3D Bioprinting Stem Cell Derived Tissues
Stem cells offer tremendous promise for regenerative medicine as they can become a variety of cell types. They also continuously proliferate, providing a renewable source of cells. Recently, it has been found that 3D printing constructs using stem cells, can generate models representing healthy or diseased tissues, as well as substitutes for diseased and damaged tissues. Here, we review the current state of the field of 3D printing stem cell derived tissues. First, we cover 3D printing technologies and discuss the different types of stem cells used for tissue engineering applications. We then detail the properties required for the bioinks used when printing viable tissues from stem cells. We give relevant examples of such bioprinted tissues, including adipose tissue, blood vessels, bone, cardiac tissue, cartilage, heart valves, liver, muscle, neural tissue, and pancreas. Finally, we provide future directions for improving the current technologies, along with areas of focus for future work to translate these exciting technologies into clinical applications.
KeywordsPluripotent stem cells Biomaterials Bioinks Regenerative medicine Tissue engineering Drug delivery Controlled Stem cell niche
The Role of 3D Printing in Tissue Engineering
3D bioprinters create cell patterns within defined spaces while simultaneously preserving cell function and viability.182 This process usually has two important components namely the ‘bioink’ or materials which mimic an extracellular matrix (ECM) environment for supporting cell adhesion, proliferation and differentiation, and biopaper.131 Normally the cells being printed are dispersed throughout the bioink, which is often generated from a hydrogel.130 ‘Biopaper’, which serves as the other major component, is the substrate or coating on which the specific patterns are deposited using the bioprinter with the bioink.130
Other commonly used techniques for bottom-up assembly of tissue-in-a-dish include 2D inkjet printing,17 which can generate a variety of tissues in vitro, such as skin190 and nerves.6,29,65,131 Soft tissue engineering has used both synthetic biodegradable polymers64,95 and natural polymers101,185 for printing using layer-by-layer freeform fabrication methods, such as 3D blotting. High concentrations of cells and cell spheroids (i.e. cellular aggregates) have been proposed as bioink for dispension-based printers, relying on the biophysical principles governing cellular self-assembly.127,137 Other than cells and their aggregates, hydrogels are often used as carriers for the former in formulating bioinks. Many different cell types have been used for 3D bioprinting engineered tissues.135 In this review, we have chosen to focus on stem cells for 3D printing tissues.
Utility of Stem Cells in 3D Bioprinting
Human stem cells possess the unique potential to develop into different cell types in the body.72 Furthermore, certain stem cells reside in many adult tissues in our bodies where they serve as an internal repair system, dividing when needed to replenish other cells during disease or injury.134 When a stem cell divides, each new cell has the potential to either remain a stem cell or become another differentiated cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. Excellent reviews exist in the field that focus on the role of stem cells in research.132 Since stem cells possess the novel capabilities of self-proliferation and targeted differentiation, they offer significant benefits compared to other cell types used, when bioprinting.88 Adult stem cells obtained from the patient have the advantage of alleviating immune rejection.
Mesenchymal stromal cells (MSCs) in tissue engineering (adapted from Ref. 141).
Relatively easy acquisition
In situ recruitment
Limited proliferative potential
Fewer cells compared to other sources
Related to age and health of donor
Donor morbidity (due to anesthesia)
Oral cavity MSCs (dental pulp, periodontal ligament)
Minimal donor morbidity
In situ recruitment
Can be co-seeded with bone marrow-derived stem cells
Cell number and activity related to donor age
Both ESCs and iPSCs can differentiate into somatic cells of all three germ layers: ecto-, meso- and endoderm.188 Thus, they offer greater multipotency than adult stem cells. ESCs originate from within the inner cell mass of a blastocyst while iPSCs represent somatic cells that have been reprogrammed into pluripotency.161,162 Concerns with ESCs include the controversy over their derivation from embryos and the limited number of cell lines, which could induce an immune response depending on the patient. iPSCs offer a feasible alternative to ESCs without the ethical and immunogenic drawbacks of the latter.188 Yamanaka et al. were the first to generate iPSCs from fibroblasts by introducing four transgenes using retroviral transfection: Oct 3/4, Sox2, Klf4, and c-Myc.162 More complex cocktails of proteins, peptides, chemicals and other factors have been developed for greater reprogramming control in recent years. The clinical use of both ESCs and iPSCs has been challenging due to the risk of in vivo teratoma formation, resulting from the presence of residual undifferentiated cells. Removing undifferentiated cells prior to implantation can help improve the anticipated outcome.128 The use of iPSCs is also linked to carcinoma generation, due to the genomic integration of a lenti-virus. Virus free iPSCs are being developed to make them a more feasible, safer option.154 Nonetheless, iPSCs have driven a paradigm shift in tissue engineering and the modelling of human disease in a dish.70,139 Additionally, the ability to reprogram patient-specific cells can enhance our understanding of disease mechanisms and phenotypic variability. 3D bioprinting has been successfully performed using multiple stem cell types of different lineages and potency.139,140
Bioinks Used in 3D Bioprinting
