Cell-compatible isotonic freezing media enabled by thermo-responsive osmolyte-adsorption/exclusion polymer matrices

Abstract

During the long-term storage of cells, it is necessary to inhibit ice crystal formation by adding cryoprotectants. Non-cell-permeable cryoprotectants have high osmotic pressure which dehydrates cells, indirectly suppressing intracellular ice crystal formation. However, the high osmotic pressure and dehydration often damage cells. Emerging polymer-type non-cell-permeable cryoprotectants form matrices surrounding cells. These matrices inhibit the influx of extracellular ice nuclei that trigger intracellular ice crystal formation. However, these polymer-type cryoprotectants also require high osmotic pressure to exert an effective cryoprotecting effect. In this study, we designed a poly(zwitterion) (polyZI) that forms firm matrices around cells based on their high affinity to cell membranes. The polyZI successfully cryopreserved freeze-vulnerable cells under isotonic conditions. These matrices also controlled osmotic pressure by adsorbing and desorbing NaCl depending on the temperature, which is a suitable feature for isotonic cryopreservation. Although cell proliferation was delayed by the cellular matrices, washing with a sucrose solution improved proliferation.