Posters presenting data for T-cells cryopreserved in CryoStor® cryopreservation solution, authored by BioLife Solutions’ Alireza Abazari, Ph.D., Scientific Applications Director and Aby J. Mathew, Ph.D., Senior Vice President & Chief Technology Officer, were featured during the International Society for Cell & Gene Therapy meeting in Melbourne, Australia, May 29 – June 1, 2019. View the posters in BioLife Solutions’ Evidence Library which includes over 425 articles, abstracts, and posters citing biopreservation efficacy of CryoStor® and HypoThermosol®.
The authors investigated the impact of cryopreservation process parameters using human pan CD3+ T-cells. Cryopreservation process parameters investigated were cryomedia formulation, DMSO content, and post-thaw processing. Cryopreserved T-cells were assessed for cell viability, recovery and expansion, and cytokine secretion, post-thaw.
The impact of processing was determined using T-cells frozen in CryoStor®CS10, 1. immediately processed post-thaw, 2. One hour wait at room temperature post-thaw prior to processing, or 3. Twenty-four hours wait in culture post-thaw prior to processing.
The authors concluded:
• “Intracellular-like CryoStor appears to be a more robust formulation for cryopreservation of human CD3+ T-cells than the conventional home-brew cocktails.”
• “In CryoStor formulations, a higher DMSO concentration (10% v/v) appeared to be more effective in preserving human T-cells than lower (5% v/v) DMSO concentration.”
• “Given a similar timeline post-thaw, expansion rate and IFN𝜸 secretion for resting culture were significantly lower than the immediate activation scenario, suggesting that for optimizing manufacturing timeline, immediate post-thaw activation of T-cells may be more beneficial.”
The authors investigated feeding regimen, cell cycle, media volume, and air/liquid contact surface area to determine the criticality of each parameter to impact cell performance post-thaw in a Jurkat T cell/ CryoStor® CS5 cryopreservation solution model.
Cell cycle investigations exposed Jurkat T-cells for 24 hours in culture medium + 10µM Ribociclib to pharmacologically arrest cells in G0/G1 of the cell cycle prior to cryopreservation. Cells were assayed 48 hours post-thaw.
The impact of feeding regimen employed a starting concentration of 2.5 x 10∧5 Jurkat T-cells in 10 mL, 20 mL or 30 mL of culture media grown in two sets of T25 flasks. The first set of flasks was incubated flat. Media was exchanged on days 3, 4, and 5 after plating. The second set was incubated upright and media was not exchanged. Control flasks containing 2.5 x 10∧5 Jurkat T-cells in 10 mL of media were incubated flat. Media was not changed prior to cryopreservation. On Day 5, culture media containing cells was removed from each flask. Cells were counted, recovered and resuspended at a density of 10∧6/mL in CS5 for cryopreservation. Following cryopreservation, cells were assayed at 0, 24, and 48 hours post-thaw.
The authors concluded:
• “In Jurkat T-cell model, feeding regimen and the timing of harvest for cryopreservation can have a significant impact on the outcome of the cryopreservation process. As such, it is suggested that feeding regimen is considered as a critical process parameter for cryopreservation process development studies. Further investigation must be conducted for specific processes and cell types to validate these results in different cells.
• While analysis of the cell cycle in the 3-day fed culture suggested cell cycle distribution was shifted toward G0/G1 phase, further experiments suggested that cell cycle per se did not have a significant impact on viability and recovery of the cells post-thaw.
• These results also suggest that viability, as assessed by membrane integrity, is not a sufficient measure of cell/process health, either during culture or post-thaw, and therefore, cells must be scrutinized and characterized with other measures as well to ensure safety, potency, and efficacy of the process and the product.”
Cryopreservation is a critical process in cell therapy manufacturing and contributes to the success of cellular therapies. Have you mapped and optimized critical cryopreservation process parameters for your cellular therapy? Seeking solutions? Present your questions to Ask the Scientists at BioLife Solutions. To place an order or for more information, visit our website at www.biolifesolutions.com. Call us at 1.866.6543 (toll-free in North America) or direct: 1.425.402.1400.