LAUSR

Background & Aim Cell-based therapies require specialized handling to maintain their potency and efficacy during ex vivo transport from the patient to the manufacturing facility and vice versa. Many current approved or in-development therapies employ a cryopreserved model for the final cell product, which allows significant logistical flexibility, including release assay testing and infusion on-demand. Nonetheless, discrete parameters within the cryopreservation process, including cryomedia formulation and freezing/thawing rates, can significantly influence post-thaw cell viability and functionality. Methods, Results & Conclusion In this study, we examined the impact of culture media refresh and cell cycle on cryopreservation efficacy in a human T-cell model. Jurkat T-cells were cryopreserved at various time points after complete media exchange with fresh complete growth media (CGM), and after removal of Ribocilib, a potent cell proliferation inhibitor which arrests the cell cycle at G0/G1 phase. The cells were suspended in CryoStor CS5 (BioLife Solutions, WA) and cryopreserved at 5 × 106 cells/mL in cryovials (1 mL) using the Asymptote ViaFreeze Duo CRF (GE Healthcare, MA). After a minimum of 24 h storage in LN2, the cells were thawed and viability and recovery was assessed using Via-1 cassettes on NC-3000 (ChemoMetec, Denmark). The cells were further cultured for up to 48 h post-thaw, and viability and proliferation rate were similarly assessed at 24h and 48 h time points. Experimental results suggested that feeding regimen had a direct impact on performance of culture post-thaw. Cell viability and recovery was directly affected by the timeline of culture media exchange, and progressively decreased with increasing the time between the last feeding and removal from culture for formulation. Follow-up experiments revealed that pharmacologic cell cycle arrest prior to cryopreservation had minimal effect on post-thaw viability and expansion. The observed differences in post-thaw viability and proliferation rate between the experimental groups cryopreserved at different growth phases could be attributed to various phases of the starting culture post-thaw rather than cryopreservation per se. This study suggests feeding regimen is not only an important factor for expansion and proliferation in culture, but is also a critical factor affecting cryopreservation efficacy. Conversly, based on these results, cell cycle did not play an important role as a process parameter impacting cryopreservation efficacy.