Reducing dimethyl sulfoxide content in Jurkat cell formulations suitable for cryopreservation

Cell-based medicinal products (CBMPs) are usually cryopreserved in formulations containing up to 10 % dimethyl sulfoxide (Me₂SO) at temperatures below -145 °C. Although Me₂SO effectively protects cells during the freezing process, it can be damaging to cells at ambient temperatures and lead to side effects in patients. The aim of this study was to reduce the amount of Me₂SO in cryopreservation formulations for an immortalized T cell line (Jurkat cells). A design of experiment (DoE) approach was applied for formulation development using seven different excipients, i.e., Me₂SO, trehalose, sorbitol, proline, ectoine, poloxamer 188 (P188) and poly vinyl pyrrolidone 40 (PVP). A DoE model was generated to predict optimal formulations resulting in a high post-thaw viability and a high glass transition temperature of the formulation to allow for frozen storage without the use of liquid nitrogen. Subsequently a stability study was performed with promising lead candidates over three months at storage temperatures of -145 °C, -80 °C, -40 °C. Three benchmark solutions were used, i.e., Cryostor CS10, CryoSOfree as well as 10 % Me₂SO in Roswell Park Memorial Institute Medium (RPMI). The excipient affecting the post-thaw viability of Jurkat cells the most was, as expected, Me₂SO, which led to increased viabilities at higher concentrations. Most formulations resulted in similar viabilities for cells stored at -145 °C and -80 °C, whereas samples stored at -40 °C did not survive. In general, benchmark formulations resulted in slightly higher viabilities than the tested formulations. Furthermore, cell samples stored at -80 °C were recultivated in cell culture and the viability was assessed after 24h. The cell viability after 24h was much lower compared to the cells analyzed directly post-thaw, indicating that freeze-thaw damages continue to unfold after thawing. In summary, several promising excipients and combinations thereof, e.g., trehalose and PVP, were identified for the cryopreservation of Jurkat cells with reduced concentrations of Me₂SO or Me₂SO-free cryopreservation. Additionally, storage at -80 °C is possible for the developed formulations.