DMSO and Serum-free cryopreservation of T-cells enabled by glycerol/betaine formulation and controlled-rate freezing with geometry-matched thermal contacts

T-Cells are key components of the adaptive immune system and engineered (e.g CAR-T) T-cells are deployed in immunotherapy, particular for haematologic malignancies. Methods to replace DMSO and serum during T-cell cryopreservation are crucial for both therapies and basic research to improve recovery and function. Glycerol and osmolytes are promising alternatives owing to their lower cytotoxicity than DMSO but often fail to give robust post-thaw recovery. Here we explore the combination of glycerol and betaine for the cryopreservation of Jurkat cells and demonstrate a significantly improved cryopreservation process that is suitable for both low-volume multi-well plate and vial-based freezing with no added serum. The optimised formulation of 10% glycerol and 2% betaine gave >90% post-thaw recovery, but crucially this was achieved when linked with a 3 °C/min freezing rate, and it was observed that faster freezing in general outperformed slower (1 °C/min) which is in contrast to standard protocols which rely on passive cooling devices. The increased rate of cooling appeared to mitigate delayed nucleation damage, which is typically associated with multi-well plate freezing, and the solutions were shown to not impact nucleation in droplet freezing assays. These results demonstrate an easily useable formulation for routine banking, particularly in multiwell-plates which is essential for assay-ready/high-throughput screening approaches as well as vials for larger-volume applications. The removal of serum may also facilitate shipping, where country-specific guidelines for animal-based serum prevent import/export as well as reducing costs.