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

Georg Damm; Gerda Schicht; Andrea Zimmermann; Christiane Rennert; Nicolas Fischer; Melanie Kießig; Tristan Wagner; Victoria Kegel; Daniel Seehofer

doi:10.17179/excli2019-1782

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

EXCLI J. 2019 Nov 18:18:1071-1091. doi: 10.17179/excli2019-1782. eCollection 2019.

Authors

Georg Damm^{1

2

3}, Gerda Schicht^{1

2}, Andrea Zimmermann^{1

2}, Christiane Rennert^{1

2}, Nicolas Fischer^{1

2}, Melanie Kießig³, Tristan Wagner¹, Victoria Kegel^{1

3}, Daniel Seehofer^{1

3}

Affiliations

¹ Department of Hepatobiliary Surgery and Visceral Transplantation, University Hospital, Leipzig University, Leipzig, Germany.
² Saxonian Incubator for Clinical Translation (SIKT), Leipzig University, Leipzig, Germany.
³ Department of General-, Visceral- and Transplantation Surgery, Charité University Medicine Berlin, 13353 Berlin, Germany.

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.

Keywords: cytochrome P450; hepatic energy metabolism; hepatocyte isolation; human liver; primary human hepatocytes.