Evaluating the Effects of Standard Cryopreservation Protocols on Human Mesenchymal Stem Cell Survival and Quality

Cell preservation will be a critical part of the manufacturing process for any allogeneic cellular therapy enabling storage and transport from manufacturing facility to bedside as well as generation of master and working cell banks. Cells are typically cryopreserved in a DMSO-containing freezing solution despite DMSO being considered cytotoxic at temperatures above 0°C. Furthermore, using an understanding of industrial-scale bioprocessing constraints; we are currently defining a realistic processing window for such DMSO-based cryopreservation processes which will not negatively impact cell survival or quality. Experiments to date using bone marrow-derived human mesenchymal stem cells (hMSCs) show that exposure to 10% (v/v) DMSO per se for up to 2 hours at ambient temperature has little impact on cells; viability remains >80%,  the number of cell doublings over 30 days in culture is unchanged and more than 80% of cells retain their CD73⁺, CD105⁺, CD23^-, HLA-DR^- phenotype (compared to 90-95% for control groups not exposed to DMSO). Similar results were obtained when cells were exposed to DMSO for up to 2 hours before being frozen at 1°C/min and subsequently rapidly thawed, although only ~60% of cells retain the correct phenotype, highlighting that prolonged processing prior to freezing does impact cell quality. A more drastic change was noted when cells were exposed to DMSO for only 5 mins, frozen and then further exposed to DMSO at 37°C for up to 2 hours post-thaw. Although after 2 hours cell viability remained ~70%, only 50% of cells were able to adhere to plastic and again, fewer than 40% of cells managed to retain the expected hMSC phenotype. This indicates that even if rapid processing times can be achieved within the manufacturing setting, processing time at the clinical end will also need to be controlled to ensure high quality cells are delivered to the patient.