Review Article
Cells Tissues Organs
DOI: 10.1159/000512792
3D Bioprinted Cardiac Tissues and
Devices for Tissue Maturation
Veronika Sedlakova a, b Christopher McTiernan a David Cortes a Erik
J. Suuronen a Emilio I. Alarcon a, c
aBEaTS
Research, Division of Cardiac Surgery, University of Ottawa Heart Institute, Ottawa, ON, Canada;
b Department
of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic;
cDepartment of Biochemistry, Microbiology, and Immunology, Faculty of Medicine, University of Ottawa,
Ottawa, ON, Canada
Keywords
3D printing · Cardiac tissue · Valve · Maturation · Bioreactor
Abstract
Cardiovascular diseases are the leading cause of mortality
worldwide. Given the limited endogenous regenerative ca-
pabilities of cardiac tissue, patient-specific anatomy, chal-
lenges in treatment options, and shortage of donor tissues
for transplantation, there is an urgent need for novel ap-
proaches in cardiac tissue repair. 3D bioprinting is a technol-
ogy based on additive manufacturing which allows for the
design of precisely controlled and spatially organized struc-
tures, which could possibly lead to solutions in cardiac tissue
repair. In this review, we describe the basic morphological
and physiological specifics of the heart and cardiac tissues
and introduce the readers to the fundamental principles un-
derlying 3D printing technology and some of the materials/
approaches which have been used to date for cardiac repair.
By summarizing recent progress in 3D printing of cardiac tis-
sue and valves with respect to the key features of cardiovas-
cular tissue (such as contractility, conductivity, and vascular-
ization), we highlight how 3D printing can facilitate surgical
planning and provide custom-fit implants and properties
karger@karger.com © 2021 S. Karger AG, Basel
www.karger.com/cto
Received: September 5, 2020
Accepted: October 27, 2020
Published online: March 5, 2021
that match those from the native heart. Finally, we also dis-
cuss the suitability of this technology in the design and fab-
rication of custom-made devices intended for the matura-
tion of the cardiac tissue, a process that has been shown to
increase the viability of implants. Altogether this review
shows that 3D printing and bioprinting are versatile and
highly modulative technologies with wide applications in
cardiac regeneration and beyond. © 2021 S. Karger AG, Basel
Introduction
Accounting for over 17 million deaths in 2016 [World
Health Organization, 2018], cardiovascular diseases
(CVDs) are the number one cause of mortality world-
wide. At the top of the list is ischemic heart disease, fol-
lowed by other conditions such as rheumatic heart dis-
ease and various cardiomyopathies. Current treatments
for CVD encompass a wide variety of pharmacological
and lifestyle interventions [Metra and Teerlink, 2017;
Waller and Sampson, 2018; Watkins et al., 2018]. How-
ever, if the damage to the heart is too severe, surgical in-
tervention may be the only option, including coronary
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Emilio I. Alarcon
Department of Biochemistry, Microbiology, and Immunology
Faculty of Medicine, University of Ottawa
40 Ruskin St, Rm H5229, Ottawa, ON K1Y 4W7 (Canada)
Glasgow Univ.Lib.
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ealarcon @ ottawaheart.ca

3D bioprinter
Fig. 1. Possible strategies for the fabrication
of functional 3D bioprinted cardiac tissue.
Created with BioRender.com.
artery bypass grafting, valve replacement and, in the case
of highly advanced disease, heart transplant. The urgency
for developing therapeutic approaches for cardiac tissue
is multifactorial and includes (1) limited endogenous re-
generative potential of cardiac tissue, (2) patient-specific
anatomical requirements, and (3) shortages of donor tis-
sue for transplantation [Prabhu and Frangogiannis, 2016;
Taylor et al., 2018].
In order to address the needs in cardiac tissue repair,
the field of tissue engineering has pushed to develop a
number of cell- and biomaterial-based therapies [Nguyen
et al., 2019]. Although there are only a few examples of
stem cell transplant therapies which have been shown
successful in regard to cardiac repair in clinical trials [Ba-
nerjee et al., 2018], recent advances in engineered tissues
presenting improved maturity [Ronaldson-Bouchard et
al., 2018], electrical integration [Tandon et al., 2009], and
lesser immune response [Boehler et al., 2011] hold great
promise that in the future functional tissue constructs,
such as myocardial tissue, valves, and even whole hearts
will provide an alternative to the current donor organ
transplant model. However, in order to fabricate implants
that best recapitulate the physical form, conductivity, and
contractility of native cardiac tissue, advanced and scal-
able fabrication techniques are required. One fabrication
technique that holds great promise in this regard is 3D
bioprinting. With the field of bioprinting coming of age
and the advent of several new modes of printing, better
cell survival, multi-material printing, and increased reso-
lution [Dey and Ozbolat, 2020], the bioprinting of car-
diac tissue seems poised to make an impact on the way
cardiac disease is treated.
2 Cells Tissues Organs
DOI: 10.1159/000512792
Biomaterial/additives
Cells
In vitro maturation
Bioink u
sit
Ex
In vivo implantation
In
sit
u
3D printed tissue
3D printing has allowed for the building of complex
3-dimensional structures [Birla and Williams, 2020].
While 3D printing has taken on many forms over the
years, bioprinting, which is a specific subdiscipline of 3D
printing that employs bioinks for the printing of artificial
tissues and tissue-engineered constructs, has rapidly
gained momentum in recent years. Bioinks are typically
either cellular (containing biomaterial and cells) or acel-
lular (with cells seeded post-printing) [Hospodiuk et al.,
2017; Deo et al., 2020], and there are examples of bioma-
terial-free 3D bioprinting, which have employed cells
only [Ong et al., 2017; Yeung et al., 2019].
