Opinion
Bioprinting scale-up tissue and organ
constructs for transplantation
Ibrahim T. Ozbolat
Biomanufacturing Laboratory, The University of Iowa, Iowa City, IA, 52242, USA
Bioprinting is an emerging field that is having a revolu- biology of natural counterparts for pharmaceutical testing
tionary impact on the medical sciences. It offers great or cancer studies [8].
precision for the spatial placement of cells, proteins, Here I present recent approaches to the bioprinting
genes, drugs, and biologically active particles to better scale-up of functional tissue and organ constructs for
guide tissue generation and formation. This emerging transplantation, including bioprinting vascularized tissue
biotechnology appears to be promising for advancing and organ constructs in vitro and in situ bioprinting tech-
tissue engineering toward functional tissue and organ nology to build tissues directly in defect sites. I discuss
fabrication for transplantation, drug testing, research major roadblocks to this approach and provide potential
investigations, and cancer or disease modeling, and has solutions and future directions.
recently attracted growing interest worldwide among
researchers and the general public. In this Opinion, I Bioprinting of vascularized tissue and organ constructs
highlight possibilities for the bioprinting scale-up of func- in vitro
tional tissue and organ constructs for transplantation and Organ bioprinting holds great promise for the future, but
provide the reader with alternative approaches, their whole-organ bioprinting has remained elusive due to
limitations, and promising directions for new research several limitations associated with biology, bioprinting
prospects. technology, bioink material, and the post-bioprinting mat-
uration process [9]. The bioprinting of functional tissues is
Bioprinting: a promising technology to revolutionize an intermediate stage toward achieving organ-level com-
medicine plexity. In vitro fabrication of functional tissues is a
Bioprinting can be defined as the spatial patterning of sophisticated phenomenon comprising a hierarchical ar-
living cells and other biologics by stacking and assembling rangement of multiple cell types, including a multiscale
them using a computer-aided layer-by-layer deposition network of vasculature in stroma and parenchyma, along
approach to develop living tissue and organ analogs for with lymphatic vessels and, occasionally, neural and mus-
tissue engineering, regenerative medicine, pharmacoki- cle tissue, depending on the tissue type. In vitro engineered
netic, and other biological studies [1]. It uses four tissue models that incorporate all of these components are
approaches to deposit living cells: inkjet [2], extrusion still far on the horizon. Bioprinting technology offers a
[3], acoustic [4] and laser [5] based. Given its great benefit great benefit in the hierarchical arrangement of cells or
in spatially arranging multiple cell types to recapitulate building tissue blocks in a 3D microenvironment, but the
tissue biology, bioprinting is a game-changer in the rapid bioink and the post-bioprinting maturation phase are as
development of tissue constructs and is receiving enor- important as the bioprinting process itself. The bioink
mous attention. Although bioprinting of functional 3D material is crucial because it should provide the spectrum
whole organs for transplantation remains in the realm of biochemical (i.e., chemokines, growth factors, adhesion
of science fiction, the field is moving forward, providing factors, or signaling proteins) and physical (i.e., interstitial
hope that shortages in tissue grafts and organ transplan- flow, mechanical and structural properties of extracellular
tation will be mitigated to some extent in the future matrix) cues to promote an environment for cell survival,
[6]. While current tissue-engineering strategies cannot motility, and differentiation [10]. In addition, the bioink
enable fabrication of fully functional tissues or organs should exhibit high mechanical integrity and structural
[7], bioprinting enables precise placement of biologics to stability without dissolving after bioprinting, enable dif-
recapitulate heterocellular tissue biology to some degree. ferentiation of autologous stem cells into tissue-specific cell
Current technology enables the development of organ or lineages, facilitate engraftment with the endogenous tis-
tissue constructs that do not require substantial vascu- sue without generating an immune response, demonstrate
larization, as well as mini-tissue models mimicking the bioprintability with ease of shear thinning, rapid solidifi-
cation and formability, and be affordable, abundant, and
commercially available with appropriate regulations for
Corresponding author: Ozbolat, I.T. ([email protected]).
