In-Space Manufacturing to Benefit the Future of Human Health on Earth and in Space

We are in the midst of a golden age of space travel with the upcoming launch of multiple reusable heavy lift rockets. These new craft will increase deliverable mass to LEO and decrease delivery costs. These rockets are essential to replacing the ISS with commercial space stations in the coming decade. These new commercial stations will enable the creation of in-space factories that leverage microgravity to improve products for use on Earth. Large-scale 3D bioprinting is one technology that will benefit from microgravity and has the potential to address the organ shortage and overreliance on animal models for drug discovery and testing.

Organ Shortage and Potential Solutions

We are in the middle of a donor organ shortage that is only getting worse. This crisis results from major factors¹ like a growing and aging population, and more addressable factors like the mismatch between belief in organ donation and the actual number of Americans who are registered organ donors. Outreach to improve education and build trust in medical professionals and potential donors could assuage some concerns about becoming a registered organ donor. But even if every American was a registered donor, there would still be a deficit because registered donors often die from conditions that leave their organs unacceptable for transplantation. To save more patients, we need more organs, and if donation is insufficient, then we must consider generating organs from scratch.

Currently two approaches are being researched as solutions to the organ shortage. One approach is the use of genetically modified animals (e.g., pigs) as bioreactors for human organs.² The second is using 3D bioprinting and biological inks (bioinks) to print replacement organs.³ The animal bioreactor approach requires the hijacking of the animal’s natural development process to create mature and implantable human organs. However, this approach has the potential for immune rejection due to animal antigens in the final organ and presents an obvious ethical dilemma. The advantage of 3D bioprinting is the ability to use induced pluripotent stem cells (iPSCs), which are created from the recipient’s own cells and not embryonic, eliminating ethical conflicts. The iPSCs are coaxed into the various cells types necessary to print the desired organ. Once all the necessary cells have been assembled and mixed into a bioink, the organ can then be printed and then implanted into the patient with zero immune rejection.

The disadvantage to 3D bioprinting organs comes from the complex and long-duration culture period required to mature the 3D print. Vascularization is the greatest hurdle to the maturation process as blood vessels are created throughout the developmental process in response to the developing organ’s requirements. Creating adult human organs in less than 18 years will require a full understanding of the developmental cues so they can be reproduced during the condensed maturation process.

The goal of tissue engineering is creating tissues and organs that can be manufactured, grown, and then implanted into humans without the need for lifelong use of immunosuppressants. We believe 3D bioprinting with iPSC-derived patient-specific cells is how to achieve that goal while eliminating ethical concerns.

3D Bioprinting and Pesky Gravity

Bioprinting terrestrially can be challenging because of the ever-present force of gravity turning your hard work into a pancake on the print platform. Freshly bioprinted organs and tissues are fragile as the cells and extracellular matrix (i.e., the interwoven natural fibers that hold us together) are not in the proper configuration. Gravity makes even “simple” blood vessel structures difficult to manufacture on Earth. Researchers have been developing methods to get around the print collapsing, including the use of crosslinking mechanisms, shear thinning bulk fluids, and high viscosity bioinks. UV or chemically induced crosslinking mechanisms increase the print’s rigidity by making chemical linkages between the components making up the print’s extracellular matrix.⁴

Shear thinning fluids are interesting materials with viscosities that can vary from nearly solid to liquid with the application of a shear force. Printing within a shear thinning bulk fluid makes it possible to print in the presence of gravity. This is accomplished by moving the dispense needle within the bulk shear thinning fluid, resulting in decreased viscosity that allows the bioink to be dispensed. After the bioink is dispensed, the shear force is removed, and the resulting increased viscosity locks the dispensed bioink in the desired shape. The final method to bioprinting large structures in gravity involves using high viscosity bioinks that incorporate self-assembling, high concentration hydrogels like gelatin.

However, none of the methods outlined for printing in gravity allow for the proper maturation and development necessary for the functional and physiological growth of the bioprint from construct to tissue. The addition of crosslinking and increased viscosity does improve the print’s structural rigidity, but at the cost of the adaptability and mobility of the cells within the print. After being printed into their new environment, cells need the ability to move in the print matrix and modify their immediate surroundings.

Crosslinking and high viscosity methods restrict the movement of cells and thus their natural ability to adapt to their new environment. If the cells cannot maneuver in their new environment, coaxing those cells to proliferate, differentiate, and mature into a functional tissue or organ becomes difficult. The number and variety of cells required to compose a single organ is likely not achievable without the help of cellular proliferation and differentiation. Therefore, we need new approaches to 3D bioprinting that use low viscosity bioinks without cross-linking, and the only place such bioinks can be used without fear of collapse is space.

3D Bioprinting and Microgravity

In microgravity, the threat of collapse from gravity is eliminated. This unique environment allows for bioprints to be created free of crosslinking mechanisms. The components, nutrients, cells, and matrix within the bioink all stay where they are placed instead of settling out under the influence of gravity. Without settling, the cell mixture will remain homogeneous during the entire printing process, resulting in a print with even cellular distribution. The absence of settling and crosslinking agents allows for full mobility of cells within the print. This free movement results in increased proliferation and self-assembly when directed by the appropriate external cues provided in an extended culture period and higher cell density. Printing at relevant cellular densities is not currently feasible due to the vascularization problem mentioned previously. It is difficult to print at such densities without a mature vascular network to provide sufficient nutrients and oxygen into the organ. For these reasons (i.e., settling, crosslinking, and self-assembly), microgravity has become increasingly viewed as the solution to the problems encountered when bioprinting large, complex structures on Earth.