3D printing has been employed in the bioprinting of
models, tissues, and organs, as well as in the development
of custom devices for tissue maturation. The various
modes of bioprinting can be subdivided into 2 main
groups:
(1) Ex situ bioprinting where the material is printed out-
side the host, matured, and then implanted [Gaetani et
al., 2015; Gao et al., 2017]; and
(2) In vivo bioprinting, where the material is printed di-
rectly in situ [Di Bella et al., 2018; Cheng et al., 2020]
within the host.
A simple scheme showing the possible routes to 3D
bioprinted cardiac tissue is shown in Figure 1. As the ma-
jority of 3D bioprinting techniques are derived from the
more classical polymer/plastic and resin-based printing
techniques, many of the principles and components un-
derlying the printing process can be similarly classified.
In general, 3D bioprinting can be thought of as a combi-
nation of the top-down and bottom-up routes to the bio-
fabrication of tissues [Shtein and Shoseyov, 2017]. Tradi-
tionally the approach to the fabrication of artificial tissues
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has taken a top-down approach whereby cells and signal-
ing molecules are introduced within a 3D biomaterial,
which can act as a scaffold that mimics the structure of
the extracellular tissue environment. More recently, there
has been an interest in bottom-up approaches, in which
larger cellular structures such as spheroids and cell-sheets
are prepared through self-assembly and then used to as-
semble larger and more complex structures [Lee et al.,
2017]. In 3D printing of tissues and organs, both the top-
down and bottom-up approaches are combined. Bioinks
comprising synthetic, natural, and mimetic polymers
along with cells, and various growth factors are deposited
in such a way as to prepare 3D structures that are not only
able to structurally mimic the tissue and organs but are
also able to recapitulate physiological functions post-
maturation. Herein, we will describe some of the different
approaches and techniques that have been employed in
the fabrication of 3D printed cardiac tissue.
Cardiac Tissue Biology
Before jumping into the 3D printing of cardiac tissue,
it is necessary to have an understanding of the morphol-
ogy, key features, and characteristics of cardiovascular
tissue, as these properties are critical in the design and
fabrication of functional biomimetic structures.
The cardiovascular system is comprised of 2 key com-
ponents, the heart and blood vessels [Ross and Pawlina,
2006; Laflamme et al., 2012], where the blood vessels (ar-
teries, veins, and capillaries) serve as transport elements,
and the heart acts as a pump to circulate the blood
throughout the body. For the heart to function in that ca-
pacity, it requires certain morphological and functional
traits. For example, it contains cardiac muscle capable of
contraction, specific cardiomyocytes that provide intrin-
sic pacemaker capacity, along with a fibrous skeleton and
valves (2 atrioventricular and 2 semilunar) that allow for
unidirectional flow. Histologically, from inside out the
heart is composed of an endocardium, myocardium (car-
diac muscle tissue), and epicardium, and is surrounded
by the pericardial sac. The constant need for oxygenation,
nutrient supply, and waste product removal from the
muscle can become a challenge if the coronary vascula-
ture is unhealthy, which can lead to a loss of metabolic
homeostasis.
If the balance is affected, cells may start failing in their
function, which marks the onset for CVDs. The current
focus of 3D bioprinting for the cardiovascular system has
been largely concentrated on the fabrication of valves and
3D Printing of Cardiac Tissues and
Devices for Their Maturation
cardiac tissue components. While the ultimate goal is the
fabrication of a whole functioning heart, advances must
be made to improve the vascularization of the constructs.
When considering that the diffusion limit of oxygen is
approximately 100–200 μm from the nearest blood vessel
[Carmeliet and Jain, 2000], there is a need to incorporate
micro- and smaller macro-vessels into 3D constructs to
provide the cells with vessel-like networks within this dis-
tance. Without such a vascular network, cell survival and
physiological function can be significantly impaired, thus
limiting the ability to fabricate functional implantable
structures that more closely recapitulate a full thickness
heart muscle.
Cardiac tissue is composed of cardiomyocytes sur-
rounded by cardiac interstitium [Ross and Pawlina, 2006;
Laflamme et al., 2012]. Cardiomyocytes are cells capable
of contraction (contain precisely organized sarcomeres
with actin and myosin) and are interconnected by spe-
cific structures called intercalated discs. These intercalat-
ed discs are comprised of desmosome junctions that pro-
vide strength, and gap junctions (composed of connex-
ins) for the conduction of electrical impulses between
cells. As a result, correct development and maturation of
sarcomeres and intercellular junctions within cardiomy-
ocytes in vitro is crucial for any in vivo tissue engineering
application [Feric and Radisic, 2016]. Cardiac intersti-
tium, the material surrounding the cardiomyocytes, is
comprised of cardiac extracellular matrix (ECM) and var-
ious other cells (e.g., macrophages, fibroblasts, endothe-
lial cells) [Ross and Pawlina, 2006; Laflamme et al., 2012].
In the native healthy ECM, glycoproteins and proteogly-
cans surround collagen type I (∼80%), collagen type III
(∼10%), elastin, and collagens type IV, V, and VI [Jour-
dan-LeSaux et al., 2010; Shamhart and Meszaros, 2010;
Horn and Trafford, 2016]. Knowledge of cardiac ECM
composition is vital in selecting an appropriate biomate-
rial for 3D printing, as the best biomaterial should pro-
vide bioactive cues to the cells, be biocompatible and bio-
degradable, and display physicochemical properties that
match those found in vivo at the site of implantation in
order to withstand the demanding pressure and volume
load present in the heart.
Heart valves are composed of dense irregular connec-
tive tissue, allowing them to withstand the flow of blood,
and are covered with endocardium [Ross and Pawlina,
2006; Laflamme et al., 2012]. While the avascular nature
of the valve leaflets would appear to simplify the design
and manufacture of biosynthetic implants, difficulties
arise when trying to produce strong yet flexible valves that
display a nonthrombogenic surface [Dangas et al., 2016].
