The previous articles in this series highlighted the current challenges to 3D bioprinting tissues and organs. The foremost challenge is that the basic building blocks of living tissues are soft and therefore difficult to manufacture precisely. This results in resolution limits that prevent us from bioprinting the capillaries that keep cells alive and tissues function in the body. By identifying key barriers to the field, we gated 3D bioprinting into four major stages which are:
Stage 1: Lacks the ability to print using cells and/or proteins with high fidelity.
Stage 2: Prints cells and/or proteins at high fidelity, but no significant tissue function upon print completion.
Stage 3: Combines an immature 3D printed tissue with a coordinated maturation system, resulting in a functional tissue for use or study.
Stage 4: Produces a tissue or organ that is functional immediately upon print completion.
Since we said previously that we are transitioning from Stage 1 to 2, talking about Stages 3 and 4 become hypothetical and philosophical at various points. Stage 3 is unique in that it is just beyond the grasp of current research capabilities, however, various elements of Stage 3 have been accomplished independently. Transitioning to Stage 3 is then a matter of concatenating separate areas of complex research together. The barriers to Stage 3 are not defined by the bioprinted tissues themselves, but bioreactors.
A bioreactor is currently defined as, “an apparatus in which a biological reaction or process is carried out, especially on an industrial scale”. This current definition is centered around producing insulin or penicillin in industrial fermentation tanks. Future bioreactors, like ones discussed here, will be far more complicated than bulk tanks. They will conduct a symphony of fluid flow, proteins, and nutrients through the tissue to act as a synthetic womb. Although our bioprinting resolution is not high enough to directly print all tissue microstructure (refer to Part 2 of the series), it is a shortsighted assumption that we must assemble every aspect of the tissue ourselves. Powerful stem cells are genetically programmed to eventually assemble into our major organs. Thanks to developmental biology everyone began life as a single cell which eventually differentiated into all the various cell types in the body with an astonishing success rate. This unmatched success means the womb is the most effective bioreactor in nature, making it the gold standard for engineered bioreactors to be compared to. With this appreciation in mind, the question then becomes, “How can we utilize the power of cell-based developmental biology to fill in the detail that is currently beyond our capability to 3D bioprint?”. To leverage biology, bioreactors must be designed to balance cellular self-assembly with an engineering control. Leaning heavily towards the former increases the risk of unintentional and uncontrolled tumor development while the latter could stymie efficient maturation. Put simply, the full biological complexity of gestation is beyond our level of comprehension, much less our ability to replicate it artificially. This means bioreactors can only be a simplified version of the body, with the new question being, “What must a bioreactor do?”.
The first answer is obvious – keep cells alive. Until now, most engineered tissues have been very small and thin to allow cells to acquire nutrients through diffusion without the need for dedicated blood vessels. The first challenge to printing large (>1cm3) tissues is delivering nutrients to the cells in the center of the construct to prevent the formation of a necrotic core. The second role of a bioreactor system is to promote the function of the specific tissue. It is not simply enough for liver tissue to contain viable liver cells. Liver tissue that cannot produce bile does not have much therapeutic potential. It is creating a tissue that produces a higher level of function close to what is seen in the body that is an even greater challenge than keeping the cells alive. Higher-level tissue function is a concerted effort between a plethora of cell types and often organ systems. Even today, the best dialysis machine is no match for a kidney which hopefully leads you to appreciate its function from an engineering standpoint as well as the fortune that yours (probably) works. That being said, a bioreactor does not have to do everything. The most appropriate role of bioreactors is to act as a bridge between immature tissues post-print (Fig. 1A) and a minimum viable tissue (Fig. 1B) suitable for implantation where it will finish maturing (Fig. 1C).
