Abstract
Cryopreservation of hepatocytes is important for use both in research and for clinical application in hepatocyte transplantation. Cryopreservation causes damage to hepatocytes with the result that cell viability and function is reduced on thawing compared to fresh cells. There are many different protocols reported for freezing human hepatocytes mainly using DMSO as cryoprotectant. In this chapter the current detailed protocols used for cryopreservation and thawing of human hepatocytes for cell transplantation at the Cell Isolation Unit at King’s College Hospital, London, are described. All procedures must be performed in a clean GMP environment using materials and reagents which are of pharmaceutical grade. The cryopreservation media is UW solution with added 300 mM glucose containing 10% DMSO and the thawing solution is EMEM containing 2% HSA. Freezing is performed in a controlled-rate freezer using a stepwise cooling programme.
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References
Terry, C., Dhawan, A., Mitry, R.R., and Hughes, R.D. (2006) Cryopreservation of isolated human hepatocytes for transplantation: state of the art. Cryobiology 53, 149–159.
Terry, C., Mitry, R.R., Lehec, S.C., Muiesan, P., Rela, M., Heaton, N.D., Hughes, R.D., and Dhawan, A. (2005) The effects of cryopreservation on human hepatocytes obtained from different sources of liver tissue. Cell Transplant. 14, 585–594.
Strom, S.C., Dorko, K., Thompson, M.T., Pisarov, L.A., and Nussler, A.K. (1982) Large scale isolation and culture of human hepatocytes, in Ilots de Langerhans et Hepatocytes (Franco, D., Boudjema, K., and Varet, B. eds.), Les Editions INSERM, Paris, pp. 195–205.
Mitry, R.R., Hughes, R.D., Aw, M.M., Terry, C., Mieli-Vergani, G., Girlanda, R., Muiesan, P., Rela, M., Heaton, N.D., and Dhawan, A. (2003) Human hepatocyte isolation and relationship of cell viability to early graft function. Cell Transplant. 12, 69–74.
Mitry, R.R. (2009) Isolation of human hepatocytes, in Hepatocyte Transplantation (Dhawan, A. and Hughes R.D., eds.), Methods Mol. Biol. 481, 3–17.
Diener, B., Utesch, D., Beer, N., Durk, H., and Oesch F. (1993) A method for the cryopreservation of liver parenchymal cells for studies of xenobiotics. Cryobiology 30, 116–127.
Steinberg, P., Fischer, T., Kiulies, S., Biefang, K., Platt, K. L., Oesch, F., Bottger, T., Bulitta, C., Kempf, P., and Hengstler, J. (1999) Drug metabolizing capacity of cryopreserved human, rat, and mouse liver parenchymal cells in suspension. Drug Metab. Dispos. 27, 1415–1422.
Freshney, R.I. (2000) Culture of Animal Cells, Wiley-Liss, New York, NY, pp. 309–328.
Terry, C., Dhawan, A., Mitry, R.R., Lehec, S.C., and Hughes, R.D. (2006) Pre-incubation of rat and human hepatocytes with cytoprotectants prior to cryopreservation can improve viability and function on thawing. Liver Transplant. 12, 165–177.
Dhawan, A., Mitry, R.R., Hughes, R.D., Lehec, C., Terry, C., Bansal, S., Arya, R., Wade, J.J., Verma, A., Heaton, N.D., Rela, M., and Mieli-Vergani, G. (2004) Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 78, 1812–1814.
Puppi, J., Tan, N., Mitry, R.R., Hughes, R.D., Lehec, S., Mieli-Vergani, G., Karani, J., Champion, M.P., Heaton, N., Rela, M., and Dhawan, A. (2008) Hepatocyte transplantation followed by auxiliary liver transplantation – a novel treatment for ornithine transcarbamylase deficiency. Am. J. Transplant. 8, 452–457.
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Hughes, R.D., Mitry, R.R., Lehec, S.C. (2010). Cryopreservation of Human Hepatocytes for Clinical Use. In: Maurel, P. (eds) Hepatocytes. Methods in Molecular Biology, vol 640. Humana Press. https://doi.org/10.1007/978-1-60761-688-7_5
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DOI: https://doi.org/10.1007/978-1-60761-688-7_5
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