. Lin, Quality of reduced-fat frankfurter modified by konjac-
starch mixed gels, J. Food Sci. (2006), doi:10.1111/j.1750-3841.2006.00003.x.
[78] L.C. Hoffman, F.D. Mellett, Quality characteristics of low fat ostrich meat pat-
ties formulated with either pork lard or modified corn starch, soya isolate
and water, Meat Sci. (2003), doi:10.1016/S0309- 1740(02)00293- 0.
[79] A.M. Herrero, P. Carmona, F. Jiménez-Colmenero, C. Ruiz-Capillas, Polysaccha-
ride gels as oil bulking agents: technological and structural properties, Food
Hydrocoll. (2014), doi:10.1016/j.foodhyd.2013.08.008.
[80] G.R. Krawczyk, A. Venables, D. Tuason, Microcrystalline cellulose, Handb. Hy-
drocoll, 2nd Ed., 2009, doi:10.1533/9781845695873.740.
[81] R.T. Heck, J.M. Lorenzo, B.A. Dos Santos, A.J. Cichoski, C.R. de Menezes,
P.C.B. Campagnol, Microencapsulation of healthier oils: an efficient strategy
to improve the lipid profile of meat products, Curr. Opin. Food Sci. (2020),
doi:10.1016/j.cofs.2020.04.010.
[82] M.A. Asgar, A. Fazilah, N. Huda, R. Bhat, A.A. Karim, Nonmeat protein alter-
natives as meat extenders and meat analogs, Compr. Rev. Food Sci. Food Saf.
(2010), doi:10.1111/j.1541-4337.2010.00124.x.
[83] N.R. Rubio, K.D. Fish, B.A. Trimmer, D.L. Kaplan, Possibilities for Engineered
Insect Tissue as a Food Source, Front. Sustain. Food Syst. (2019), doi:10.3389/
fsufs.2019.0 0 024.
[84] G. Forgacs, F. Marga, K.R. Jakab, ENGINEERED COMESTIBLE MEAT, 2012.
[85] R. Osen, U. Schweiggert-Weisz, High-Moisture Extrusion : meat Analogues,
Refence Modul. Food Sci. 1 (2016) 1–7, doi:10.1016/j.elspec.20 09.07.0 01.
[86] G.A. Krintiras, J. Gadea Diaz, A.J. Van Der Goot, A.I. Stankiewicz, G.D. Stefani-
dis, On the use of the Couette Cell technology for large scale production of
textured soy-based meat replacers, J. Food Eng. (2016), doi:10.1016/j.jfoodeng.
2015.08.021.
[87] Y. Ren, Texturized Vegetable Proteins: the Challenges and Solutions to Mim-
icking Meat Texture from Plant Proteins, 2011.
[88] S. Samard, B.Y. Gu, G.H. Ryu, Effects of extrusion types, screw speed and addi-
tion of wheat gluten on physicochemical characteristics and cooking stability
of meat analogues, J. Sci. Food Agric. (2019), doi:10.1002/jsfa.9722.
[89] K.S. Liu, F.H. Hsieh, Protein-protein interactions during high-moisture extru-
sion for fibrous meat analogues and comparison of protein solubility meth-
ods using different solvent systems, J. Agric. Food Chem. (2008), doi:10.1021/
jf073343q.
[90] V.L. Pietsch, M.A. Emin, H.P. Schuchmann, Process conditions influencing
wheat gluten polymerization during high moisture extrusion of meat analog
products, J. Food Eng. (2017), doi:10.1016/j.jfoodeng.2016.10.027.
[91] V. Rampon, P. Robert, N. Nicolas, E. Dufour, Protein structure and network
orientation in edible films prepared by spinning process, J. Food Sci. (1999),
doi:10.1111/j.1365-2621.1999.tb15890.x.
[92] M. Nieuwland, P. Geerdink, P. Brier, P. Van Den Eijnden, J.T.M.M. Henket,
M.L.P. Langelaan, N. Stroeks, H.C. Van Deventer, A.H. Martin, Food-grade elec-
trospinning of proteins, Innov. Food Sci. Emerg. Technol. (2013), doi:10.1016/
j.ifset.2013.09.004.
[93] R. Boyer, High protein food product and process for its preparation,
US2682466A, 1954.
[94] M.M. Sternberg, C.Y. Kim, Process for preparing wet spun proteinaceous fila-
ments, US3956514A, 1976.
[95] F.K.G. Schreuders, B.L. Dekkers, I. Bodnár, P. Erni, R.M. Boom, A.J. van der Goot,
Comparing structuring potential of pea and soy protein with gluten for meat
analogue preparation, J. Food Eng. (2019), doi:10.1016/j.jfoodeng.2019.04.
022.
[96] S. Oguz, N. Tam, A. Aydogdu, G. Sumnu, S. Sahin, Development of novel pea
16
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
flour-based nanofibres by electrospinning method, Int. J. Food Sci. Technol. 53
(2018) 1269–1277, doi:10.1111/ijfs.13707.
[97] M. Wiebe, Myco-protein from fusarium venenatum: a well-established prod-
uct for human consumption, Appl. Microbiol. Biotechnol. (2002), doi:10.1007/
s0 0253-0 02-0931-x.
[98] W.J. Means, G.R. Schmidt, Algin/Calcium Gel as a Raw and Cooked Binder
in Structured Beef Steaks, J. Food Sci. (1986), doi:10.1111/j.1365-2621.1986.
tb10836.x.
[99] S.K. WILLIAMS, J.L. OBLINGER, R.L. WEST, EVALUATION OF A CALCIUM AL-
GINATE FILM FOR USE ON BEEF CUTS, J. Food Sci. (1978), doi:10.1111/j.
1365-2621.1978.tb02288.x.
[100] K.G. WANSTEDT, S.C. SEIDEMAN, L.S. DONNELLY, N.M. QUENZER, Sensory At-
tributes of Precooked, Calcium Alginate-Coated Pork Patties, J. Food Prot. 44
(1981) 732–735, doi:10.4315/0362- 028x- 44.10.732.
[101] M.E. Danoviz, Z. Yablonka-Reuveni, Skeletal muscle satellite cells: background
and methods for isolation and analysis in a primary culture system, Methods
Mol. Biol. (2012), doi:10.1007/978- 1- 61779- 343- 1_2.
[102] R. Chen, L. Li, L. Feng, Y. Luo, M. Xu, K.W. Leong, R. Yao, Biomaterial-assisted
scalable cell production for cell therapy, Biomaterials 230 (2020) 119627,
doi:10.1016/j.biomaterials.2019.119627.
[103] G. Molnar, N.A. Schroedl, S.R. Gonda, C.R. Hartzell, Skeletal muscle satellite
cells cultured in simulated microgravity, Vitr. Cell. Dev. Biol. - Anim. (1997),
doi:10.1007/s11626- 997- 0010- 9.
[104] K.J.M. Boonen, K.Y. Rosaria-Chak, F.P.T. Baaijens, D.W.J. Van Der Schaft,
M.J. Post, Essential environmental cues from the satellite cell niche: opti-
mizing proliferation and differentiation, Am. J. Physiol. - Cell Physiol. (2009),
doi:10.1152/ajpcell.0 0 015.20 09.
[105] K. Gwon, E. Kim, G. Tae, Heparin-hyaluronic acid hydrogel in support of cel-
lular activities of 3D encapsulated adipose derived stem cells, Acta Biomater.
49 (2017) 284–295, doi:10.1016/j.actbio.2016.12.001.
[106] L.E. Flynn, G.D. Prestwich, J.L. Semple, K.A. Woodhouse, Proliferation and dif-
ferentiation of adipose-derived stem cells on naturally derived scaffolds, Bio-
materials 29 (2008) 1862–1871, doi:10.1016/j.biomaterials.2007.12.028.
[107] C. Yu, A. Kornmuller, C. Brown, T. Hoare, L.E. Flynn, Decellularized adipose tis-
sue microcarriers as a dynamic culture platform for human adipose-derived
stem/stromal cell expansion, Biomaterials (2017), doi:10.1016/j.biomaterials.
2016.12.017.
[108] V. Jossen, C. van den Bos, R. Eibl, D. Eibl, Manufacturing human mesenchymal
stem cells at clinical scale: process and regulatory challenges, Appl. Microbiol.
Biotechnol. (2018), doi:10.10 07/s0 0253- 018- 8912- x.
[109] E. Swartz, Meeting the Needs of the Cell-Based Meat Industry, Chem. Eng.
(CEP), AIChE Mag. (2019) 41–45 https://ezproxy.usim.edu.my:2056/docview/
2310613647/fulltextPDF/AF713AB89EE54383PQ/1?accountid=33993.
[110] G. Zhang, X. Zhao, X. Li, G. Du, J. Zhou, J. Chen, Challenges and possibilities for
bio-manufacturing cultured meat, Trends Food Sci. Technol. 97 (2020) 443–
450, doi:10.1016/j.tifs.2020.01.026.
[111] S.J. Allan, P.A. De Bank, M.J. Ellis, Bioprocess Design Considerations for Cul-
tured Meat Production With a Focus on the Expansion Bioreactor, Front. Sus-
tain. Food Syst. 3 (2019), doi:10.3389/fsufs.2019.0 0 044.
[112] OECDOECD Environmental Outlook to 2030, OECD, 2008, doi:10.1787/
9789264040519-en.
[113] P. Nold, C. Brendel, A. Neubauer, G. Bein, H. Hackstein, Good manufac-
turing practice-compliant animal-free expansion of human bone marrow
derived mesenchymal stroma cells in a closed hollow-fiber-based biore-
actor, Biochem. Biophys. Res. Commun. (2013), doi:10.1016/j.bbrc.2012.11.
001.
[114] S.A. Tuin, B. Pourdeyhimi, E.G. Loboa, Fabrication of novel high surface area
mushroom gilled fibers and their effects on human adipose derived stem
cells under pulsatile fluid flow for tissue engineering applications, Acta Bio-
mater. (2016), doi:10.1016/j.actbio.2016.03.025.
[115] S. Khan, L. Davila, M. Salkhordeh, D.J. Stewart, S.H. Mei, L. McIntyre,
D.W. Courtman, cGMP-compatible large-scale production of mesenchymal
stem cells (MSCs) in Xeno- and serum-free media for allogeneic cell thera-
pies, Cytotherapy (2018), doi:10.1016/j.jcyt.2018.02.107.
[116] I. Roberts, S. Baila, R.B. Rice, M.E. Janssens, K. Nguyen, N. Moens, L. Ruban,
D. Hernandez, P. Coffey, C. Mason, Scale-up of human embryonic stem cell
culture using a hollow fibre bioreactor, Biotechnol. Lett. (2012), doi:10.1007/
s10529- 012- 1033- 1.
[117] N.M.S. Bettahalli, J. Vicente, L. Moroni, G.A. Higuera, C.A. Van Blitterswijk,
M. Wessling, D.F. Stamatialis, Integration of hollow fiber membranes im-
proves nutrient supply in three-dimensional tissue constructs, Acta Biomater.
(2011), doi:10.1016/j.actbio.2011.06.012.
[118] Y. Yamamoto, A. Ito, H. Jitsunobu, K. Yamaguchi, Y. Kawabe, H. Mizumoto,
M. Kamihira, Hollow fiber bioreactor perfusion culture system for magnetic
force-based skeletal muscle tissue engineering, J. Chem. Eng. Japan. (2012),
doi:10.1252/jcej.11we237.
[119] J.C. Gerlach, Y.C. Lin, C.A. Brayfield, D.M. Minteer, H. Li, J.P. Rubin, K.G. Marra,
Adipogenesis of human adipose-derived stem cells within three-dimensional
hollow fiber-based bioreactors, Tissue Eng. - Part C Methods. (2012), doi:10.
1089/ten.tec.2011.0216.