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Cells Tissues Organs 3
DOI: 10.1159/000512792
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 Given the limited capacity of cardiomyocyte regenera-
tion in humans, damaged heart tissue is substituted with
a collagenous scar upon injury [Bergmann et al., 2009;
Steenbergen and Frangogiannis, 2012]. While this fibrotic
region significantly hinders normal cardiac function
[Steenbergen and Frangogiannis, 2012], several cell-based
tissue engineering approaches have been developed to
limit scar formation and allow the tissue to retain its elas-
ticity. These cell-based therapies have been applied either
alone (single cell suspensions, cell sheets, spheroids) or in
combination with biomaterials (injectable hydrogels,
patches, 3D printed scaffolds) [Atala, 2004; He and Chen,
2020]. Typically, cell-based therapies for cardiac applica-
tion employ human embryonic stem cells (hESCs), hu-
man induced pluripotent stem cells (hiPSCs), mesenchy-
mal stromal cells, hematopoietic stem cells, cardiac stem
cells, cardiac progenitor cells (CPCs), primary cardiomy-
ocytes, skeletal myoblasts, fibroblasts, or endothelial pro-
genitor cells [Jover et al., 2018; Müller et al., 2018; Wang
et al., 2018; He and Chen, 2020]. Despite the seemingly
large number of available cell types, there are many limita-
tions to these cells, which represents a major bottleneck
toward cardiac tissue engineering [Huang NF et al., 2018;
Müller et al., 2018; He and Chen, 2020]. One of the first
issues to arise relates to difficulties obtaining a clinically
relevant number of cells [Huang NF et al., 2018]. In par-
ticular, primary cells have limited proliferative potential
in vitro before entering senescence [Kengla et al., 2017],
while differentiation protocols for hESCs and hiPSCs and
other stem/stromal cells struggle with yield and purity of
the differentiated target cell type [He and Chen, 2020].
Moreover, undifferentiated cell populations may pose a
risk of forming teratomas or tumors [He and Chen, 2020].
The second major obstacle that must be addressed relates
to difficulties in obtaining cells of sufficient maturity. For
example, surrogate cells may have a more amorphous
shape compared to the rectangular one found in mature
cardiomyocytes. Further, these cells may lack proper sar-
comere organization and alignment, as well as calcium
handling mechanisms, all key properties for efficient con-
tractile function. Metabolism and transcriptional profile
also change with maturation, and such immaturity could
influence successful engraftment. Many approaches have
been proposed to promote maturation; these include the
use of hormones, mechanical strain stimulation, electrical
stimulation, patterned surfaces and substrate stiffness (6–
10 kPa), and interactions with other cells or ECM [Huang
NF et al., 2018; Ahmed et al., 2020; Guo and Pu, 2020; He
and Chen, 2020]. However, even with these methods, cell
maturation into adult cardiomyocytes is still poor. An-
4 Cells Tissues Organs
DOI: 10.1159/000512792
other issue that must be addressed is cell survival and re-
tention at the site of implantation, as well as immunoge-
nicity [Huang NF et al., 2018; He and Chen, 2020]. Fol-
lowing implantation, improper electrical coupling
between engineered and native tissue can cause life-
threatening arrhythmias [Huang NF et al., 2018]. Lastly,
regulatory policies on research and clinical application of
stem cells are different all over the world, and this may
restrict their therapeutic application [Gopalan et al.,
2018].
The cell types that have most commonly been used in
the design of 3D printed cardiac and vascular tissue in-
clude primary cardiomyocytes, CPCs, cardiac fibroblasts,
hiPSC-derived cardiomyocytes, smooth muscle cells, en-
dothelial cells, hESC-derived cardiomyocytes, valvular
interstitial cells, and human umbilical vein endothelial
cells (HUVECs) (Table 1) [Duan et al., 2013; Gao et al.,
2017; Bejleri et al., 2018; Wang et al., 2018; Lee et al.,
2019]. As for the biomaterials, the most commonly used
include collagens, gelatin, alginate, hyaluronic acid, and
decellularized cardiac ECM [Pati et al., 2014; Gaetani et
al., 2015; Zhang et al., 2016; Izadifar et al., 2017]. To in-
crease electroconductivity of biomaterials, carbon nano-
tubes or gold nanorods have been used [Ho et al., 2017;
Zhu et al., 2017; Hosoyama et al., 2018], as well as other
substances that have been shown to increase electrocon-
ductivity, such as graphene oxide, aniline, or polypyrrole
[Sedlakova et al., 2019].
Fundamental Concepts of 3D Printing
3D printing, also known as additive manufacturing, is
the process through which 3D objects are created from
3D models using computer-aided design (CAD) software
or from medical imaging devices (magnetic resonance
imaging [MRI], computerized axial tomography scan,
functional MRI, among others). Whether one is interest-
ed in printing plastic models and devices or bioprinting
functional tissues and organs, one needs to begin with a
digital 3D model of the object to be printed, or a pattern
to be recapitulated, as it is this model or pattern that pro-
vides the cartesian coordinates as to where the printed
material is to be deposited or crosslinked. While there are
a number of software suites available for CAD design
(both free and proprietary), in order to generate biomi-
metic tissues and organs that can imitate the features and
functions of the desired targets, it is necessary to intro-
duce advanced imaging modalities within the 3D model-
ing process [Squelch, 2018]. Through the use of image
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Table 1. 3D bioprinted cardiac tissue, implants, and components of the heart

Target tissue Bioink Cells used In vivo model Achievement Reference

or implant
Cardiac Fibrin Primary rat cardiomyocytes N/A Drug testing Wang et al. 2018)
Cardiac Gelatin Human mesenchymal stem cells N/A Enzymatic crosslinking Tijore et al. 2018)
Cardiac patch Hyaluronic acid; Human cardiac-derived progenitor Implantation onto Reduced adverse remodeling Gaetani et al.
