Synthetic Polyampholytes as Macromolecular Cryoprotective Agents

Abstract

A series of polyampholytes based on different molar ratios on N, N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA), and optionally, N- tert-butylacrylamide ( t-BuAAm), were prepared by free radical copolymerization, and tested as DMSO-free cryoprotective agents for 3T3 fibroblast cells by using a standard freeze-rethaw protocol. Polybetaines prepared by reaction of DMAPMA homo and copolymers with 1,3-propane sultone were used as additional controls. Results showed strong effects of copolymer composition, molecular weight, polymer and NaCl concentrations, on post-thaw cell viability. Binary (DMAPMA/AA) copolymers showed best post-thaw cell viability of 70% at a 30/70 mol % ratio of DMAPMA/AA, which increased to 90% upon introduction of 9 mol % t-BuAAm while maintaining the 30/70 mol % cation/anion ratio. The use of acrylamide linkages in DMAPMA ensures absence of hydrolytic loss of cationic side chains. These polyampholytes were found to decrease ice crystal size and to form a polymer-rich, ice-free layer around cells, reducing damage from intercellular ice crystals during both freezing and thawing steps. These polyampholytes also dehydrate cells during freezing, which helps protect cells from intracellular ice damage. While cell viability immediately after thawing was high, subsequent culturing revealed poor attachment and long-term viability, which is attributed to residual cell damage from intracellular ice formation. Addition of 2 wt % DMSO or 1% BSA to the polymer-based freeze medium was found to mitigate this damage and result in post-thaw viabilities matching those achieved with 10 wt % DMSO.

Natural Cryoprotective and Cytoprotective Agents in Cryopreservation: A Focus on Melatonin

“…Polyampholytes have also been reported to cryopreserve cells by strongly interacting with the cell membrane, with hydrophobicity increasing the cytoprotective effect by increasing both the affinity for membrane interaction and affinity for ice recrystallization inhibition [ 19 ]. This type of NPA can be of either synthetic [ 19 , 20 ] or semisynthetic nature, such as carboxylated ε-poly-ʟ-lysine polymers [ 18 ] that have successfully been implemented into the cryopreserved human mesenchymal stem cells and induced pluripotent stem cells. In addition, temperature-responsive physical gels and zwitterionic polymers have been used in the cryoprotection of 3D cell cultures in which these materials can act at the same time as the 3D scaffolds and CPA in the composite cell system [ 21 ].…”

Low DMSO Cryopreservation of Stem Cells Enabled by Macromolecular Cryoprotectants

“…However, the majority of cells died after 24 h in culture. 17 Similarly, Naaldijk et al reported immediate post-thaw viabilities of MSC frozen with DMSO and hydroxyethyl starch to be ∼85%, but this plummeted to <30% after 3 days in culture for samples containing <4% DMSO. 24 These studies highlight that immediate post-thaw viability is not a good predictor of cell health.…”

Size and Shape Control of Ice Crystals by Amphiphilic Block Copolymers and Their Implication in the Cryoprotection of Mesenchymal Stem Cells

“…Even when cell viability is excellent just after thawing the cells, the final cell recovery can be low . The post-thaw growth rate of 3T3 fibroblast cells was significantly suppressed, even though immediate post-thaw cell viability was as good as 70 to 90% when using cryoprotecting polyampholytes consisting of N , N -dimethylaminopropyl methacryl amide and acrylic acid . In addition, cell adhesion and long-term cell viability were poor.…”

“…57 The post-thaw growth rate of 3T3 fibroblast cells was significantly suppressed, even though immediate post-thaw cell viability was as good as 70 to 90% when using cryoprotecting polyampholytes consisting of N,N-dimethylaminopropyl methacryl amide and acrylic acid. 58 In addition, cell adhesion and long-term cell viability were poor. A similar trend was observed for carboxylated poly(L-lysine) for cryopreservation of human mesenchymal stem cells.…”