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The Emerging Role of Microfluidics in Multi-Material 3D Bioprinting

Cynthia Richard, a,b Adrian Neild, a Victor J. Cadarso b,c

Lab on a Chip Accepted Manuscript

To assist the transition of 3D bioprinting technology from simple lab-based tissue fabrication, to
fully functional and implantable organs, the technology must not only provide shape control, but
also functional control. This can be accomplished by replicating the cellular composition of the
native tissue at the microscale, such that cell types interact to provide the desired function. There is
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therefore a need for precise, controllable, multi-material printing that could allow for high, possibly
even single cell, resolution. This paper aims to draw attention to technological advancements made
in 3D bioprinting that target the lack of multi-material, and/or multi cell-type, printing capabilities
of most current devices. Unlike other reviews in the field, which largely focus on variations in
single-material 3D bioprinting involving the standard methods of extrusion-based, droplet-based,
laser-based, or stereolithographic methods; this review concentrates on sophisticated multi-material
3D bioprinting using multi-cartridge printheads, co-axial nozzles and microfluidic-enhanced printing
nozzles.

Research into the development of new tissue engineering tech-

nologies may solve the under supply of transplant organs 1–3 . The
U.S. Department of Health and Human Services estimates that a
patient is added to the national transplant waiting list every ten
minutes. Unfortunately, whilst donor numbers are slowly rising,
the demand is too high to be matched by donors and alterna-
tives need to be developed 4 . Tissue engineering has the potential
to address this deficiency in organ donation through the fabrica-
tion of artificial organs, which, if constructed from patient derived
cells, could also lead to a reduction of adverse immune response
upon implantation. However, implantation is not the only moti-
vation for complex tissue engineering, there are also highly com-
pelling in vitro physiological applications. For example, engineer-
ing physiological or pathological tissue models could, eventually,
eliminate the use of animals in pharmacology and pharmaceuti-
cal research for drug efficacy, toxicity, delivery and disease de-
tection 5–7 . Furthermore, the efficient development of complex
tissues made from patient samples will have a positive impact on
personalized medicine, including predictive drug screening, and
is expected to lead to advances in regenerative therapies 8 .
In order to create complex tissues and even fully functional or-
gans, emphasis must be placed on the fabrication of biomimetic
tissues. In nature, living organisms are assembled in a bottom-up
a
Laboratory for Micro Systems, Department of Mechanical and Aerospace Engineering,
Monash University, Clayton, VIC 3800, Australia. Email: adrian.neild@monash.edu
b
Applied Micro- and Nanotechnology Laboratory, Department of Mechanical and
Aerospace Engineering, Monash University, Clayton, VIC 3800, Australia. Email: vic-
tor.cadarso@monash.edu
c
The Melbourne Centre for Nanofabrication, Australian National Fabrication Facility
– Victorian Node, Clayton, Victoria 3800, Australia
approach; firstly by expression of genetic information on a cellu-
lar level, followed by proliferation, differentiation and lastly by
the self-assembly of various cell types with different functions to
form specific tissues, which then come together to form organs 9 .
Bottom-up biofabrication processes aim to produce macroscopic
biological tissues by attempting to imitate and utilize the self-
assembly of biomaterials, and particularly of cells. In tissue en-
gineering, the self-assembling process involves a system where
spontaneous cell organization occurs, relying on the cell’s abil-
ity to produce extracellular matrix (ECM) naturally 10 . Currently
however, the construction of tissues require the use of a scaffold,
meaning that cell seeding/adherence is needed to encourage the
formation of the ECM.
Researchers are working to realise the full potential of the op-
portunities in tissue engineering through advancing biofabrica-
tion technologies (Fig 1. Among other methods, bioprinting has
emerged as an ideal candidate to manufacture complex 3D tissues
and organs 11 . Much like polymer or metallic 3D printing 10,11 ,
3D bioprinting is a form of additive manufacturing where 3D
structures are produced in a layer-by-layer procedure with the
use of computer-aided transfer (e.g. reading in data from CAD
drawings) and build-up processes 5,12 . 3D printing is currently al-
ready used in a variety of applications in the medical industry, in-
cluding surgical instruments, prostheses, anatomical models, and
customized implants using inert materials (metals, ceramics, or
polymers) for medical devices like dental implants and hearing
aids 13,14 , as well as for other components for instance in me-
chanical, aerospace and structural engineering 15 . However, 3D
bioprinting involves the use of biological components (e.g. pro-
teins, peptides, DNA, cells, hormones, etc.) together with bioma-

