Optimization of Zwitterionic Polymers for Cell Cryopreservation

Cryopreservation techniques are valuable for the preservation of genetic properties in cells, and the development of this technology contributes to various fields. In a previous study, an isotonic freezing medium composed of poly(zwitterion) (polyZI) has been reported, which alleviates osmotic shock, unlike typical hypertonic freezing media. In this study, the primitive freezing medium composed of emerging polyZI is optimized. Imidazolium/carboxylate-type polyZI (VimC3C) is the optimal chemical structure. The molecular weight and degree of ion substitution (DSion) are not significant factors. There is an impediment with the primitive polyZI freezing media. While the polyZI forms a matrix around the cell membrane to protect cells, the matrix is difficult to remove after thawing, resulting in low cell proliferation. Unexpectedly, increasing the poly(VimC3C) concentration from 10% to 20% (w/v) improves cell proliferation. The optimized freezing medium, 20% (w/v) poly(VimC3C)_DSion(100%)/1% (w/v) NaCl aqueous solution, exhibited a better cryoprotective effect. Isotonic freezing media composed of polyzwitterions, which alleviates osmotic shock during cryopreservation, have been developed but not optimized. In this study, the emerging polyzwitterion media, from viewpoints of molecular weight, anion, cation, degree of ion substitution, and concentration, is optimized. The optimized freezing medium well cryopreserved cells, including improved cell proliferation, after thawing. image