N-Methylacetamide for Cryopreservation - An Alternative to Dimethyl Sulfoxide

Cell death due to cryopreservation is caused by insufficient protection from freezing-induced damage and cytotoxicity of the cryopreservation agent, acting both before freezing and after thawing. Compared to dimethyl sulfoxide (DMSO), Nmethylacetamide (NMA) was previously suggested to have lower cytotoxicity in cryopreserved cells because the sulfinyl group, which has high polarity and d-orbital effect, is replaced by an amide group, which has moderate polarity. Furthermore, NMA retains the two methyl groups present in DMSO, which are known to confer permeation ability to DMSO. Therefore, NMA is expected to have lower cytotoxicity than that of DMSO, while retaining both the cryopreservative activity and permeation ability of DMSO. We performed cell viability experiments in cells incubated with DMSO and NMA and then cryopreserved. We found that, after thawing and complete removal of cryopreservatives, cells cryopreserved with NMA exhibited a higher recovery rate than those cryopreserved with DMSO. Moreover, after 1 h of incubation with 5% cryopreservative at room temperature, the cell survival rate was higher for NMA than for DMSO, indicating that NMA has lower cytotoxicity under conditions corresponding to the pre-freezing state and after thawing. No significant differences between NMA and DMSO were observed regarding the colony-forming ability per cell that survived after thawing and complete removal of cryopreservatives. Finally, to achieve similar cryopreservation action(post-thawing recovery rate, 75.5%), the necessary NMA concentration was estimated to be lower than the DMSO concentration (3% vs. 5%). Our findings support the use of NMA for cryopreservation.