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DOI: 10.1039/C9LC01184F

Lab on a Chip Accepted Manuscript

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Fig. 1 The current biofabrication techniques that have been developed (top: 3D Bioprinting and bottom: Microfluidic biofabrication) along with a
new method currently under development (middle: Microfluidic-enhanced 3D bioprinting). 3D cell-laden hydrogel scaffolds have been 3D bioprinted
using single-material printheads to print with a single cell-type (SCT3D – single cell-type 3D bioprinting) for simple tissue fabrication or multi-cartridge
printheads for multi-material printing (MCT3D – multi cell-type 3D bioprinting) for slightly more complex hydrogel scaffolds. Examples of such
tissues can be seen in Fig 2 and Fig 3. Microfluidic biofabrication (MFBF) has been used for organ-on-a-chip applications and to create biological
constructs such as organoids. Organ-on-a-chip methods are used for cell culture within microlfuidic chips and are not used in MF3D. Methods such
as multiple-phase microfluidics, t-junctions and emulsions, or double emulsions are used to create cells in droplets, cell-laden filaments and multi-layer
cell droplets/filaments, respectively. Such microfluidic capabilities can be combined with 3D bioprinting methods allowing for increased fluid and cell
handling capabilities outside of the chip and can offer enhanced 3D bioprinting methods (MF3D – microfluidic-enhanced 3D printing) for the fabrication
of highly organized, multi-cell-type/multi-material tissue constructs. The methods that can be incorporated into microfluidic-enhanced bioprinting
nozzles include utilizing the laminar flow regime, flow-focusing, or co-axial flow focusing, fluid mixing, as well as the previously mentioned methods
for the fabrication of organoids. Organoid fabrication in MFBF methods are produced within the chip and would be extruded in MF3D methods to
produce 3D constructs of multi-layered organoids containing different cell types in order to construct complex, organized tissues. Examples of tissues
fabricated using these methods can be seen in Fig 4, Fig 5, and Fig 6. Further examples are tabulated in Table 1.

terials, such as hydrogels, to construct living tissue 3 . 3D bioprint-
ing has the potential to generate specific structures and shapes
with materials that can be tailored to deliver the desired physi-
cal, chemical and biological properties 5,12,16 , hence developing
and mastering these techniques has become a major focus in the
tissue engineering community.
Most of the current 3D bioprinting technologies available use
standard 3D printing platforms to deposit biological material
(Fig 1). As such, 3D bioprinting can be divided into the same
four main categories as 3D printing: laser-based methods, stere-
olithography, extrusion-based (EB) methods and droplet-based
(DB) (also known as printer-based) methods. For instance inkjet
printing (IJP), one of the main printing methods for the addi-
tive manufacturing of ceramics 15 , is also a commonly used sin-
gle cell-type 3D bioprinting method, known as droplet-based 3D
bioprinting. Stereolithography, one of the earliest methods of ad-
ditive manufacturing used for the effective fabrication of com-
plex nanocomposites 15 , is now also being used to construct tis-
sues from cell-laden scaffolds in 3D bioprinting. A more detailed
overview of the capabilities of these different single cell-type 3D
bioprinting methods, we will term this SCT3D, can be seen in Ta-
ble 1 of the SI. For a detailed overview of the use of standard
additive manufacturing processes for SCT3D, and of their use in
printing tissues and organs, please refer to the review paper by
Vijayavenkataraman et al. 3 . There are also numerous other arti-
cles offering detailed reviews of the full range of SCT3D Bioprint-
ing methods and their applications in tissue engineering 3,18–23 ,
as well as method specific reviews 24–27 , and tissue specific re-
views 7,11,16,28–30 , just to name a few.
The progress of bioprinting processes has allowed tissues and
anatomical structures, from several of the eleven organ systems
present in the human body, to be printed and tested 3 . These

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eleven organ systems consist of the skeletal 31–34 , muscular 35–37 ,
nervous 38–40 , lymphatic 41 , endocrine 42,43 , reproductive 44 , in-
tegumentary 45–47 , respiratory 48 , digestive 49,50 , urinary 51 , and
circulatory systems 52–54 . Nonetheless, there is still a long way
to go before 3D bioprinting can be moved from lab-based ex-
periments to full-scale manufacturing of functional tissues and
organs 55 . The anatomical arrangement of human tissues at the
micro-, meso-, and macro-scales to form complex 3D hierarchi-
cal structures is integral to the proper operation of the individual
tissues and organs and thus to the organism as a whole 3 . But,
one of the major limiting factors of current 3D bioprinting tech-
nologies is the lack of microscale precision and regulation when it
comes to cellular composition or arrangement using multiple cell
lenges, each organ system pose tissue-specific issues which must
be addressed 3 .
The loss of microscale precision during mesoscale fabrication
hinders the arrangement of different cell types within a tissue
assembly. But, it is the microenvironment created by signalling
between neighbouring cells that can influence primary cell differ-
entiation, and lead to spontaneous morphogenesis 56 . Thus, lack
of microscale accuracy placement in turn affects the macroscale
organoid morphology and therefore limits the reproducibility and
application of artificial tissues 56 . In order to fabricate artificial
biological tissues that function just as well as natural ones, it
is crucial to position specific cell types in multiscale structures
with hierarchical features that replicate the natural microscale tis-

Lab on a Chip Accepted Manuscript

types, or single cell resolution, during the printing process. Ex- sue environment 9 . For that reason, a multi-material, bottom-up
isting technologies only have control over the macroscale geom- biofabrication approach would be highly beneficial. Researchers
etry of cell-laden bioinks and hydrogel scaffolds. However, print- have therefore begun addressing these issues by combining the
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ing multiple materials, such as heterogeneous cell populations, cell and fluid handling capabilities offered by microfluidics with
growth factors and nutrients, in a well-timed and orderly manner, the biofabrication potential of 3D bioprinting.
or recreating micro-scale structure by controlling the placement In order to improve microscale precision, microfluidic biofab-
of individual cells, is still under development. Beyond these chal- rication techniques utilise the offered cell and biomaterial han-