gelatin cells infarcted murine hearts 2015)
Cardiac patch Methacrylated gelatin Human induced pluripotent stem Implantation onto Improved cardiac function; Gao et al. 2017)
cell-derived cardiomyocytes, smooth infarcted murine hearts reduced scar size
muscle cells and endothelial cells
Cardiac Decellularized cardiac Rat myoblast cells N/A Decellularized cardiac ECM Pati et al. 2014)
ECM
Cardiac patch Decellularized cardiac Human cardiac progenitor cells Implantation onto rat Decellularized cardiac ECM Bejleri et al. 2018)
ECM; gelatin hearts
methacrylate
Cardiac Alginate; Human umbilical vein endothelial Murine subcutaneous Formation of vessel-like Maiullari et al.,
PEG-fibrinogen cells; induced pluripotent stem implantation structures 2018
cell-derived cardiomyocytes
Cardiac Gelatin; alginate; Human umbilical vein endothelial N/A Perfusable hollow vessels Jia et al., 2016
poly(ethylene glycol)- cells; human mesenchymal stem
tetra-acrylate cells
Cardiac Alginate; gelatin Human umbilical vein endothelial N/A Toxicity testing; bioreactor Zhang et al., 2016
methacryloyl cells; neonatal rat cardiomyocytes;
human induced pluripotent stem
cell-derived cardiomyocytes
Cardiac patch Decellularized cardiac Human c-kit+ cardiac progenitor Implantation onto Decellularized cardiac ECM Jang et al., 2017
ECM cells infarcted rat hearts
Cardiac Gelatin methacryloyl; Cardiomyocytes; cardiac fibroblasts N/A Gold nanorods Zhu et al., 2017
gold nanorods (electroconductivity)
Cardiac patch Methacrylated Human coronary artery endothelial N/A Carbon nanotubes (electro- Izadifar et al.,
collagen; carbon cells conductivity); UV-integrated 2017
nanotubes 3D bioprinting technique
Cardiac Polycaprolactone; Rat H9c2 cells N/A Carbon nanotubes Ho et al., 2017
carbon nanotubes (electroconductivity)
Cardiac patch No Human induced pluripotent stem Implantation onto rat Biomaterial-free 3D Ong et al., 2017
cell-derived cardiomyocytes; hearts bioprinting
fibroblasts; endothelial cells
Cardiac patch No Human induced pluripotent stem Implantation onto rat Biomaterial-free 3D Yeung et al., 2019
cell-derived cardiomyocytes; hearts bioprinting
fibroblasts; endothelial cells
Valve Alginate; gelatin Smooth muscle cells; aortic valvular N/A Heterogenous cell Duan et al., 2013
interstitial cells encapsulation
Valve Methacrylated Human aortic valvular interstitial N/A Deposition of collagen and Duan et al., 2014
hyaluronic acid; cells glycosaminoglycans
methacrylated gelatin
Valve Polyethylene glycol- Porcine aortic valvular interstitial N/A Photocrosslinking Hockaday et al.,
diacrylate; alginate cells 2012
Heart Alginate N/A N/A FRESH method (freeform Hinton et al., 2015
reversible embedding of
suspended hydrogels)
Coronary Collagen type I; C2C12 cells; human embryonic Murine subcutaneous FRESH method (freeform Lee et al., 2019
artery; alginate stem cell-derived cardiomyocytes; vascularization model reversible embedding of
ventricle; cardiac fibroblasts; human umbilical suspended hydrogels)
valve; heart vein endothelial cells

ECM, extracellular matrix.

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3D Printing of Cardiac Tissues and Cells Tissues Organs 5

Devices for Their Maturation DOI: 10.1159/000512792
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and geometry data obtained from computed tomography
and MRI scanning, one can generate patient-specific
models that are of high quality and accuracy, which is
valuable considering the current interest and push to-
ward personalized health care. Once the 3D model has
been created, it is then processed in slicing software that
generates a 3D map with coordinates and specifications
that direct the 3D bioprinter on how and where to print
the desired construct [Bücking et al., 2017].
While there are many different types and principles
behind the currently available printers for bioprinting,
there has been much interest in the development of hand-
held devices and robotically-assisted devices aimed at
printing tissues, organs, and scaffolds in situ [Singh et al.,
2020]. Although there has been some progress toward
this goal [Keriquel et al., 2010; Skardal et al., 2012; Di
Bella et al., 2018; Hakimi et al., 2018; Albanna et al., 2019;
Adib et al., 2020; Cheng et al., 2020; Urciuolo et al., 2020],
it remains challenging due to the unavailability of bioinks
that precisely match mechanical and biological properties
needed to print tissues with intricate geometric features
and complex biomechanical properties, such as cardiac
tissue and blood vessels. For these reasons there have
been no examples of in situ printed cardiac tissue to date.
As such, in the next sections, we will focus on ex situ bio-
printing. Many of the current ex situ bioprinting devices
and techniques are derived from traditional 3D printing
as they have been thoroughly studied and developed.
During ex situ bioprinting, the scaffold, tissue, or organ
is printed outside of the body under sterile aseptic condi-
tions and then transplanted to the target site (with or
without the inclusion of exogenous cells) or used in vitro
in the study of tissue development, disease, and drug
treatments. Considering that the bioprinting of cell-laden
and cell-free constructs is most frequently accomplished
through the use of extrusion-based printers, we will focus
on them. However, there are a number of other tech-
niques which have been developed such as freeform re-
versible embedding of suspended hydrogels (FRESH)
[Hinton et al., 2015; Lee et al., 2019], inkjet [Angelopou-
los et al., 2020], laser/light-induced [Guillotin et al., 2013],
and other less explored techniques such as ultrasonic
standing bulk acoustic waves (SBAW) [Chansoria and
Shirwaiker, 2019], aspiration-assisted [Ayan et al., 2020],
3D sacrificial molding [Miller et al., 2012], optogenetic
manipulation [Huang Y et al., 2018], Kenzan method
[Aguilar et al., 2019], and pick-and-place [Blakely et al.,
2015] based bioprinting.
