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.