The effect of Me2SO overexposure during cryopreservation on HOS TE85 and hMSC viability, growth and quality

Timothy J Morris,Andrew Picken,Duncan M.C Sharp,Nigel K.H Slater,Christopher J Hewitt,Karen Coopman

doi:10.1016/j.cryobiol.2016.09.004

Timothy J Morris, Andrew Picken + Show 4 more

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https://doi.org/10.1016/j.cryobiol.2016.09.004

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Abstract

With the cell therapy industry continuing to grow, the ability to preserve clinical grade cells, including mesenchymal stem cells (MSCs), whilst retaining cell viability and function remains critical for the generation of off-the-shelf therapies. Cryopreservation of MSCs, using slow freezing, is an established process at lab scale. However, the cytotoxicity of cryoprotectants, like Me2SO, raises questions about the impact of prolonged cell exposure to cryoprotectant at temperatures >0 °C during processing of large cell batches for allogenic therapies prior to rapid cooling in a controlled rate freezer or in the clinic prior to administration. Here we show that exposure of human bone marrow derived MSCs to Me2SO for ≥1 h before freezing, or after thawing, degrades membrane integrity, short-term cell attachment efficiency and alters cell immunophenotype. After 2 h's exposure to Me2SO at 37 °C post-thaw, membrane integrity dropped to ∼70% and only ∼50% of cells retained the ability to adhere to tissue culture plastic. Furthermore, only 70% of the recovered MSCs retained an immunophenotype consistent with the ISCT minimal criteria after exposure. We also saw a similar loss of membrane integrity and attachment efficiency after exposing osteoblast (HOS TE85) cells to Me2SO before, and after, cryopreservation.Overall, these results show that freezing medium exposure is a critical determinant of product quality as process scale increases. Defining and reporting cell sensitivity to freezing medium exposure, both before and after cryopreservation, enables a fair judgement of how scalable a particular cryopreservation process can be, and consequently whether the therapy has commercial feasibility.

Highlights

Cell therapies hold the potential to revolutionise healthcare as regenerative medicines, replicating the success of the human therapeutic protein industry
Preventing changes to phenotype and metabolism is vital for cell therapeutics including human mesenchymal stem cell (hMSC), which have been described by Caplan and Correa [3] as a “drugstore” for injury with the ability to establish a regenerative microenvironment through secreted protein factors, which regulate the local immune response at the injury site
Working cell banks for manufacturing can contains hundreds of vials and with cellular therapeutics requiring >109 cells per dose [31] and with these increased volumes the cellcryoprotectant exposure time will increase as the process scales from a laboratory to manufacturing environment

Summary

Introduction

Cell therapies hold the potential to revolutionise healthcare as regenerative medicines, replicating the success of the human therapeutic protein industry. Cell therapies are more complex than protein therapeutics, which to the makes the preservation, long-term storage and shipment of cellular therapies a challenging prospect. Cryopreservation methods for long-term storage are described only in brief when reported in cell therapy clinical trial protocols. While a cooling rate may be Abbreviations: CPA, cryoprotective agent; hMSC, human mesenchymal stem cell; ISCT, International Society for Cellular Therapy; pNNP, p-nitrophenyl phosphate; MoA, mechanism of action. Provided, processing times before freezing or after thawing are rarely given [13]. T.J. Morris et al / Cryobiology 73 (2016) 367e375 consistent cell therapy product, which can be globally distributed and administered at an affordable cost

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