l-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation

Cryopreservation of biological samples is an important technology for expanding their applications in the biomedical field. However, the quality and functionality of samples after rewarming are limited by the toxicity of commonly used cryoprotectant agents (CPAs). Here, we developed a novel preservation system by combining the natural amino acid L-proline (L-Pro) with gelatin methacryloyl (GelMA) hydrogels. Compared with dimethyl sulfoxide (DMSO), L-Pro and GelMA demonstrated excellent biocompatibility when co-culturing with cells. Cryopreservation procedures were optimized using 3T3 as model cells. The results showed that rapid cooling was the most suitable cooling procedure for L-Pro and GelMA among the three cooling procedures. Co-culturing with cells for 3 h before cryopreservation, 6% L-Pro + 7% GelMA had the highest survival rate, reaching up to 80%. Differential Scanning Calorimetry (DSC) analysis showed that 6% L-Pro + 7% GelMA lowered the freezing point of the solution to -4.2 °C and increased the unfrozen water content to 20%. To the best of our knowledge, this is the first report of cell cryopreservation using a combination of L-Pro and GelMA hydrogels, which provides a new strategy for improving cell cryopreservation.