title: High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers.
abstract: 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.
authors.affiliation: Department of Medicine, Columbia University, New York, New York, USA.
authors.forename: Ingmar
authors.initials: I
authors.lastname: Mederacke
authors.affiliation: Institute of Human Nutrition, Columbia University, New York, New York, USA.
authors.forename: Dianne H
authors.initials: DH
authors.lastname: Dapito
authors.affiliation: Department of Medicine, Columbia University, New York, New York, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Affò
authors.affiliation: Department of Gastroenterological Surgery, Akita University School of Medicine, Akita, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uchinami
authors.affiliation: 1] Department of Medicine, Columbia University, New York, New York, USA. [2] Institute of Human Nutrition, Columbia University, New York, New York, USA.
authors.forename: Robert F
authors.initials: RF
authors.lastname: Schwabe
mesh_terms: D000818:Animals; D002499:Centrifugation, Density Gradient; D017364:Collagenases / Q000737:chemistry; D003584:Cytological Techniques / Q000295:instrumentation / Q000379:methods*; D005434:Flow Cytometry; D055166:Hepatic Stellate Cells; D008099:Liver / Q000166:cytology; D008103:Liver Cirrhosis / Q000473:pathology*; D008822:Mice, Transgenic; D010477:Perfusion
chemical_list: D017364:Collagenases