[120] B. Cunha, T. Aguiar, S.B. Carvalho, M.M. Silva, R.A. Gomes, M.J.T. Carrondo,
P. Gomes-Alves, C. Peixoto, M. Serra, P.M. Alves, Bioprocess integration for hu-
man mesenchymal stem cells: from up to downstream processing scale-up to
cell proteome characterization, J. Biotechnol. (2017), doi:10.1016/j.jbiotec.2017.
01.014.
[121] N. Wung, S.M. Acott, D. Tosh, M.J. Ellis, Hollow fibre membrane bioreac-
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
tors for tissue engineering applications, Biotechnol. Lett. (2014), doi:10.1007/
s10529- 014- 1619- x.
[122] S.B.M. Usuludin, X. Cao, M. Lim, Co-culture of stromal and erythroleukemia
cells in a perfused hollow fiber bioreactor system as an in vitro bone mar-
row model for myeloid leukemia, Biotechnol. Bioeng. (2012), doi:10.1002/bit.
24400.
[123] V. Bodiou, P. Moutsatsou, M.J. Post, Microcarriers for Upscaling Cultured Meat
Production, Front. Nutr. 7 (2020) 1–16, doi:10.3389/fnut.2020.0 0 010.
[124] J.H. Lee, H.W. Jung, I.K. Kang, H.B. Lee, Cell behaviour on polymer sur-
faces with different functional groups, Biomaterials (1994), doi:10.1016/
0142-9612(94)90169-4.
[125] G.B. Schneider, A. English, M. Abraham, R. Zaharias, C. Stanford, J. Keller, The
effect of hydrogel charge density on cell attachment, Biomaterials (2004),
doi:10.1016/j.biomaterials.2003.09.084.
[126] A.K.L. Chen, X. Chen, A.B.H. Choo, S. Reuveny, S.K.W. Oh, Critical microcarrier
properties affecting the expansion of undifferentiated human embryonic stem
cells, Stem Cell Res. (2011), doi:10.1016/j.scr.2011.04.007.
[127] S. Guo, X. Zhu, M. Li, L. Shi, J.L.T. Ong, D. Jańczewski, K.G. Neoh, Parallel Con-
trol over Surface Charge and Wettability Using Polyelectrolyte Architecture:
effect on Protein Adsorption and Cell Adhesion, ACS Appl. Mater. Interfaces.
(2016), doi:10.1021/acsami.6b09481.
[128] L.C. Xu, C.A. Siedlecki, Effects of surface wettability and contact time on
protein adhesion to biomaterial surfaces, Biomaterials (2007), doi:10.1016/j.
biomaterials.2007.03.032.
[129] C.J. Wilson, R.E. Clegg, D.I. Leavesley, M.J. Pearcy, Mediation of biomaterial-
cell interactions by adsorbed proteins: a review, Tissue Eng. (2005), doi:10.
1089/ten.2005.11.1.
[130] M.C. Morán, G. Ruano, F. Cirisano, M. Ferrari, Mammalian cell viability on hy-
drophobic and superhydrophobic fabrics, Mater. Sci. Eng. C. (2019), doi:10.
1016/j.msec.2019.01.088.
[131] S. Verbruggen, D. Luining, A. van Essen, M.J. Post, Bovine myoblast cell pro-
duction in a microcarriers-based system, Cytotechnology 70 (2018) 503–512,
doi:10.1007/s10616- 017- 0101- 8.
[132] M. Hervy, J.L. Weber, M. Pecheul, P. Dolley-Sonneville, D. Henry, Y. Zhou,
Z. Melkoumian, Long term expansion of bone marrow-derived hMSCs on
novel synthetic microcarriers in xeno-free, defined conditions, PLoS ONE
(2014), doi:10.1371/journal.pone.0092120.
[133] S. Derakhti, S.H. Safiabadi-Tali, G. Amoabediny, M. Sheikhpour, Attachment
and detachment strategies in microcarrier-based cell culture technology: a
comprehensive review, Mater. Sci. Eng. C. (2019), doi:10.1016/j.msec.2019.
109782.
[134] J. Dong, Q. Sun, J.Y. Wang, Basic study of corn protein, zein, as a biomaterial
in tissue engineering, surface morphology and biocompatibility, Biomaterials
(2004), doi:10.1016/j.biomaterials.2003.10.084.
[135] S. Tansaz, A.K. Durmann, R. Detsch, A.R. Boccaccini, Hydrogel films and micro-
capsules based on soy protein isolate combined with alginate, J. Appl. Polym.
Sci. (2017), doi:10.1002/app.44358.
[136] M.A. Benjaminson, J.A. Gilchriest, M. Lorenz, In vitro edible muscle protein
production system (MPPS): stage 1, fish, Acta Astronaut. 51 (2002) 879–889,
doi:10.1016/S0 094-5765(02)0 0 033-4.
[137] F.S. Marga, B.P. Purcell, G. Forgacs, A. Forgacs, Edible and animal-product-free
microcarriers for engineered meat, US 9,752,122 B2, 2017.
[138] D. Choquet, D.P. Felsenfeld, M.P. Sheetz, Extracellular matrix rigidity causes
strengthening of integrin- cytoskeleton linkages, Cell (1997), doi:10.1016/
S0 092-8674(0 0)81856-5.
[139] K. Ghosh, Z. Pan, E. Guan, S. Ge, Y. Liu, T. Nakamura, X.D. Ren, M. Rafailovich,
R.A.F. Clark, Cell adaptation to a physiologically relevant ECM mimic with dif-
ferent viscoelastic properties, Biomaterials (2007), doi:10.1016/j.biomaterials.
2006.09.038.
[140] E.A. Klein, L. Yin, D. Kothapalli, P. Castagnino, F.J. Byfield, T. Xu, I. Levental,
E. Hawthorne, P.A. Janmey, R.K. Assoian, Cell-Cycle Control by Physiological
Matrix Elasticity and In Vivo Tissue Stiffening, Curr. Biol. (2009), doi:10.1016/
j.cub.2009.07.069.
[141] Y.H. Bae, K.L. Mui, B.Y. Hsu, S.L. Liu, A. Cretu, Z. Razinia, T. Xu, E. Puré,
R.K. Assoian, A FAK-Cas-Rac-lamellipodin signaling module transduces extra-
cellular matrix stiffness into mechanosensitive cell cycling, Sci. Signal. (2014),
doi:10.1126/scisignal.2004838.
[142] A.J. Engler, M.A. Griffin, S. Sen, C.G. Bönnemann, H.L. Sweeney, D.E. Dis-
cher, Myotubes differentiate optimally on substrates with tissue-like stiff-
ness: pathological implications for soft or stiff microenvironments, J. Cell Biol.
(2004), doi:10.1083/jcb.200405004.
[143] P.C. Georges, P.A. Janmey, Cell type-specific response to growth on soft mate-
rials, J. Appl. Physiol. (2005), doi:10.1152/japplphysiol.01121.2004.
[144] P.M. Gilbert, K.L. Havenstrite, K.E.G. Magnusson, A. Sacco, N.A. Leonardi,
P. Kraft, N.K. Nguyen, S. Thrun, M.P. Lutolf, H.M. Blau, Substrate elasticity reg-
ulates skeletal muscle stem cell self-renewal in culture, Science (80-.) (2010),
doi:10.1126/science.1191035.
[145] T. Boontheekul, E.E. Hill, H.J. Kong, D.J. Mooney, Regulating myoblast phe-
notype through controlled gel stiffness and degradation, Tissue Eng. (2007),
doi:10.1089/ten.2006.0356.
[146] G. Lacraz, A.J. Rouleau, V. Couture, T. Söllrald, G. Drouin, N. Veillette,
M. Grandbois, G. Grenier, Increased stiffness in aged skeletal muscle impairs
muscle progenitor cell proliferative activity, PLoS ONE (2015), doi:10.1371/
journal.pone.0136217.
[147] L.-.S. Wang, J.E. Chung, P.P.-Y. Chan, M. Kurisawa, Injectable biodegrad-
able hydrogels with tunable mechanical properties for the stimulation of
17
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
neurogenic differentiation of human mesenchymal stem cells in 3D cul-
ture, Biomaterials 31 (2010) 1148–1157, doi:10.1016/j.biomaterials.2009.10.
042.
[148] Z. Li, Y. Gong, S. Sun, Y. Du, D. Lü, X. Liu, M. Long, Differential regulation of
stiffness, topography, and dimension of substrates in rat mesenchymal stem
cells, Biomaterials (2013), doi:10.1016/j.biomaterials.2013.06.059.
[149] J.S. Park, J.S. Chu, A.D. Tsou, R. Diop, Z. Tang, A. Wang, S. Li, The effect of
matrix stiffness on the differentiation of mesenchymal stem cells in response
to TGF-β , Biomaterials (2011), doi:10.1016/j.biomaterials.2011.02.019.
[150] M. Kim, Y.H. Kim, G. Tae, Human mesenchymal stem cell culture on heparin-
based hydrogels and the modulation of interactions by gel elasticity and hep-
arin amount, Acta Biomater. 9 (2013) 7833–7844, doi:10.1016/j.actbio.2013.04.
041.
[151] J.M. Banks, L.C. Mozdzen, B.A.C. Harley, R.C. Bailey, The combined effects of
matrix stiffness and growth factor immobilization on the bioactivity and dif-
ferentiation capabilities of adipose-derived stem cells, Biomaterials 35 (2014)
8951–8959, doi:10.1016/j.biomaterials.2014.07.012.
[152] M.J. Osiecki, T.D. Michl, B.K. Babur, M. Kabiri, K. Atkinson, W.B. Lott,
H.J. Griesser, M.R. Doran, Packed bed bioreactor for the isolation and expan-
sion of placental-derived mesenchymal stromal cells, PLoS ONE 10 (2015) 1–
18, doi:10.1371/journal.pone.0144941.
[153] D.W. Hutmacher, H. Singh, Computational fluid dynamics for improved biore-
actor design and 3D culture, Trends Biotechnol. (2008), doi:10.1016/j.tibtech.
2007.11.012.
[154] T. Ben-Arye, S. Levenberg, Tissue Engineering for Clean Meat Production,
Front. Sustain. Food Syst. 3 (2019) 1–19, doi:10.3389/fsufs.2019.0 0 046.
[155] H.M. Leinonen, S. Lepola, E.M. Lipponen, T. Heikura, T. Koponen, N. Parker,
S. Ylä-Herttuala, H.P. Lesch, Benchmarking of Scale-X Bioreactor System in
Lentiviral and Adenoviral Vector Production, Hum. Gene Ther. 31 (2020) 376–
384, doi:10.1089/hum.2019.247.
[156] R. Portner, O.B. Platas, D. Fassnacht, D. Nehring, P. Czermak, H. Markl, Fixed
Bed Reactors for the Cultivation of Mammalian Cells: design, Performance
and Scale-Up, Open Biotechnol. J. (2007), doi:10.2174/187407070701014107.
[157] S. Levenberg, J. Rouwkema, M. Macdonald, E.S. Garfein, D.S. Kohane, D.C. Dar-
land, R. Marini, C.A. Van Blitterswijk, R.C. Mulligan, P.A. D’Amore, R. Langer,
Engineering vascularized skeletal muscle tissue, Nat. Biotechnol. (2005),
doi:10.1038/nbt1109.
[158] S. Torgbo, P. Sukyai, Bacterial cellulose-based scaffold materials for bone tis-
sue engineering, Appl. Mater. Today. 11 (2018) 34–49, doi:10.1016/j.apmt.
2018.01.004.
[159] R.J. Hickey, A.E. Pelling, Cellulose biomaterials for tissue engineering, Front.
Bioeng. Biotechnol. (2019), doi:10.3389/fbioe.2019.0 0 045.
[160] M. Hu, R. Deng, K.M. Schumacher, M. Kurisawa, H. Ye, K. Purnamawati,
J.Y. Ying, Hydrodynamic spinning of hydrogel fibers, Biomaterials. 31 (2010)
863–869, doi:10.1016/j.biomaterials.2009.10.002.
