Neural progenitor cells are usually derived from pluripotent stem cells (PSCs) through the formation of embryoid bodies (EBs), the three-dimensional (3D) aggregate-like structure mimicking embryonic development. Cryo-banking of EBs is a critical step for sample storage, process monitoring, and preservation of intermediate cell populations during the lengthy differentiation procedure of PSCs. However, the impact of microenvironment (including 3D cell organization and biochemical factors) of EBs on neural lineage commitment postcryopreservation has not been well understood. In this study, intact EBs (I-E) and dissociated EBs (D-E) were compared for the recovery and neural differentiation after cryopreservation. I-E group showed the enhanced viability and recovery upon thaw compared with D-E group due to the preservation of extracellular matrix, cell–cell contacts, and F-actin organization. Moreover, both I-E and D-E groups showed the increased neuronal differentiation and D-E group also showed the enhanced astrocyte differentiation after thaw, probably due to the modulation of cellular redox state indicated by the expression of reactive oxygen species. In addition, mesenchymal stem cell secretome, known to bear a broad spectrum of protective factors, enhanced EB recovery. Taken together, EB microenvironment plays a critical role in the recovery and neural differentiation postcryopreservation.
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