Post-thaw application of ROCK-inhibitors increases cryopreserved T-cell yield†

Emerging cell-based therapies such as CAR-T (Chimeric Antigen Receptor T) cells require cryopreservation to store and deliver intact and viable cells. Conventional cryopreservation formulations use DMSO to mitigate cold-induced damage, but do not address all the biochemical damage mechanisms induced by cold stress, such as programmed cell death (apoptosis). Rho-associated protein kinases (ROCK) are a key component of apoptosis, and their activation contributes to apoptotic blebbing. Here we demonstrate that the ROCK inhibitor fasudil hydrochloride, when supplemented into the thawing medium of T-cells increases the overall yield of healthy cells. Cell yield was highest using 5 or 10% DMSO cryopreservation solutions, with lower DMSO concentrations (2.5%) leading to significant physical damage to the cells. After optimisation, the post-thaw yield of T-cells increased by approximately 20% using this inhibitor, a significant increase in the context of a therapy. Flow cytometry analysis did not show a significant reduction in the relative percentage of cell populations undergoing apoptosis, but there was a small reduction in the 8 hours following thawing. Fasudil also led to a reduction in reactive oxygen species. Addition of fasudil into the cryopreservation solution, followed by dilution (rather than washing) upon thaw also gave a 20% increase in cell yield, demonstrating how this could be deployed in a cell-therapy context, without needing to change clinical thawing routines. Overall, this shows that modulation of post-thaw biochemical pathways which lead to apoptosis (or other degradative pathways) can be effectively targeted as a strategy to increase T-cell yield and function post-thaw.