Synthetic Polyampholytes as Macromolecular Cryoprotective Agents

Area of Science:

• Biomaterials Science
• Cell Biology
• Cryobiology

Background:

• Cryoprotective agents are crucial for preserving cells during freezing.
• Dimethyl sulfoxide (DMSO) is a common cryoprotectant but can be cytotoxic.
• Developing DMSO-free alternatives is essential for improved cell viability.

Purpose of the Study:

• To synthesize and evaluate novel polyampholytes as DMSO-free cryoprotective agents for 3T3 fibroblast cells.
• To investigate the impact of copolymer composition, molecular weight, and salt concentration on cryoprotection.
• To understand the mechanism of cryoprotection offered by these polyampholytes.

Main Methods:

• Free radical copolymerization was used to synthesize polyampholytes from N, N-dimethylaminopropyl methacrylamide (DMAPMA), acrylic acid (AA), and N-tert-butylacrylamide (t-BuAAm).
• A standard freeze-thaw protocol was employed to assess cell viability of 3T3 fibroblast cells.
• Polybetaines were prepared and used as control cryoprotective agents.

Main Results:

• Binary DMAPMA/AA copolymers achieved 70% post-thaw viability, increasing to 90% with the addition of t-BuAAm.
• Polyampholytes reduced ice crystal size and formed an ice-free layer around cells, mitigating freezing and thawing damage.
• While immediate post-thaw viability was high, long-term culturing showed issues attributed to intracellular ice damage, which was improved by adding DMSO or BSA.

Conclusions:

• Polyampholytes demonstrate significant potential as effective DMSO-free cryoprotective agents.
• Copolymer composition and formulation are critical factors for optimizing cryoprotection.
• Further research and formulation adjustments are needed to fully overcome residual intracellular ice damage for long-term cell survival.