[161] Z.J. Meng, W. Wang, R. Xie, X.J. Ju, Z. Liu, L.Y. Chu, Microfluidic generation of
hollow Ca-alginate microfibers, Lab Chip. (2016), doi:10.1039/c6lc00640j.
[162] U.H.T. Pham, M. Hanif, A. Asthana, S.M. Iqbal, A microfluidic device approach
to generate hollow alginate microfibers with controlled wall thickness and
inner diameter, J. Appl. Phys. (2015), doi:10.1063/1.4919361.
[163] K. Tuzlakoglu, I. Pashkuleva, M.T. Rodrigues, M.E. Gomes, G.H. Van Lenthe,
R. Müller, R.L. Reis, A new route to produce starch-based fiber mesh scaffolds
by wet spinning and subsequent surface modification as a way to improve
cell attachment and proliferation, J. Biomed. Mater. Res. - Part A. (2010),
doi:10.1002/jbm.a.32358.
[164] X. Shi, S. Ostrovidov, Y. Zhao, X. Liang, M. Kasuya, K. Kurihara, K. Nakajima,
H. Bae, H. Wu, A. Khademhosseini, Microfluidic spinning of cell-responsive
grooved microfibers, Adv. Funct. Mater. (2015), doi:10.1002/adfm.201404531.
[165] Y. Yang, J. Sun, X. Liu, Z. Guo, Y. He, D. Wei, M. Zhong, L. Guo, H. Fan,
X. Zhang, Wet-spinning fabrication of shear-patterned alginate hydrogel mi-
crofibers and the guidance of cell alignment, Regen. Biomater. 4 (2017) 299–
307, doi:10.1093/rb/rbx017.
[166] Y. Wang, R.K. Kankala, K. Zhu, S. Bin Wang, Y.S. Zhang, A.Z. Chen, Coaxial
Extrusion of Tubular Tissue Constructs Using a Gelatin/GelMA Blend Bioink,
ACS Biomater. Sci. Eng. (2019), doi:10.1021/acsbiomaterials.9b00926.
[167] Y. Yu, L. Shang, J. Guo, J. Wang, Y. Zhao, Design of capillary microflu-
idics for spinning cell-laden microfibers, Nat. Protoc. (2018), doi:10.1038/
s41596- 018- 0051- 4.
[168] D. Wei, J. Sun, J. Bolderson, M. Zhong, M.J. Dalby, M. Cusack, H. Yin, H. Fan,
X. Zhang, Continuous Fabrication and Assembly of Spatial Cell-Laden Fibers
for a Tissue-Like Construct via a Photolithographic-Based Microfluidic Chip,
ACS Appl. Mater. Interfaces. (2017), doi:10.1021/acsami.7b0 0 078.
[169] M. Hu, M. Kurisawa, R. Deng, C.M. Teo, A. Schumacher, Y.X. Thong,
L. Wang, K.M. Schumacher, J.Y. Ying, Cell immobilization in gelatin-
hydroxyphenylpropionic acid hydrogel fibers, Biomaterials. 30 (2009) 3523–
3531, doi:10.1016/j.biomaterials.20 09.03.0 04.
[170] Y. Cheng, Y. Yu, F. Fu, J. Wang, L. Shang, Z. Gu, Y. Zhao, Controlled Fabrica-
tion of Bioactive Microfibers for Creating Tissue Constructs Using Microfluidic
Techniques, ACS Appl. Mater. Interfaces. (2016), doi:10.1021/acsami.5b11445.
[171] S. Jana, S.K.L. Levengood, M. Zhang, Anisotropic Materials for Skeletal-Muscle-
Tissue Engineering, Adv. Mater. (2016), doi:10.10 02/adma.20160 0240.
[172] M.F. Leong, J.K.C. Toh, C. Du, K. Narayanan, H.F. Lu, T.C. Lim, A.C.A. Wan,
J.Y. Ying, Patterned prevascularised tissue constructs by assembly of polyelec-
trolyte hydrogel fibres, Nat. Commun. (2013), doi:10.1038/ncomms3353.
[173] N.F. Huang, S. Patel, R.G. Thakar, J. Wu, B.S. Hsiao, B. Chu, R.J. Lee, S. Li,
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
Myotube assembly on nanofibrous and micropatterned polymers, Nano Lett.
(2006), doi:10.1021/nl060060o.
[174] J.S. Choi, S.J. Lee, G.J. Christ, A. Atala, J.J. Yoo, The influence of electro-
spun aligned poly(ε -caprolactone)/collagen nanofiber meshes on the forma-
tion of self-aligned skeletal muscle myotubes, Biomaterials (2008), doi:10.
1016/j.biomaterials.2008.03.031.
[175] K.J. Aviss, J.E. Gough, S. Downes, Aligned electrospun polymer fibres for skele-
tal muscle regeneration, Eur. Cells Mater. (2010), doi:10.22203/eCM.v019a19.
[176] M.T. Lam, Y.C. Huang, R.K. Birla, S. Takayama, Microfeature guided skeletal
muscle tissue engineering for highly organized 3-dimensional free-standing
constructs, Biomaterials 30 (2009) 1150–1155, doi:10.1016/j.biomaterials.
2008.11.014.
[177] S. Chen, T. Nakamoto, N. Kawazoe, G. Chen, Engineering multi-layered skele-
tal muscle tissue by using 3D microgrooved collagen scaffolds, Biomaterials
(2015), doi:10.1016/j.biomaterials.2015.09.010.
[178] J.M. Dugan, R.F. Collins, J.E. Gough, S.J. Eichhorn, Oriented surfaces of ad-
sorbed cellulose nanowhiskers promote skeletal muscle myogenesis, Acta Bio-
mater. (2013), doi:10.1016/j.actbio.2012.08.050.
[179] H. Takahashi, T. Shimizu, M. Nakayama, M. Yamato, T. Okano, The use of
anisotropic cell sheets to control orientation during the self-organization of
3D muscle tissue, Biomaterials (2013), doi:10.1016/j.biomaterials.2013.06.033.
[180] V. Hosseini, S. Ahadian, S. Ostrovidov, G. Camci-Unal, S. Chen, H. Kaji, M. Ra-
malingam, A. Khademhosseini, Engineered contractile skeletal muscle tissue
on a microgrooved methacrylated gelatin substrate, Tissue Eng. - Part A.
(2012), doi:10.1089/ten.tea.2012.0181.
[181] H.S. Yang, N. Ieronimakis, J.H. Tsui, H.N. Kim, K.Y. Suh, M. Reyes, D.H. Kim,
Nanopatterned muscle cell patches for enhanced myogenesis and dystrophin
expression in a mouse model of muscular dystrophy, Biomaterials (2014),
doi:10.1016/j.biomaterials.2013.10.067.
[182] S. Jana, A. Cooper, M. Zhang, Chitosan scaffolds with unidirectional micro-
tubular pores for large skeletal myotube generation, Adv. Healthc. Mater.
(2013), doi:10.10 02/adhm.20120 0177.
[183] V. Kroehne, I. Heschel, F. Schügner, D. Lasrich, J.W. Bartsch, H. Jockusch,
Use of a novel collagen matrix with oriented pore structure for muscle
cell differentiation in cell culture and in grafts, J. Cell. Mol. Med. (2008),
doi:10.1111/j.1582-4934.20 08.0 0238.x.
[184] Y.S. Choi, L.G. Vincent, A.R. Lee, K.C. Kretchmer, S. Chirasatitsin, M.K. Dobke,
A.J. Engler, The alignment and fusion assembly of adipose-derived stem
cells on mechanically patterned matrices, Biomaterials (2012), doi:10.1016/j.
biomaterials.2012.06.057.
[185] Y.-.W. Cheng, D.J. Shiwarski, R.L. Ball, K.A. Whitehead, A.W. Feinberg,
Engineering Aligned Skeletal Muscle Tissue Using Decellularized Plant-
Derived Scaffolds, ACS Biomater. Sci. Eng. 6 (2020) 3046–3054, doi:10.1021/
acsbiomaterials.0c0 0 058.
[186] T. Matsumoto, J.I. Sasaki, E. Alsberg, H. Egusa, H. Yatani, T. Sohmura, Three-
dimensional cell and tissue patterning in a strained fibrin gel system, PLoS
ONE (2007), doi:10.1371/journal.pone.0001211.
[187] H.H. Vandenburgh, P. Karlisch, Longitudinal growth of skeletal myotubes in
vitro in a new horizontal mechanical cell stimulator, Vitr. Cell. Dev. Biol.
(1989), doi:10.1007/BF02623630.
[188] T. Okano, T. Matsuda, Tissue engineered skeletal muscle: preparation of
highly dense, highly oriented hybrid muscular tissues, Cell Transplant. (1998),
doi:10.1016/S0963-6897(97)0 0 067-5.
[189] P. Yilgor Huri, C.A. Cook, D.L. Hutton, B.C. Goh, J.M. Gimble, D.J. DiGiro-
lamo, W.L. Grayson, Biophysical cues enhance myogenesis of human adi-
pose derived stem/stromal cells, Biochem. Biophys. Res. Commun. (2013),
doi:10.1016/j.bbrc.2013.07.049.
[190] H. Egusa, M. Kobayashi, T. Matsumoto, J.I. Sasaki, S. Uraguchi, H. Yatani, Ap-
plication of cyclic strain for accelerated skeletal myogenic differentiation of
mouse bone marrow-derived mesenchymal stromal cells with cell alignment,
Tissue Eng. - Part A. (2013), doi:10.1089/ten.tea.2012.0164.
[191] M. Levy-Mishali, J. Zoldan, S. Levenberg, Effect of scaffold stiffness on my-
oblast differentiation, Tissue Eng. - Part A. (2009), doi:10.1089/ten.tea.2008.
0111.
[192] A.J. Engler, S. Sen, H.L. Sweeney, D.E. Discher, Matrix Elasticity Directs Stem
Cell Lineage Specification, Cell. (2006), doi:10.1016/j.cell.2006.06.044.
[193] K. Ren, T. Crouzier, C. Roy, C. Picart, Polyelectrolyte multilayer films of con-
trolled stiffness modulate myoblast cell differentiation, Adv. Funct. Mater.
(2008), doi:10.1002/adfm.200701297.
[194] T. Zhang, S. Lin, X. Shao, S. Shi, Q. Zhang, C. Xue, Y. Lin, B. Zhu, X. Cai, Reg-
ulating osteogenesis and adipogenesis in adipose-derived stem cells by con-
trolling underlying substrate stiffness, J. Cell. Physiol. (2018), doi:10.1002/jcp.
26193.
[195] J. Oliver-De La Cruz, G. Nardone, J. Vrbsky, A. Pompeiano, A.R. Pere-
strelo, F. Capradossi, K. Melajová, P. Filipensky, G. Forte, Substrate mechan-
ics controls adipogenesis through YAP phosphorylation by dictating cell
spreading, Biomaterials 205 (2019) 64–80, doi:10.1016/j.biomaterials.2019.03.
009.
[196] J. Xie, D. Zhang, C. Zhou, Q. Yuan, L. Ye, X. Zhou, Substrate elasticity reg-
ulates adipose-derived stromal cell differentiation towards osteogenesis and
adipogenesis through β -catenin transduction, Acta Biomater. 79 (2018) 83–
95, doi:10.1016/j.actbio.2018.08.018.
[197] D.A. Young, Y.S. Choi, A.J. Engler, K.L. Christman, Stimulation of adipoge-
nesis of adult adipose-derived stem cells using substrates that mimic the
stiffness of adipose tissue, Biomaterials 34 (2013) 8581–8588, doi:10.1016/j.
biomaterials.2013.07.103.
18
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
[198] K. Comley, N.A. Fleck, A micromechanical model for the Young’s modulus of
adipose tissue, Int. J. Solids Struct. 47 (2010) 2982–2990, doi:10.1016/j.ijsolstr.
2010.07.001.