The BioFabrication Facility

To capitalize on bioprinting in microgravity, a pneumatic-based extrusion bioprinter called the BioFabrication Facility (BFF) was created and flown to the ISS in 2019.⁵ BFF is an L-shaped payload (Figure 1) composed of a double-locker print chamber coupled to a single-locker electronics compartment. BFF is combined with the ADvanced Space Experiment Processor (ADSEP), Figure 2, which houses up to three finished prints and maintains the environment during the extended culture period.

Tissue culture cassettes (TCC) that can interface with the print chamber and ADSEP are used to house the bioreactor (print platform), media and waste bags, cleaning station, camera, and lighting. The bioreactor within the TCC contains all the necessary hardware to stimulate the print biochemically, mechanically, or electrically to direct tissue development. The current print platform allows for a 31 mm by 31 mm by 24 mm, printed construct. BFF consists of four pneumatic extrusion pumps (SmartPumps) that were designed in collaboration with nScrpyt. The Smart-Pumps can be loaded independently, allowing for up to four unique bioinks (10 mL each) in the creation of a single bioprint. The system allows for quick change out of print tips, enabling control of the extrusion orifice diameter. Fine pneumatic controls allow for precise extrusion of materials with viscosities ranging from that of water to that of peanut butter.

A crucial upgrade enables temperature control of bioinks during extended printing operations. Bioink formulations can start solidifying upon warming, and cooler temperatures also boost cell viability by generally slowing enzymatic reactions in and around the cell. The cooling was accomplished by interfacing BFF with the ISS payload liquid cooling system that can provide (~16 – 18 °C) cool liquid to the payload. A second interior cooling loop was developed for the BFF print volume as well. With these upgrades in place, BFF was returned to the ISS in November 2022 to begin full-scale experimentation, starting with the BFF-Meniscus project.

BFF-Meniscus

In 2023, Redwire successfully created the first 3D bioprinted meniscus on the ISS (Figure 3). The principal investigators for this project were from the Uniformed Services University (USU) and its 4DBio3 program. Meniscal injuries are a great test case because of their prevalence, lack of treatment options, and avascular nature (i.e., meniscus does not have a lot of blood vessels). Meniscal injuries are one of the most common injuries in military personnel, which motivated USU to investigate 3D bioprinting as a solution. In July 2023, the meniscus was printed on the ISS using BFF, and the meniscus was returned to Earth for analysis in September 2023.

BFF-Cardiac

Aboard SpX-30, which was launched on March 21, 2024, were all the ingredients to create a bioprint composed of iPSC-derived cardiomyocytes and fibroblasts. The cells were all created in house within Redwire’s Biological Laboratory. The iPSCs were derived from fibroblasts from a single donor and then differentiated into cardiomyocytes. The resulting prints were composed entirely of cells from the same individual, marking a solid step toward creating a patient-specific bioprint that can be implanted without immune rejection. The cardiac constructs were cultured over a two-week period on the ISS before returning ALIVE to the ground.

On April 30, 2024, we received the cardiac constructs at the Space Station Processing Facility at Kennedy Space Center following the successful return of SpX-30 using an insert for University of Alabama at Birmingham’s Microgravity Experiment Research Locker Incubator (MERLIN). By loading the TCCs into the MERLIN insert, we were able to maintain media perfusion to the tissue and a temperature of 37 °C during the multiday return trip to Earth.

Once on the ground, the cardiac tissue samples were collected and stained using a calcein AM/ethidium homodimer-1 (live/dead staining) prior to fixation in 4 percent paraformal-dehyde (Figure 4). A small portion of the cardiac print was stained to confirm the presence of live cells in the tissue. The stain confirmed the presence of live cells and the homogeneous distribution of the cells, representing a significant improvement in homogeneity of the construct compared to previous ground validation prints. This effort demonstrated the feasibility of creating patient-specific bioprints on the ISS, culturing and returning a print that was alive and, in theory, ready for implantation.

Future Work

Much work remains before bioprinting can address the organ shortage. As we move toward that solution, there are many way-points where bioprinting is poised to advance medicine. Opportunities on this journey, like the creation of small, organ-like structures known as organoids, will be explored. Organoids are perfect models for testing pharmaceuticals. Animal models are often poor substitutes for human trials, whether for ethical concerns or comparability. However, early drugs cannot be tested in humans, so a human analog is required. Small organoids could be produced to create models for the human condition, commonly called microphysiological systems. These systems consist of multiple different organ models that can be challenged by drugs, with the response having greater relevance to humans than current animal models. These models are a natural stepping stone to creating fully functional organs for human transplantation.

We may not be able to create a human organ for transplantation today; however, we get closer to that goal, and generate ancillary discoveries that result in better therapeutics, with each step. We have only begun to scratch the surface of what microgravity can offer to tissue engineering, but with BFF, we can now dig deep.

This article was written by Aaron J. Rogers, Senior Scientist, Redwire (Jacksonville, FL). For more information, visit here  .

References

1. The Strong Financial Case for Regenerative Medicine and the Regen Industry, Chris Mason and Peter Dunnil, Regen. Med., 2008, 3(3), 351-363.
2. First Pig Kidney Transplant in a Person: What it Means for the Future, Smriti Mallapaty and Max Kozlov, Nature.
3. 3D-printed Organs May Be a Reality. Looking ahead, we’ll no need donor hearts, Carolyn Barber, Fortune, February 15, 2023.
4. Recent Advances in Bioprinting Techniques: Approaches, Applications and Future Prospects. Li, J., Chen, M., Fan, X., Zhou, H., Journal of Translational Medicine, 14, 271, 2016.
5. 3D Printer Capable of Printing Human Tissue Set to Launch to the ISS, Patrick O’Neill, November 3, 2022.

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