Extrusion-based bioprinters work in a very similar
fashion to fused-deposition modeling (FDM) 3D print-
6 Cells Tissues Organs
DOI: 10.1159/000512792
ers, with the main difference being that the printing fila-
ment found in traditional FDM printers is replaced with
cell-laden and cell-free biocompatible inks (bioinks). The
largest driving factor making extrusion-based printers a
go-to in the bioprinting community is that the printing
technique can be easily achieved through the conversion/
modification of conventional extrusion-based 3D print-
ers, a process that reduces the cost and time spent devel-
oping the printing device [Pusch et al., 2018]. Having said
that, there are a variety of commercially available, modi-
fied and custom-made extrusion-based 3D printers being
employed throughout the literature.
Even though there still remain challenges to overcome
in order to produce more physiologically relevant 3D
printed models via extrusion-based bioprinting, the re-
search community has made, and continues to make,
great advancements in the development of better equip-
ment, techniques, and printing material. The efforts to
reduce printing time, the development of equipment to
print with multiple materials to create more complex
structures, and the use of sacrificial materials to create
channels and vasculature within the printed constructs
still make extrusion-based 3D bioprinting a promising
technique to advance the field of tissue engineering.
One of the main limitations encountered with extru-
sion-based bioprinting techniques is the inability to
print complex 3D structures due to the need for support
materials and the low rigidity and stiffness of the bio-
inks. During FRESH bioprinting, a liquid hydrogel is
extruded within a thermoreversible bath, which be-
haves as a Bingham fluid [Hinton et al., 2015]. The sup-
port bath acts as a gelatin-like solid at rest and as a liquid
when sufficient shear stress is generated within the flu-
id. During 3D printing, the motion of the needle that
extrudes the bioink generates shear stress of enough
magnitude such that the bath behaves like a liquid dur-
ing the motion of the needle, but it acts as a solid sup-
port to the extruded bioink. Once the printing process
is completed, the temperature of the support bath is
raised such that it becomes a liquid, and the crosslinked
3D printed structure can be easily removed. This behav-
ior of the support bath, coupled with cross-linking reac-
tions between the bath and the bioink, allow for the 3D
printing of constructs with more complex structures
and higher fidelity to the employed CAD model. Even
though it is still in its early stages, this technology has
shown potential to produce geometrically complex
structures using both cell-free and cell-laden bioinks,
solving some of the limitations of conventional extru-
sion-based bioprinting.
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 Another printing technique that also made use of pre-
viously developed technologies and adapted them to
serve the purpose of tissue engineering and bioprinting is
inkjet bioprinting. In inkjet bioprinting, the same con-
cept of droplet printing is applied, but instead of ink, the
modified printers eject bioinks. The droplets are ejected
using thermal [Cui et al., 2012a], microvalve (pressur-
ized) [Ng et al., 2017], or piezoelectric components
[Hoehne et al., 2020]. This technique allows for a non-
contact dispersal of cell-loaded bioinks with high accu-
racy and, in some cases, the possibility to determine how
many cells are encapsulated and dispersed in every drop-
let at each location [Li et al., 2018]. In spite of these ad-
vancements, this bioprinting method has not gained as
much attention as other methods due to low cell viability
after the printing process, and the inability to generate
geometrically complex structures, which hinders its ap-
plication in clinically relevant settings [Li et al., 2018].
Laser/light-induced bioprinting has also gained sub-
stantial interest among researchers and companies in re-
cent years. Although within the concept of utilizing light
to bioprint 3D structures there are various different de-
vices and techniques, they all use light and/or lasers to
initiate the crosslinking of reactive functionalities, or to
eject with high accuracy droplets of bioink in specific pat-
terns. In the former, researchers apply concepts of photo-
chemistry to induce the photo-crosslinking of bioinks. To
accomplish this, various teams have applied concepts re-
lated to stereolithography and digital light processing,
where lasers and reflective surfaces are used to photo-
crosslink a liquid into very complex shapes in a layer-by-
layer manner [Connell et al., 2013; Cui et al., 2019; Grigo-
ryan et al., 2019; Hong et al., 2020]. This approach allows
for the rapid printing of complex and vascularized con-
structs reducing the need for support material and avoid-
ing the risk of cell death due to shear forces created as the
bioink would typically pass through the nozzles of an ex-
trusion-based system. However, one of the main con-
cerns and limitations of this technique is the toxicity of
some of the photoinitiator molecules themselves, or the
reactive intermediates and byproducts that are generated
during the photo-crosslinking process [Zhu et al., 2015;
Bagheri and Jin, 2019]. Other devices that combine extru-
sion-based bioprinting and light-induced techniques to
create complex structures with different materials have
also been developed [Cui et al., 2012b; Hockaday et al.,
2012; Shao et al., 2018]. This method allows for the use of
more readily accessible 3D bioprinters, as well as the gen-
eration of multi-material constructs, where some of these
materials are intended as support or sacrificial materials
3D Printing of Cardiac Tissues and
Devices for Their Maturation
that are washed away or degraded after the printing pro-
cess, generating vascularized and/or more geometrically
complex structures.
Yet another 3D printing technique that has gained
some interest among research groups is the application of
laser-induced forward transfer techniques for bioprint-
ing [Gruene et al., 2011; Kingsley et al., 2013; Koch et al.,
2013]. During this process, also referred to as laser-assist-
ed bioprinting, a cell-loaded hydrogel is propelled for-
ward by the pressure generated by a laser-induced vapor
bubble. Usually, there is a laser-absorbing layer and a cell-
loaded hydrogel layer. Upon irradiation with the laser, a
droplet of hydrogel is ejected onto another surface. Using
this technique, 3D printed structures with fine patterning
can be generated [Koch et al., 2013].
