npj | microgravity Article Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA https://doi.org/10.1038/s41526-024-00435-y Surface tension enables induced pluripotent stem cell culture in commercially available hardware during spaceflight Check for updates Maedeh Mozneb 1,2,3,4 , Madelyn Arzt 1,2,3,4 , Pinar Mesci 5 , Dylan M. N. Martin 6 , Stephany Pohlman 1,2,3,4 , George Lawless 1,2 , Shankini Doraisingam 6 , Sultan Al Neyadi 7 , Rayyanah Barnawi 5,8 , Ali Al Qarni 5,8 , Peggy A. Whitson 5 , John Shoffner 5 , Jana Stoudemire 5 , Stefanie Countryman 6 , Clive N. Svendsen 1,2 & Arun Sharma 1,2,3,4 Low Earth Orbit (LEO) has emerged as a unique environment for evaluating altered stem cell properties in microgravity. LEO has become increasingly accessible for research and development due to progress in private spaceflight. Axiom Mission 2 (Ax-2) was launched as the second all-private astronaut mission to the International Space Station (ISS). Frozen human induced pluripotent stem cells (hiPSCs) expressing green fluorescent protein (GFP) under the SOX2 promoter, as well as fibroblasts differentiated from SOX2-GFP hiPSCs, were sent to the ISS. Astronauts then thawed and seeded both cell types into commercially available 96-well plates, which provided surface tension that reduced fluid movement out of individual wells and showed that hiPSCs or hiPSC-derived fibroblasts could survive either in suspension or attached to a Matrigel substrate. Furthermore, both cell types could be transfected with red fluorescent protein (RFP)-expressing plasmid. We demonstrate that hiPSCs and hiPSC-fibroblasts can be thawed in microgravity in off-the-shelf, commercially-available cell culture hardware, can associate into 3D spheroids or grow adherently in Matrigel, and can be transfected with DNA. This lays the groundwork for future biomanufacturing experiments in space. Stem cell research on a variety of stem cell types in low Earth orbit (LEO) has revealed multiple opportunities for understanding fundamental changes to stem cell properties and potentially harnessing these changes for bioma- nufacturing applications in microgravity 1 . Investigations into the effects of culturing stem cells in microgravity could identify novel mechanisms cap- able of improving biomanufacturing of cellular therapeutics on Earth, or serve as a catalyst for future large-scale, on-orbit biomanufacturing of stem cell-derived products 2,3 . Experiments in simulated microgravity have shown alterations in the differentiation capacity, viability, and proliferative potential of progenitor cells derived from pluripotent stem cells 4,5 . Later disease modeling experi- ments in microgravity have indicated that the unique environment on the International Space Station (ISS) can lead to the identification of novel targets for enhancing the therapeutic benefit of heart muscle cells (cardiomyocytes) 6 , and have demonstrated the feasibility of long-term microgravity culture of human induced pluripotent stem cell (hiPSC)- derived cardiomyocytes 7 . Neural stem cells, mouse embryonic stem cells, and cardiac progenitor cells have also shown enhanced proliferative capacity in spaceflight experiments 8–11 . Another area of discussion is whether a sustained microgravity environment can elicit a beneficial effect on stem cell differentiation. Certain pilot data have demonstrated that microgravity may facilitate the differentiation of specialized cell types from pluripotent stem cells, such as cardiomyocytes 12 . However, given the limited data and diffi- culty of reproducing experimental results in LEO, it has yet to be confirmed if the production or differentiation of other cell types from pluripotent stem cells can also be enhanced in a microgravity environment. Indeed, there are 1 Board of Governors Regenerative Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA. 2 Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA. 3 Smidt Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA. 4 Cancer Institute, Cedars-Sinai Medical Center, Los Angeles, CA, USA. 5 Axiom Space, Inc., Houston, TX, USA. 6 BioServe Space Technologies, Boulder, CO, USA. 7 Mohammed bin Rashid Space Centre, Dubai, UAE. 8 Saudi Space Commission, Riyadh, Saudi Arabia. e-mail: Clive.svendsen@cshs.org; Arun.sharma@cshs.org npj Microgravity | (2024)10:97 1 1234567890():,; 1234567890():,;