Fig. 2 Multi-Cartridge 3D Bioprinting of human-scale tissues. (a) The ITOP system. (b) Mandible bone construct where (i) shows the 3D architecture
using CAM software developed by Kang et al. (the lines of red, blue and green show the dispensing paths of the cell-laden hydrogel, Pluronic F-127
and PCL respectively), (ii) demonstrates the patterning of one layer of the 3D printing process, (iii) is a photograph of the 3D printed bone defect
construct and (iv) shows the osteogenic differentiation of hAFSCs in the printed construct by Alizarin Red S staining, indicating the deposition of
calcium. (c) Calvarial bone construct where the dispensing paths of the PCL/TCP mixture and the cell-laden hydrogel are shown. (d) (i) photographs
of the printed bone implanted at day 0 (top) and at 5 months (bottom), and (ii – iv) show the histological and immunohistological images of the
nontreated construct (top) and the hAFSCs-printed construct after 5 months of implantation (bottom). (iii) H&E staining, (iv) modified tetrachrome
staining (Tetrachrome staining: red, mature bone: blue), and (v) vWF immunostaining (blood vessels: red). In the bottom images, the bioprinted
construct shows newly formed vascularized bone tissue throughout the implant, including in the central region, whereas in the untreated implant (top),
there is minimal bone tissue formation around the periphery of the implant. NB: new bone, PCL/TCP: remaining scaffold. (Reprinted with permission
from 17 )