clinical use [11]. A variety of bioinks, including naturally
Keywords: bioprinting; bioprinting for transplantation; vascularized-tissue printing;
in situ bioprinting. and synthetically derived materials, has been used for
0167-7799/ tissue regeneration, as detailed in the literature
ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2015.04.005 [12,13]. The post-bioprinting process is also crucial and
necessitates mechanical and chemical stimulation and
Trends in Biotechnology, July 2015, Vol. 33, No. 7 395
Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7
signaling to regulate tissue remodeling and growth, the vascular tubes [19] using tissue spheroids as building
development of new bioreactor technologies enabling rapid blocks [26] that are printed inside a mold pattern; and
maturation of tissues, multiscale vascularization for sur- (ii) bioprinting of vasculature using direct extrusion of a
vivability of tissues, and mechanical integrity and inner- tubular network [16–18]. A recent study [3] demonstrated
vation for transplantation. hybrid biofabrication of vasculature in tandem with tissue
Although several researchers have studied bioprinting strands, where fibroblast tissue strands quickly fuse to
of tissue constructs, the fabrication of scale-up tissues with each other, maturate, and form the tissue around the
a high volumetric oxygen-consumption rate, such as cardi- vasculature (Figure 1C–E). Tissue strands were made
ac, pancreas, or liver tissue, is still a challenge. One major scaffold free and used as building blocks to construct the
roadblock is associated with the integration of the vascular scale-up tissue due to their quick fusion, folding, and
hierarchical network spanning arteries and veins down to maturation capabilities. This approach demonstrated the
capillaries. To bioprint vascularized thick tissues, highly proof of concept toward larger-scale perfusable tissues;
repeatable and straightforward technologies and protocols further work is needed to demonstrate a complex vascular
should be developed in logical steps, from simple to com- network within larger tissues with vascularization on
plex. Since it is difficult to print capillaries at the submi- multiple scales that can be envisioned using a Multi-
cron scale using current technology, an alternative could be Arm BioPrinter [27]. Although vascularization is impor-
to bioprint the macrovasculature and then leave nature to tant for larger-scale tissue constructs for transplantation,
create the capillaries. To this end, two alternative anastomosis to the circulatory system and functionality
approaches have been considered: (i) indirect bioprinting post-transplantation should also be considered. The vas-
by utilizing a fugitive ink that is removed by thermally cular network should be designed and bioprinted so that it
induced decrosslinking, leaving a vascular network behind can be sutured to a vascular network easily, and it should
[14,15]; and (ii) direct bioprinting of a vasculature network have certain properties, such as enough mechanical prop-
in a tubular shape [16–19] (Figure 1). erties to satisfy suture retention and burst pressure, suffi-
Several recent attempts have been made to bioprint a cient intactness of endothelium to prevent thrombosis, and
fugitive bioink to create vascular channels [15,20–22]. Cell a high patency rate to support occlusion-free circulation
laden hydrogels were used to fabricate the tissue construct, [28]. Compared with indirect bioprinting of a vascular
and integration of the vascular network demonstrated network, the direct bioprinting of vasculature can be more
increased cell viability inside the construct; regions near convenient, suturing to the host at the time of implanta-
channels exhibited significant differences compared with tion.
regions away from channels. Although most researchers
have attempted to create a vascular network on a macro- From in vitro to in situ: regenerating tissues through
scale and generate an endothelium lining inside the lumen direct bioprinting into defect sites
via gluing endothelial cells through perfusion, Lee et al. Bioprinting living tissue constructs or cell laden scaffolds
[21] took one step forward and successfully achieved an- in vitro has been well studied, and thin tissues or tissues
giogenesis by sprouting endothelial cells within a fibrin that do not need vascularization, including skin, cartilage,
network loaded with other pericyte-like supporting cells and blood vessels, have been grown [12]. However, in situ
(Figure 1A,B). Their study demonstrated that endothelial bioprinting can enable the growth of thick tissues in critical
spouting generated a considerable increase in the perme- defects with the help of vascularization driven by nature in
ability of the tissue construct. More advanced angiogenesis lesions. Therefore, it is a promising direction for the bio-
and vasculogenesis have already been developed in micro- printing of porous tissue analogs that can engraft with
fluidic devices; several supporting cells have been endogenous tissue and regenerate new tissue along with
attempted and used in cancer metastasis studies [23]. De- vasculogenesis through the migration of progenitor cells
spite the flexibility in bioprinting channels and the ability into the tissue construct and sprouting of capillaries from
to create angiogenesis, this technology still faces major the endogenous tissue.
