3D culture and cryopreservation/thawing strategy of human induced pluripotent stem cells for spaceflight experiments

Although current space-based cell biology experiments predominantly rely on two-dimensional (2D) culture systems combined with in-orbit chemical fixation protocols, the development of a stem cell three-dimensional (3D) culture strategy with integrated long-term cryopreservation capabilities remains a significant technical challenge in space.

To design 3D culture, cryopreservation, and thawing methods of human induced pluripotent stem cells (hiPSCs) suitable for spaceflight experiments.

A biocompatible PDMS material was utilized to construct a porous 3D culture chamber, which was combined with VitroGel ® Hydrogel Matrix for hiPSCs 3D culture. An automated culture module system was validated, integrating cell culture, imaging, perfusion of cryoprotectant, -80 °C freezing, and thawing processes. Ground-based matching experiments were conducted to confirm the stability of the culture chamber, its mechanical tolerance, and the compatibility of the entire process.

The developed 3D culture system successfully supported the stable growth of hiPSCs and maintained pluripotency in space cell culture units. Both cells and culture chambers demonstrated tolerance to mechanical vibration stress. The established cryopreservation method enabled the direct long-term storage of 3D stem cell spheroids from culture boxes in a -80 °C freezer, achieving a cell survival rate exceeding 85% after thawing. Module-level end-to-end matching experimental confirmed that the 3D culture system established in this study supports cell growth, imaging, medium exchange, fixation, and cryopreservation.

The 3D growth and cryopreservation system developed in this study is highly reliable and adaptable to the conditions of the Chinese Space Station. It is capable of supporting research on the 3D growth of hiPSCs both in space and post-flight on Earth.