115 Exploring the improvement of human cell cryopreservation – Benchmarking the “gold standard”

Regenerative medicine is an emerging industry and there is currently limited knowledge related to the long term storage, shipment and preservation of cell therapies. The most commonly used method for long term storage is cryopreservation, but there are problems associated with protocol definition and reagent toxicity. Cryopreservation is a peripheral but vital process in the development of allogeneic cell-based products. With a number of human cell products currently in phase 3 trials, there is a need for a more regulated, fully characterized and efficient preservation process which may entail moving away from the current “gold standard” of 10% v/v Me2SO in serum, growth medium or other solutions. Me2SO is considered cytotoxic to cells and serum is commonly extracted from cattle. To meet regulatory and clinical needs, a safer, more characterized and efficient process for cryopreservation is needed. As part of a larger project, we have therefore started by fully characterizing this “gold standard” protocol. Early work has focused on benchmarking the overall efficacy of 10% v/v Me2SO in comparison to other common cryoprotective agents (CPAs) when used with HOS TE-85 cells, both immediately post thaw and long after. Compared to the other CPAs used – 5% v/v Me2SO, Glycerol 10% v/v and 1,2-propanediol 10% v/v, data shows that Me2SO 10% v/v is indeed the best (all in FBS as vehicle solution). (n=3). Me2SO is considered toxic, methods specify the need to reduce the time that cells are exposed to Me2SO, at room temperature as much as possible. However protocols and methods using Me2SO are often undefined, with ambiguous terms such as “work quickly”. Therefore, current work is focused on the time sensitive cytotoxicity of Me2SO to cells in order to determine a realistic time frame for processing of cells in freezing solution before and after freezing. Exposure to Me2SO at room temperature decreases viability to ∼90%. Similar exposure after freezing decreases viability to ∼70%. Cells over-exposed to Me2SO, frozen and then analyzed retain 85% viability immediately post thaw, however these cells recover to ∼95% viability after one passage. Decreased (70%) cell attachment to tissue culture surface is exhibited in cells which had been overexposed to Me2SO immediately before seeding- normally, attachment of >90% is expected after 1h. Cells over-exposed to Me2SO prior to freezing recover at normal rates, whereas cells over-exposed after freezing grow slower, those exposed for two hours had some variability slowed growth or complete death. The alkaline-phosphatase assay is currently being performed to assess cell quality (n=3). The data gathered from these experiments will eventually be used to construct an improved process based on design of experiment parameters. These experiments will also be repeated with hMSCs extracted from fresh bone marrow aspirate and defined under the ISCT parameters.Source of funding: Project funded by UK Government Engineering and Physical Sciences Research Council (EPSRC) and Bioprocessing Research Industry Club (BRIC) part of the Biotechnology and Biological Sciences Research Council (BBSRC).Conflict of interest: None declared.Acknowledgements: Prof Nigel Slater, Dr Duncan Sharp, Dr Andy Picken, Karina Brosnan, Kirsty-Louise Marrow, Nathalie Robinson, Alex [email protected]