[199] A. Samani, J. Zubovits, D. Plewes, Elastic moduli of normal and patho-
logical human breast tissues: an inversion-technique-based investigation
of 169 samples, Phys. Med. Biol. (2007), doi:10.1088/0031-9155/52/6/
002.
[200] E.M. Chandler, C.M. Berglund, J.S. Lee, W.J. Polacheck, J.P. Gleghorn, B.J. Kirby,
C. Fischbach, Stiffness of photocrosslinked RGD-alginate gels regulates adi-
pose progenitor cell behavior, Biotechnol. Bioeng. 108 (2011) 1683–1692,
doi:10.1002/bit.23079.
[201] M.J. Post, Cultured beef: medical technology to produce food, J. Sci. Food
Agric. (2014), doi:10.1002/jsfa.6474.
[202] U. Cheema, S.Y. Yang, V. Mudera, G.G. Goldspink, R.A. Brown, 3-D in vitro
model of early skeletal muscle development, Cell Motil. Cytoskeleton. (2003),
doi:10.1002/cm.10095.
[203] A.S.T. Smith, S. Passey, L. Greensmith, V. Mudera, M.P. Lewis, Characterization
and optimization of a simple, repeatable system for the long term in vitro
culture of aligned myotubes in 3D, J. Cell. Biochem. (2012), doi:10.1002/jcb.
23437.
[204] H. Vandenburgh, J. Shansky, M. Del Tatto, J. Chromiak, Organogenesis of
Skeletal Muscle in Tissue Culture, Tissue Eng. Methods Protoc. (1999) 217–
225, doi:10.1385/0- 89603- 516- 6:217.
[205] L. Thorrez, J. Shansky, L. Wang, L. Fast, T. VandenDriessche, M. Chuah,
D. Mooney, H. Vandenburgh, Growth, differentiation, transplantation and sur-
vival of human skeletal myofibers on biodegradable scaffolds, Biomaterials
(2008), doi:10.1016/j.biomaterials.2007.09.014.
[206] M. Post, C. van der Weele, Principles of Tissue Engineering for Food, Princ.
Tissue Eng, 4th Ed., 2013, doi:10.1016/B978- 0- 12- 398358- 9.0 0 078-1.
[207] H. Vandenburgh, S. Kaufman, In vitro model for stretch-induced hypertrophy
of skeletal muscle, Science (80-.) (1979), doi:10.1126/science.569901.
[208] C.A. Powell, B.L. Smiley, J. Mills, H.H. Vandenburgh, Mechanical stimulation
improves tissue-engineered human skeletal muscle, Am. J. Physiol. - Cell
Physiol. (2002), doi:10.1152/ajpcell.00595.2001.
[209] D.G. Moon, G. Christ, J.D. Stitzel, A. Atala, J.J. Yoo, Cyclic mechanical precondi-
tioning improves engineered muscle contraction, Tissue Eng. - Part A. (2008),
doi:10.1089/tea.2007.0104.
[210] S. Rangarajan, L. Madden, N. Bursac, Use of flow, electrical, and mechanical
stimulation to promote engineering of striated muscles, Ann. Biomed. Eng.
(2014), doi:10.1007/s10439- 013- 0966- 4.
[211] D.C. Turner, A.M. Kasper, R.A. Seaborne, A.D. Brown, G.L. Close, M. Murphy,
C.E. Stewart, N.R.W. Martin, A.P. Sharples, Exercising bioengineered skeletal
muscle in vitro: biopsy to bioreactor, Methods Mol. Biol. (2019), doi:10.1007/
978- 1- 4939- 8897- 6_5.
[212] P. Juffer, A.D. Bakker, J. Klein-Nulend, R.T. Jaspers, Mechanical Loading by
Fluid Shear Stress of Myotube Glycocalyx Stimulates Growth Factor Expres-
sion and Nitric Oxide Production, Cell Biochem. Biophys. (2014), doi:10.1007/
s12013-013- 9812- 4.
[213] S. Naskar, V. Kumaran, B. Basu, On the Origin of Shear Stress Induced Myo-
genesis Using PMMA Based Lab-on-Chip, ACS Biomater. Sci. Eng. (2017),
doi:10.1021/acsbiomaterials.7b00206.
[214] A. Ramey-Ward, H. Su, K. Salaita, Mechanical Stimulation of Adhesion Re-
ceptors Using Light-Responsive Nanoparticle Actuators Enhances Myogenesis,
ACS Appl. Mater. Interfaces. (2020), doi:10.1021/acsami.0c08871.
[215] C.A. Cezar, E.T. Roche, H.H. Vandenburgh, G.N. Duda, C.J. Walsh, D.J. Mooney,
Biologic-free mechanically induced muscle regeneration, Proc. Natl. Acad. Sci.
U. S. A. (2016), doi:10.1073/pnas.1517517113.
[216] M.L.P. Langelaan, K.J.M. Boonen, K.Y. Rosaria-Chak, D.W.J. van der Schaft,
M.J. Post, F.P.T. Baaijens, Advanced maturation by electrical stimulation: dif-
ferences in response between C2C12 and primary muscle progenitor cells, J.
Tissue Eng. Regen. Med. (2011), doi:10.1002/term.345.
[217] I.-.C. Liao, J.B. Liu, N. Bursac, K.W. Leong, Effect of Electromechanical Stimula-
tion on the Maturation of Myotubes on Aligned Electrospun Fibers, Cell. Mol.
Bioeng. (2008), doi:10.1007/s12195- 008- 0021- y.
[218] S. Ahadian, J. Ramón-Azcón, S. Ostrovidov, G. Camci-Unal, V. Hosseini, H. Kaji,
K. Ino, H. Shiku, A. Khademhosseini, T. Matsue, Interdigitated array of Pt elec-
trodes for electrical stimulation and engineering of aligned muscle tissue, Lab
Chip. (2012), doi:10.1039/c2lc40479f.
[219] H. Kaji, T. Ishibashi, K. Nagamine, M. Kanzaki, M. Nishizawa, Electrically in-
duced contraction of C2C12 myotubes cultured on a porous membrane-based
substrate with muscle tissue-like stiffness, Biomaterials (2010), doi:10.1016/j.
biomaterials.2010.05.071.
[220] H. Park, R. Bhalla, R. Saigal, M. Radisic, N. Watson, R. Langer, G. Vunjak-
Novakovic, Effects of electrical stimulation in C2C12 muscle constructs, J. Tis-
sue Eng. Regen. Med. (2008), doi:10.1002/term.93.
[221] E. Serena, M. Flaibani, S. Carnio, L. Boldrin, L. Vitiello, P. De Coppi, N. El-
vassore, Electrophysiologic stimulation improves myogenic potential of mus-
cle precursor cells grown in a 3D collagen scaffold, Neurol. Res. (2008),
doi:10.1179/174313208X281109.
[222] A. Khodabukus, L. Madden, N.K. Prabhu, T.R. Koves, C.P. Jackman, D.M. Muoio,
N. Bursac, Electrical stimulation increases hypertrophy and metabolic flux in
tissue-engineered human skeletal muscle, Biomaterials (2019), doi:10.1016/j.
biomaterials.2018.08.058.
[223] K. Donnelly, A. Khodabukus, A. Philp, L. Deldicque, R.G. Dennis, K. Baar, A
Novel bioreactor for stimulating skeletal muscle in vitro, Tissue Eng. - Part C
Methods. (2010), doi:10.1089/ten.tec.2009.0125.
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
[224] R. Dong, P.X. Ma, B. Guo, Conductive biomaterials for muscle tissue engineer-
ing, Biomaterials (2020), doi:10.1016/j.biomaterials.2019.119584.
[225] I. Jun, S. Jeong, H. Shin, The stimulation of myoblast differentiation by
electrically conductive sub-micron fibers, Biomaterials (2009), doi:10.1016/j.
biomaterials.2008.12.063.
[226] K.J. Gilmore, M. Kita, Y. Han, A. Gelmi, M.J. Higgins, S.E. Moulton, G.M. Clark,
R. Kapsa, G.G. Wallace, Skeletal muscle cell proliferation and differentiation
on polypyrrole substrates doped with extracellular matrix components, Bio-
materials (2009), doi:10.1016/j.biomaterials.2009.06.059.
[227] R.D. Breukers, K.J. Gilmore, M. Kita, K.K. Wagner, M.J. Higgins, S.E. Moulton,
G.M. Clark, D.L. Officer, R.M.I. Kapsa, G.G. Wallace, Creating conductive struc-
tures for cell growth: growth and alignment of myogenic cell types on poly-
thiophenes, J. Biomed. Mater. Res. - Part A. (2010), doi:10.1002/jbm.a.32822.
[228] S. Ribeiro, A.C. Gomes, I. Etxebarria, S. Lanceros-Méndez, C. Ribeiro, Electroac-
tive biomaterial surface engineering effects on muscle cells differentiation,
Mater. Sci. Eng. C. (2018), doi:10.1016/j.msec.2018.07.044.
[229] M.C. Chen, Y.C. Sun, Y.H. Chen, Electrically conductive nanofibers with highly
oriented structures and their potential application in skeletal muscle tissue
engineering, Acta Biomater (2013), doi:10.1016/j.actbio.2012.10.024.
[230] S.H. Ku, S.H. Lee, C.B. Park, Synergic effects of nanofiber alignment and
electroactivity on myoblast differentiation, Biomaterials (2012), doi:10.1016/
j.biomaterials.2012.05.018.
[231] L.S. Moreira Teixeira, J. Feijen, C.A. van Blitterswijk, P.J. Dijkstra, M. Karperien,
Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue en-
gineering, Biomaterials (2012), doi:10.1016/j.biomaterials.2011.10.067.
[232] P. Bajaj, R.M. Schweller, A. Khademhosseini, J.L. West, R. Bashir, 3D biofabri-
cation strategies for tissue engineering and regenerative medicine, Annu. Rev.
Biomed. Eng. (2014), doi:10.1146/annurev- bioeng- 071813- 105155.
[233] R.J. Wade, J.A. Burdick, Engineering ECM signals into biomaterials, Mater. To-
day. (2012), doi:10.1016/S1369-7021(12)70197-9.
[234] O. Chaudhuri, Viscoelastic hydrogels for 3D cell culture, Biomater. Sci. (2017),
doi:10.1039/c7bm00261k.
[235] M. Geerligs, G.W.M. Peters, P.A.J. Ackermans, C.W.J. Oomens, F.P.T. Baai-
jens, Linear viscoelastic behavior of subcutaneous adipose tissue, Biorheology
(20 08), doi:10.3233/BIR-20 08-0517.
[236] D.D. McKinnon, D.W. Domaille, J.N. Cha, K.S. Anseth, Biophysically defined
and cytocompatible covalently adaptable networks as viscoelastic 3d cell cul-
ture systems, Adv. Mater. (2014), doi:10.1002/adma.201303680.
[237] A. Bauer, L. Gu, B. Kwee, W.A. Li, M. Dellacherie, A.D. Celiz, D.J. Mooney, Hy-
drogel substrate stress-relaxation regulates the spreading and proliferation of
mouse myoblasts, Acta Biomater. (2017), doi:10.1016/j.actbio.2017.08.041.
[238] C. Fan, D.A. Wang, Macroporous Hydrogel Scaffolds for Three-Dimensional
Cell Culture and Tissue Engineering, Tissue Eng. - Part B Rev. (2017), doi:10.
1089/ten.teb.2016.0465.
[239] A.R. Kang, J.S. Park, J. Ju, G.S. Jeong, S.H. Lee, Cell encapsulation via microtech-
nologies, Biomaterials (2014), doi:10.1016/j.biomaterials.2013.12.073.
[240] H.P. Lee, L. Gu, D.J. Mooney, M.E. Levenston, O. Chaudhuri, Mechanical con-
finement regulates cartilage matrix formation by chondrocytes, Nat. Mater.
(2017), doi:10.1038/nmat4993.
