Synthesis of Poly(allyl glycidyl ether)-Derived Polyampholytes and Their Application to the Cryopreservation of Living Cells

Through the postpolymerization modification of poly­(allyl glycidyl ether) (PAGE), a functionalizable polyether with a poly­(ethylene oxide) backbone, we engineered a new class of highly tunable polyampholyte materials. These polyampholytes can be synthesized to have several useful properties, including low cytotoxicity and pH-responsive coacervate formation. In this study, we used PAGE-based polyampholytes (PAGE-PAs) for the cryopreservation of mammalian cell suspensions. Typically, dimethyl sulfoxide (DMSO) is the cryoprotectant used for preserving mammalian cells, but DMSO suffers from key drawbacks including toxicity and difficult post-thaw removal that motivates the development of new materials and methods. Toxicity and post-thaw survival were dependent on PAGE-PA composition with the highest immediate post-thaw survival for normal human dermal fibroblasts occurring for the least toxic PAGE-PA at a cation/anion ratio of 35:65. With low toxicity, the PAGE-PA concentration could be increased in order to increase immediate post-thaw survival of the immortalized mouse embryonic fibroblasts (NIH/3T3). While immediate post-thaw viability was achieved using only the PAGE-PAs, long-term cell survival was low, highlighting the challenges involved with the design of cryoprotective polyampholytes. An environment utilizing both PAGE-PAs and DMSO in a cryoprotective solution offered promising post-thaw viabilities exceeding 70%, with long-term metabolic activities comparable to unfrozen cells.