challenges. First, loading cells in hydrogels does not sup- The idea of in situ bioprinting was first proposed by
port cell–cell interactions, because those take place in vivo, Weiss using inkjet technology [29]; however, translating
and limited phenotypic stability and activity of cells are bioprinters into operating rooms was considered to be
observed during prolonged in vitro incubation. Second, challenging due to the perception that surgeons can be
while fibrin is a suitable environment for angiogenesis considered artists and prefer off-the-shelf solutions, such
because it has a crucial role in blood clotting [24], it is as using prefabricated tissue constructs and cutting or
not a convenient environment for tissue-specific cells, such carving them into a form to be implanted into the defect
as islets in a pancreas or follicles in a lymph node; a site. Limited research has been performed on in situ bio-
scaffold-free environment should be considered for these. printing since Weiss proposed this technology. Inkjet-
A recent article [25] demonstrated contiguous vasculariza- based bioprinting of skin cells has been tested for burn
tion of cell aggregates in tumor spheroid models and robust wounds [30], and laser-assisted bioprinting has been per-
angiogenesis into the fibrin matrix where spheroids were formed to test the feasibility of printing nanohydroxyapa-
encapsulated, showing the possibility of generating anas- tite particles on a mouse model [31]. The idea of bioprinting
tomosis of vascular networks of stromal and parenchymal skin cells (fibroblasts and keratinocytes zonally) has been
tissues in vitro. considered feasible for transitioning the technology to
The other approach is direct bioprinting of a vascular clinical settings, with the hope of repairing major wound
network via: (i) bioprinting of scaffold-free branched defects of soldiers on the battlefield.
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Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7
(A) Vascular channels (C) Stage 1
Detachable Fibrin nozzle Collagen Co-axial Embed endothelial nozzle and suppor ng cells
Stage 2
Capillary network is formed (D) within fibrin scaffold Tissue
strands
Stage 1 Stage 2 Stage 3 Stage 3
Angiogenic sprou ng is formed Vasculature from vascular channels to the Key: capillary network Endothelial cells Assembly through Par al fusion of ssue Complete fusion of ssue Suppor ng cells hybrid bioprin ng strands around the strands ghtly adhering vasculature to the vasculature (B) (E)
Capillary network Sprou ng
Endothelium
100µm
TRENDS in Biotechnology
Figure 1. Vascularized tissue construct bioprinting in vitro. In the first approach, (A) a fugitive ink is bioprinted to create vascular channels inside a collagen support
structure along with deposition of endothelial and fibroblast cells in a fibrin scaffold, resulting in capillary network formation in fibrin scaffold followed by endothelial
sprouting from vascular channel to capillary network in 9 days, where (B) a immunohistochemistry image shows integration of large sprouts from the parental vascular
channel (red) to the capillary network (green). In the second approach, (C) a hybrid bioprinting approach can be applied using a Multi-Arm BioPrinter, where (D) the tissue
assembly is created by placing the vasculature in the middle, resulting in fusion and maturation of scaffold-free tissue strands around the vasculature, further resulting in
(E) the tight fusion of the fibroblast tissue to the vasculature in 1 week [3]. The co-axial nozzle used in that study can enable continuous printing of a single lumen
vasculature throughout the fabrication of larger-scale hybrid tissue constructs. Reproduced and adapted, with permission, from [21] (B).
In situ bioprinting is challenging, and further system- and regulatory issues related to animals or humans neces-
atic research is required to take the technology into a sitating safe delivery of the tissue construct under anes-
robust state. There are major limitations associated with thesia. In addition, in situ bioprinting can sometimes
its biological, biomaterial, and engineering aspects, such as increase the duration and cost of surgery.