[241] O. Chaudhuri, L. Gu, D. Klumpers, M. Darnell, S.A. Bencherif, J.C. Weaver,
N. Huebsch, H.P. Lee, E. Lippens, G.N. Duda, D.J. Mooney, Hydrogels with tun-
able stress relaxation regulate stem cell fate and activity, Nat. Mater. (2016),
doi:10.1038/nmat4489.
[242] R. Myhan, M. Markowski, T. Daszkiewicz, P. Zapotoczny, P. Sadowski, Non-
linear stress relaxation model as a tool for evaluating the viscoelastic proper-
ties of meat products, J. Food Eng. (2015), doi:10.1016/j.jfoodeng.2014.09.006.
[243] M.M. Fitzgerald, K. Bootsma, J.A. Berberich, J.L. Sparks, Tunable stress re-
laxation behavior of an alginate-polyacrylamide hydrogel: comparison with
muscle tissue, Biomacromolecules (2015), doi:10.1021/bm501845j.
[244] J. Carballo, J. Ayo, F.J. Colmenero, Microbial transglutaminase and caseinate as
cold set binders: influence of meat species and chilling storage, LWT - Food
Sci. Technol. (2006), doi:10.1016/j.lwt.2005.03.020.
[245] C.B.B. Cazarin, G.C. Lima, J.K. da Silva, M.R. Maróstica Jr, Enzymes in Meat
Processing, in: M. Chandrasekaran (Ed.), Enzym. Food Beverage Process, 2015,
p. 337, doi:10.21048/ijnd.2019.56.2.23505.
[246] R. Lantto, K. Kruus, E. Puolanne, K. Honkapaa, K. Roininen, J. Buchert, En-
zymes in Meat Processing, in: R.J. Whitehurst, M. van Oort (Eds.), Enzym.
Food Technol, 2009, p. 264, doi:10.1002/9781444309935. Second Ed..
[247] S. Rourou, N. Riahi, S. Majoul, K. Trabelsi, H. Kallel, Development of an in
situ detachment protocol of Vero cells grown on Cytodex1 microcarriers un-
der animal component-free conditions in stirred bioreactor, Appl. Biochem.
Biotechnol. (2013), doi:10.1007/s12010- 013- 0307- y.
[248] S.R. Caruso, M.D. Orellana, A. Mizukami, T.R. Fernandes, A.M. Fontes,
C.A.T. Suazo, V.C. Oliveira, D.T. Covas, K. Swiech, Growth and functional har-
vesting of human mesenchymal stromal cells cultured on a microcarrier-
based system, Biotechnol. Prog. (2014), doi:10.1002/btpr.1886.
[249] Q.A. Rafiq, K. Coopman, A.W. Nienow, C.J. Hewitt, Systematic microcar-
rier screening and agitated culture conditions improves human mesenchy-
mal stem cell yield in bioreactors, Biotechnol. J. (2016), doi:10.1002/biot.
201400862.
[250] Q.A. Rafiq, M.P. Hanga, T.R.J. Heathman, K. Coopman, A.W. Nienow,
D.J. Williams, C.J. Hewitt, Process development of human multipotent stromal
cell microcarrier culture using an automated high-throughput microbioreac-
tor, Biotechnol. Bioeng. (2017), doi:10.1002/bit.26359.
[251] A.W. Nienow, C.J. Hewitt, T.R.J. Heathman, V.A.M. Glyn, G.N. Fonte,
M.P. Hanga, K. Coopman, Q.A. Rafiq, Agitation conditions for the culture and
19
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
detachment of hMSCs from microcarriers in multiple bioreactor platforms,
Biochem. Eng. J. (2016), doi:10.1016/j.bej.2015.08.003.
[252] M. Sponchioni, U. Capasso Palmiero, D. Moscatelli, Thermo-responsive poly-
mers: applications of smart materials in drug delivery and tissue engineering,
Mater. Sci. Eng. C. (2019), doi:10.1016/j.msec.2019.04.069.
[253] B. Chen, J. Dang, T.L. Tan, N. Fang, W.N. Chen, K.W. Leong, V. Chan, Dynam-
ics of smooth muscle cell deadhesion from thermosensitive hydroxybutyl chi-
tosan, Biomaterials (2007), doi:10.1016/j.biomaterials.2006.11.027.
[254] A. Tamura, M. Nishi, J. Kobayashi, K. Nagase, H. Yajima, M. Yamato, T. Okano,
Simultaneous enhancement of cell proliferation and thermally induced har-
vest efficiency based on temperature-responsive cationic copolymer-grafted
microcarriers, Biomacromolecules (2012), doi:10.1021/bm300256e.
[255] C.H. Chen, C.C. Tsai, W. Chen, F.L. Mi, H.F. Liang, S.C. Chen, H.W. Sung, Novel
living cell sheet harvest system composed of thermoreversible methylcellu-
lose hydrogels, Biomacromolecules (2006), doi:10.1021/bm0506400.
[256] A.K.A. Silva, C. Richard, G. Ducouret, M. Bessodes, D. Scherman, O.W. Merten,
Xyloglucan-derivatized films for the culture of adherent cells and their ther-
mocontrolled detachment: a promising alternative to cells sensitive to pro-
tease treatment, Biomacromolecules (2013), doi:10.1021/bm3017737.
[257] D. Billig, J.M. Clark, A.J. Ewell, C.M. Carter, C. Gebb, The separation of har-
vested cells from microcarriers: a comparison of methods, Dev. Biol. Stand.
(1983).
[258] R. Moloudi, S. Oh, C. Yang, K.L. Teo, A.T.L. Lam, M.E. Warkiani,
M.W. Naing, Inertial-Based Filtration Method for Removal of Micro-
carriers from Mesenchymal Stem Cell Suspensions, Sci. Rep. (2018),
doi:10.1038/s41598- 018- 31019- y.
[259] B. Cunha, C. Peixoto, M.M. Silva, M.J.T. Carrondo, M. Serra, P.M. Alves, Fil-
tration methodologies for the clarification and concentration of human mes-
enchymal stem cells, J. Memb. Sci. (2015), doi:10.1016/j.memsci.2014.12.041.
[260] C.Y. Lin, C.H. Huang, Y.K. Wu, N.C. Cheng, J. Yu, Maintenance of human adi-
pose derived stem cell (hASC) differentiation capabilities using a 3D culture,
Biotechnol. Lett. (2014), doi:10.1007/s10529- 014- 1500- y.
[261] H.A. Grabitske, J.L. Slavin, Low-Digestible Carbohydrates in Practice, J. Am.
Diet. Assoc. (2008), doi:10.1016/j.jada.2008.07.010.
[262] K.I. Hilgendorf, C.T. Johnson, A. Mezger, S.L. Rice, A.M. Norris, J. Demeter,
W.J. Greenleaf, J.F. Reiter, D. Kopinke, P.K. Jackson, Omega-3 Fatty Acids Ac-
tivate Ciliary FFAR4 to Control Adipogenesis, Cell 179 (2019) 1289–1305 e21,
doi:10.1016/j.cell.2019.11.005.
[263] L. Specht, An Analysis of Culture Medium Costs and Production Volumes For
Cultivated Meat, Good Food Inst., Washington, DC, USA, 2020.
[264] A.W. Xie, W.L. Murphy, Engineered biomaterials to mitigate growth fac-
tor cost in cell biomanufacturing, Curr. Opin. Biomed. Eng. 10 (2019) 1–10,
doi:10.1016/j.cobme.2018.12.004.
[265] C. Borselli, H. Storrie, F. Benesch-Lee, D. Shvartsman, C. Cezar, J.W. Lichtman,
H.H. Vandenburgh, D.J. Mooney, Functional muscle regeneration with com-
bined delivery of angiogenesis and myogenesis factors, Proc. Natl. Acad. Sci.
U. S. A. (2010), doi:10.1073/pnas.0903875106.
[266] M.J. Worrallo, R.L.L. Moore, K.E. Glen, R.J. Thomas, Immobilized hematopoietic
growth factors onto magnetic particles offer a scalable strategy for cell ther-
apy manufacturing in suspension cultures, Biotechnol. J. (2017), doi:10.1002/
biot.201600493.
[267] S. Rinker, Y. Ke, Y. Liu, R. Chhabra, H. Yan, Self-assembled DNA nanostructures
for distance-dependent multivalent ligand-protein binding, Nat. Nanotechnol.
(2008), doi:10.1038/nnano.2008.164.
[268] D.G. Belair, N.N. Le, W.L. Murphy, Design of growth factor sequestering bio-
materials, Chem. Commun. (2014), doi:10.1039/c4cc04317k.
[269] S. Anastasi, S. Giordano, O. Sthandier, G. Gambarotta, R. Maione, P. Comoglio,
P. Amati, A natural hepatocyte growth factor/scatter factor autocrine loop in
myoblast cells and the effect of the constitutive met kinase activation on
myogenic differentiation, J. Cell Biol. (1997), doi:10.1083/jcb.137.5.1057.
[270] G.R. Adams, Invited Review: autocrine/paracrine IGF-I and skeletal muscle
adaptation, J. Appl. Physiol. (2002), doi:10.1152/japplphysiol.01264.2001.
[271] M. Hemmingsen, S. Vedel, P. Skafte-Pedersen, D. Sabourin, P. Collas, H. Bruus,
M. Dufva, The Role of Paracrine and Autocrine Signaling in the Early Phase of
Adipogenic Differentiation of Adipose-derived Stem Cells, PLoS ONE (2013),
doi:10.1371/journal.pone.0063638.
[272] M.R.I. Shishir, L. Xie, C. Sun, X. Zheng, W. Chen, Advances in micro and
nano-encapsulation of bioactive compounds using biopolymer and lipid-
based transporters, Trends Food Sci. Technol. 78 (2018) 34–60, doi:10.1016/
j.tifs.2018.05.018.
[273] B. Ghorani, N. Tucker, Fundamentals of Electrospinning As a Novel Delivery
Vehicle For Bioactive Compounds in Food Nanotechnology, Food Hydrocoll.,
2015, doi:10.1016/j.foodhyd.2015.05.024.
[274] G. Liu, X. Wang, X. Sun, C. Deng, A. Atala, Y. Zhang, The effect of urine-derived
stem cells expressing VEGF loaded in collagen hydrogels on myogenesis and
innervation following after subcutaneous implantation in nude mice, Bioma-
terials (2013), doi:10.1016/j.biomaterials.2013.07.077.
[275] J. Zou, W. Wang, A.T. Neffe, X. Xu, Z. Li, Z. Deng, X. Sun, N. Ma, A. Lendlein,
Adipogenic differentiation of human adipose derived mesenchymal stem cells
in 3D architectured gelatin based hydrogels (ArcGel), Clin. Hemorheol. Micro-
circ. (2017), doi:10.3233/CH-179210.
[276] C.A. Rossi, M. Flaibani, B. Blaauw, M. Pozzobon, E. Figallo, C. Reggiani, L. Vi-
tiello, N. Elvassore, P. De Coppi, In vivo tissue engineering of functional skele-
tal muscle by freshly isolated satellite cells embedded in a photopolymeriz-
able hydrogel, FASEB J (2011), doi:10.1096/fj.10-174755.
[277] W. Wang, M. Fan, L. Zhang, S.H. Liu, L. Sun, C.Y. Wang, Compatibility of
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
hyaluronic acid hydrogel and skeletal muscle myoblasts, Biomed. Mater.
(2009), doi:10.1088/1748-6041/4/2/025011.
[278] F.S. Kamelger, R. Marksteiner, E. Margreiter, G. Klima, G. Wechselberger,
S. Hering, H. Piza, A comparative study of three different biomaterials in the
engineering of skeletal muscle using a rat animal model, Biomaterials (2004),
doi:10.1016/S0142- 9612(03)00520- 9.
[279] M. Halbleib, T. Skurk, C. De Luca, D. Von Heimburg, H. Hauner, Tissue engi-
neering of white adipose tissue using hyaluronic acid-based scaffolds. I: in
vitro differentiation of human adipocyte precursor cells on scaffolds, Bioma-
terials 24 (2003) 3125–3132, doi:10.1016/S0142-9612(03)00156-X.
