Evaluation of dimethyl sulfoxide-free formulations for cryopreservation of human T cells

Chimeric antigen receptor (CAR) T cells are stored cryopreserved at temperatures below -145 °C. Cryopreservation formulations typically contain dimethyl sulfoxide (DMSO) at concentrations between 5 % and 10 % DMSO, which effectively protects cells during the freezing process. However, DMSO occasionally leads to adverse effects in patients and is detrimental for cells in liquid formulations. We evaluated formulation candidates containing a minimal DMSO amount for their ability to cryopreserve T cells. After storage T cells were thawed and their quality was determined via Trypan blue and Annexin V staining. Additionally, cells in selected formulations were cultured for 1 week and the cell proliferation was determined. Apparent post-thaw survival rates differed depending upon the assay used. Trypan blue staining led to higher apparent survival for all formulations compared to Annexin V staining. Cells in the benchmark formulations Cryostor CS10 and fetal calf serum/10 % DMSO (FCS10) and the formulations containing ectoine and proline were able to proliferate under culture conditions post-thaw. However, this ability was highly donor dependent. Neither high apparent survival rates for Trypan blue staining nor Annexin V staining were indicative of a high proliferation rate. Thus, formulations containing no or low concentrations of DMSO did not achieve comparable results with respect to post-thaw quality, i.e., membrane-intact and non-apoptotic cells, and post-thaw proliferation when compared to T cells frozen in benchmark formulations with 10 % DMSO. Nevertheless, promising formulation candidates were identified, and further studies are needed to optimize the cryopreservation process itself.