An ideal 3D printed construct promotes growth while attracting cells, allowing them to migrate and proliferate to form functional tissues. The micro-environmental niche serves as the foremost important factor for influencing cell fate, as seen in developmental biology.122 The ECM delivers mechanical and chemical cues, which can be assembled bottom–up in vitro. Hydrogels, which are commonly used as bioinks, possess several of these micro-environmental cues required for cell adhesion and growth. Bioinks distinguish themselves from traditional biomaterials such as polymer networks and foam scaffolds, due to their ability to be deposited as filaments during an additive manufacturing process. However, unlike other additive manufacturing materials such as thermoplastic polymers, ceramics and metals, they are processed under milder conditions, retaining their properties to prevent the degradation of bioactive molecules and the viability of living cells.189 An ideal bioink possesses high shear thinning ability (printability), viscosity, gelation kinetics, biocompatibility, hydration ability and viscoelasticity.48 Most bioinks are often adopted from existing hydrogel biomaterials, being derivatives of natural polymers like gelatin, alginate and fibrin.48 Hydrogels are degradable, absorbable and most importantly biocompatible, providing them with significant advantages over other materials.38,119 These materials exhibit epitopes (e.g. the peptide sequence—RGD) that promote cell adhesion and other related effects. For example, Das et al. cultured tissue-derived mesenchymal progenitor cells encapsulated in a unique silk fibroin–gelatin based bioink. The effect of optimized rheology, favorable amino acid sequences of silk–gelatin bioink known to promote cell adhesion, temporally controllable gelation strategies and printing parameters led to maximum cell viability and multi-lineage differentiation of the encapsulated human mesenchymal progenitor cells.34
Alginate hydrogels have been used extensively as bioinks for 3D bioprinting.90 However, native alginates possess limited biodegradation capability. Accordingly, Jia et al. explored the applicability of oxidized alginates with controlled degradation in bioprinting of human adipose derived stem cells (hADSCs).90 They investigated the effects of two key material properties (i.e. viscosity and density) of alginate solutions on their printability to identify a suitable range of material properties to be applied to bioprinting. Four different formulations of alginate solutions with varied biodegradability were printed with into lattice-structured, cell-laden hydrogels. As a result, these alginate-based bioinks could modulate proliferation and spreading of hADSCs without affecting the structure integrity of the lattice structures. This research laid a foundation for the development of alginate-based bioinks for tissue-specific applications.90 Wust et al. formulated a hydrogel composite including alginate and gelatin precursors which included different concentrations of hydroxyapatite (HA). It was characterized in terms of rheology, swelling behavior and mechanical properties to assess the versatility of the material towards bioprinting.183 Human mesenchymal stem cells (hMSCs) mixed into these bioinks showed high cell viability of ~ 85% viability after 3 days of subsequent in vitro culture with the inclusion of HA, making it useful for tissue engineering applications.183
Other bioinks with unique thermogelling properties, including carboxylated agarose, allow the fabrication of scaffolds with tunable mechanical properties. Tunable bioinks can reproduce natural mechanical aspects of functional tissues.50 Similarly, peptide bioinks containing lysine-hexapeptides self-assembled into stable, nanofibrous three-dimensional hydrogels with high stiffness values.116 These biocompatible scaffolds were shown to support the three-dimensional culture of human stem cells. Recently, Anil Kumar et al. used a novel visible light induced crosslinkable gelatin-based bioink for engineering cardiac tissue.3 The integration of such naturally derived and biodegradable polymers and stem cells with existing bioprinting techniques offers immense opportunities for tissue repair, organ transplantation, therapeutics and diagnosis.89,110 While most bioinks are hydrophilic, Campos et al. developed a hydrophobic high-density fluid, perfluorotributylamine (C(12)F(27)N) bioink, which allowed hMSCs and MG-63 cells placed within 3D constructs to remain viable allowing for cell proliferation and production of ECM.23 Thus, a wide range of biomaterials have been developed for bioprinting applications.
Current Status and Translational Concerns in 3D Bioprinting
Bioprinting combines cells with various natural and synthetic bioinks to fabricate scaffold-based or scaffold-free constructs. The desired tissue geometries are generated using computer aided design, which is then realized by using various bioprinting modalities including droplet-, extrusion-, or laser-based bioprinting to create tissue constructs. Further, some of these techniques, such as laser-based bioprinting, allow the printing of uniform sized embryoid bodies (cell clusters) that are necessary for targeted differentiation of ESCs.37,142 Laser-induced jet formation used for bioprinting of MSCs showed high viability and high resolution of encapsulated cells.2 Microfluidic/valve-based extrusion bioprinting methods can produce uniform sized cell aggregates.43,186 A microvalve-based bioprinting system for the manufacturing of high-resolution, multi-material 3D-structures was reported by Blaesar et al.16 This fluid-dynamics based system controlled the shear stress at the nozzle site as different levels of shear stress influenced cell viability and proliferation potential during bioprinting.16 Distinct 3D patterns of ESCs were achieved using a combination of dielectrophoresis and stereolithographic techniques.5 Computer assisted biofabrication of fully functional living tissue for regenerative medicine provides unique possibilities for the deposition of different living cells and biomaterials in a well-defined 3D structure.98 The application of robotics in 3D printing allowed direct delivery of cells submerged in a hydrogel bioink using an automated robotic dispensing system which resulted in them adopting an elongated morphology in alignment with the patterned grooved substrate.13 Since each specific bioink has its own set of advantages and disadvantages,26 various materials and techniques are combined to maximize the benefits. Researchers have been successful in bioprinting cartilage, bone, cardiac, nervous, liver, and vascular tissues. We have chosen not to discuss skin in this review as it is often considered to be a 2D construct. One major limitation to clinical translation is building large-scale vascularized constructs. Many challenges must be overcome before this technology becomes routine in a clinical setting.25,28,54,83,86,88,102,163, 164, 165 Here, we review specific, relevant examples of 3D printed tissues using stem cells.