[280] L. Flynn, G.D. Prestwich, J.L. Semple, K.A. Woodhouse, Adipose tissue engi-
neering with naturally derived scaffolds and adipose-derived stem cells, Bio-
materials 28 (2007) 3834–3842, doi:10.1016/j.biomaterials.20 07.05.0 02.
[281] J.P. Beier, J. Stern-Straeter, V.T. Foerster, U. Kneser, G.B. Stark, A.D. Bach, Tissue
engineering of injectable muscle: three-dimensional myoblast-fibrin injection
in the syngeneic rat animal model, Plast. Reconstr. Surg. (2006), doi:10.1097/
01.prs.0 0 0 02210 07.97115.1d.
[282] G.H. Borschel, D.E. Dow, R.G. Dennis, D.L. Brown, Tissue-engineered axially
vascularized contractile skeletal muscle, Plast. Reconstr. Surg. (2006), doi:10.
1097/01.prs.0 0 0 0224295.54073.49.
[283] J. Liu, H.H.K. Xu, H. Zhou, M.D. Weir, Q. Chen, C.A. Trotman, Human umbilical
cord stem cell encapsulation in novel macroporous and injectable fibrin for
muscle tissue engineering, Acta Biomater (2013), doi:10.1016/j.actbio.2012.08.
009.
[284] F. Mehta, R. Theunissen, M.J. Post, Adipogenesis from bovine precursors,
Methods Mol. Biol. (2019), doi:10.1007/978- 1- 4939- 8897- 6_8.
[285] V. Guneta, Q.L. Loh, C. Choong, Cell-secreted extracellular matrix formation
and differentiation of adipose-derived stem cells in 3D alginate scaffolds with
tunable properties, J. Biomed. Mater. Res. - Part A. 104 (2016) 1090–1101,
doi:10.1002/jbm.a.35644.
[286] S.W. Kang, B.H. Cha, H. Park, K.S. Park, K.Y. Lee, S.H. Lee, The Effect of Con-
jugating RGD into 3D Alginate Hydrogels on Adipogenic Differentiation of
Human Adipose-Derived Stromal Cells, Macromol. Biosci. 11 (2011) 673–679,
doi:10.1002/mabi.201000479.
[287] W.S. Kim, D.J. Mooney, P.R. Arany, K. Lee, N. Huebsch, J. Kim, Adipose tissue
engineering using injectable, oxidized alginate hydrogels, Tissue Eng. - Part
A. (2012), doi:10.1089/ten.tea.2011.0250.
[288] E. Zakhem, S. Raghavan, R.R. Gilmont, K.N. Bitar, Chitosan-based scaffolds for
the support of smooth muscle constructs in intestinal tissue engineering, Bio-
materials (2012), doi:10.1016/j.biomaterials.2012.03.051.
[289] M.H. Kim, H.N. Hong, J.P. Hong, C.J. Park, S.W. Kwon, S.H. Kim, G. Kang,
M.J. Kim, The effect of VEGF on the myogenic differentiation of adipose tis-
sue derived stem cells within thermosensitive hydrogel matrices, Biomateri-
als (2010), doi:10.1016/j.biomaterials.2009.10.057.
[290] J.H. Elisseeff, A.T. Hillel, S. Unterman, Z. Nahas, B. Reid, J.M. Coburn, J. Axel-
man, J.J. Chae, Q.Y. Guo, R. Trow, A. Thomas, Z.P. Hou, S. Lichtsteiner, D. Sut-
ton, C. Matheson, P. Walker, N. David, S. Mori, J.M. Taube, Photoactivated
Composite Biomaterial for Soft Tissue Restoration in Rodents and in Humans,
Sci. Transl. Med. (2011).
[291] E.M. Cronin, F.A. Thurmond, R. Bassel-Duby, R.S. Williams, W.E. Wright,
K.D. Nelson, H.R. Garner, Protein-coated poly(L-lactic acid) fibers provide a
substrate for differentiation of human skeletal muscle cells, J. Biomed. Mater.
Res. - Part A. (2004), doi:10.1002/jbm.a.30009.
[292] C.W. Patrick, P.B. Chauvin, J. Hobley, G.P. Reece, Preadipocyte seeded PLGA
scaffolds for adipose tissue engineering, Tissue Eng (1999), doi:10.1089/ten.
1999.5.139.
[293] C. Brännmark, A. Paul, D. Ribeiro, B. Magnusson, G. Brolén, A. Enejder,
A. Forslöw, Increased adipogenesis of human adipose-derived stem cells on
polycaprolactone fiber matrices, PLoS ONE 9 (2014) 1–27, doi:10.1371/journal.
pone.0113620.
[294] B. Perniconi, A. Costa, P. Aulino, L. Teodori, S. Adamo, D. Coletti, The pro-
myogenic environment provided by whole organ scale acellular scaffolds
from skeletal muscle, Biomaterials (2011), doi:10.1016/j.biomaterials.2011.07.
016.
[295] M.T. Wolf, K.A. Daly, J.E. Reing, S.F. Badylak, Biologic scaffold composed
of skeletal muscle extracellular matrix, Biomaterials (2012), doi:10.1016/j.
biomaterials.2011.12.055.
[296] B.M. Sicari, J. Peter Rubin, C.L. Dearth, M.T. Wolf, F. Ambrosio, M. Boninger,
N.J. Turner, D.J. Weber, T.W. Simpson, A. Wyse, E.H.P. Brown, J.L. Dziki,
L.E. Fisher, S. Brown, S.F. Badylak, An acellular biologic scaffold promotes
skeletal muscle formation in mice and humans with volumetric muscle loss,
Sci. Transl. Med. (2014), doi:10.1126/scitranslmed.3008085.
[297] J.A. Werkmeister, J.A.M. Ramshaw, Recombinant protein scaffolds for tissue
engineering, Biomed. Mater. (2012), doi:10.1088/1748-6041/7/1/012002.
[298] Q. Wang, C. Zhong, H. Xiao, Genetic Engineering of Filamentous Fungi for Ef-
ficient Protein Expression and Secretion, Front. Bioeng. Biotechnol. 8 (2020)
1–8, doi:10.3389/fbioe.2020.00293.
[299] T. Wang, J. Lew, J. Premkumar, C.L. Poh, M.Win Naing, Production of recom-
binant collagen: state of the art and challenges, Eng. Biol. 1 (2017) 18–23,
doi:10.1049/enb.2017.0 0 03.
[300] S. Gomes, I.B. Leonor, J.F. Mano, R.L. Reis, D.L. Kaplan, Natural and genetically
engineered proteins for tissue engineering, Prog. Polym. Sci. (2012), doi:10.
1016/j.progpolymsci.2011.07.003.
[301] W. Suntornsuk, P. Pochanavanich, L. Suntornsuk, Fungal chitosan produc-
tion on food processing by-products, Process Biochem. (2002), doi:10.1016/
S0 032-9592(01)0 0265-5.
20
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
[302] G. Blüml, Microcarrier Cell Culture Principles and Methods, in: Handbooks
from GE Healthc, 2021, doi:10.1007/978- 1- 59745- 399- 8_5.
[303] W. Li, Y. Han, H. Yang, G. Wang, R. Lan, J.Y. Wang, Preparation of microcarriers
based on zein and their application in cell culture, Mater. Sci. Eng. C. (2016),
doi:10.1016/j.msec.2015.09.045.
[304] C. Wang, Y. Gong, Y. Lin, J. Shen, D.A. Wang, A novel gellan gel-based micro-
carrier for anchorage-dependent cell delivery, Acta Biomater. (2008), doi:10.
1016/j.actbio.20 08.03.0 08.
[305] B. Sarker, A.R. Boccaccini, Alginate Utilization in Tissue Engineering and Cell
Therapy, in: B.H.A. Rehm, M.F. Moradali (Eds.), Alginates Their Biomed, Appl.,
2018, pp. 121–155, doi:10.1007/978- 981- 10- 6910- 9_5.
[306] A.B. Bello, D. Kim, D. Kim, H. Park, S.H. Lee, Engineering and functionalization
of gelatin biomaterials: from cell culture to medical applications, Tissue Eng.
- Part B Rev. (2020), doi:10.1089/ten.teb.2019.0256.
[307] E.G. Popa, M.E. Gomes, R.L. Reis, Cell delivery systems using alginate-
carrageenan hydrogel beads and fibers for regenerative medicine applications,
Biomacromolecules (2011), doi:10.1021/bm200965x.
[308] S.M. Mihaila, A.K. Gaharwar, R.L. Reis, A.P. Marques, M.E. Gomes,
A. Khademhosseini, Photocrosslinkable kappa-carrageenan hydrogels for
tissue engineering applications, Adv. Healthc. Mater. 2 (2013) 895–907,
doi:10.10 02/adhm.20120 0317.
[309] J.V. Araujo, N. Davidenko, M. Danner, R.E. Cameron, S.M. Best, Novel porous
scaffolds of pH responsive chitosan/carrageenan-based polyelectrolyte com-
plexes for tissue engineering, J. Biomed. Mater. Res. - Part A. (2014), doi:10.
1002/jbm.a.35128.
[310] L. Tytgat, M. Vagenende, H. Declercq, J.C. Martins, H. Thienpont, H. Ottevaere,
P. Dubruel, S. Van Vlierberghe, Synergistic effect of κ -carrageenan and gelatin
blends towards adipose tissue engineering, Carbohydr. Polym. 189 (2018) 1–9,
doi:10.1016/j.carbpol.2018.02.002.
[311] R. Yegappan, V. Selvaprithiviraj, S. Amirthalingam, R. Jayakumar, Carrageenan
based hydrogels for drug delivery, tissue engineering and wound healing, Car-
bohydr. Polym. 198 (2018) 385–400, doi:10.1016/j.carbpol.2018.06.086.
[312] R. Yegappan, V. Selvaprithiviraj, S. Amirthalingam, A. Mohandas, N.S. Hwang,
R. Jayakumar, Injectable angiogenic and osteogenic carrageenan nanocompos-
ite hydrogel for bone tissue engineering, Int. J. Biol. Macromol. 122 (2019)
320–328, doi:10.1016/j.ijbiomac.2018.10.182.
[313] S.M. Mihaila, E.G. Popa, R.L. Reis, A.P. Marques, M.E. Gomes, Fabrication of
endothelial cell-laden carrageenan microfibers for microvascularized bone
tissue engineering applications, Biomacromolecules 15 (2014) 2849–2860,
doi:10.1021/bm50 0 036a.
[314] E.G. Popa, S.G. Caridade, J.F. Mano, R.L. Reis, M.E. Gomes, Chondrogenic po-
tential of injectableκ -carrageenan hydrogel with encapsulated adipose stem
cells for cartilage tissue-engineering applications, J. Tissue Eng. Regen. Med.
(2015), doi:10.1002/term.1683.
[315] P.M. Rocha, V.E. Santo, M.E. Gomes, R.L. Reis, J.F. Mano, Encapsula-
tion of adipose-derived stem cells and transforming growth factor-β 1 in
carrageenan-based hydrogels for cartilage tissue engineering, J. Bioact. Com-
pat. Polym. (2011), doi:10.1177/0883911511420700.
[316] Y. Deng, M. Huang, D. Sun, Y. Hou, Y. Li, T. Dong, X. Wang, L. Zhang, W. Yang,
Dual Physically Cross-Linked κ -Carrageenan-Based Double Network Hydro-
gels with Superior Self-Healing Performance for Biomedical Application, ACS
Appl. Mater. Interfaces. (2018), doi:10.1021/acsami.8b15385.
[317] X. Qi, T. Su, M. Zhang, X. Tong, W. Pan, Q. Zeng, Z. Zhou, L. Shen, X. He,
J. Shen, Macroporous Hydrogel Scaffolds with Tunable Physicochemical Prop-
erties for Tissue Engineering Constructed Using Renewable Polysaccharides,
ACS Appl. Mater. Interfaces. (2020), doi:10.1021/acsami.9b20794.