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 Lab on a Chip
dling and manipulation capabilities (Fig 1). Since microfluidic
devices offer the geometrical constraint of fluids in the micro-
scale, where lower dimensional forces such as viscous forces and
surface tension dominate, the laminar flow can be utilized to gen-
erate well-defined patterns by controlling the fluids containing
biomolecules, cells, organism, or chemical agents 57 . For instance,
different fluidic mechanisms such as focusing 58–61 , concentrat-
ing 62–64 , mixing 65–68 or separating can be utilized to produce
various constructs, within the microfluidic device, such as cells in
droplets, microfibers, or cell sheets 9,69,70 . These constructs can
be composed of different biomaterials or cell-laden bioinks. In
addition, the microfluidic environment with multiple inlets could
allow for growth factors and nutrients to be provided during the
printing process, increasing cell viability and guiding primary cell
differentiation. Furthermore, a seamless transition between ma-
terials is possible. We will term microfluidic biofabrication as
Published on 18 May 2020. Downloaded on 5/19/2020 7:38:13 AM.
MFBF. These systems do not include bioprinting, rather the con-
structs are internal to the chip. However, MFBF techniques could
be easily combined with current 3D bioprinting platforms by mod-
ifying the nozzles to incorporate microfluidic chips, allowing for
multi-material printing with greater control over material con-
centration, positioning and composition. Microfluidic-enhanced
3D bioprinting (MF3D) could be used to print various organoids
in a 3D manner that would better represent the structural and
functional integrity of natural tissues. For example, in order to
achieve a 3D multi-material organized tissue construct as demon-
strated in the (Fig 1) MF3D, cell-laden multi-layer filaments could
be used to print vascularized hollow tissue to serve as blood ves-
sel, interlaced with cardiac cells printed as cells in droplets along
with a hydrogel to serve as a scaffold to achieve functional cardiac
tissue.
There have been several recent review papers that have
focused solely on compiling and describing the differences
between traditional 3D bioprinting technologies (laser-based,
droplet-based, extrusion-based and stereolithographic methods)
and their applications in tissue engineering and regenerative
medicine 3,5,7,14,18–21,71 . These reviews focus on printing of sin-
gle cell types, SCT3D. The review of Sodupe-Ortega et al. cov-
ers multi-cartridge 3D bioprinting 72 , lets call this MCT3D – or
multiple cell-type 3D bioprinting, whilst that of Costantini et al.
includes co-axial 3D bioprinting 73 . The use of microfluidics to
enhance microstructural control has been reviewed by Ma et al.
in 2018, but the emphasis is on printing cells within microfluidic
systems to form organ-on-a-chip constructs and layer-based bio-
printing methods that form built-in microchannels 70 , rather than
on the use of microfluidic technologies to form structures external
to the chip.
In this review we cover the recent technological advancements
made in 3D bioprinting that address the need for multi cell-
type and/or multi-material bioprinting. Emphasis is placed on
3D bioprinting using multi-cartridge printheads and microfluidic-
enhanced printing nozzles which transfer the sample and cell
handling control offered by microfluidics to complex tissue print-
ing (microfluidic 3D bioprinting or MF3D). As such, the focus
is on the technological developments enabling replication of the
type of microscale cellular compositions that are transitioning the
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field from shape control to functional control. Meaning that the
juxtaposition of different cell types causes interactions to occur
which deliver the desired function, for example, in the case of
heart tissue the appropriate location of cardiomyocytes and car-
diac pacemaker cells is required.
Advanced Multiple Cell-type 3D Bioprinting by Multi-
cartridge
Tissues normally have complex architectures, with distinct layers
having different cell densities and mechanical properties, often
composed of different cell types 3 . This is of critical relevance for
bioprinting as cell behaviour and function are strongly affected
Lab on a Chip Accepted Manuscript
by their neighbouring cells. Having different cells within close
proximity to one another in an uncontrolled way could lead to
differentiation into an undesired cell type or to negatively af-
fecting proliferation. For example, bone tissue has a solid outer
part but an inner porous structure and would thus require both a
permeable scaffold structure and cell-laden hydrogels during the
printing process. Other tissues in the human body, such as lymph
nodes, are intricate structures with highly organized stromal and
lymphoid structures with a large variety of cell types, functional
skin also has many specialized cell types including sweat glands,
sweat ducts, hair follicles, and a multitude of other skin cells
in the epidermis and dermis 3 . So to fabricate biomimetic tis-
sues, with zone-specific heterogeneity, multi-material and multi
cell-type bioprinting with control of the exact cell-type in each
location is crucial. To this end, some bioprinters have incorpo-
rated multiple cartridges to allow for material switching during
the printing process, while others use co-axial extruders which
allow for a bioink and a crosslinking agent to be printed simulta-
neously.
A simple approach for multi-material bioprinting is to mod-
ify current extrusion-based or droplet-based 3D bioprinting plat-
forms to incorporate multiple cartridges where controlled switch-
ing between materials is possible. Kang et al. used an inte-
grated tissue-organ printing (ITOP) extrusion-based system with
a multi-cartridge module to simultaneously deliver different cell
types embedded in hydrogels, reinforcing polymers and sacri-
ficial hydrogels to fabricate human-scale tissue constructs such
as mandible and calvarial bone, cartilage and skeletal muscle 17 .
Material dispensing and switching is accomplished via XYZ stage
movements and air pressure actuation which is computer con-
trolled (Fig. 2(a)). A mandible bone fragment was fabricated
in a size and shape that could be suitable for facial reconstruc-
tion using human amniotic fluid-derived stem cells (hAFSCs) in
a bioink (a mixture of poly(ε-caprolactone) (PCL) and trical-
cium phosphate (TCP)), a composite hydrogel (Gelatin, fibrino-
gen, hyaluronic acid (HA) and glycerol), and a Pluronic F127
temporary support (Fig. 2(b)i). After 28 days of osteogenic dif-
ferentiation, calcium deposition was observed through staining of
the 3D bone structure using Alizarin Red S, in the hAFSC-laden
hydrogel (Fig. 2(b)iv). The team also fabricated a rat calvarial
bone construct (Fig. 2(c)) to study in vivo maturation of the bio-
printed bone 17 . The artificial bone was cultured in osteogenic
medio for 10 days and then implanted. Following 5 months of
 Page 5 of 13 Lab on a Chip
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Fig. 3 Characterization and comparison of manually seeded and bio-
printed cultures by LSM and brightfield microscopy for manually seeded
(left) and bioprinted (right) tissue. (a) – (d) Immunofluoresnce labelling
of Factin cytoskeleton (green), nuclei (gray) and labelling of endothelial
cells with PECAM-1 (blue), in mono-cultures. (e) & (f) Laser scanning
micrographs of the two-layer co-cultures after 3 days. Cells stained for
F-actin (red) and nuclei (white). A549 cells are in green and endothelial
cells are labelled with VE-cadherin (pink). Yellow arrow heads in (e)
shows the thick MatrigelTM layer. (g) & (h) Brightfield micrographs
of embedded histological cross sections stained with Masson-Goldner
trichrome coloration. Cytoplasm is red, collagen fibers of the ECM Ma-
trigelTM are green and cell nuclei are brown. It is seen that the cells
seeded manually (left) typically grow in patches, and often in multi-
layered clusters, whereas the bioprinted cells (right) spread uniformly
over the available surface to form thin monolayers. This is observed for
both cell types and for single cell and and two cell layers. Scale bars (e
– f) 50 µm, 10 µm for the close-up, (g – h) 100 µm. (Reprinted with
permission from 48 )
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DOI: 10.1039/C9LC01184F
implantation, the bioprinted construct was analysed and showed
newly formed vascularized bone tissue throughout the implant
(Fig. 2(d)iii-v upper) and in the central portion. The untreated
defect without cells (only the scaffold) showed fibrotic tissue in-
growth and minimal bone tissues formation (Fig. 2(d)iii-v lower)
only along the periphery of the implant. In a more recent work,
Seol et al. used the same ITOP system for the 3D bioprinting
of a skin substitute combined with a wound dressing layer for
facial skin reconstruction 74 . A custom ‘BioMask’ was fabricated
by depositing three layers; a porous polyurathan (PU) layer, a
keratincyte-laden hydrogel layer, and a fibroblast-laden hydrogel
layer. The researchers were able to develop patient-specific fa-
cial skin composed of two different skin cell types for the epi-
Lab on a Chip Accepted Manuscript
dermis and dermis layers. After 14 days of implantation onto a
face-shaped construct, the BioMask showed promising skin tissue
regeneration and integration into the surround tissue.
Using a more multifaceted bioprinting platform, with a valve-
based inkjet and an extrusion-based printing system incorporat-
ing a crosslinking laser unit, Horváth et al. engineered an in
vitro human alveolar air-blood barrier 48 . The two cell-layer bar-
rier system was printed over two days with extracellular matrix
(ECM) being printed first on a porous membrane (serving merely
as a support for the printing process), followed by a layer of
EA.hy926 endothelial cells suspended in cell culture medium on
MatrigelTM. On the second day, a second ECM layer was printed
on the endothelial cells, subsequently a layer of A549 epithelial
cells suspended in cell culture medium was printed. The printed
construct was designed to attain close physiological resemblance
to the microenvironment of the native tissue. The bioprinted tis-
sue was compared to manually seeded cultures to demonstrate
the benefit of precise cell and biomaterial positioning on the de-
velopment of biomimetic tissue. The cellular morphology be-
tween the manually seeded and the bioprinted methods were
compared using laser scanning microscopy (LMS) and the results
are shown in Fig 3 (a) - (d), where the nuclei are depicted in
white, and the F-actin of immunostained cells are in green. It can
be seen that the cells seeded manually typically grow in patches,
and often in multi-layered clusters, whereas the bioprinted cells
spread uniformly over the available surface to form thin mono-
layers. This was seen for both the endothelial (EA.hy926) and
epithelial (A549) cells. Similar results were obtained for the two
cell-layers, as shown in Fig 3 (e) and (f), and in the brightfield
images obtained in Figures (g) and (h). This demonstrates the
need for organized cell placement for successful tissue growth.
Merceron et al. utilized a four-cartridge bioprinter for the as-
sembly of different components into a single integrated muscle-
tendon unit (MTU) construct 35 . For the formation of muscle tis-
sue thermoplastic polyurethane (PU) was co-printed on one side
of the construct with C2C12 cell-laden hydrogel-based bioink,
while poly(ε-caprolactone) (PCL) was cp-printed with NIH/3T3
cell-laden hydrogel-based bioink on the other side for tendon de-
velopment. The overall construct had >80 % cell viability 7 days
after printing and demonstrated early stage tissue development,
cell differentiation, and interaction of the different cell types at
the junction.
For high resolution bioprinting (approximately 50 µm) of
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Fig. 4 (a) Schematic of the co-axial printing nozzle where the bioink is delivered from the core and the crosslinking agent (CaCl2 ) is delivered from the
outer sheath. (b) Diagram showing the two-step crosslinking process, alginate is first crosslinked during the printing process by CaCl2 (left), followed
by crosslinking the GelMA by UV illumination. (c) Confocal fluorescence micrograph showing the distribution of HUVECs in a single printed microfiber
at day 14. (d) Confocal fluorescence micrograph showing the GFP-HUVECs in a single fibre for CD31, GFP, and nuclei. (e) Schematic of the scaffold
seeded with the cardiomyocytes. (f) F-actin (green) staining showing the location where two microfiber layers intersect with the cardiomyocytes can
be seen on the surface, this expression is an indicator of proper contractile and intercellular conductive function. (Reprinted with permission from 75 )

