Under
the right conditions, some biological systems can maintain
high viability after being frozen and thawed, but many others (e.g.,
organs and many mammalian cells) cannot. To increase the rates of
post-thaw viability and widen the library of living cells and tissues
that can be stored frozen, an improved understanding of the mode of
action of polymeric cryoprotectants is required. Here, we present
a polymeric cryoprotectant, poly(methyl glycidyl sulfoxide) (PMGS),
that achieved higher post-thaw viability for fibroblast cells than
its small-molecule analogue dimethyl sulfoxide. By limiting the amount
of water that freezes and facilitating cellular dehydration after
ice nucleation, PMGS mitigates the mechanical and osmotic stresses
that the freezing of water imparts on cells and facilitates higher-temperature
vitrification of the remaining unfrozen volume. The development of
PMGS advances a fundamental physical understanding of polymer-mediated
cryopreservation, which enables new material design for long-term
preservation of complex cellular networks and tissue.
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.
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