[318] F. Munarin, S.G. Guerreiro, M.A. Grellier, M.C. Tanzi, M.A. Barbosa, P. Petrini,
P.L. Granja, Pectin-based injectable biomaterials for bone tissue engineering,
Biomacromolecules (2011), doi:10.1021/bm101110x.
[319] N. Ninan, M. Muthiah, I.K. Park, A. Elain, S. Thomas, Y. Grohens,
Pectin/carboxymethyl cellulose/microfibrillated cellulose composite scaffolds
for tissue engineering, Carbohydr. Polym. 98 (2013) 877–885, doi:10.1016/j.
carbpol.2013.06.067.
[320] R.F. Pereira, C.C. Barrias, P.J. Bártolo, P.L. Granja, Cell-instructive pectin hy-
drogels crosslinked via thiol-norbornene photo-click chemistry for skin tis-
sue engineering, Acta Biomater 66 (2018) 282–293, doi:10.1016/j.actbio.2017.
11.016.
[321] M.M. Fares, E. Shirzaei Sani, R. Portillo Lara, R.B. Oliveira, A. Khademhos-
seini, N. Annabi, Interpenetrating network gelatin methacryloyl (GelMA) and
pectin-g-PCL hydrogels with tunable properties for tissue engineering, Bio-
mater. Sci. (2018), doi:10.1039/c8bm00474a.
[322] M. Mehrali, A. Thakur, F.B. Kadumudi, M.K. Pierchala, J.A.V. Cordova,
M.A. Shahbazi, M. Mehrali, C.P. Pennisi, G. Orive, A.K. Gaharwar,
A. Dolatshahi-Pirouz, Pectin Methacrylate (PEMA) and Gelatin-Based Hy-
drogels for Cell Delivery: converting Waste Materials into Biomaterials, ACS
Appl. Mater. Interfaces. 11 (2019) 12283–12297, doi:10.1021/acsami.9b00154.
[323] A. Lapomarda, A. De Acutis, I. Chiesa, G.M. Fortunato, F. Montemurro, C. De
Maria, M. Mattioli Belmonte, R. Gottardi, G. Vozzi, Pectin-GPTMS-Based
Biomaterial: toward a Sustainable Bioprinting of 3D scaffolds for Tissue
Engineering Application, Biomacromolecules (2020), doi:10.1021/acs.biomac.
9b01332.
[324] S. Chen, S. Cui, H. Zhang, X. Pei, J. Hu, Y. Zhou, Y. Liu, Cross-Linked Pectin
Nanofibers with Enhanced Cell Adhesion, Biomacromolecules (2018), doi:10.
1021/acs.biomac.7b01605.
[325] F. Chen, Y. Ni, B. Liu, T. Zhou, C. Yu, Y. Su, X. Zhu, X. Yu, Y. Zhou, Self-
crosslinking and injectable hyaluronic acid/RGD-functionalized pectin hydro-
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
gel for cartilage tissue engineering, Carbohydr. Polym. (2017), doi:10.1016/j.
carbpol.2017.02.059.
[326] J.G. Martins, S.E.A. Camargo, T.T. Bishop, K.C. Popat, M.J. Kipper, A.F. Martins,
Pectin-chitosan membrane scaffold imparts controlled stem cell adhesion and
proliferation, Carbohydr. Polym. (2018), doi:10.1016/j.carbpol.2018.05.062.
[327] Y. Zhao, M. He, L. Zhao, S. Wang, Y. Li, L. Gan, M. Li, L. Xu, P.R. Chang,
D.P. Anderson, Y. Chen, Epichlorohydrin-Cross-linked Hydroxyethyl Cellu-
lose/Soy Protein Isolate Composite Films as Biocompatible and Biodegrad-
able Implants for Tissue Engineering, ACS Appl. Mater. Interfaces. (2016),
doi:10.1021/acsami.5b11152.
[328] K. Rodríguez, J. Sundberg, P. Gatenholm, S. Renneckar, Electrospun nanofi-
brous cellulose scaffolds with controlled microarchitecture, Carbohydr. Polym.
100 (2014) 143–149, doi:10.1016/j.carbpol.2012.12.037.
[329] J.R. Gershlak, S. Hernandez, G. Fontana, L.R. Perreault, K.J. Hansen, S.A. Lar-
son, B.Y.K. Binder, D.M. Dolivo, T. Yang, T. Dominko, M.W. Rolle, P.J. Weath-
ers, F. Medina-Bolivar, C.L. Cramer, W.L. Murphy, G.R. Gaudette, Crossing king-
doms: using decellularized plants as perfusable tissue engineering scaffolds,
Biomaterials. 125 (2017) 13–22, doi:10.1016/j.biomaterials.2017.02.011.
[330] Y. Huang, J. Wang, F. Yang, Y. Shao, X. Zhang, K. Dai, Modification and eval-
uation of micro-nano structured porous bacterial cellulose scaffold for bone
tissue engineering, Mater. Sci. Eng. C. (2017), doi:10.1016/j.msec.2017.02.174.
[331] J. Ran, P. Jiang, S. Liu, G. Sun, P. Yan, X. Shen, H. Tong, Con-
structing multi-component organic/inorganic composite bacterial cellulose-
gelatin/hydroxyapatite double-network scaffold platform for stem cell-
mediated bone tissue engineering, Mater. Sci. Eng. C. (2017), doi:10.1016/j.
msec.2017.04.062.
[332] Y. Zhao, M. He, H. Jin, L. Zhao, Q. Du, H. Deng, W. Tian, Y. Li, X. Lv, Y. Chen,
Construction of highly biocompatible hydroxyethyl cellulose/soy protein iso-
late composite sponges for tissue engineering, Chem. Eng. J. (2018), doi:10.
1016/j.cej.2018.02.046.
[333] R.J. Hickey, D.J. Modulevsky, C.M. Cuerrier, A.E. Pelling, Customizing the
Shape and Microenvironment Biochemistry of Biocompatible Macroscopic
Plant-Derived Cellulose Scaffolds, ACS Biomater. Sci. Eng. (2018), doi:10.1021/
acsbiomaterials.8b00178.
[334] S. Ahn, C.O. Chantre, A.R. Gannon, J.U. Lind, P.H. Campbell, T. Grevesse,
B.B. O’Connor, K.K. Parker, Soy Protein/Cellulose Nanofiber Scaffolds Mim-
icking Skin Extracellular Matrix for Enhanced Wound Healing, Adv. Healthc.
Mater. (2018), doi:10.1002/adhm.201701175.
[335] A. Tiwari, J.J. Grailer, S. Pilla, D.A. Steeber, S. Gong, Biodegradable hydrogels
based on novel photopolymerizable guar gum-methacrylate macromonomers
for in situ fabrication of tissue engineering scaffolds, Acta Biomater. (2009),
doi:10.1016/j.actbio.20 09.06.0 01.
[336] M. Ragothaman, T. Palanisamy, C. Kalirajan, Collagen-poly(dialdehyde) guar
gum based porous 3D scaffolds immobilized with growth factor for tissue en-
gineering applications, Carbohydr. Polym. (2014), doi:10.1016/j.carbpol.2014.
08.045.
[337] S. Kundu, A. Das, A. Basu, D. Ghosh, P. Datta, A. Mukherjee, Carboxymethyl
guar gum synthesis in homogeneous phase and macroporous 3D scaffolds
design for tissue engineering, Carbohydr. Polym. (2018), doi:10.1016/j.carbpol.
2018.03.007.
[338] D. Anandan, G. Madhumathi, N.A. Nambiraj, A.K. Jaiswal, Gum based 3D com-
posite scaffolds for bone tissue engineering applications, Carbohydr. Polym.
214 (2019) 62–70, doi:10.1016/j.carbpol.2019.03.020.
[339] L.R. Stevens, K.J. Gilmore, G.G. Wallace, M. In het Panhuis, Tissue engi-
neering with gellan gum, Biomater. Sci. 4 (2016) 1276–1290, doi:10.1039/
c6bm00322b.
[340] C.A. Vilela, C. Correia, A. da Silva Morais, T.C. Santos, A.C. Gertrudes,
E.S. Moreira, A.M. Frias, D.A. Learmonth, P. Oliveira, J.M. Oliveira, R.A. Sousa,
J.D. Espregueira-Mendes, R.L. Reis, In vitro and in vivo performance of
methacrylated gellan gum hydrogel formulations for cartilage repair∗ , J.
Biomed. Mater. Res. - Part A. (2018), doi:10.1002/jbm.a.36406.
[341] Y. Gong, C. Wang, R.C. Lai, K. Su, F. Zhang, D.A. Wang, An improved injectable
polysaccharide hydrogel: modified gellan gum for long-term cartilage regen-
eration in vitro, J. Mater. Chem. 19 (2009) 1968–1977, doi:10.1039/b818090c.
[342] C.J. Ferris, L.R. Stevens, K.J. Gilmore, E. Mume, I. Greguric, D.M. Kirchmajer,
G.G. Wallace, M. In Het Panhuis, Peptide modification of purified gellan gum,
J. Mater. Chem. B. (2015), doi:10.1039/c4tb01727g.
[343] N.A. Silva, M.J. Cooke, R.Y. Tam, N. Sousa, A.J. Salgado, R.L. Reis, M.S. Shoichet,
The effects of peptide modified gellan gum and olfactory ensheathing glia
cells on neural stem/progenitor cell fate, Biomaterials (2012), doi:10.1016/j.
biomaterials.2012.05.050.
[344] J.T. Koivisto, C. Gering, J. Karvinen, R. Maria Cherian, B. Belay, J. Hyttinen,
K. Aalto-Setälä, M. Kellomäki, J. Parraga, Mechanically Biomimetic Gelatin-
Gellan Gum Hydrogels for 3D Culture of Beating Human Cardiomyocytes, ACS
Appl. Mater. Interfaces. (2019), doi:10.1021/acsami.8b22343.
[345] J.T. Oliveira, L. Martins, R. Picciochi, P.B. Malafaya, R.A. Sousa, N.M. Neves,
J.F. Mano, R.L. Reis, Gellan gum: a new biomaterial for cartilage tissue en-
gineering applications, J. Biomed. Mater. Res. - Part A. 93 (2010) 852–863,
doi:10.1002/jbm.a.32574.
[346] D. Dyondi, T.J. Webster, R. Banerjee, A nanoparticulate injectable hydrogel as
a tissue engineering scaffold for multiple growth factor delivery for bone re-
generation, Int. J. Nanomedicine. (2012), doi:10.2147/IJN.S37953.
[347] L.P. Da Silva, M.T. Cerqueira, R.A. Sousa, R.L. Reis, V.M. Correlo, A.P. Marques,
Engineering cell-adhesive gellan gum spongy-like hydrogels for regenerative
medicine purposes, Acta Biomater (2014), doi:10.1016/j.actbio.2014.07.009.
[348] D.F. Coutinho, A.F. Ahari, N.N. Kachouie, M.E. Gomes, N.M. Neves, R.L. Reis,
21
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
A. Khademhosseini, An automated two-phase system for hydrogel microbead
production, Biofabrication (2012), doi:10.1088/1758-5082/4/3/035003.
[349] A.C. Mendes, E.T. Baran, R.C. Pereira, H.S. Azevedo, R.L. Reis, Encapsulation
and Survival of a Chondrocyte Cell Line within Xanthan Gum Derivative,
Macromol. Biosci. (2012), doi:10.1002/mabi.201100304.
[350] A. Kumar, K.M. Rao, S.E. Kwon, Y.N. Lee, S.S. Han, Xanthan gum/bioactive
silica glass hybrid scaffolds reinforced with cellulose nanocrystals: morpho-
logical, mechanical and in vitro cytocompatibility study, Mater. Lett. (2017),
doi:10.1016/j.matlet.2017.01.143.