multi-component hydrogels, Zimmermann et al. used an
inkjet printer with a dual printhead composed of piezoelectric
pipettes 76 . The platform allowed for two reactive hydrogel pre-
cursor solutions to be dispensed simultaneously to create spatially
structured multi-component hydrogels as well as cell-laden hy-
drogel scaffolds using human bone-marrow derived mesenchymal
stem cells (hMSCs).
Multi-cartridge 3D bioprinting platforms have had many suc-
cesses in the past few years by offering the capability of simul-
taneously dispensing different cell-laden hydrogels, reinforcing
polymers and sacrificial elements 77 . The method also allows con-
tinuous, smooth, and rapid switching between different material
types 78 . The result is a technology which researchers have used
to print complex tissue constructs with regional differences in cell
types, while also providing structural integrity 35,77 . An outcome
which is not viable with single-material 3D bioprinting devices.
Microfluidic Biofabrication
Microfluidics provide accurate control of fluids carrying
biomolecules, cells, organisms or chemical agents and are ideal
for performing a multitude of tasks and are thus highly benefi-
cial for integration into modified 3D bioprinting nozzles. Fur-
thermore, the laminar flow-regime offered by microfluidic devices
also allows for multiple materials to be input simultaneously with-
out mixing, or provides a seamless transition between these dif-
ferent materials (Fig 1). The regulation of minute amounts of
liquid confined to microchannels allows for fine-tuning of phys-
ical and chemical compositions of the tissues being synthesized
by allowing for chemical reactions to be triggered at specific lo-
cations, for instance through the use of controlled mixing (Fig 1),
with precise timing, inside the fluidic channel during the printing
process 9 .
Whilst MCT3D can print a range of individual cell types, mi-
crofluidic biofabrication (MFBF) methods can be used to produce
small constructs often referred to as microtissues or organoids.
Once created these micrometer-scale cellular building blocks 59
these microtissues can be assembled into off-chip 3D constructs.
Microtissues can be categorized into point-, line-, and plane-
shaped and they provide high cell-density monolayer sheets,
spheroids, or cell-laden hydrogel blocks which are then spatially
organized to produce heterogeneous macrotissues, to replicate
those produced naturally 72 .
Different fluidic mechanisms have been applied for the genera-
tion of these different shaped-microtissues (Fig 1). For example,
in order to generate line-shape (fibrous) organoids flow-focusing
or multiple-phase flows are commonly used. Whereas for point-
shaped microtissues ( droplets) T-junctions or emulsions (Fig 1)
are needed 9 . Microfluidic biofabrication methods have been used
to prepare wide range of biological building blocks, including li-
posomes 86,87 , cell-laden microfibers 88–90 , hydrogel sheets for co-
culture 91 , and protein microbeads 92 . In addition, in their recent
work Kang et al. explored the use of microfluidics and acoustics

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to create 3D cell patterns to construct extrudable functional blood
vessels 93 , a key aspect required for all tissues. For further read-
ing, reviews of bottom-up microfluidic biofabrication techniques
are given by Nie et al. 9 and Wang et al. 69 . Such MFBF methods
can be used to generate the initial building blocks that can then
be bioprinted.
Microfluidic-Enhanced 3D Bioprinting
The major limiting factors of current bioprinting techniques are
the lack of multi-material printing and regional material control,
damage imparted to cells during the printing process and noz-
zle clogging due to viscous bioinks. There is therefore a need
ents 97–100 , switching between flow regimes 101,102 and changing
suspension medium 103,104 . The inclusion of microfluidics within
a bioprinter’s nozzle opens this range of controls over the fluidic
environment and the suspended matter. One clever example, util-
ising ultrasonic methods of manipulating suspended matter, by
Collino et al., addressed both the issues with nozzle clogging and
multi-material bioprinting 105 . They incorporated an acoustically-
excited microfluidic printing nozzle where acoustic focusing was
used to deposit a two-phase ordered structure using a single noz-
zle 105 . Microfluidic devices have been integrated into bioprinting
nozzles to provide improved regulation and precision for multi-
material, or multi cell-type printing.