Different Types of Tissues Engineered by Bioprinting Stem Cells
Adipose tissues, the loose connective tissue that store lipids and fat in the body, contains a significant amount of stem cells as discussed previously.51 As 3D tissue culture mimics the natural tissue architecture more accurately, 3D bioprinted adipose tissue using stem cells, can provide a deeper insight into metabolic diseases and can potentially serve as a replacement for damaged tissues. A group of German researchers used laser-assisted bioprinter (LaBP) to generate 3D grid-shaped grafts using hADSCs and an alginate-blood plasma bioink, which had no negative effect on stem cell behavior, proliferation, and differentiation, opening up the possibility for autologous tissue replacement.66 These engineered tissues were kept in culture for 21 days and they produced lipids and expressed the adipogenic markers LPL, aP2 and PPAR-γ 2. The ability of a direct-write, pen-based bioprinter to fabricate cellular spheroid constructs using multipotent adipose-derived stromal vascular fraction (SVF) cell has also been studied.180 These spheroids were created by crosslinking printed droplets of alginate bioink. The gauge of the needle and pressure of the direct-write printer system can be tuned to precisely control the size of the generated spheroids. The adipose SVF cell population maintained their viability and integrity for 16 days in suspension. This direct-write bioprinter can also print more complex structures by varying the needle size and the extrusion pressure, forming tube-like structures in cell suspension culture. Using 3D bioprinting to generate cell laden constructs derived from mouse MSCs and a bioink consisting of nanocellulose, alginate, and hyaluronic acid (HYA) can produce functional adipose tissues.74 This process yields more mature tissues compared to traditional 2D culture methods. The cells showed increased lipid accumulation and gene expression of adipogenic markers PPARγ and FABP4 in comparison to cells cultured using conventional 2D methods. Adipose tissue occupies a large percentage of the salivary glands.157 Patients suffer from dry mouth or xerostomia when the salivary glands do not function properly and it is particularly severe after radiotherapy for head and neck cancers.47,174 These glands contain stem cells capable of producing adipose tissue as well as organoids knows as salispheres.148 Further improvement in nanotechnology based bioprinting can develop therapeutic approaches for repair of damaged salivary glands.
A summary of the vascularization techniques and cells used to promote both vasculogenesis and angiogenesis using 3D bioprinting technology.
Pluronic F-127 fugitive ink extrusion bioprinting
Formation of microvascular network
Endothelial cell migration
3D multicellular array by laser-assisted bioprinting
Cellular response to 3D environment
Blend bionk using double-nozzle extrusion
Creation of perfusable vasculature
Fugitive porcine gelatin bioink with fibrin matrix
hUVECs, Normal Human Lung Fibroblasts
Capillary network formation
3D perfused microstructure with biologically inspired smart release nanocoating
Smart-release system for defect reconstruction
Tubular channel construction using coaxial nozzle extrusion bioprinting
Formation of cell-laden and perfusable tubular channels
Bourget et al. co-printed human umbilical vein endothelial cells (hUVECs) and human bone marrow mesenchymal stem cells (hBMSCs) onto a collagen biopaper using a LaBP for vascularization applications.19 Specifically, the study focused on the influence of cell dispersion relative to islets of deposited ECs and MSCs. The cells were printed either alone or as a 1:1 mixture in collagen droplets about 250 μm apart to form a composite 1000 μm line. ECs printed homogeneously spread throughout the collagen matrix after 24 h. Bioprinted MSCs showed negligible migration in the collagen gel. hUVECs stayed in the printed area instead of migrating when a suspension of mixed cell types was printed with the same parameters. These results suggested that ECs in their printed area would eventually form capillaries due to the stabilization of MSCs on capillaries. In another study, a multicellular array of adipose derived stem cells (ADSCs) and human endothelial colony-forming cells (ECFCs) were printed on a HYA-fibrinogen hydrogel using a LaBP.67 Adipose stem cells migrated toward the ECFCs after 3 days, making contact. The ECFCs formed vascular networks and matured into large networks after a week. A vascular tree formed due to these cell–cell interactions. The flexibility of this bioprinting technique enables the investigation of multicellular arrays on other cell types.
Smart release systems that intelligently regulate angiogenesis and osteogenesis have been developed using 3D fabrication techniques followed by functionalization procedures to create vascularized and dynamic bone growth.33 A bone model with blood vessels was 3D printed using a fused deposition modeling printer. Recombinant human vascular endothelial growth factor was coated on the top 5 layers and recombinant human bone morphogenetic protein was deposited on the bottom 15 layers, followed by crosslinking to enhance the bioactivity of the scaffold. This growth distribution ensured that blood vessel formation occurred before osteogenic differentiation when co-culturing hMSCs and hUVECs, and it successfully induced angiogenesis and osteogenesis.
The flexibility of 3D bioprinting can use coaxial nozzles to create tubular constructs when engineering blood vessels.35,76,195 Yu et al. demonstrated proof of principle by co-printing cartilage progenitor cells in alginate around a coaxial crosslinker material that stabilizes a hollow cylindrical structure upon printing.194 Not only did the structures have a homogenous structural integrity, but they could be perfused with oxygenated media with no leakage. These observations can be extended to a triaxial nozzle to construct larger vessels by surrounding an inner endothelial layer with smooth muscle cells. However, this technique cannot form vessel-like structures in a submicron or nano scale. Additional challenges now lie in refining these techniques for clinical settings.
3D bioprinting can treat complex elbow fractures as reviewed recently.192 3D printing bone has potential advantages compared with the conventional surgery, such as lower blood loss, shorter surgical duration and higher elbow functionality. In a relevant example, 3D bioprinted structures consisting of 5% GelMa were generated with perfusable blood vessels to enhance cell survival and proliferation during in vitro maturation into bone over 21 days.22 3D bioprinting technology can produce biomimetic bone matrix for studying the interaction between hBMSCs and breast cancer (BrCa) cells. This model showed that the BrCa cells prevented proliferation of MSCs while MSCs improved the growth of BrCa cells.196 In other work, a 3D Bio-plotter dispensing system printed hADSCs into 3D bone tissue constructs.176 High levels of cell viability and noticeable formation of bone matrix were observed in these 3D constructs.
