Investigation into antifreeze performances of natural amino acids for novel CPA development

Cryopreservation can prolong the viability of cells and help meet the demand for biosamples of high medical value. Cryoprotectants (CPAs) can mitigate unavoidable cell cryoinjury caused by the formation and growth of ice crystals during freeze-thaw cycles. Therefore, the development of efficient and biocompatible CPAs has attracted extensive attention. In this work, the antifreeze performances of 18 water-soluble natural amino acids were systematically investigated by wet experiments and molecular dynamics simulations to explore their potentials as CPAs. Phenylalanine has an amphipathic structure with excellent ice recrystallization inhibition (IRI) activity, and methionine exhibits optimal depressing freezing point ability. Combining phenylalanine or methionine with osmolyte (proline) as CPAs can maintain normal morphology of sheep red blood cells (RBCs), and the recovery of sheep RBCs reaching 85 and 71%, respectively, while only 32% in the 20% (w/v) glycerol solution. This study demonstrates the potential of amino acid-based CPAs and reveals the synergistic effect of IRI activity and osmotic pressure regulation in cryopreservation.