Lab on a Chip Accepted Manuscript

to incorporate more appropriate biomaterial and cell handling In comparison to standard printing nozzles, microfluidic mod-
methods into current devices to address and eliminate these is- ified printheads would not only provide access to multiple bio-
sues. Examples of microfluidic suspended cell handling meth- materials or different cell-laden bioinks simultaneously, but they
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ods include systems using forcefields generated from magnetic,
electrical, optical and acoustical effects, which can operate even
down to the single cell level if required. In addition, a range
of methods have been described for fluid control in the form
of, for example, mixing 94–96 , generating concentration gradi-
would also provide a micro/nano-scale reaction chamber for
increased structural complexity and micro-architecture control.
Furthermore, the microfluidic environment can help to protect
cells from damage induced by the printing process by hydrody-
namically focusing cells in the centre of the channel; shear-stress

Table 1 Overview of Microfluidic-enhanced 3D bioprinting technologies refereing to the standard bioprinting platform used as well as the general
microfluidic method employed.

Bioprinting Method Microfluidic Component Application Advantages Reference

Extrusion-based & Stere- Laminar flow (Fig. 1)
olithography
Extrusion-based • Laminar flow (Fig. 1)
• Flow-focusing (Fig. 1)
• Herringbone micro-mixer
(Fig. 1)
• T-junction (Fig. 1)
• Cross-flow-filter for cell
concentration
Extrusion-based Multi-axial channel
Co-axial Laminar flow (Fig. 1)
Heterogeneous cellularized
constructs (Fig. 7)
Complex heterogenous cellu-
larized constructs
Bi- and tri-layered hollow
channel constructs of differ-
ent cell types (endothelial
cell and fibroblasts)
Hydrogel fibres laden with
muscle precursor cells
• Multi-material
• Fast switching 79
• High spatial resolution
• Multi-material
• Vary ratio between core 80
and sheath materials
• Printing of reactive com-
ponents
• Cells in droplets
• Removes excess liquid
right before dispensing
• Multi-material
• Seamless transition 81
• Hollow channel constructs
• Multi-material
• On the fly crosslinking 82

Extrusion-based Laminar flow (Fig. 1) Hetero-cellular model of hu- • Multi-material

man liver • Print cell-laden bioinks 83
and structural materials
simultaneously
Co-axial Serpentine-based mixing Osteochondral tissue (Fig. 5) • Layers or continuous
(Fig. 1) gradients of chemical, 84
mechanical and biological
cues
• Simultaneous fabrication
of scaffolds
Co-axial Laminar flow (Fig. 1) Glioblastoma tumor model • Multi-material
using fibrin-based bioink for • On the fly crosslinking 85
drug screening (Fig. 6) • Minimizes shear-stress