3D scaffolds with highly interconnected microvascular channels and supporting structures have been successfully printed to enhance vascular cell growth and regeneration of the osteogenic bone.79 Cross-communication of bone progenitor cells and ECs enabled the construction of a mature vascularized bone.9 For instance, culturing human hMSCs, together with ECs in collagen gels, promoted the formation of the vasculature bone.168 3D bioprinting was used to fabricate function-control modules into stem cell laden methacrylamide gelatin scaffolds with high cell viability (> 90%) post printing.39 hMSCs were printed layer-by-layer in polymerized poly(ethylene glycol) (PEG) -GelMA to develop a mechanically strong bone.56 Acrylated peptides and PEG hydrogels were printed simultaneously with hMSCs using photo-polymerization, and the resulting constructs were able to form bone tissue.57
Thermal inkjet bioprinting has shown promise for bone tissue engineering. hMSCs suspended in poly(ethylene glycol) dimethacrylate (PEGDMA) were printed with bioactive ceramic nanoparticles with accurate locations in the resulting 3D structure.55 In a different approach, bioprinted constructs consisting of MSCs encapsulated in poly(lactic acid) (PLA) and GelMA-gellan gum bioinks cultured inside spinner flasks, yield viable constructs.111 Indirect 3D printing creates complex structures by injecting liquids into powder biomaterials, yielding biomimetic scaffolds.105 These scaffolds supported high levels of MSC proliferation. Inkjet-based bioprinting can generate accurate low-dose presentation of modified protein patterns for directed differentiation of stem cells. For example, presentation of BMP-2 using acellular dermal matrix directs osteoprogenitors, resulting in complex bioprinted osseous structures.158 3D printing of drug releasing particles, such as those that deliver BMP-2, promotes osteogenic differentiation of stem cells both in vitro and in vivo.147
In another study, patterning human osteoprogenitors and nano-HA using LaBP was performed to study the effect of this bioprinting method on the cell micro-environment.24 LaBP printing did not alter the physio-chemical properties of the scaffold and cell viability. Grafts containing MSCs were generated using laser-induced forward transfer (LIFT) technique.68 These printed MSC grafts differentiated into cartilage and bone with high levels of viability. Inkjet bioprinting technology can mimic naturally occurring stem cell micro-environments, including growth factors.146 Using this approach, printed patterns of BMP-2 seeded with muscle-derived stem cells (MDSCs) were able to differentiate into osteogenic lineages. Overall, bioprinting enables the replication of important parameters, like the distribution of mineralized and non-mineralized regions, protein fibers formation, and the interaction between interface cells, resulting in the generation of complex structures found in bone.
Cardiovascular disease remains the leading cause of morbidity and mortality in the world despite recent advances in cardiology and cardiac surgery.10 Myocardial infarction, the most common cause of heart failure, occurs when plaque build-up within the arteries and veins constricts blood flow to the heart wall, resulting in ischemia and then necrosis of the cardiac muscle cells.170 Cardiac cells possess a limited ability to regenerate, making the loss of these cells particularly devastating in terms of reducing function. These events also produce a collagenous scar tissue, compromising the contractile ability of the heart. Accordingly, there is huge interest in developing regenerative medicine that restores normal cardiac function following heart damage. Organ transplantation remains the most popular method for the treating heart disease, but donor organ supples remain limited. Mechanical methods, like ventricular assist devices, can restore the lost function, but poses the risks of strokes, excessive bleeding, infection, and device malfunction over the lifespan of the implant. Cardiomyoplasty, the process of injecting viable cells into the damaged region of the myocardium to replace necrotic cardiomyocytes, had achieved significant improvement in cardiac performance, but the lack of retention and vascularization and the migration of cells from the target location limits cell viability.
Cardiac Valve Replacements
The aortic valve ensures the smooth and proper functioning of the cardiovascular system, including the heart, by directing the flow of blood through the aorta and coronary arteries. Common heart valve ailments include valvular stenosis and insufficiency. Stenosis occurs when one more of the heart valves do not fully open due to fused or stiff leaflets, resulting in extra strain on the heart when pumping blood that leads to heart failure. Vascular insufficiency occurs when heart valves do not close fully resulting in regurgitation or the backward flow of blood. Trans catheter aortic valve replacement serves as the most popular treatment strategy for people suffering from these issues. However, paravalvular leakage can occur if the prosthetic implant does not perfectly fit. Efficiency of valve performance depends on its complex anatomical geometry and heterogeneous tissue biomechanics.21 Congenital defects and acquired valve diseases can compromise the valve shape and tissue mechanics resulting in stenosis or regurgitation. Although surgical repair of aortic valves is possible, prosthetic replacement remains the only viable option for a majority of patients suffering from aortic valve disease.49,173 Tissue engineering of the aortic valve offers an alternative strategy to provide a living valve replacement that can grow and also effectively integrate with the host tissue.53 Hockaday et al. produced tissue engineered bioprinted valves using encapsulated MSCs that were evaluated using in vitro testing and mechanistic studies.78 One of the main challenges of developing a valve tissue is the lack of biomaterials that can effectively withstand the physiologic pressure while providing a micro-environment that can promote cell survival and growth.40,77 Although cardiac valve tissue engineering has a lot of exciting potential, several technical challenges must be overcome before clinical translations. It is not yet possible to fabricate a cell laden heart valve, especially ones with thin leaflets that can withstand the physiological pressures that a native heart is subjected to, even after static culture. There needs to be significant effort in this field to successfully develop a 3D bioprinted heart valve.
