The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection sur
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The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus Charlotte J. Green . Catriona A. Charlton . Lai-Mun Wang . Michael Silva . Karl J. Morten . Leanne Hodson
Received: 2 February 2017 / Accepted: 7 July 2017 Ó The Author(s) 2017. This article is an open access publication
Abstract Two-step perfusion is considered the gold standard method for isolating hepatocytes from human liver tissue. As perfusion may require a large tissue specimen, which is encapsulated and has accessible vessels for cannulation, only a limited number of tissue samples may be suitable. Therefore, the aim of this work was to develop an alternative method to isolate hepatocytes from non-encapsulated and small samples of human liver tissue. Healthy tissue from 44 human liver resections were graded for steatosis and tissue weights between 7.8 and 600 g were used for hepatocyte isolations. Tissue was diced and underwent a two-step digestion (EDTA and collagenase). Red cell lysis buffer was used to prevent red blood cell C. J. Green (&) C. A. Charlton L. Hodson Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), University of Oxford, Churchill Hospital, Headington, Oxford OX3 7LE, UK e-mail: [email protected] L.-M. Wang Department of Cellular Pathology, Oxford University Hospitals, Oxford, UK M. Silva Department of Hepatobiliary and Pancreatic Surgery, Oxford University Hospital NHS Trust, Churchill Hospital, Oxford OX3 7LE, UK K. J. Morten Nuffield Department of Obstetrics and Gynaecology, The Women’s Centre, University of Oxford, John Radcliffe Hospital, Headley Way, Headington, Oxford, UK
contamination and toxicity. Isolated hepatocyte viability was determined by trypan blue exclusion. Western blot and biochemical analyses were undertaken to ascertain cellular phenotype and function. Liver tissue that weighed C50 g yielded significantly higher (P \ 0.01) cell viability than tissue \50 g. Viable cells secreted urea and displayed the phenotypic hepatocyte markers albumin and cytochrome P450. Presence of steatosis in liver tissue or intrahepatocellular triglyceride content had no effect on cell viability. This methodology allows for the isolation of viable primary human hepatocytes from small amounts of ‘‘healthy’’ resected liver tissue which are not suitable for perfusion. This work provides the opportunity to increase the utilisation of resection surplus tissue, and may ultimately lead to an increased number of in vitro cellular studies being undertaken using the gold-standard model of human primary hepatocytes. Keywords
Hepatocyte Liver Human Isolation
Introduction A variety of cellular models have been used to investigate and understand human liver function and metabolism in health and disease. These models are important for delineating mechanisms of disease initiation and progression. Primary human
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Cell Tissue Bank
hepatocytes are considered the gold standard human liver cell model; although availabilit
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