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 Lab on a Chip
Fig. 5 (a) Microfluidic extrusion system composed of (b) the microfluidic printing head and (c) the co-axial adapter. (d) Zonal structure of the
osteochondral (OC) interface and the 3D bioprinted design of the layered scaffold for the regeneration of the cartilaginous part of the OC region. (e)
Published on 18 May 2020. Downloaded on 5/19/2020 7:38:13 AM.
Hematoxylin and eosin staining with insets showing higher magnification. (i) Shows the 3D bioprinted structure where the black arrows in the inset
point to cartilage cell differentiation in the trochlea of the knee implant. In the control implant (ii) this differentiation is missing and a white region
is seen. (Reprinted with permission from 84 )
is at a minimum at the centre of the flow profile. Microfluidic-
enhanced 3D bioprinting (MF3D) methods have thus become a
new focus in tissue printing applications, with a multitude of pa-
pers being published in recent years (Table 1).
In a commonly used approach, co-axial wet-spinning extrusion
3D bioprinting delivers a bioink from the core of the printing
nozzle and an ionic crosslinking solution is sheathed on the side
(Fig 4(a)), allowing the crosslinking to occur only at the noz-
zle tip. The solutions can also be inversed so that hollow hy-
drogel fibres are produced. An advantage of this method is that
it permits for lower viscosity bioinks to be used which is benefi-
cial for reducing nozzle clogging 73,106 . For instance, Zhang et al.
generated endothelialized myocardium for blood vessels through
the use of co-axial printing technology 75 . They first generated
multilayer hydrogel microfibrous scaffolds, where the bioink was
composed of a mixture of hydrogel precursors (alginate, GelMA,
and a photo-initiator) and encapsulating human umbilical vein
endothelial cells (HUVECs). The crosslinking agent (CaCl2 ) was
simultaneously dispensed through the outer sheath flow of the
printing needle to crosslink the alginate component in the bioink
(Fig 4(b) left). A second chemical crosslinking step through UV
illumination was then used to crosslink the GelMA component
(Fig 4(b) right). Following two weeks of culture, the HUVECs in
the bioprinted scaffold migrated to the peripheries of the microfi-
bres leading to the formation of a pattern similar to blood vessel
walls (Fig 4(c & d)). The cardiomyocytes could then be seeded
into the endothelialized scaffold structure to form engineered en-
dothelialized myocardium that structurally resembles native my-
ocardium (Fig 4(e & f)). They found that the cardiomyocytes that
grew on the bioprinted microfibrous scaffold strongly expressed
proteins required for proper contractile function as well as proper
inter-cellular conductive function. Not only did the microfluidic
nozzle allow for the structure of the native tissue to be replicated,
it demonstrated the possibility of becoming a functional tissue.
In a more recent work, Pi et al. produced tubular urothelial
tissues composed of inner human muscle cells and outer human
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DOI: 10.1039/C9LC01184F
Lab on a Chip Accepted Manuscript
bladder smooth muscle cells in a single step using a multi-channel
co-axial extrusion system 107 . In their setup, the crosslinking
agent (CaCl2 ) was delivered from the core of the printing needle,
whereas two bioinks were simultaneously extruded from concen-
tric rings.
In their recent paper, Idaszek et al. developed a microfluidic
printing head with a serpentine-based passive mixer with two-
inlets and a co-axial adapter for precise material crosslinking
(Fig. 5(a-c)) 84 . Their device was designed to address the problem
of long-term stability in regenerated osteochondral (OC) tissue.
By allowing for multiple bioinks to be delivered either individ-
ually, or at the same time and rapidly mixed, it enables layers
or continuous gradients of chemical, mechanical and biological
cues to be extruded such that scaffolds with very high shape fi-
delity and cell viability can be fabricated to mimic the compart-
mentalized zonal microstructure and composition of OC tissue
(Fig. 5(d)). In comparison to the SCT3D methods, the microflu-
idic component provided additional control of chemical reactions
and provided higher resolution due to a better control over cell
type positioning. Furthermore, they were able to develop bioinks
that mimic the composition of different zones that are present
in the native articular cartilage and the 3D bioprinted construct
demonstrated heterogenous differentiation of stem cells, show-
ing promise for the regeneration of the interfacial tissue in vitro
and in vivo. The presence of differentiating cartilaginous cells at
the implantation site in the knees, was shown through H and E
staining (Fig. 5(e) i). The differentiation was not present in the
control group (Fig. 5(e) ii).
Lee et al. utilized a multi-inlet microfluidic printhead with a
co-axial flow-focusing extruder giving them the choice of three
different cell-types, or biomaterials, during the printing process,
and crosslinking of the bioink at the nozzle tip (Fig. 6(a)) 85 . They
used this technology to fabricate a fibrin-based 3D printed model
of glioblastoma multiforme (GBM), a deadly primary brain can-
cer, for cellular characterization and drug screening. Over a 12-
day culture period, the 3D bioprinted glioblastoma cells displayed
 Page 9 of 13 Lab on a Chip
high levels of cell viability and demonstrated the ability to spon-
taneously form increasingly large and dense spheroids (Fig. 6(b))
with high levels of protein markers associated with cancer stem
cells and metastatic invasiveness. The cells ability to naturally
form spheroids provides accurate in vivo tumor propagation char-
acteristics that can be used to study cell-cell interactions within
tumors, as well as drug penetration, response and resistance.
In order to package multiple cell types into heterotypic arrays,
Snyder et al. developed an improved multi-material bioprinter by
incorporating a microfluidic printing nozzle with multiple inlets
and one outlet to exploit the laminar flow to print several ma-
terials without mixing 83 . Their droplet-based MF3D platformed
allowed for packaging of multiple cell types to be printed into
200 nl droplets to model human liver cells for pharmacokinetic
studies. They were also able to operate their device in extrusion
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mode to fabricate 3D hetero-cellular scaffolds. Miri et al. created
a unique microfluidic chip for the consecutive printing of different
bioinks in a digital micromirror device (DMD) -based projection
bioprinting setup (Fig. 7(a)) 79 . An elastomeric membrane was
also included in the channel design to seal the microfluidic cham-
ber during the exchange of bioinks and the transitional channel
washing steps. The device was used to create a variety of multi
cell-type models, for example a tumour model was fabricated by
printing scattered breast cancer cells (MCF7) in GelMA and the
structure was further seeded with HUVECS (Fig. 7(b)). A muscle
model and a tendon-to-bone insertion model were also carried
out to demonstrate the diversity and success of applications using
this bioprinting platform (Fig. 7(a)). The post-printing cell viabil-
ity for all models indicated all cell types maintained satisfactory
proliferation and metabolic activity after seven days of culture.
In a very detailed work, Serex et al. demonstrated how mi-
crofluidics can be used to increase the functionality of current
extrusion-based 3D printers by integrating various microfluidic
components into the printhead 80 . Multi-inlet microfluidic chips
were designed for material switching, hydrodynamic focusing for
controlled material thickness, a herringbone micro-mixer and a
concentrator using a cross-flow filter. The implementation of hy-
drodynamic focusing introduces core width adjustability during
the printing process by simply varying the ratio between the core
and lateral flows. This allows for increased resolution of the de-
sired material at specific locations in printed structure.
The Herringbone micro-mixer allows even very viscous materi-
als (glycerol with a viscosity of approximately 1.412 Pa s 108 ) to be
mixed right before being extruded. This represents an advance on
a previous work in which Serex et al. had used a meander-based
static micro-mixer to perform mixing of slow reacting materials
for the 3D printing of carboxymethylcellulose-based cryogels 109 .
Lastly, the concentrator uses a crossflow filter to remove excess
fluid while keep particles/cells within the centre of the channel.
Concentrating the particles in a composite material right before
printing makes it possible to work with diluted solutions, which
have lower viscosities, right until the printing happens, reducing
the risk of clogging. This simplifies the printing process but still
produces constructs with the desired composition and reduces the
amount of unnecessary fluid.
View Article Online
DOI: 10.1039/C9LC01184F
Lab on a Chip Accepted Manuscript
Fig. 6 Microfluidic-enhanced co-axial bioprinter nozzle. (a) Depicts the
microfluidic chip and the reaction between the bioink and cross-linker.
(b) Is the 3D bioprinted glioblastoma cells at day 0 (top) and at day 12
(bottom) of culture. Over time they observed a spontaneous formation of
increasingly large and dense spheroids. The formation of such spheroids
within the scaffolds demonstrates not only high cell viability but also the
cells ability to naturally form spheroids despite being printed as single
cells. (Reprinted with permission from 85 )
Conclusions and Outlook
3D bioprinting is a rapidly growing technology for the develop-
ment of engineered tissues and organs. In this review, we summa-
rized the development of multi-material 3D bioprinting methods
with an emphasis on multi-cartridge systems, and more specif-
ically on microfluidic-enhanced printing nozzles. Despite many
advancements made in recent years, there are still many limita-
tions and hurdles that need to be overcome in order to create
completely functional, biomimetic tissues and organs that can be
used for human implantation and for reliable drug modelling and
testing.
One of the major enduring restrictions is the inability to pro-
duce tissue constructs with highly complex microarchitectures
with precise control over local cellular composition. Being able
to precisely place individual cells at specific locations to perfectly
mimic the micro-scale hierarchical structures of the natural tis-
sue is the next stage that researchers should focus on to improve
the current technology. In order for this to be achieved, a plat-
form allowing for multiple cell types to be printed in a controlled,
and patterned way, is critical. Furthermore, a built-in method to
provide nutrients, growth factors, or buffer changes, during the
printing process would be advantageous. In addition, since most
current methods use devices with nozzles, strategies to avoid noz-
zle clogging and to reduce shear stress on cell populations during
the printing process are of great importance.
Microfluidic-enhanced bioprinting methods are a promising ap-
proach that may allow the development of hybrid 3D bioprinting
platforms capable of printing multiple materials, and/or multiple
cell types, and to create constructs with controlled heterogenous
cell populations in more of a bottom-up approach. Some first re-
sults regarding these advantages have been reviewed here; for
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Lab on a Chip Accepted Manuscript