Cartilage tissues possess a limited regenerative capacity, often requiring significant healing time with larger injuries and degenerative osteoarthritis treatments requiring prosthetics or grafts.129 The current accepted treatment for osteoarthritis and chondral injuries uses autologous cartilage implantation where cartilage tissues or cells are harvested from the patient and implanted at the treatment site.133,138 Articular cartilage provides a smooth surface for joint movement that maintains low friction even when load bearing, allowing the tissue to withstand compressive and shear forces.30,138 3D bioprinting enables the manufacturing of cartilage replacements with precise geometry, like replacement joint pads or superficial constructs.106,129
Hwang et al. showed ESC derived embryoid bodies expressed more cartilage and bone related transcription factors when cultured in 3D hydrogels compared to monolayer cultures.85 Articular cartilage-derived progenitor cells (CPCs), found at low concentrations in the cartilage, exhibit higher rates of self-renewal than chondrocytes. Levato et al. bioprinted GelMA hydrogels seeded with CPCs, MSCs, and chondrocytes alone and in combination to generate cartilage.112 The CPC-derived tissues contained more cells than the MSC samples after 56 days. All cell types underwent chondrogenic differentiation indicated by glycosaminoglycan (GAG) production with MSCs outperforming the chondrocytes and, to a lesser extent, the CPCs.
Gao et al. overexpressed the transcription factor NR2F2 in MSCs to promote chondrogenesis.59 These 3D bioprinted constructs containing NR2F2 over-expressing cells along with control tissues were implanted subcutaneously in mice. Safranin O staining showed higher proteoglycan production in the NR2F2 over-expressed constructs, which were also much stiffer than the controls on day 21, due to the accumulation of cartilage ECM. Stichler et al. functionalized poly(glycidol) (PG) scaffolds with HYA to generate a novel bioink for printing the cartilage tissue.160 The hydrogel formulations showed to promote cartilage specific ECM production, including cartilage specific collagen II and aggrecan. Soybean oil epoxidized acrylate, a novel bioink with many desirable properties, is biocompatible and photo-curable without heating or the addition of photo-initiators, making it easy to bioprint. MSCs have higher attachment and proliferation in the soybean bioink than the more commonly used PEG-based bioinks.125 Pati et al. used a novel decellularized ECM (dECM) bioink to print MSCs for generating cartilage.143 The dECM bioink was printed along-side pre-printed 200 μm polymeric supports to produce the desired structure, capable of withstanding the compressive forces subjected on cartilage. The GAG and collagen II decreased after decellularizing cartilage ECM due to increased trypsinization time for the denser matrix. The group speculated that factors were still present that triggered chondrogenesis due to the morphology of seeded MSCs and their tendency to create a mesh structure. The bioinks produced with dECM were designed heat sensitive to gel as temperature increased from working temperature (15 °C) to body temperature (37 °C).143
Costantini et al. used GelMA based bioinks, either solely or modified with HYAMA or chondroitin sulfate amino ethyl methacrylate (CS-AEMA), along with Ca2+ crosslinkable alginate to print their 3D chondrogenic constructs.30 Alginate allowed for rapid gelation at the time of extrusion. while GelMA was cured after printing to create stable hydrogels for long-term culture. The alginate in the constructs dissolved after a few days, leaving the covalently crosslinked GelMA to persist. While all the constructs maintained good cell viability, the bioink consisting of alginate, GelMA and CS-AEMA had the highest collagen type II/collagen type X ratio and equal expression of collagen type I to type II, making it the most suitable for chondrogenesis. In other work, Gao et al. demonstrated the production of cartilage tissues from hMSCs using PEG in a modified inkjet bioprinting system. Their constructs showed homogeneous cell distribution, good mechanical properties, chondrogenic differentiation and ECM production.60 PG has a similar structure to PEG but has additional side groups that can be functionalized to improve its properties as a bioink.160 PG, like PEG, is bio-inert and must be modified to support cell adhesion. Stichler et al. functionalized PG scaffolds with HYA to increase the bioactivity of the gel, producing mechanically stable scaffolds that supported MSC viability for 21 days post print.160 Nguyen et al. used nanofibrillated cellulose (NFC) in combination with alginate and HYA as a collagen biomimetic bioink to support the differentiation of iPSCs into cartilage.133 NFC/HYA bioinks induced iPSC differentiation while the NFC/alginate bioink maintained their pluripotency after printing. The cells differentiated into cartilage tissue after 5 weeks of culture.133 Möller et al. printed NFC/alginate constructs seeded with hMSCs and implanted these constructs subcutaneously into mice.129 The implanted constructs were integrated into the subcutaneous tissues after 14 days and show improved stiffness after day 60. These samples expressed GAGs and collagen II production, indicating production of cartilage.
The Multi-head Tissue/Organ Building System, an extrusion based bioprinter, offers multiple dispensing heads, which are individually controlled to allow the printing of various biomaterials in one structure.106 Temperature controlled extrusion heads make printing support polymers, like poly(caprolactone) (PCL), along with cell-seeded hydrogels possible.106,143 Lee et al. used the system to print a PCL support structure as well as PEG sacrificial layers, producing the complex geometry of the outer-ear.106 The PEG layers created a negative of the ear shape for the PCL. The PEG is then dissolved, leaving the ear structure onto which chondrocyte and adipocyte laden alginate hydrogels were bioprinted, to create the cartilage and lobe fat of the ear respectively. Both chondrogenesis and adipogenesis occurred with the tissues being confined to their deposition location.106
Graham et al. used droplet bioprinting to produce millimeter scale cartilage tissues consisting of bovine MSCs in an agarose bioink.63 These constructs were cultured in the presence of TGFβ3 and expressed SOX9, an early chondrogenic differentiation factor, after 3 days. Gao et al. modified an HP Deskjet 500 thermal inkjet printer and cartridges, generating a bioprinting system.60 Like droplet-based printing, inkjet printing can precisely deposit cells, scaffolds and growth factors in 2D and 3D arrangements. The group used the modified inkjet printer to print constructs of hMSCs in PEG diacrylate (PEGDA) with high levels of viability. The cultures were maintained for 4 weeks and showed increased proteoglycan production, indicative of chondrogenesis. Overall, an extensive amount of work has been done in the field of bioprinting cartilage.
