Dimethyl-Sulfoxide-Free\nCell Cryoprotectant Derived\nfrom Amino Acids

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