Abstract
Isolated human primary hepatocytes are an essential in vitro model for basic and clinical research. For successful application as a model, isolated hepatocytes need to have a good viability and be available in sufficient yield. 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. Remnant liver pieces from tissue designated as surgical waste were collected from 1034 donors with informed consent. Human hepatocytes were isolated by a two-step collagenase perfusion technique with modifications and hepatocyte yield and viability were subsequently determined. The accompanying patient data was collected and entered into a database. 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. 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. Further, if the viability and yield of the isolated hepatocytes is lower than expected, this will highlight a methodological problem that can be addressed.
Highlights
The liver carries out a diverse range of necessary functions, such as homeostasis, metabolism and detoxification
A total of 1034 hepatocyte isolations were done with an average viability of 78610% and average yield of 13611 million viable hepatocytes per gram liver with the values represented in means 6 standard deviation
In the case of the yield of hepatocytes, it was found that the yield was decreased by increases in age, body mass index (BMI), liver fat, alkaline phosphatase (AP) activity, GOT activity, GGT activity, GPT activity, bilirubin content in the blood, partial thromboplastin time (PTT), warm ischemia time in vivo and weight of resected or perfused liver (Table S2)
Summary
The liver carries out a diverse range of necessary functions, such as homeostasis, metabolism and detoxification. One of the main uses of a human in vitro hepatocyte model is for the validation of studies done using animal models due to species differences. Olson et al, [1] showed that when 150 drugs that cause human toxicity are tested, the concordance between toxicity found in animal studies and that observed in clinical practice is 70%. Brambilla and Martelli found that when 42 compounds from various chemical families were tested for their toxicity in rat or human hepatocytes, 28 had similar toxicities, 10 were more toxic for rats, 3 were moderately more toxic for human hepatocytes and 1 was lethal for rat hepatocytes at a concentration 30-fold lower than that toxic for human hepatocytes [2]. Animal models could have less genetic variation than humans; Brambilla and Martell [2] found
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