The liver, an essential organ made up mainly of hepatocytes, metabolizes foreign chemicals, produces proteins, and generates bile. Liver failure often results from drug induced toxicity.44,108,118 Better liver tissue models would significantly lower the risk of drug induced side effects going undetected in pharmaceutical development.44,108 Hepatocytes make up most of the cells in the liver, performing most of its functions. Sinusoidal ECs line the hepatic sinusoid, providing filtration. Kupffer cells, macrophages present in the liver, mediate inflammatory response. Hepatic stellate cells contain an abundance of intracytoplasmic fat droplets and branched processes which, along with ECs create the sinusoid lining where they regulate sinusoid contraction.97 Liver biofabrication often focuses on hepatocytes as they perform the most functions and are the most abundant cells in the organ.
Lee et al. used a dECM bioink for printing liver tissues.108 The constructs consisted of this bioink seeded with BMSCs as well as liver cancer cells (human hepatocellular carcinoma) along with PCL for structural support with control constructs prepared with a collagen bioink. The dECM group had higher expression of liver specific transcription factors (HNF1A, HNF3B, HNF4A, HNF) particularly HNF4A. However, low expression of the HNF6 marker was observed for both bioinks. Analysis of liver-specific functions of these constructs by assessing albumin and urea levels showed that the dECM bioink enhanced the cell functions.108 Ma et al. developed 3D models consisting of mature hiPSC-derived hepatic progenitor cells (HPCs) cultured with endothelial and mesenchymal derived supporting cells.118 The different cells types were arranged by a digital light processing 3D printer in precise patterns through masking and unmasking areas based on photo-polymerization of a GelMa based bioink. The patterns created vascularized liver lobule like prints. Higher expression of mature hepatic markers (HNF4A, TTR, ALB) was found in 3D multicellular model in comparison to 2D and 3D HPC only cultures. The multicellular constructs maintained the highest levels of albumin and urea production, making it physiologically similar to native liver tissues.
Faulkner-Jones et al. differentiated hiPSCs and hESCs into hepatocyte-like cells (HLCs) using a nanolitre droplet dispensing system to generate 3D constructs.44 The viability of printed human hESCs and hiPSCs remained high post printing. hiPSCs viability was comparable to the un-printed control with hESCs viability higher than the control. The differentiation process was not disrupted by the printing process as the constructs expressed hepatic markers. The group then printed taller constructs consisting of hESC-derived HLCs in 20 or 40-layer tubes. Viability decreased in the first 24 h, resulting in low cell density in the prints. These 3D printed constructs took longer to differentiate in comparison to 2D culture.44 In conclusion, 3D printed liver tissues would greatly improve drug testing models as they would provide an accurate response to toxicity as chemicals are metabolized. However, significant issues remain when engineering such tissues.
Skeletal muscle tissue engineering holds great promise for replacing diseased or injured muscle tissue.45 Researchers used inkjet printing to print scaffolds using recombinant human BMP immobilized in fibrin as a bioink for differentiating MDSCs into muscle.146 Another group of researchers fabricated a 3D muscle tissue construct containing multiple cell types including canine SMCs, hAFSCs, and bovine aortic ECs and an alginate bioink using ink-jet printer.187 The cells were separately mixed with calcium chloride (CaCl2) which acts a crosslinker and loaded into three different ink cartridges and printed layer-by-layer, creating a sodium alginate-collagen composite. 3D ink-jet printing has the ability and mechanism to overcome the challenge of incorporating vasculature while printing thick tissues as it allows organizing multiple cell types as well as different growth factors inside an appropriate scaffold. The research group cultured the cells in the printed construct for three days and then implanted the samples into the backs of three mice for up to 18 weeks.187 The printed cells proliferated, retained phenotypic characteristics and physiological properties within the 3D constructs in vitro and survived with adequate vascularization when implanted in vivo.
As 3D environment accelerates smooth muscle cell differentiation, extrusion-based bioprinting is a potential technique for printing muscle tissue construct. Another group bioprinted hADSC-derived smooth muscle cells evenly mixed with a gelatin bioink using an extrusion-based 3D bioprinter.193 Their results showed enhancement in smooth muscle cell differentiation and maintained more robust cell viability and cell proliferation compared to other conventional bioprinting methods. Higher expression of smooth muscle actin and smoothelin was observed after 3 days and western blotting analysis confirmed the differentiation of smooth muscle cells. Though skeletal muscle tissue has the ability of self-healing in case of small injury, it cannot restore the function of lost tissues after severe injury. For this reason, skeletal muscle tissue engineering holds great promise for the replacement of severely diseased or injured muscle tissue. In other work, an innovative microfluidic-enhanced high-resolution 3D bioprinting technique for printing skeletal muscle tissue with functional morphologies was developed.31 They custom-built a bioprinter with microfluidic nozzle head coupled to a co-axial needle extruder for printing aligned hydrogel fibers since aligned muscle cells generate efficient contraction in order to promote enhanced myogenic differentiation from myoblast cells to parallel aligned long-range myotubes formation. They also optimized the 3D bioprinting process combining the rheological behavior of the extruded bioink consisting of PEG-fibrinogen and alginate biopolymer laden with muscle precursor cells (C2C12). The 3D bioprinted structures with C2C12 cells were cultured up to 21 days forming highly aligned multinucleated myotubes in vitro. After 7 days of culture, some of the constructs were implanted in the backs of mice along with bulk-hydrogel constructs used as controls for in vivo studies and both types of grafts were collected from the bodies after 28 days of implantation. After analyzing both kinds of samples, the bioprinted samples revealed a better organized muscle-like tissue compared to bulk hydrogels. These samples were completely striated confirming the advanced degree of myotube maturation and partially degraded and substituted by arising myofibers as well. A similar study was done by another group 93 where they used a different bioprinting technique known as an integrated tissue–organ printer (ITOP) for fabricating human-scale tissue constructs of any shape and structural integrity. They performed both in vitro and in vivo studies using supporting PCL layers, cell-laden hydrogel, pluronic F127 as a sacrificial material and thrombin solution as a cross-linker. After 7 days of in vitro culture the differentiated aligned myotube formation was observed. These bioprinted tissues promoted adequate innervation of implanted muscle tissue in vivo.
