Cryopreservation and CO₂-independent culture of 3D cardiac progenitors for spaceflight experiments

Space experimentation of cardiomyocyte differentiation from human induced pluripotent stem cells offers an exciting opportunity to explore the potential of these cells for disease modeling, drug discovery and regenerative medicine. Previous studies on the International Space Station were done with 2D non-cryopreserved cultures of cardiomyocytes being loaded and cultivated in spaceflight culture modules with CO₂. Here we report the development of methods of cryopreservation and CO₂-independent culture of 3D cardiac progenitors. The cryopreservation allows preparation and pretesting of the cells before spaceflight, makes it easier to transport the cell culture, reduces the impact of strong gravitational force exerted on the cells during the launch of spaceflight, and accommodates a more flexible working schedule for the astronauts. The use of CO₂-independent medium with supplements supports cell growth and differentiation without a CO₂ incubator. With these methods, we conducted a spaceflight experiment through the SpaceX-20 mission to evaluate the effect of microgravity on the survival and differentiation of 3D cardiac progenitors. Our cryopreserved cardiac progenitor spheres were successfully cultivated in a spaceflight culture module without CO₂ for 3 weeks aboard the International Space Station. Beating cardiomyocytes were generated and returned to the earth for further study.