There are at least two critical features that must develop to produce this minimum viable tissue: (i) a nutrient delivery network and (ii) the minimization of the immune response. It is important to note that the minimum functional requirements for tissues are organ-specific such as burst pressure for valves, a contractile force for muscle, and protein secretome for GI organs. To transplant larger tissues a blood inlet and outlet system should be present as constructs cannot rely on bulk passive diffusion like 2D cell culture can. This interaction with blood, therefore, means interaction with the immune system, creating immediate and long-term threats. The immediate threat to the engineered tissue is clotting on foreign surfaces. The endothelial cells that line the inside of blood vessels can be thought of as a Teflon barrier that prevents blood platelets and proteins from attaching to healthy blood vessels. A crucial role in bioreactor maturation is the production of a coherent endothelium that acts as a barrier to the body’s natural clotting cascade. Much exciting work has been done on smaller scale models to endothelialize tissue-engineered constructs 1,2, as the path to implantation is predicated on the presence of endothelium. The long-term immunological threat is rejection whereby the body recognizes the cells or materials in the tissue as foreign and either wall off or attacks the implanted tissue. To avoid this potential threat, it is preferable to use the patient’s own cells, however obtaining, growing, differentiating, and printing numerous patient-specific cell lines adds an additional layer of technical and logistical complexity. It is not very feasible to expect a bioreactor to be as coordinated as the womb. When it comes to a bioreactor, the only resources available to developing the tissue are what is put into it. There is no full immune, endocrine, or any organ system already present to help mature the tissue. Instead, we provide exogenous chemical cocktails to act as nascent in vitro substitutes for these systems such as antibiotics and growth factors to stand in for the immune and endocrine systems, respectively.
Bioreactor development is a burgeoning field that has already produced very promising results that hint at the potential to eventually transplant bioreactor-matured tissues. Notably, Syedain et al. placed a fibrinogen gel of Ovine and human dermal fibroblasts in a tubular mold inside a bioreactor3. After pulsing the molds for five weeks, the cells produced a cylindrical tube of the extracellular matrix that could then be sewn onto a traditional heart valve frame. The engineered heart valves were successfully transplanted into sheep for the entire six months of the study with impressive function and minimal immune response. Other studies have also shown the ability for bioprinted vessel networks to maintain cell viability for weeks, however, these tissues have yet to be transplanted into a subject after bioreactor maturation 4,5. The current tissue development pipeline relies on passing the baton of a nascent bioprinted tissue to a bioreactor system. Improving the quality and resolution of a bioprinted tissue will ease the workload on the bioreactor system, meaning bioprinting research will remain a critical endeavor in the future. Although implanting a bioreactor-matured functional tissue that was also bioprinted has yet to be demonstrated, both projects have been published separately. Joining the two halves together remains sizable, but a surmountable task. The first demonstration of a functional tissue production pipeline would provide substantial de-risking of tissue-engineered therapies to large outside investment, making it likely that bioreactor technology will become a linchpin in ushering in the next generation of regenerative medicine.
1. Miller, J. S. et al. Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat. Mater. 11, 768–774 (2012).
2. Nguyen, D. H. T. et al. Biomimetic model to reconstitute angiogenic sprouting morphogenesis in vitro. Proc. Natl. Acad. Sci. U. S. A. 110, 6712–6717 (2013).
3. Syedain, Z. et al. 6-Month Aortic Valve Implantation of an Off-the-Shelf Tissue- engineered Valve in Sheep. 612–626 (2017) doi:10.1016/j.biomaterials.2015.09.016.6-Month.
4. Kolesky, D. B., Homan, K. A., Skylar-Scott, M. A. & Lewis, J. A. Three-dimensional bioprinting of thick vascularized tissues. Proc. Natl. Acad. Sci. U. S. A. 113, 3179–3184 (2016).
5. Lee, A. et al. 3D bioprinting of collagen to rebuild components of the human heart. Science (80-. ). 365, 482–487 (2019).
About the Author:
Chief Operations Officer, Fluidform
Andrew is a co-founder of FluidForm and leads the development, manufacturing, and scale-up efforts of LifeSupport™. His research focuses on developing the next generation of techniques for vascularizing 3D-bioprinted tissues to improve the clinical translational potential of tissue-engineered therapies.