AbstractCryopreservation of cells is necessary for long periods of storage. However, some cell lines cannot be efficiently cryopreserved, even when optimized commercial cryoprotectants are employed. Previously, we found that a low-toxic synthetic zwitterion aqueous solution enabled good cryopreservation. However, this zwitterion solution could not cryopreserve some cells, such as human kidney BOSC cells, with good efficiency. Therefore, details of the cryoprotective effect of the zwitterions and optimization based on its mechanisms are required. Herein, we synthesized 18 zwitterion species and assessed the effects of the physical properties of water/zwitterion mixtures. Non-cell-permeable zwitterions can inhibit ice crystal formation extracellularly via direct interaction with water and intracellularly via dehydration of cells. However, cells that could not be cryopreserved by zwitterions were insufficiently dehydrated in the zwitterion solution. Dimethyl sulfoxide (DMSO) was combined as a cell-permeable cryoprotectant to compensate for the shortcomings of non-cell-permeable zwitterions. The water/zwitterion/DMSO (90/10/15, v/w/w) could cryopreserve different cells, for example freezing-vulnerable K562 and OVMANA cells; yielding ~1.8-fold cell viability compared to the case using a commercial cryoprotectant. Furthermore, molecular dynamics simulation indicated that the zwitterions protected the cell membrane from the collapse induced by DMSO.
Synthetic zwitterions as efficient non-permeable cryoprotectants