Dimethyl-Sulfoxide-Free Cell Cryoprotectant Derived from Amino Acids

With the large-scale applications of cryopreservation technology in the cell therapy fields, traditional permeable cryoprotectants (CPAs) have led to serious issues, such as cell cycle arrest, inhibition of cell proliferation and differentiation, apoptosis, altered gene expression, etc. Development of green, non-toxic cryoprotectants is critically needed. Amino acids could serve as substrates for protein and cellular metabolism and as cryoprotectants with non-toxicity, balancing the intracellular water osmotic pressure. Current research on amino acids as cryoprotectants is hindered by several limitations, including unclear protection mechanisms, cryopreservation methods, and poor efficacy of individual formulations. Therefore, three specific amino acids and derivatives, including l-proline, l-carnitine, and betaine, as cryoprotectants were used for two types of cell cryopreservation. Single-factor experiments were conducted to obtain the optimal concentration range for each of the three amino acid cryoprotectants. On the basis of the key thermophysical parameters, the ability to inhibit ice crystals, and the effect after cryopreservation, multivariate orthogonal experiments were carried out to evaluate the actual effect of the three-component mixed cryoprotectant on cell cryopreservation. In comparison to the gold standard of 10% dimethyl sulfoxide (DMSO) for cell cryopreservation, the mixed cryoprotectant derived from amino acids achieves comparable preservation efficacy at lower concentrations with a convenient application method, which offers guidance for DMSO-free cryoprotectants.