Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes

Authors

  • Georg Damm Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, Leipzig, Germany; Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
  • Gerda Schicht Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, Leipzig, Germany https://orcid.org/0000-0002-2799-6210
  • Andrea Zimmermann Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, Leipzig, Germany https://orcid.org/0000-0001-6603-8704
  • Christiane Rennert Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, Leipzig, Germany https://orcid.org/0000-0001-8753-8441
  • Nicolas Fischer Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, Leipzig, Germany
  • Melanie Kießig Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
  • Tristan Wagner Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany
  • Victoria Kegel Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany
  • Daniel Seehofer Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany; Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany

DOI:

https://doi.org/10.17179/excli2019-1782

Keywords:

human liver, primary human hepatocytes, hepatocyte isolation, hepatic energy metabolism, cytochrome P450

Abstract

Primary human hepatocytes (PHHs) remain the gold standard for in vitro investigations of xenobiotic metabolism and hepatotoxicity. However, scarcity of liver tissue and novel developments in liver surgery has limited the availability and quality of tissue samples. In particular, warm ischemia shifts the intracellular metabolism from aerobic to anaerobic conditions, which increases glycogenolysis, glucose depletion and energy deficiency. Therefore, the aim of the present study was to investigate whether supplementation with glucose and insulin during PHH isolation could reconstitute intracellular glycogen storage and beneficially affect viability and functionality. Furthermore, the study elucidated whether the susceptibility of the tissue’s energy status correlates with body mass index (BMI). PHHs from 12 donors were isolated from human liver tissue obtained from partial liver resections using a two-step EDTA/collagenase perfusion technique. For a direct comparison of the influence of glucose/insulin supplementation, we modified the setup, enabling the parallel isolation of two pieces of one tissue sample with varying perfusate. Independent of the BMI of the patient, the glycogen content in liver tissue was notably low in the majority of samples. Furthermore, supplementation with glucose and insulin had no beneficial effect on the glycogen concentration of isolated PHHs. However, an indirect improvement of the availability of energy was shown by increased viability, plating efficiency and partial cellular activity after supplementation. The plating efficiency showed a striking inverse correlation with increasing lipid content of PHHs. However, 60 h of cultivation time revealed no significant impact on the maintenance of albumin and urea synthesis or xenobiotic metabolism after supplementation. In conclusion, surgical procedures and tissue handling may decrease hepatic energy resources and lead to cell stress and death. Consequently, PHHs with low energy resources die during the isolation process without supplementation of glucose/insulin or early cell culture, while their survival rates are improved with glucose/insulin supplementation.

Published

2019-11-18

How to Cite

Damm, G., Schicht, G., Zimmermann, A., Rennert, C., Fischer, N., Kießig, M., … Seehofer, D. (2019). Effect of glucose and insulin supplementation on the isolation of primary human hepatocytes. EXCLI Journal, 18, 1071–1091. https://doi.org/10.17179/excli2019-1782

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Section

Original articles

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