4 in 1: Antibody‐free protocol for isolating the main hepatic cells from healthy and cirrhotic single rat livers

Abstract

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 [HMΦ] and hepatic stellate cells [HSC]) from a single rat liver. Control and cirrhotic (CCl4 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 + HMΦ. Further LSEC and HMΦ 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, HMΦ 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.

Transcriptomic Profiling of the Liver Sinusoidal Endothelium during Cirrhosis Reveals Stage-Specific Secretory Signature

“…LSECs were isolated from rats at the three stages of CCl 4 -CLD progression, from TAA-cirrhotic rats, from cBDL-cirrhotic rats, and from healthy rats as previously described [ 19 ]. For detailed procedures see supplementary information .…”

Nuclear deformation mediates liver cell mechanosensing in cirrhosis

“…LSEC fenestration was assessed by scanning electron microscopy. 14 LSECs were fixed overnight with 2% glutaraldehyde dissolved in 0.1 M cacodylate buffer (pH 7.4). After 3 washes with cacodylate buffer for 5 min, the cover glasses were incubated for 1 h with 1% tannic acid and subsequently incubated with 2% osmic acid for 2 h. The samples were dehydrated with an ethanol battery (50%, 70%, 90%, 95%, and 100%), critical-point dried with carbon dioxide, sputter coated with gold, and examined by scanning electron microscopy.…”

“…Isolation of primary cells was performed as previously described. 14 Please see the supplementary information for further details regarding cell isolation and pharmacological treatments.…”

Combination of phosphodiesterase‐5‐inhibitors and beta blockers improves experimental portal hypertension and erectile dysfunction

“…The subsets of liver cells (hepatic stellate cells, liver sinusoidal endothelial cells) were isolated from healthy and BDL rats, as described previously 25,27‐30 . Briefly, primary liver cells were isolated in a two‐step pronase‐collagenase perfusion fractionated by density gradient centrifugation.…”