3D Printing of Cardiac Tissue and Its Applications
Cardiac Tissue
Contractility is the most fundamental function of car-
diac tissue. There are several examples of 3D printed con-
structs displaying this property. For example, Wang et al.
[2018] developed a spontaneously contractile cardiac tis-
sue from a fibrin-based bioink and primary rat cardio-
myocytes, which maturated in vitro and responded phys-
iologically to drugs. Tijore et al. [2018] generated a pat-
terned 3D bioprinted gelatin hydrogel with microchannels
that promoted differentiation of human mesenchymal
stem cells and beating of cardiomyocytes. While Gaetani
et al. [2015] developed a patch from hyaluronic acid, gel-
atin and human cardiac-derived progenitor cells, which,
upon implantation to the infarcted murine heart, pre-
served cardiac function and reduced adverse ventricular
remodeling. Similarly, Gao et al. [2017] showed that a car-
diac patch derived from printed methacrylated gelatin,
hiPSC-derived cardiomyocytes, smooth muscle cells, and
endothelial cells improved cardiac function and reduced
scar formation in infarcted murine hearts.
The importance of cardiac-specific ECM was recog-
nized by Pati et al. [2014], who used decellularized por-
cine ventricles to develop a cardiac-specific decellularized
ECM bioink that resulted in printed constructs that al-
lowed for better maturation of rat myoblast cells as com-
pared to collagen printed counterparts. Another ap-
proach by Bejleri et al. [2018] involved the combination
of decellularized cardiac ECM, gelatin methacrylate, and
human CPCs to generate cardiac patches. Incorporation
of cardiac ECM within these patches led to greater matu-
ration of human CPCs as confirmed by increased expres-
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sion of cardiogenic genes, while patches implanted with-
in the rat model showed good vascularization.
Given the limited oxygen diffusion [Gershlak et al.,
2017] and exceptionally high demand of cardiac tissue for
oxygen and nutrients, the incorporation of vessel-like
structures into a 3D bioprinted construct is a necessity.
Maiullari et al. [2018] exploited the endogenous ability of
HUVECs to form monolayers and tubular structures and
combined this with the idea of 3D bioprinted constructs
consisting of perpendicularly printed fibers. In particular,
they used 2 cell-laden bioinks, the first one combined algi-
nate and poly(ethylene glycol)-fibrinogen (PEG-fibrino-
gen) hydrogel with HUVECs, whereas the second com-
bined induced pluripotent stem cell (iPSC)-derived car-
diomyocytes with the hydrogel. A variety of 3D printed
structures consisting of perpendicular fibers were pre-
pared, each with a different pattern of incorporated cell
types. For example, there were Janus constructs, which
consisted of HUVECs and iPSC-derived cardiomyocytes
within each fiber layer, as well as 2:2:2:2:2 structures, which
consisted of alternating double layers of fibers comprising
either HUVECS or iPSC-derived cardiomyocytes, and
4:2:4 structures where the pattern of cell deposition was
interrupted by a double layer of unique cells every fifth
layer. Upon subcutaneous implantation in NOD-SCID
mice, the Janus constructs showed the highest formation
of vessel-like structures originating from HUVECs as well
as host vascularization, while the incorporated cardiomy-
ocytes displayed the best alignment. Jia et al. [2016] have
also developed a method for incorporating hollow vessels
within 3D printed constructs through the use of a multi-
layer coaxial extrusion system employing gelatin methac-
ryloyl, sodium alginate, and poly(ethylene glycol)-tetra-
acrylate derived bioinks which are amenable to both ionic-
and photo-crosslinking. This method allowed for the
bioprinting of perfusable hollow tubes as well as the incor-
poration of HUVECs and human mesenchymal stem cells
into the constructs. A more stepwise approach to this goal
was developed by Zhang et al. [2016]. First, a microfibrous
alginate/gelatin methacryloyl scaffold with HUVECs was
printed. Next, the HUVECs were allowed to form lumen-
like structures. Finally, neonatal rat cardiomyocytes or
hiPSC-derived cardiomyocytes were seeded into the inter-
stitial space to form a beating endothelialized cardiac tis-
sue. These constructs were intended to be used as in vitro
models to reveal drug side effects and potential cardiotox-
icity. As a proof-of-concept, constructs were exposed to
doxorubicin and showed dose-dependent toxicity. Lastly,
Jang et al. [2017] looked at improving the vascularization
of constructs by using a decellularized porcine cardiac
8 Cells Tissues Organs
DOI: 10.1159/000512792
ECM bioink, hoping there would be synergistic effects.
The resulting patches with human c-kit+ cells and human
mesenchymal stem cells were implanted onto infarcted rat
hearts and were found to promote cardiac function, vas-
cularization, and new muscle formation.
Another key feature of cardiac tissue is its ability to
propagate electrical signals owing to its intrinsic electro-
conductivity. There are several examples of 3D bioprint-
ed constructs that have been printed using bioinks con-
taining electroconductive components such as metallic
nanoparticles, graphene-like materials, and synthetic
conductive polymers. For example, Zhu et al. [2017] em-
bedded gold nanorods into a gelatin methacryloyl bioink.
The gold nanorods were found to promote cardiomyo-
cyte coupling and synchronized contraction. Carbon
nanotubes have also found use in the design of conductive
constructs. In one study, a UV-integrated 3D bioprinting
technique was employed to create nanoreinforced and
conductive cardiac patches [Izadifar et al., 2017]. Through
the combination of human coronary artery endothelial
cells with methacrylated collagen and carbon nanotubes,
the authors demonstrated that one can significantly im-
prove electrical conductivity in these types of materials.