Decellularized ECM (dECM) derived from porcine skeletal muscle tissue can also be used as a bioink for printing skeletal muscle.27 This bioink remained at solution state at temperatures below 15 °C and transformed into a gel state after incubating at 37 °C for 30 min. They printed parallel, diamond and chain-types of 3D cell-printed muscle constructs using the muscle dECM bioink and the 3D bioprinting system. The results of the experiments revealed that the bioink had sufficient printability for creating various geometries of 3D muscle constructs, with mechanical properties and which exhibited good cell viability, proliferation, myogenic differentiation and myotube formation. Thus, such bioinks show promise for bioprinting physiologically relevant muscle tissues.
ECM molecules can serve as scaffolds for 3D printing neural tissue as studied by Lee et al.104 They used a type 1 collagen gel as bioink for printing astrocytes and neurons from day 18 embryonic rats using a layer-by layer 3D printing A total of eight layers of collagen were printed for the multilayer-cell constructs by previously crosslinking the hydrogel precursor layer with sodium bicarbonate, a nebulized crosslinking solution. The cells were suspended and printed on the partially-crosslinked hydrogel and then coated with the sodium bicarbonate solution with the cells showing high levels of viability post printing. Immunostaining was also performed after 12 days, indicating the presence of neurons and astrocytes.
The controlled release of morphogens and bioactive proteins serves as an important tool when engineering neural tissue from stem cells.179 The combination of collagen and vascular endothelial growth factor (VEGF) releasing fibrin scaffolds for 3D printing applications was studied by Lee et al. Changes in cell morphology and migration were observed for VEGF-releasing fibrin gels, including elongated neurite-like shapes after 2 days of culture. Scaffolds containing higher collagen concentrations showed more proliferation. Furthermore, increased cell viability was observed at the lowest collagen concentration. Zhang et al. combined graphene and GelMA, producing a novel nano-bioink called “G-GelMA” for 3D printing neural tissue.197 A stereolithography based 3D bioprinter was used to 3D print neural constructs containing NSCs. These tissues contained neurons as indicated by β-tubulin III expression and formed neurite extension. Huang et al. prepared a bioink consisting of PU and graphene for 3D printing adult murine NSCs.84 Constructs with dimensions of 15 mm × 15 mm × 15 mm (W × D × H) were printed using a Regenovo printer. The composite bioink of PU with a low graphene concentration of 25 ppm resulted favorable for 3D printed NSCs survival and differentiation into neurons and astrocytes.84
3D printed tissues can be combined with other technologies to improve the functionality of these tissues, such as low level light therapy (LLLT).1,169 LLLT, also known as ‘biostimulation”, has beneficial effects on cell proliferation, metabolic processes, wound healing, inflammation and neurological disorders.1 Accordingly, Zhang et al. exposed 3D printed mice NSCs to LLLT to promote cellular proliferation and differentiation.177 They used a self-developed 3D printer based on the Printrbot® rapid prototype platform and a combination of 10% GelMA and 15% PEGDA for the bioink, generating a transparent scaffold enabling light transmission.198 The bioprinted tissues were stimulated for different periods of time with a 15 s exposure resulting in the highest proliferation rate with 60s and 90s reducing cell proliferation rates. Increased reactive oxygen species (ROS) synthesis was observed following light stimulation in comparison with non-stimulated cells. β-tubulin III expression increased for the stimulated cells.
Electrospun fibers can also be combined with 3D printing. One group 3D printed hydrogels containing PCL electrospun fibers and NSCs as a way to engineer neural tissue.109 They used a stereolithographic based 3D printing platform in combination with the Printerbot. Electrospun fibers were placed at the bottom of the dish, and then the neural tissue was bioprinted on top of these scaffolds. The addition of PCL and PCL/gelatin fibers onto the printed scaffold increased the Young’s modulus and the attachment of NSCs. Increased neuronal differentiation was observed for tissues grown on PCL/gelatin fibers after 11 days. This group also presented the greatest degree of neurite alignment with the electrospun fibers.
Interestingly, 3D printed constructs can serve as tumor models for screening chemotherapy agents for brain cancer treatment. Xu et al. 3D printed SU3 glioma cells using gelatin, alginate, and fibrinogen bioink to mimic the in vivo tumor environment.184 These tumor spheroids replicated their in vivo functions, including secretion of VEGF and expression of nestin, a glioblastoma marker. These tissues were then exposed to temozolomide, one of the most common brain tumor chemotherapeutics with the 3D printed glioma cells being more resistant to this drug than 2D cultures. These results show the importance of the 3D architecture and physiology in making such models more relevant for drug screening applications.184 Mimicking the real architecture and complexity of the brain and spinal cord enables the construction of accurate artificial neural tissues for disease modeling, performing drug screening and tissue transplantation. 3D printing can be used to generate such neural tissues as detailed in this section.
The pancreas is composed of many cell types in a complex, vascularized 3D structure, which performs both endocrine and exocrine functions. Pancreatic islets contain the endocrine cells of the pancreas, α, β, δ, γ, and ε-cells, which all function to produce required hormones. α and β-cells control blood glucose levels by the secretion of glucagon and insulin respectively. δ-cells produce somatostatin which inhibits α and β-cell action. ε-cells release ghrelin to regulate energy consumption and appetite.149 Type 1 diabetes results in the loss of beta cells, the insulin producers in the islets of Langerhans. Current treatment strategies require the transplantation of β-cells or pancreatic tissues. Associated challenges include a lack of donor cells and tissues and immune response causing the cells to stop functioning.
