Abstract
Nonalcoholic steatohepatitis (NASH) is characterized by accumulation of lipids in the hepatocytes (steatosis) and chronic inflammation. Liver resident macrophages (Kupffer cells) play a pivotal role in inducing inflammation. Cross-talk between hepatocytes and Kupffer cells (KCs) regulate both steatosis and inflammation during the pathogenesis of NASH. Isolated hepatocytes and KC serve as important tools to study mechanistic events during NASH in an in vitro setting. Because mice and humans share identical genes, primary mouse hepatocytes and KC are valuable ex vivo models for NASH studies. However, isolation of mouse liver cells is challenging and requires specific technical procedure and skills. Here, we elaborate a method for effective isolation of both primary hepatocytes and KC from adult liver of the same mouse. This protocol can be used for isolation of liver cells from both wild-type (WT) and genetically-engineered mice. The principle of the method is based on a two-step collagenase perfusion technique in which the liver is washed by perfusion, liver cells are segregated by collagenase treatment, and hepatocytes and KC are then purified and cultured. We optimized this protocol in terms of reproducibility, yield of different population of liver cells, and viability.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Anstee QM, Reeves HL, Kotsiliti E et al (2019) From NASH to HCC: current concepts and future challenges. Nat Rev Gastroenterol Hepatol 16:411–428
Ibrahim SH, Hirsova P, Gores GJ (2018) Non-alcoholic steatohepatitis pathogenesis: sublethal hepatocyte injury as a driver of liver inflammation. Gut 67:963–972
Zhou Z, Xu MJ, Gao B (2016) Hepatocytes: a key cell type for innate immunity. Cell Mol Immunol 13:301–315
Lefere S, Tacke F (2019) Macrophages in obesity and non-alcoholic fatty liver disease: crosstalk with metabolism. JHEP Rep 1:30–43
Krenkel O, Tacke F (2017) Liver macrophages in tissue homeostasis and disease. Nat Rev Immunol 17:306–321
Oses C, Aouadi M (2020) Kupffer cell and hepatocyte isolation from a single mouse liver by gradient centrifugation. Methods Mol Biol 2164:1–10
Severgnini M, Sherman J, Sehgal A et al (2012) A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development. Cytotechnology 64:187–195
Berry MN, Friend DS (1969) High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol 43:506–520
Schreiber G, Schreiber M (1973) The preparation of single cell suspensions from liver and their use for the study of protein synthesis. Subcell Biochem 2:307–353
Li WC, Ralphs KL, Tosh D (2010) Isolation and culture of adult mouse hepatocytes. Methods Mol Biol 633:185–196
Howard RB, Christensen AK, Gibbs FA, Pesch LA (1967) The enzymatic preparation of isolated intact parenchymal cells from rat liver. J Cell Biol 35:675–684
Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83
Troyanovsky S (2005) Cadherin dimers in cell-cell adhesion. Eur J Cell Biol 84:225–233
Acknowledgments
This work was supported in part by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) under Grant 1R01DK107451-01A1, the National Cancer Institute (NCI) under Grants 1R01CA230561-01A1, 1R01CA240004-01, and 1R01CA244993-01, and the Department of Defense (DOD) under Grant CA170048.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Mendoza, R., Banerjee, I., Reghupaty, S.C., Yetirajam, R., Manna, D., Sarkar, D. (2022). Isolation and Culture of Mouse Hepatocytes and Kupffer Cells (KCs). In: Sarkar, D. (eds) Non-Alcoholic Steatohepatitis. Methods in Molecular Biology, vol 2455. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2128-8_7
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2128-8_7
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2127-1
Online ISBN: 978-1-0716-2128-8
eBook Packages: Springer Protocols