Low-Molecular-Weight PEGs for Cryopreservation of Stem Cell Spheroids

Stem cell spheroids (SCSs) are a valuable tool in stem cell research and regenerative medicine. SCSs provide a platform for stem cell behavior in a more biologically relevant context with enhanced cell-cell communications. In this study, we investigated the recovery of SCSs after cryopreservation at -196 °C for 7 days. Prior to cryopreservation, the SCSs were preincubated for 0 h (no preincubation), 2 h, 4 h, and 6 h at 37 °C in the presence of low-molecular-weight poly(ethylene glycol) (PEG) with molecular weights of 200, 400, and 600 Da. The recovery rate of SCSs was markedly affected by both the PEG molecular weight and the preincubation time. Specifically, when SCSs were preincubated with a PEG200 solution for 2 to 6 h, it significantly enhanced the recovery rate of the SCSs. Internalization of PEG200 through simple diffusion into the SCSs may be the cryoprotective mechanism. The PEG200 diffuses into the SCSs, which not only suppresses osmotic pressure development inside the cell but also inhibits ice formation. The recovered SCSs demonstrated both fusibility and capabilities for proliferation and differentiation comparable to SCSs recovered after dimethyl sulfoxide 10% cryopreservation. This study indicates that PEG200 serves as an effective cryoprotectant for SCSs. A simple preincubation procedure in the presence of the polymer greatly improves the recovery rate of SCSs from cryopreservation.