3D printing offers the ability to create physiologically relevant pancreatic models with precise cell and spatial arrangements. Xu et al. printed ADSCs using a gelatin, alginate and fibrinogen bioink with pancreatic islets added post printing to generate an in vitro model of pancreatic tissue.185 Pancreatic progenitor cells (PPCs) can differentiate into cell types in the pancreas, both endocrine and exocrine. Cell–cell contact may improve the function of PPCs, so the cells are often cultured in small aggregates to allow sufficient nutrient diffusion to the interior. Yang et al. developed a high throughput 3D printing method to control the size of generated cell clusters to produce transplantable β-cells.191 Tobacco mosaic virus (TMV) particles, modified with arginine-glycine-aspartate (RGD) tripeptides, were patterned using an inkjet printer. The group seeded PPCs on to their printed TMV constructs and showed controllably and reproducibly sized clusters. The RGD improved cell adhesion to the 3D printed TMV constructs. Song et al. bioprinted hESCs encapsulated in fibrin gel, then embedded in a PLA scaffold as a way to generate stem cell derived beta cells (SC-β).159 In vivo experiments were run to assess the level of stimulated insulin secretion. SC-β seeded devices were implanted into mice; insulin secretion was measured at 0 and 30 min after mice were fasted for 16 h then injected with glucose. Mice with the devices implanted had detectable human insulin with glucose injections increasing levels of human insulin approximately 2.5 times. Overall, 3D printed pancreatic tissues provide much needed models for pathology response during drug testing as well as for studying pancreatic disorders like metabolic syndrome. This technology can be applied to high-throughput generation of transplantable β-cells for the treatment of Type I diabetes.
Future Work and Outlook
This review has summarized a large body of work detailing the use of 3D printing techniques for generating tissues from stem cells. These techniques include fuse deposition modeling, inkjet bioprinting, and extrusion-based printing methods. Current methods are limited in terms of the types of materials that can be printed, the resolution of the printed constructs, and the number of materials being incorporated into a construct. The need to incorporate vasculature structures serves as one of these important issues to be addressed when engineering stem cell-based constructs. These limitations can be addressed by using more sophisticated printing technologies. For example, the Feinberg group at Carnegie Mellon University developed a novel 3D printing method using the freeform reversible embedding of suspended hydrogels (FRESH).75 This novel printing process generates intricate structures that mimic the properties of native tissues found in vivo, including the structures found in bone and brain. Another interesting new bioprinting technology has been developed by the Vancouver based company—Aspect Biosystems. Their novel Lab-on-a-Printer system enables rapid switching between materials and cell types.11,12,20 They currently market a novel engineered tissue—the 3D Bioring Airway—which replicates the physiological function of this muscle tissue. Often, maintaining cell viability and ensuring proper differentiation of stem cells can be challenging. One possibility for addressing these challenges is through the fabrication and characterization of more sophisticated bioinks that deliver the necessary cues for promoting cell survival and the desired differentiation. Thus, bioink development remains a high priority for future studies. In particular, the use of decellularized matrix provides an interesting opportunity for additional research.
Organ damage can result from genetic conditions, accidents, and diseases, representing a critical medical problem in our society.32 Current treatment for major organ damage depends completely on organ transplants, a complex and expensive process. There is an acute shortage of organ transplantation as there is not enough donor to satisfy the needs of the patients and also the huge cost of transplant surgery which is more than $300 billion in 2012.32 Again, organ transplantation involves the risk of immune-rejection, meaning the patients must find a donor who is a tissue match.153 These issues could be addressed by 3D bioprinting the tissues or organs using stem cells taken from patient’s own body to build a replacement of the damaged organ. Thus, 3D bioprinting holds great promise providing excellent outcomes to treat various serious diseases in a simple and cost-effective way. Bioprinting of layer-by-layer organs or tissue is possible using cost-effective bioprinter and bioink materials. Markets and Markets reported in April 2016 the overall 3D printing market will reach $30.19 billion by 2022.153 Many of the modern 3D printers cost about $2,500 to $3,000, and even simpler models might be bought for as little as $300 to $400.172 Therefore, compared to drugs and surgical therapy, bioprinting organs from stem cells would become more cost-effective in a few decades.
Finally, the advent of 3D bioprinting has also raised certain ethical issues.61 These issues include considering whether there should be limitations placed on what structures can be generated and examining the key risks of significant harm associated with testing 3D bioprinting for humans. Additional concerns will require the investigation and development of appropriate clinical trial paradigms for testing 3D bioprinter tissues along with developing a specific framework for the regulation and testing of 3D bioprinting treatments. Also, analysis should be performed that examines the ethical questions of irreversibility, loss of treatment opportunity and replicability associated with 3D bioprinting. Overall, the use of 3D bioprinting using stem cells to generate tissues has grown in popularity and future work will translate these promising technologies for a wide variety of applications from drug screening to clinical use.
The Willerth lab would like to acknowledge funding from the Stem Cell Network, the Canada Research Chairs program, the NSERC Discovery Grant program, MITACS, and the British Columbia Innovation Council. Dr. Willerth has a collaborative research agreement with Aspect Biosystems. Laura De la vega has received graduate support from MITACS. Dr. Joddar acknowledges NIH BUILD Pilot 8UL1GM118970-02, NIH 1SC2HL134642-01 and the NSF-PREM program (DMR 1205302). Nishat Tasnim acknowledges the Anita Mochen Loya fellowship at UTEP. Matthew Alonzo acknowledges the Eloise E. and Patrick B. Wieland fellowship at UTEP. Schweta Anil Kumar, Laila Abelseth, and Meitham Amereh have no other funding sources to declare outside of those mentioned earlier.
This article does not contain any studies with human participants or animals performed by any of the authors.
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