Similarly, Ho et al. [2017] used carbon nanotubes and
combined them with polycaprolactone and showed bio-
compatibility with rat H9c2 cells.
A biomaterial-free 3D bioprinting approach has also
been developed by Ong et al. [2017]. Spheroids derived
from hiPSC-derived cardiomyocytes, fibroblasts, and en-
dothelial cells were bioprinted onto a needle array and
allowed to fuse. The resulting fused cardiac tissue was re-
moved from the array and allowed to mature. In vivo im-
plantation of the cardiac patches for 1 week onto the
hearts of rats resulted in their engraftment and vascular-
ization. This work was then expanded by Yeung et al.
[2019], who investigated the consequences of in vivo im-
plantation for 4 weeks. After 4 weeks, cardiac function
trended toward improvement, while scar size was re-
duced, and vascularization was promoted.
Heart Valves
Several approaches have been taken toward the fabri-
cation of 3D printed valves. Duan et al. [2013] prepared
heterogeneously populated aortic valve conduits based
on a alginate/gelatin hydrogel. The root region was popu-
lated by human aortic root smooth muscle cells, whereas
the leaflets contained porcine aortic valve interstitial cells.
The authors then went on to further improve their ap-
proach for printing tri-leaflet valves by utilizing methac-
rylated hyaluronic acid and gelatin along with human
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aortic valvular interstitial cells [Duan et al., 2014]. The
cells actively remodeled the initial scaffold through col-
lagen and glycosaminoglycan deposition. Hockaday et al.
[2012] printed an aortic valve via photocrosslinking of
printed polyethylene glycol-diacrylate supplemented
with alginate and seeded with porcine aortic valve inter-
stitial cells. While Lee et al. [2019] printed a collagen-de-
rived tri-leaflet heart valve at adult human scale using the
FRESH method and showed mechanical functionality in
a flow system as well as biocompatibility with HUVECs
that eventually formed a monolayer on the surface.
Heart
As previously mentioned, the FRESH method of 3D
printing, developed by Hinton et al. [2015], allows for the
3D printing of hydrated materials with elastic modulus
<500 kPa using a secondary hydrogel support bath. Using
this method, the authors successfully printed a chick em-
bryonic heart model from alginate with complex trabecu-
lar architecture. The FRESH method has also been used
to bioprint collagen type I under pH-driven control of
gelation into various scale model structures [Lee et al.,
2019]. Starting from small vessels and microporous scaf-
folds, they proceeded to print a model ventricle seeded
with human stem cell-derived cardiomyocytes and car-
diac fibroblasts, which was capable of contraction. The
resulting ventricle was capable of spontaneous beating at
a rate of ∼0.5 Hz and could also be paced at 1 and 2 Hz.
Additionally, they designed a human heart model with
valves, trabeculae, and vessels and demonstrated vessel
patency in a segment if the vasculature was down to ∼100
μm.
3D printed models have also found use in helping to
better understand patient-specific anatomical conditions
and in planning the best surgical approach, especially in
complex or borderline cases, such as patients with con-
genital heart defects, patients requiring valve replace-
ment, ventricular assist device implantation or heart
transplant [Ripley et al., 2016; Valverde et al., 2017; An-
war et al., 2018; Farooqi et al., 2019; Forte et al., 2019;
Tuncay and van Ooijen, 2019]. A summary of the afore-
mentioned achievements can be found in Table 1.
3D Printing of Devices for Culture and Maturation of
Cardiac Tissue
While there have been significant advances in the bio-
printing of tissue constructs, in order for these materials
to be used as accurate models for the study of drugs and
3D Printing of Cardiac Tissues and
Devices for Their Maturation
disease or as replacements of diseased or damaged tissue,
they need to be matured/trained in an environment which
recapitulates the in vivo environment they are to mimic
or be employed. Considering the complexity of cardio-
vascular tissue, there is a clear need to design universal
bioreactors which can accommodate a variety of geome-
tries and allow for the culture of cells, perfusion with me-
dia and growth factors, stimulation (mechanical, electri-
cal, electromechanical), visualization, and real-time
monitoring of tissue health, maturity, and physical prop-
erties, all while maintaining a sterile/aseptic environment
[Borovjagin et al., 2017]. While the advantages of 3D bio-
reactors over 2D culture are well-known, and there exist
a number of different designs and approaches developed
over the years, many of the devices developed to date are
quite simple, with the majority of them failing to incor-
porate features that would allow for accurate mimicry of
in vivo environments [Murphy et al., 2020]. Also, they
typically involve multi-step, complex, and destructive
transfer of bioprinted constructs into and out of the bio-
reactor, and have unintuitive interfaces and limited con-
trol over stimulation parameters or regimes [Murphy et
al., 2020]. As the availability of both autoclavable and bio-
compatible filaments and resins has increased, and both
FDM and stereolithography printing technologies have
matured to the point where printers are readily accessible
by most research groups, there is now the possibility of
rapidly generating 3D printed prototypes of complex bio-
reactors in-house. Using CAD software and the direct in-
put from scientists, researchers, and clinicians that are
developing, studying, and implanting these constructs, it
is relatively simple to develop improved prototypes with
features that could possibly enhance the translation and
efficacy of the fabricated tissues. In the design of 3D bio-
reactors for cardiac tissues, there are a number of features
that the device must allow for control of such as cell-cell
interactions, cell-ECM interactions, mechanical stimula-
tion, electrical stimulation, tissue stiffness, and vessel for-
mation [Borovjagin et al., 2017]. Considering these re-
quirements, there have been relatively few 3D printed re-
actors reported for cardiac tissue, with the majority of
reactors focused primarily on mechanical stimulation
alone, a property which has been used for the condition-
ing of cardiac tissue but should ideally be accompanied
with electrochemical cues as these combinations have
been demonstrated to improve maturation of cell and tis-
sue cultures toward more functional phenotypes [Lu et
al., 2013; Miklas et al., 2014; Morgan and Black, 2014;
Ruan et al., 2016; Kroll et al., 2017; Besser et al., 2018].
