Polyampholytes as Emerging Macromolecular Cryoprotectants

Abstract

Cellular cryopreservation is a platform technology which underpins cell biology, biochemistry, biomaterials, diagnostics, and the cold chain for emerging cell-based therapies. This technique relies on effective methods for banking and shipping to avoid the need for continuous cell culture. The most common method to achieve cryopreservation is to use large volumes of organic solvent cryoprotective agents which can promote either a vitreous (ice free) phase or dehydrate and protect the cells. These methods are very successful but are not perfect: not all cell types can be cryopreserved and recovered, and the cells do not always retain their phenotype and function post-thaw. This Perspective will introduce polyampholytes as emerging macromolecular cryoprotective agents and demonstrate they have the potential to impact a range of fields from cell-based therapies to basic cell biology and may be able to improve, or replace, current solvent-based cryoprotective agents. Polyampholytes have been shown to be remarkable (mammalian cell) cryopreservation enhancers, but their mechanism of action is unclear, which may include membrane protection, solvent replacement, or a yet unknown protective mechanism, but it seems the modulation of ice growth (recrystallization) may only play a minor role in their function, unlike other macromolecular cryoprotectants. This Perspective will discuss their synthesis and summarize the state-of-the-art, including hypotheses of how they function, to introduce this exciting area of biomacromolecular science.

Figure 1

Schematic showing conventional cryopreservation process and sites of damage. Pathways for vitrification and slow cooling processes are indicated. Point where ice recrystallization inhibiting polymers (outside of context of this Perspective) can impact is shown, and the range of processes where membrane damage (a possible mechanism of action of polyampholytes) is also shown.

Figure 2

Common synthetic strategies used to access polyampholytes. (A) Copolymerization of methacrylic acid (MAA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA). (B) Postpolymerization modification of poly(ε-lysine) using succinic anhydride.

Figure 3

(A) Synthetic scheme for the copolymerization of maleic anhydride with styrene to produce a perfectly alternating copolymer, followed by ring opening of the anhydride to produce a polyampholyte. (B) Synthetic scheme for the synthesis of a poly(DMAEMA-MAA) ampholyte, and a schematic showing how 50:50 monomer incorporation across all chains may not be evenly distributed within a single chain.

Figure 4

(A) IRI (ice recrystallization inhibition) activity and viability of a carboxylated poly(ε-lysine) with varying ratios of carboxylation. (B) IRI activity represented as mean largest grain size for varying ratios of anionic to cationic monomers in a vinyl-based polymer.

Figure 5

(A) Synthesis and application of a polyampholyte derived from the commodity polymer poly(methyl vinyl ether-alt-maleic anhydride). (B) Suspension cryopreservation of A549 cells using the polyampholyte showing total cells recovered. (C) Monolayer cryopreservation of A549 cells using the polyampholyte showing total cells recovered. Results show total cell recovery as a function of both polyampholyte and DMSO concentration. Reproduced with permission from Bailey et al. Copyright 2019 American Chemical Society.

Figure 6

Chemical structures of polyampholytes referred to in Table 2.