Migrating to Mars is the next exploration of human space missions and adapting to the extreme environment for a long period of time needs to be studied in-prior. Studies on cells in space may promote advancements in our understanding of the human body. The aim of this study is to optimize the protocol for the preparation of EndoCβH1 cells alone in the space chamber and co-culturing EndoCβH1 cells and BCs for microgravity experiments. We analyzed the cell survival and biocompatibility of β- cells with different parameters and the best concentration was used for the co-culture test. A 1:1 ratio of β-cells and BCs were cultured in the space chamber for viability and survival test. Co-culture experiment showed a significant increase in β-cell viability in the space chamber relative to in vitro tests. The effects of mitochondrial health of the cell inside the chamber was evaluated using the mitochondrial membrane test using the fluorescent probe 5,5,6,6’- tetrachloro-1,1’,3,3’ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1). We determined that the β-cell fitness inside the chamber was two times higher than the 24-well plate. To assess the viability of β-cells in a bioprinted gelatin scaffold we used the Live-dead fluorescence microscopy assay. Scaffolds were stained with Calcein AM (CAM) and propidium iodide (PI) and checked for survival. The result of scaffold staining showed that β- cells in gelatin and BCs in the media had more living cells compared to β-cells without BCs. Thus, space flight (SF) exposure to this culture system can be a platform for further studies in the treatment of diabetes.