Table 2 lists some of the 3D bioreactors which have been
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 Table 2. 3D printed bioreactors for tissue maturation and testing

Device name Printer/material Tissue(s) Stimulation Reference

FABRICA SLA Liver Perfusion Smith et al., 2018

Dental SG resin Kidney
Bone
BEaTS-α FDM Cardiac Electromechanical Cortes et al., 2020
Nylon 680
– SLA Tendon Mechanical Banik and Brown, 2020
Not described
– FDM Cardiac Mechanical Putame et al., 2019
ABS plus-P430
SR30 soluble
– FDM Tendon Mechanical Raveling et al., 2018
ABS
– FDM Cardiac Mechanical Putame et al., 2019
ABS plus-P430
SpinΩ FDM Neural Mechanical Qian et al., 2016
ULTEM 9085
– PµSL Vascularized Vascularization Xia and Fang, 2009
Poly(ethylene glycol)

ABS, acrylonitrile butadiene styrene; FDM, fused deposition modeling; SLA, stereolithography; SG, surgical
guide; PµSL, projection micro-stereolithography.

fabricated using 3D printing technologies; while this list
is not meant to be all-encompassing, it does highlight dif-
ferent types of bioreactors using these techniques and
gives an idea of the kind of stimulation regimes that can
be incorporated in these devices. Recently, we have re-
ported on the development of the BEaTS-α bioreactor, an
open-source system for the simultaneous electro and me-
chanical stimulation of cells in vitro (hiPSC-derived car-
diomyocytes) [Cortes et al., 2020]. The BEaTS-α device
was designed with CAD software, and most components
were 3D printed in-house via FDM using autoclavable
and FDA-approved materials. Combined with a com-
mercially available C-PACE EP system, our device is ca-
pable of electromechanically stimulating cells cultured on
flexible silicone membranes fitted to a standard 6-well
plate. The compact and open-access system is capable of
partially recapitulating the natural contraction and signal
propagation found within the heart. Our experiments in-
dicated that hiPSC-derived cardiomyocytes cultured un-
der electromechanical stimulation with the device showed
a more mature cardiomyocyte phenotype than non-stim-
ulated cells after only 7 days of stimulation [Cortes et al.,
2020].
In addition to in-house fabricated bioreactors, a num-
ber of companies have begun using 3D printing technol-
ogies to speed up development of new and custom biore-
actors. Having the ability to share design files between
sites and print locally means that devices do not have to
be shipped between manufacturing and testing sites, sav-
ing time and money, and allowing for quicker optimiza-
tion as slight modifications to the design files can be made
by those deploying the device or, if necessary, shared dig-
itally with the manufacturer who can quickly make the
changes and send back the file for printing [Formlabs,
2019].
Future Directions
Cardiac tissue capable of contraction and spontaneous
beating and functional valves have been successfully cre-
ated using 3D printing technology. While this is promis-
ing, in order to proceed to cell-containing large-scale
constructs or even to whole hearts of human size, solu-
tions to problems facing the vascularization of theses con-
structs and artificial tissues will need to be developed.
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DOI: 10.1159/000512792 Alarcon
Glasgow Univ.Lib.
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Given the diffusion limit of oxygen [Carmeliet and Jain,
2000], an intricate network of capillaries supplied by ar-
terioles is needed. However, the current 3D printing tech-
nology is not able to print such microvascular structures
[Gershlak et al., 2017]. Increases in printing resolution
and advances in the use of sacrificial materials might en-
able us to overcome this limitation of printing micro-
sized hollow structures. Concurrently, the use of biocom-
patible and biomimetic materials and reduced use of cy-
totoxic photocrosslinkers and other initiators might
significantly increase cell viability. Furthermore, the de-
velopment of even more sophisticated printers may en-
able simultaneous printing of multiple bioinks, thus lead-
ing to more geometrically and physicochemically com-
plex constructs allowing for higher spatiotemporal
specificity of biomimetic cues and heterogenous cell colo-
nization of the construct. Miniaturization would also en-
able in situ bioprinting of cardiac tissue, as so far only in
situ printing for skin or cartilage repair was achieved [Di
Bella et al., 2018; Cheng et al., 2020]. An improvement of
reactors to encompass multiple stimulation modalities
and regimes, automated transfer of bioprinted constructs
in and out (to achieve the highest standard of aseptic con-
ditions), or more user-friendly interfaces will also likely
lead to materials with greater translational success.
Conclusion
most frequently used. Contractile and electroconductive
tissue constructs/patches with vessel-like structures have
been successfully tested both in vitro and in vivo with very
promising outcomes. Custom-made bioprinted and cell-
containing valves, as well as 3D printed models of the
heart hold great promise for treating cardiac disease.
Maturation of the printed tissues can be further enhanced
by cultivation in custom-made bioreactors that allow for
perfusion and/or electromechanical stimulation. Novel
advancements in the bioprinting of cardiac tissue are also
expected with improvements to currently available print-
ing techniques as well as through the combination of pre-
viously developed strategies.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This work was made possible by funding from the Natural Sci-
ences and Engineering Research Council (NSERC) RG-
PIN-2015-0632 to E.I.A. and Canadian Institutes of Health Sci-
ences (CIHR) to E.I.A. and E.J.S. E.I.A. also thanks the New Fron-
tiers Research Program Exploration fund and the Government of
Ontario for an Early Career Research Award.

3D bioprinting is a versatile technology enabling the Author Contributions

development of cell-laden and cell-free constructs. Cur- The manuscript was written through contributions from all au-
rently, in the field of cardiovascular tissue engineering, thors. All authors have given approval to the final version of the
extrusion-based printing and the FRESH method are manuscript.

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