115 Exploring the improvement of human cell cryopreservation - Benchmarking the 鈥済old standard鈥?/span>

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• 作者：Tim Morris ; Christopher J. Hewitt ; Karen Coopman
• 刊名：Cryobiology
• 出版年：December, 2013
• 年：2013
• 卷：67
• 期：3
• 页码：430-431
• 全文大小：53 K

文摘

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 鈥済old standard鈥?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 鈥済old standard鈥?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 鈥渨ork quickly鈥? 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 鈭?0%. Similar exposure after freezing decreases viability to 鈭?0%. Cells over-exposed to Me₂SO, frozen and then analyzed retain 85% viability immediately post thaw, however these cells recover to 鈭?5% 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 1 h. 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 Chan.