printing difficulties on nonhorizontal surfaces, the need for Bioprinting ex vivo on explants can be considered a
highly advanced robotics bioprinters coupled with comput- transitional stage (Figure 2A) in which tissue constructs
er-aided design technologies (i.e., scanning the defect site can be built and engineered inside explants [32]. In situ
and providing an interactive user interface for surgeons), bioprinting is promising for developing tissue analogs
the requirement for a highly effective extrudable bioink directly on the defect model in operating rooms
enabling instant solidification in a living body (without the (Figure 2B), which paves the way to developing associated
need for an external solidifier, such as a ultraviolet light or enabling technologies for humans in the future. When the
a chemical crosslinker), the need for a biologically appeal- technology is translated into clinics, it will have several
ing ink for enhanced tissue formation, the requirement for benefits: (i) direct bioprinting of tissue constructs into
technologies that do not interfere with in vivo conditions defects can eliminate the need for preshaping or reshaping
397
Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7 (A) Cells migra ng (B) from defect
Printed cells In situ bioprin ng Bioprinter nozzle
Defect
Bioprinter Explant
Bioprinter Fixture Bioprinter nozzle nozzle Stem cells Plasmid DNA Calvarial defects Rat
Bioink Printed ssue inside a car lage defect Biodegradable micropar cle
(C)
In situ hybrid bioprin ng in opera ng rooms Large defects
Dura subs tute Printed Printed frame(biological or hydrogel made of syneth c) composite biodegradable loaded with hard polymer stem cells and genes
TRENDS in Biotechnology
Figure 2. In situ bioprinting. (A) Bioprinting cells into explants in situ can be considered an intermediate step toward (B) bioprinting tissue constructs directly into animal
models, where DNA can also be printed for bioprinting mediated gene therapy to transfect and differentiate printed stem cells into multiple lineages. This will have a great
impact on (C) transitioning in situ bioprinting technology into operating rooms for humans in the near future; for example, large, deep calvarial defects can be regenerated
on a synthetic or biological Dura substitute, where a hard polymeric biodegradable frame can be printed along with stem cells and genes loaded in hydrogels to generate
the vascularized bone tissues. Image in (C) reproduced courtesy of Christopher Barnatt.
the scaffold based on the defect geometry. This can avoid When bioprinting stem or progenitor cells in situ, cells are
the laborious nature of scaffold preparation and the risks exposed to the natural environment with growth factors that
associated with contamination and limited activity of cells can induce their differentiation into the desired lineages;
in vitro; (ii) for bioprinting of cell-laden tissue constructs (iii) in situ bioprinting can enable the precise deposition of
for critical- or large-size defects, in situ bioprinting can cells, genes, or cytokines inside the defect, unlike manual
eliminate the need for differentiation of stem or progenitor interventions, such as prefabricated scaffolds, in which the
cells in vitro, which might be expensive and time consuming. shape can alter due to swelling, contraction, or deformations.
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Opinion Trends in Biotechnology July 2015, Vol. 33, No. 7
Localized control via bioprinting, such as printing different Two approaches are discussed: (i) in vitro bioprinting of
cytokines at different layers, is an asset for future bioprint- tissue and organ constructs with a focus on alternative
ing research; (iv) standard defects made by surgery tools are technologies in bioprinting a multiscale vascular network
easily addressed by manipulation and bioprinting of tissue in tandem with the rest of the tissue construct, and (ii) in situ
analogs. However, naturally occurring defects resulting bioprinting of tissue construct for regeneration of large
from trauma, surgical excision, or other issues are random defects that will one day enable the repair of body parts
in morphology and geometry and need to be captured pre- directly in patients in operating rooms. Although the tech-
cisely; laser- or image-based scanning systems can overcome nology shows a great deal of promise for bioprinting at
this challenge. In situ bioprinting can eliminate the need for organ-level complexity, there is still a long way to go to
multiple operations because the tissue constructs can be realize this ambitious vision. There is a need for advance-
bioprinted into the defect immediately; and (v) twisting ment in several areas, including new cell sources and stem
multi-axis robotic arms can enable angled deposition and cell technology, novel bioinks, and advanced fully automat-
printing of the bioink into nonhorizontally oriented defects. ed bioprinter technologies and bioprinting processes. Over-
Defects on the live model can be at random locations, and it is coming current impediments in these technologies along
not convenient for the surgeon to change the position of the with advancements in in vivo integration is essential for
anesthetized model during bioprinting. developing seamlessly automated technology from autolo-
As a result of these benefits, in situ bioprinting of tissue gous stem cell isolation to tissue or organ construct trans-
analogs can be applied to various sites on the body, such as plantation.
deep dermal or extremity injuries and calvarial or cranio-
facial defects during maxillofacial or neurosurgeries. In the Acknowledgments
future, in situ bioprinting technology could be considered This work has been supported by National Science Foundation Awards
CMMI 1462232, CAREER 1349716, Diabetes in Action Research and
for humans (Figure 2C). For large calvarial defects that
Education Foundation, and the Grow Iowa Values Funds. The author
need a great deal of microvascularization along with struc-
would like to express his gratitude to G. Dai, Christopher Barnatt, Yin
tural support, one can envision bioprinting a frame (using
Yu, and Kerim Moncal for providing some of the high-quality images in
hard osteoconductive polymers, such as a blend of poly- the figures.
caprolactone, copolymers, and hydroxyapatite) in tandem
with stem cells (loaded in blends of hydrogels, such as References
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