An algorithm that predicts the viability and the yield of isolated human hepatocytes from surgical remnant tissues

Abstract

Introduction: The liver is an organ that carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of hepatocytes is especially important in the fields of pharmacokinetics, toxicology and translational research. In order to obtain hepatocytes suitable for use as a model, hepatocytes with high viability and yield must be isolated. Therefore, this study aims to identify donor characteristics, intra-operative factors, tissue processing and cell isolation parameters that affect the viability and yield of human hepatocytes. Methods: Remnant liver pieces from tissue designated as surgical waste was collected from 1034 donors with informed consent. Corresponding data on donor characteristics, medical histories, operations, tissue processing and cell isolation parameters were collected and entered into a database. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications. Hepatocyte yield and viability were then subsequently determined using a hemocytometer-based trypan blue exclusion assay. Univariate and multivariate analyses were carried out using Software R. Results: Univariate analyses found that the viability and the yield of hepatocytes were affected by many of the variables examined. Multivariate analyses were then carried out to confirm the factors that have a significant relationship with the viability and the yield. It was found that the viability of hepatocytes was significantly decreased by the presence of fibrosis, liver fat and with increasing gamma-glutamyltranspeptidase activity and bilirubin content. Yield was significantly decreased by the presence of liver fat, septal fibrosis, with increasing aspartate aminotransferase activity, cold ischemia times and weight of perfused liver. However, yield was significantly increased by chemotherapy treatment. Discussion: In conclusion, this study determined the variables that have a significant effect on the viability and the yield of isolated human hepatocytes. These variables have been used to generate an algorithm that can calculate projected viability and yield of isolated human hepatocytes. In this way, projected viability can be determined even before isolation of hepatocytes, so that donors that result in high viability and yield can be identified as candidates for isolating hepatocytes. These isolated hepatocytes can be then used for studying cellular metabolism, pharmacokinetics, toxicology of xenobiotics, translational research etc. The use of human hepatocytes also remains an important model for validating research done in animal models and can also reduce the number of animals used for research in line with the 3R ethical framework. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.