Open Access4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers
Author(s) -
FernándezIglesias Anabel,
OrtegaRibera Martí,
GuixéMuntet Sergi,
GraciaSancho Jordi
Publication year - 2019
Publication title -
journal of cellular and molecular medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.44
H-Index - 130
eISSN - 1582-4934
pISSN - 1582-1838
DOI - 10.1111/jcmm.13988
Subject(s) - collagenase , hepatic stellate cell , biology , differential centrifugation , in vivo , decellularization , liver cytology , albumin , antibody , microbiology and biotechnology , immunology , biochemistry , extracellular matrix , endocrinology , enzyme , liver metabolism
Liver cells isolated from pre‐clinical models are essential tools for studying liver (patho)physiology, and also for screening new therapeutic options. We aimed at developing a new antibody‐free isolation method able to obtain the four main hepatic cell types (hepatocytes, liver sinusoidal endothelial cells [ LSEC ], hepatic macrophages [ HM Φ] and hepatic stellate cells [ HSC ]) from a single rat liver. Control and cirrhotic ( CC l 4 and TAA ) rat livers (n = 6) were perfused, digested with collagenase and mechanically disaggregated obtaining a multicellular suspension. Hepatocytes were purified by low revolution centrifugations while non‐parenchymal cells were subjected to differential centrifugation. Two different fractions were obtained: HSC and mixed LSEC + HM Φ. Further LSEC and HM Φ enrichment was achieved by selective adherence time to collagen‐coated substrates. Isolated cells showed high viability (80%‐95%) and purity (>95%) and were characterized as functional: hepatocytes synthetized albumin and urea, LSEC maintained endocytic capacity and in vivo fenestrae distribution, HM Φ increased expression of inflammatory markers in response to LPS and HSC were activated upon in vitro culture. The 4 in 1 protocol allows the simultaneous isolation of highly pure and functional hepatic cell sub‐populations from control or cirrhotic single livers without antibody selection.