Abstract
Background & Aim Cellular therapies are living drugs that have proven highly effective in the treatment of a broad range of human ailments. Logistical considerations for manufacturing living drugs necessitate incorporation of strategies to preserve their potency and efficacy. Many commercialization models employ a frozen cell product to be delivered to the clinic and stored, thawed and infused into patients on demand. Factors influencing post-cryopreservation cell viability and function include, but are not limited to, the selection of an appropriate biopreservation solution and cryoprotective agents, pre-freeze processing time, the temperature of cryomedia addition, ice nucleation temperature, and freezing and warming rates. Methods, Results & Conclusion In this study, CD4+ and CD8+ human T-cells from healthy donors were isolated from fresh PBMC (isolated by using CD4- or CD8-magnetically labelled microbeads) and cryopreserved separately at ∼ 20e6 cells/mL in either a home-brew formulation (10% DMSO, 20% Normosol, and 20% of a 25% Human Serum Albumin solution), or in commercially-available, serum-free, protein-free CryoStor® (BioLife Solutions, Bothell, WA) with 5% v/v or 10% v/v DMSO (CryoStor CS5, and CryoStor CS10, respectively). The cells were subject to varying pre-freeze incubation times at 2-8°C before the freezing process. The vials containing 1 mL of the cell suspension were placed inside a Mr. Frosty type device and were placed at -80°C for a minimum of 2 hours before transfer to LN2. On thaw, cells were processed using different thawing practices (fast vs. slow, and cold vs. warm dilution, among others). Post-thaw cellular viability and count was assessed using the NC-3000 imaging cytometer (ChemoMetec, Denmark) immediately post-thaw. To investigate delayed onset cell death, thawed samples were cultured in complete growth medium (RPMI+ 10%FBS) and cell viability was assessed at 24 h and 48 h post-thaw. Mitochondrial membrane potential was assessed to investigate the potential of this assay as a predictive analytic for delayed onset cell death. Our results suggest that cryopreservation media formulation and post-thaw dilution practice can significantly influence cell viability and recovery, and demonstrate how these parameters contribute to increased variability in observed post-thaw results. Our findings indicate that optimization of the cryopreservation process should be a major focus during early development as a practical means to improve the clinical efficacy of cellular therapies.
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