Fig. 7 Microfluidic-enhanced multi-material bioprinter. Schematic showing the bioprinter setup with the microfluidic device (left) with different tissue
models showing, in order, for the tumor (a) and muscle (b) models the schematic of the tissue model, the mask for printing, the bioprinted construct,
Published on 18 May 2020. Downloaded on 5/19/2020 7:38:13 AM.

and lastly the bright-field optical stained images depicting the cells present in the construct. In the case of the tendon model (c), the schematic of the
tissue model, the mask for printing, the bright-field optical image showing the different cells present, and lastly a measurement of the cell proliferation.
The MCF7 cells are stained in blue and the HUVECs in green for the tumor model in (a). For the muscle model in (b), the C2C12 cells are red and the
fibroblasts are blue. In the tendon model in (c), the osteoblasts are blue, the MSCs red, and the fibroblasts in green; the inset shows a magnified image
of the region hosting fibroblasts, where the cells were stained for f-actin (green) and nuclei (blue). All cell types maintained satisfactory proliferation
and metabolic activity post-bioprinting. (Reprinted with permission from 79 )

example in the successful fabrication of osteochondral (OC) tis-
sue by Idaszek et al., where microlfuidics allowed for the com-
partmentalized zonal microstructure and composition of the OC
tissue to be reconstructed. Additionally, the integration of mi-
crofluidic devices allows for quick and easy medium change as
well as the delivery of growth factors and other nutrients during
the printing process. This would be extremely beneficial to min-
imize cell damage and increase cell survival. For example, Miri
et al. utilized a pneumatic valve in their microchannel to change
between materials, however this technology could be utilized to
provide nutrients in a controlled and timely manner. This could
not be incorporated into the traditional printing nozzles but re-
quires the addition of a microfluidic component. Another advan-
tage of a multi-material microfluidic-enhanced printer rather than
a multi-cartridge system, is that the transition between materials
in the latter is not smooth and rather slow, whereas microfluidic
printing would allow for a seamless transition.
Although there has been creditable progress in developing new
multi-material 3D bioprinting platforms which have been success-
fully used to fabricate tissue constructs, taking the technology
from the laboratory bench to real world applications still requires
focused efforts, specifically towards achieving functional form.
For example current research demonstrates cell growth and dif-
ferentiation over a series of days in the 3D bioprinted constructs
however, the published works do not provide data about cell func-
tionality. Researchers will therefore not only need to work on ad-
vancing the technology, but also on insuring that the cells have
not undergone any functional changes during the printing pro-
cess, which would negatively impact the fabricated tissue.
Significant advantages have already been gained through the
use of passive microfluidic elements within 3D bioprinting, the
control of the microenvironment and the delivery of multiple cell
types has led to the fabrication of a range of functional tissues. We
expect that future developments will include further use of active
microfluidic capabilities, in which external force fields, including
but not limited to, electric, acoustic, or magnetic approaches, of-
fer further control of fluid and cell motion within the microfluidic
printer head, furthering our ability to accurately mimic natural
tissues in both microstructure and microenvironment.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors are grateful for their support through the Monash -
Warwick Alliance Accelerator scheme.
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