Size Effect of Stem Cell Spheroids in Their Cryopreservation Using Low-Molecular-Weight PEGs as a Cryoprotectant

Cryopreservation is a crucial procedure to maintain quality of stem cell spheroids (SCSs) for a long period. It is affected by spheroid size because only internalized cryoprotectants can effectively play their roles within SCSs. Here, small, medium, and large SCSs with diameters of 30 to 80, 80 to 150, and 100 to 200 μm, respectively, were prepared using tonsil-derived stem cells. SCSs were preincubated in the presence of poly(ethylene glycol)s (PEGs) (10.0 wt % in a medium) with molecular weights of 200, 400, or 600 Da at 37 °C for 2 h, and then cryopreserved at -196 °C for 7 d. SCS recovery rate from cryopreservation was significantly affected by their size as well as molecular weight of PEGs; excellent recovery was observed for the small SCSs that were preincubated in PEG200 solutions. Population density of PEGs in the SCSs was 2.0 to 4.5 times higher in small SCSs than in large SCSs, which contributed to the survival of the SCSs during cryopreservation. The small SCSs recovered from cryopreservation showed innate activities of stem cells including fusion, proliferation, and differentiation much better than medium or large SCSs. The small SCSs employing the PEG200 preincubation protocol exhibited a cell recovery rate of >60% for 1 month of cryopreservation. These findings provide valuable insights into size-dependent cryopreservation strategies for high-level complex cellular systems.