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GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process.

Massie, I; Selden, C; Hodgson, H; Fuller, B; Gibbons, S; Morris, GJ; (2014) GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process. Tissue Eng Part C Methods 10.1089/ten.TEC.2013.0571. Green open access

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Abstract

Cryopreservation protocols are increasingly required in regenerative medicine applications but must deliver functional products at clinical scale and comply with Good Manufacturing Process (GMP). Whilst GMP cryopreservation is achievable on a small scale using a Stirling cryocooler-based controlled rate freezer (EF600), successful large scale GMP cryopreservation is more challenging due to heat transfer issues and control of ice nucleation, both complex events which impact on success. We have developed a large scale cryocooler based CRF (VIA Freeze) which can process larger volumes and have evaluated it using alginate encapsulated liver cell (HepG2) spheroids (ELS). It is anticipated that ELS will comprise the cellular component of a bioartificial liver and will be required in volumes of approximately 2L for clinical use. Sample temperatures and Stirling cryocoolerpower consumption were recorded throughout cooling runs for both small (500ul) and large (200ml) volume samples. ELS recoveries were assessed using viability (FDA/PI staining with image analysis), cell number (nuclei count) and function (protein secretion), along with cryo-SEM and freeze substitution techniques to identify possible injury mechanisms. Slow cooling profiles were successfully applied to samples in both the EF600 and the VIA Freeze, and a number of cooling and warming profiles were evaluated. An optimised cooling protocolwith a non-linear cooling profile from ice nucleation to -60ºCwas implemented in both the EF600 and VIA Freeze. In the VIA Freeze the nucleation of ice is detected by the control software, allowing both non-invasive detection of the nucleation event for quality control (QC) purposes and the potential to modify the cooling profile following ice nucleation in an active manner. When processing 200ml of ELS in the VIA Freeze - viabilities at 93.4+/-7.4%, viable cell numbers at 14.3+/-1.7 million nuclei/ml alginate and protein secretion at 10.5+/-1.7 ug/ml/24h were obtained which, compared well with control ELS (viability - 98.1+/- 0.9%; viable cell numbers - 18.3+/-1.0 million nuclei/ml alginate, protein secretion- 18.7+/-1.8 ug/ml/24h). Large volume GMP cryopreservation of ELS is possible with good functional recovery using the VIA Freeze and may also be applied to other regenerative medicine applications.

Type: Article
Title: GMP Cryopreservation of Large Volumes of Cells for Regenerative Medicine: Active Control of the Freezing Process.
Open access status: An open access version is available from UCL Discovery
DOI: 10.1089/ten.TEC.2013.0571
Publisher version: http://dx.doi.org/10.1089/ten.TEC.2013.0571
Additional information: © Mary Ann Liebert, Inc.This work is licensed under a Creative Commons Attribution 3.0 United States License. You are free to copy, distribute, transmit and adapt this work, but you must attribute this work as ‘‘Tissue Engineering, Part C. Copyright 2014 Mary Ann Liebert, Inc. http://liebertpub.com/tec, used under a Creative Commons Attribution License: http://creativecommons.org/licenses/by/3.0/us/’’
UCL classification: UCL
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Brain Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Medicine > Inst for Liver and Digestive Hlth
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Surgery and Interventional Sci
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Div of Surgery and Interventional Sci > Department of Surgical Biotechnology
URI: https://discovery.ucl.ac.uk/id/eprint/1418512
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