[351] A. Kumar, K.M. Rao, S.S. Han, Application of xanthan gum as polysaccha-
ride in tissue engineering: a review, Carbohydr. Polym. (2018), doi:10.1016/
j.carbpol.2017.10.009.
[352] A.C. Mendes, E.T. Baran, C. Nunes, M.A. Coimbra, H.S. Azevedo, R.L. Reis,
Palmitoylation of xanthan polysaccharide for self-assembly microcapsule for-
mation and encapsulation of cells in physiological conditions, Soft Matter
(2011), doi:10.1039/c1sm05594a.
[353] A.C. Mendes, E.T. Baran, R.L. Reis, H.S. Azevedo, Fabrication of phospholipid-
xanthan microcapsules by combining microfluidics with self-assembly, Acta
Biomater. (2013), doi:10.1016/j.actbio.2013.01.035.
[354] V.B. Bueno, R. Bentini, L.H. Catalani, L.R.S. Barbosa, D.F.S. Petri, Synthesis and
characterization of xanthan-hydroxyapatite nanocomposites for cellular up-
take, Mater. Sci. Eng. C. (2014), doi:10.1016/j.msec.2014.01.002.
[355] Z. Li, Y. Su, B. Xie, X. Liu, X. Gao, D. Wang, A novel biocompatible double
network hydrogel consisting of konjac glucomannan with high mechanical
strength and ability to be freely shaped, J. Mater. Chem. B. (2015), doi:10.
1039/c4tb01653j.
[356] J. Tang, J. Chen, J. Guo, Q. Wei, H. Fan, Construction and evaluation of fibrillar
composite hydrogel of collagen/konjac glucomannan for potential biomedical
applications, Regen. Biomater. (2018), doi:10.1093/rb/rby018.
[357] H. Kanniyappan, P. Thangavel, S. Chakraborty, V. Arige, V. Muthuvijayan, De-
sign and evaluation of Konjac glucomannan-based bioactive interpenetrating
network (IPN) scaffolds for engineering vascularized bone tissues, Int. J. Biol.
Macromol. (2020), doi:10.1016/j.ijbiomac.2019.12.012.
[358] H. Zhang, S. Cui, H. Lv, X. Pei, M. Gao, S. Chen, J. Hu, Y. Zhou, Y. Liu, A
crosslinking strategy to make neutral polysaccharide nanofibers robust and
biocompatible: with konjac glucomannan as an example, Carbohydr. Polym.
(2019), doi:10.1016/j.carbpol.2019.03.075.
[359] L. Sun, Z. Xiong, W. Zhou, R. Liu, X. Yan, J. Li, W. An, G. Yuan, G. Ma, Z. Su,
Novel konjac glucomannan microcarriers for anchorage-dependent animal
cell culture, Biochem. Eng. J. (2015), doi:10.1016/j.bej.2014.12.012.
[360] J. Chen, Z. Cai, Q. Wei, D. Wang, J. Wu, Y. Tan, J. Lu, H. Ai, Proanthocyanidin-
crosslinked collagen/konjac glucomannan hydrogel with improved mechani-
cal properties and MRI trackable biodegradation for potential tissue engineer-
ing scaffolds, J. Mater. Chem. B. (2020), doi:10.1039/c9tb02053e.
[361] Q. Jiang, N. Reddy, Y. Yang, Cytocompatible cross-linking of electrospun zein
fibers for the development of water-stable tissue engineering scaffolds, Acta
Biomater (2010), doi:10.1016/j.actbio.2010.04.024.
[362] S. Gong, H. Wang, Q. Sun, S.T. Xue, J.Y. Wang, Mechanical properties and in
vitro biocompatibility of porous zein scaffolds, Biomaterials (2006), doi:10.
1016/j.biomaterials.2006.02.019.
[363] D. Dippold, M. Tallawi, S. Tansaz, J.A. Roether, A.R. Boccaccini, Novel electro-
spun poly(glycerol sebacate)-zein fiber mats as candidate materials for car-
diac tissue engineering, Eur. Polym. J. (2016), doi:10.1016/j.eurpolymj.2015.12.
030.
[364] J. Tu, H. Wang, H. Li, K. Dai, J. Wang, X. Zhang, The in vivo bone formation by
mesenchymal stem cells in zein scaffolds, Biomaterials (2009), doi:10.1016/j.
biomaterials.2009.04.054.
[365] H. Xu, S. Cai, A. Sellers, Y. Yang, Intrinsically water-stable electrospun three-
dimensional ultrafine fibrous soy protein scaffolds for soft tissue engineering
using adipose derived mesenchymal stem cells, RSC Adv. (2014), doi:10.1039/
c3ra47547f.
[366] N. Reddy, Y. Yang, Soyprotein fibers with high strength and water stability for
potential medical applications, Biotechnol. Prog. (2009), doi:10.1002/btpr.244.
[367] K.B. Chien, R.N. Shah, Novel soy protein scaffolds for tissue regeneration: ma-
terial characterization and interaction with human mesenchymal stem cells,
Acta Biomater. (2012), doi:10.1016/j.actbio.2011.09.036.
[368] K.B. Chien, E. Makridakis, R.N. Shah, Three-dimensional printing of soy pro-
tein scaffolds for tissue regeneration, Tissue Eng. - Part C Methods. (2013),
doi:10.1089/ten.tec.2012.0383.
[369] H. Olami, M. Zilberman, Microstructure and in vitro cellular response to novel
soy protein-based porous structures for tissue regeneration applications, J.
Biomater. Appl. (2016), doi:10.1177/0885328215614713.
[370] J. Huang, K. Huang, X. You, G. Liu, G. Hollett, Y. Kang, Z. Gu, J. Wu, Evaluation
of tofu as a potential tissue engineering scaffold, J. Mater. Chem. B. (2018),
doi:10.1039/c7tb02852k.
[371] K. Huang, Z. Gu, J. Wu, Tofu-Incorporated Hydrogels for Potential Bone
Regeneration, ACS Biomater. Sci. Eng. (2020), doi:10.1021/acsbiomaterials.
9b01997.
[372] C. Liu, Y. Chen, X. Wang, J. Huang, P.R. Chang, D.P. Anderson, Improvement
in physical properties and cytocompatibility of zein by incorporation of pea
protein isolate, J. Mater. Sci. (2010), doi:10.1007/s10853-010-4774-z.
[373] K. Tuzlakoglu, N. Bolgen, A.J. Salgado, M.E. Gomes, E. Piskin, R.L. Reis, Nano-
and micro-fiber combined scaffolds: a new architecture for bone tissue engi-
neering, J. Mater. Sci. Mater. Med. (2005), doi:10.1007/s10856- 005- 4713- 8.
[374] M.E. Gomes, H.L. Holtorf, R.L. Reis, A.G. Mikos, Influence of the porosity of
starch-based fiber mesh scaffolds on the proliferation and osteogenic differ-
JID: ACTBIO
ARTICLE IN PRESS
S. Ng and M. Kurisawa
entiation of bone marrow stromal cells cultured in a flow perfusion bioreac-
tor, Tissue Eng. (2006), doi:10.1089/ten.2006.12.801.
[375] M.I. Santos, S. Fuchs, M.E. Gomes, R.E. Unger, R.L. Reis, C.J. Kirkpatrick,
Response of micro- and macrovascular endothelial cells to starch-based
fiber meshes for bone tissue engineering, Biomaterials (2007), doi:10.1016/
j.biomaterials.20 06.08.0 06.
[376] I. Van Nieuwenhove, A. Salamon, S. Adam, P. Dubruel, S. Van Vlierberghe,
K. Peters, Gelatin- and starch-based hydrogels. Part B: in vitro mesenchymal
stem cell behavior on the hydrogels, Carbohydr. Polym. (2017), doi:10.1016/j.
carbpol.2017.01.010.
[377] M.A. Da Silva, A. Crawford, J. Mundy, A. Martins, J.V. Araújo, P.V. Hat-
ton, R.L. Reis, N.M. Neves, Evaluation of extracellular matrix formation in
polycaprolactone and starch-compounded polycaprolactone nanofiber meshes
when seeded with bovine articular chondrocytes, Tissue Eng. - Part A. (2009),
doi:10.1089/ten.tea.2007.0327.
[378] I. Silva, M. Gurruchaga, I. Goñi, M. Fernández-Gutiérrez, B. Vázquez,
J.S. Román, Scaffolds based on hydroxypropyl starch: processing, morphol-
ogy, characterization, and biological behavior, J. Appl. Polym. Sci. (2013),
doi:10.1002/app.37551.
[379] D. Anandan, G. Madhumathi, N.A. Nambiraj, A.K. Jaiswal, Gum based 3D com-
posite scaffolds for bone tissue engineering applications, Carbohydr. Polym.
(2019), doi:10.1016/j.carbpol.2019.03.020.
[380] N. Pettinelli, S. Rodríguez-Llamazares, V. Abella, L. Barral, R. Bouza, Y. Farrag,
F. Lago, Entrapment of chitosan, pectin or κ -carrageenan within methacry-
late based hydrogels: effect on swelling and mechanical properties, Mater.
Sci. Eng. C. (2019), doi:10.1016/j.msec.2018.11.071.
[381] A. Ali, S. Ahmed, Recent Advances in Edible Polymer Based Hydrogels as a
Sustainable Alternative to Conventional Polymers, J. Agric. Food Chem. (2018),
doi:10.1021/acs.jafc.8b01052.
22
View publication stats
[m5G;February 21, 2021;17:50]
Acta Biomaterialia xxx (xxxx) xxx
[382] A.M. Hermansson, E. Eriksson, E. Jordansson, Effects of potassium, sodium
and calcium on the microstructure and rheological behaviour of kappa-
carrageenan gels, Carbohydr. Polym. (1991), doi:10.1016/0144-8617(91)
90115-S.
[383] Z.H. Qu, H.J. Wang, T.T. Tang, X.L. Zhang, J.Y. Wang, K.R. Dai, Evaluation of
the zein/inorganics composite on biocompatibility and osteoblastic differen-
tiation, Acta Biomater. 4 (2008) 1360–1368, doi:10.1016/j.actbio.2008.03.006.
[384] D. Yang, Y. Yuan, L. Wang, X. Wang, R. Mu, J. Pang, J. Xiao, Y. Zheng, A review
on konjac glucomannan gels: microstructure and application, Int. J. Mol. Sci.
18 (2017), doi:10.3390/ijms18112250.
[385] A. Noreen, Z. i. H. Nazli, J. Akram, I. Rasul, A. Mansha, N. Yaqoob, R. Iqbal,
S. Tabasum, M. Zuber, K.M. Zia, Pectins functionalized biomaterials; a new
viable approach for biomedical applications: a review, Int. J. Biol. Macromol.
(2017), doi:10.1016/j.ijbiomac.2017.03.029.
[386] A. George, P.A. Shah, P.S. Shrivastav, Guar gum: versatile natural polymer for
drug delivery applications, Eur. Polym. J. (2019), doi:10.1016/j.eurpolymj.2018.
10.042.
[387] T. Hemamalini, V.R. Giri Dev, Comprehensive review on electrospinning of
starch polymer for biomedical applications, Int. J. Biol. Macromol. (2018),
doi:10.1016/j.ijbiomac.2017.08.079.
[388] S. Tansaz, A.R. Boccaccini, Biomedical applications of soy protein: a brief
overview, J. Biomed. Mater. Res. - Part A. (2016), doi:10.1002/jbm.a.35569.
[389] H.J. Wang, J.X. Fu, J.Y. Wang, Effect of water vapor on the surface character-
istics and cell compatibility of zein films, Colloids Surfaces B Biointerfaces
(2009), doi:10.1016/j.colsurfb.2008.11.015.
[390] R. Paliwal, S. Palakurthi, Zein in controlled drug delivery and tissue engineer-
ing, J. Control. Release. 189 (2014) 108–122, doi:10.1016/j.jconrel.2014.06.036.