Here, we report the features of a novel closed cell culture chamber (DCC) for use in experimental settings in space, and the results of subsequent validation tests. The DCC consists of a basal plate, a film and two medium ports. The basal plate and film are made from clear polystyrene. Oxygen and carbon dioxide gas exchange is performed through the thin film. The two medium ports are made from silicone in which syringe needles are inserted, and the culture medium is exchanged. The corona discharge treatment is applied on the basal plate to change the nature of the surface depending on the requirements for cell adhesion. Here, cell culture was performed using a human hepatoma cell line (HepG2), a hamster pancreatic β-cell line (HIT-T15) and a mouse fibroblast cell line (L929) in both DCCs and regular culture dishes. We confirmed that there were no differences in cell shape, growth rate, and dissolved oxygen concentration. Although the DCC was developed for use in space, it can also be used for cell transportation on the ground.