Exploring potential FDA-approved components for DMSO-free T-cell cryopreservation solutions

Background & Aim The cryopreservation process is an integral part of cellular therapies. However, current DMSO-containing cryomedia are toxic and cause many cyto- and genotoxic such as causing stress at the membrane interface, oxidizing free thiol groups on proteins, and alters the epigenetic DNA methylation profile etc. These has prompted an urgent need for the development of a non-toxic, DMSO-free cryomedia. Our goal is to develop DMSO-free cryomedia with similar or superior cell preservation capacity and post-thaw viability than the leading DMSO-containing cryomedia currently use. We explored the cryoprotectant capabilities and other parameters regarding prevention of cryo-damage of components including anti-apoptosis molecules, antioxidants, and molecules related to cell proliferation. These components offer various functionalities by working against intra- and extracellular ice formation as well as against cell damage during post-thaw recovery. All components are approved by the FDA and under regulatory compliance. Aim:The current T-cell capacity for CAR-T DMSO cryomedia is over 3 × 106 cells per mL of cryomedia; our objective goal is to develop a solution that will successfully preserve 5 × 106 cells per mL Methods, Results & Conclusion Methods: We used Jurkat T-cells, an immortalized human suspension cell line, as the initial model system during cryomedia optimization. During the optimization process, we compared the cryopreservation performance of our DMSO-free cryomedia to DMSO-containing cryomedia using Jurkat T-cells. The tested cryomedia components were rapidly screened by evaluating initial post-thaw viability; when the post-thaw viability was higher than 70%, they were further evaluated after a 72-hour recovery period to ensure cell viability and proliferation were not impacted by the cryomedia. Results Many promising candidates were observed based on the relative post-thaw viability and performance on recovery viability. Finally, we evaluate the potential candidates using PBMC-derived T-cells to verify the success of the final cryomedia We found T cell can preserve > 90% superior than traditional DMSO cryopreservation solution at great post-thaw viability.. Conclusion This study exhibits an accelerated cryomedia screening system which has the potential to be a valuable paradigm in future development of cellular therapeutic products as it can be applied to many cell types and cell therapies including CAR-T, CAR-NK etc. The cryopreservation process is an integral part of cellular therapies. However, current DMSO-containing cryomedia are toxic and cause many cyto- and genotoxic such as causing stress at the membrane interface, oxidizing free thiol groups on proteins, and alters the epigenetic DNA methylation profile etc. These has prompted an urgent need for the development of a non-toxic, DMSO-free cryomedia. Our goal is to develop DMSO-free cryomedia with similar or superior cell preservation capacity and post-thaw viability than the leading DMSO-containing cryomedia currently use. We explored the cryoprotectant capabilities and other parameters regarding prevention of cryo-damage of components including anti-apoptosis molecules, antioxidants, and molecules related to cell proliferation. These components offer various functionalities by working against intra- and extracellular ice formation as well as against cell damage during post-thaw recovery. All components are approved by the FDA and under regulatory compliance. Aim:The current T-cell capacity for CAR-T DMSO cryomedia is over 3 × 106 cells per mL of cryomedia; our objective goal is to develop a solution that will successfully preserve 5 × 106 cells per mL Methods: We used Jurkat T-cells, an immortalized human suspension cell line, as the initial model system during cryomedia optimization. During the optimization process, we compared the cryopreservation performance of our DMSO-free cryomedia to DMSO-containing cryomedia using Jurkat T-cells. The tested cryomedia components were rapidly screened by evaluating initial post-thaw viability; when the post-thaw viability was higher than 70%, they were further evaluated after a 72-hour recovery period to ensure cell viability and proliferation were not impacted by the cryomedia.