Spheroidal formation preserves human stem cells for prolonged time under ambient conditions for facile storage and transportation

Human stem cells are vulnerable to unfavorable conditions, and their transportation relies on costly and inconvenient cryopreservation. We report here that human mesenchymal stem cells (MSC) in spheroids survived ambient conditions (AC) many days longer than in monolayer. Under AC, the viability of MSC in spheroids remained >90% even after seven days, whereas MSC in monolayer mostly died fast. AC-exposed MSC spheroids, after recovery under normal monolayer culture conditions with controlled carbon dioxide and humidity contents, resumed typical morphology and proliferation, and retained differentiating and immunosuppressive capabilities. RNA-sequencing and other assays demonstrate that reduced cell metabolism and proliferation correlates to the enhanced survival of AC-exposed MSC in spheroids versus monolayer. Moreover, AC-exposed MSC, when injected as either single cells or spheroids, retained therapeutic effects in vivo in mouse colitis models. Spheroidal formation also prolonged survival and sustained pluripotency of human embryonic stem cells kept under AC. Therefore, this work offers an alternative and relatively simple method termed spheropreservation versus the conventional method cryopreservation. It shall remarkably simplify long-distance transportation of stem cells of these and probably also other types within temperature-mild areas, and facilitate therapeutic application of MSC as spheroids without further processing.