High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers

PROTOCOL

High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers

Ingmar Mederacke1,4, Dianne H Dapito2,4, Silvia Aff1, Hiroshi Uchinami3 & Robert F Schwabe1,2

1Department of Medicine, Columbia University, New York, New York, USA. 2Institute of Human Nutrition, Columbia University, New York, New York, USA. 3Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, Japan. 4These authors contributed equally to this work. Correspondence should be addressed to R.F.S. ([email protected]).

Published online 22 January 2015; http://www.nature.com/doifinder/10.1038/nprot.2015.017

Web End =doi:10.1038/nprot.2015.017

Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic

fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has

subsequently been adapted to mice, thus allowing the study of fibrogenesis by genetic approaches in transgenic mice. However,

mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity

and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver

and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal

and fibrotic livers via subsequent flow cytometric sorting, thus providing a validated method to determine gene expression

changes during HSC activation devoid of cell culture artifacts or contamination with other cells. The described isolation procedure

takes ~4 h to complete.

2015 Nature America, Inc. All rights reserved.

INTRODUCTION

Fibrosis, defined as replacement of healthy parenchyma with extracellular matrix (ECM)- and myofibroblast-rich scar tissue, has been estimated to contribute to up to 45% of deaths in the developed world1. In the liver, fibrosis develops in response to hepatic injury, and it is common to virtually all liver diseases with hepatocellular damage2. Of note, liver fibrosis has been implicated in many of the grave complications of liver disease, such as the development of portal hypertension, progression to liver cirrhosis and the associated occurrence of hepatic failure, as well as the development of hepatocellular carcinoma2,3. At a cellular level, multiple cell populations have been considered as contributors to fibrosis, including HSCs, portal fibroblasts, bone marrowderived fibrocytes and hepatocytes2,4. Recent cell fate tracing studies have positively identified HSCs as the dominant contributor to the myofibroblast...