Characterization and Optimization of a Novel Bioreactor for Biomanufacturing and Cryopreservation

1 PROJECT SUMMARY 2 Organoids derived from stem cells are valuable tools that have potential to unlock cellular-based regenerative 3 medicine applications for otherwise incurable illness. Severe liver dysfunction, which affects millions of people 4 and is the 10th leading cause of death in the United States per year, can only be treated with a liver transplant, 5 but a critical shortage of organ donors means many patients die while on the transplant waiting list. Creating 6 functional and transplantable bioengineered liver tissue that can treat severe liver dysfunction would address a 7 critical unmet need in modern-day medicine. Achieving closed system operation and effective cryopreservation 8 in tissue culture are critical requirements for producing clinical grade bioengineered tissue that meets 9 regulatory requirements. With the goal of producing highly functional bioengineered liver tissue for cell-based 10 regenerative medicine applications, we developed a custom bioreactor to biomanufacturer induced pluripotent 11 stem cell (IPSC)-derived liver organoids in the simulated microgravity of low shear rotational suspension 12 culture. Our custom bioreactor, Tissue Orb, supports isochoric supercooling cryopreservation and is the 13 world’s first isochoric supercooling chamber that can support sterile tissue culture. Isochoric supercooling uses 14 constant volume to maintain aqueous solutions in a liquid state at sub-zero temperatures without the harmful 15 formation of ice. Importantly, the constant volume confinement has been shown to impart stability to the 16 supercooled condition, which decreases the need of toxic cryoprotectants that are required in other 17 cryopreservation methods. Furthermore, isochoric supercooling is simple to implement, involving a rigid 18 container with no moving parts, and was recently demonstrated to be a promising cryopreservation method 19 that can be easily translated to clinical or research settings to preserve biological material. Our lab is the first to 20 apply isochoric cryopreservation to liver organoids. We are now combining this technology with simulated 21 microgravity tissue culture in efforts to produce high quality liver organoids for regenerative medicine therapies. 22 The objective of this proposal is to improve isochoric supercooling protocols for liver organoids and complete 23 the design of the Tissue Orb to allow tissue culture and cryopreservation to occur in a single, closed system 24 and is a critical step towards achieving scalability and repeatability that is needed for high quality, clinical grade 25 products. We will expand on our prior work through a biological aim which looks to improve the protocol for 26 isochoric supercooling of liver organoids (Aim 1) and an engineering aim which looks to complete the design 27 for the heat exchange system to allow integrated isochoric supercooling in the Tissue Orb (Aim 2). The 28 proposed aims will result in information and technology that can lead to substantial improvements in our ability 29 to produce and preserve liver organoids, which, in turn, can drastically increase the availability of high-quality 30 bioengineered liver tissues to researchers and healthcare facilities around the world.