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.
Highlights
The liver is the largest internal organ in humans being the main site for macromolecule synthesis and storage, blood clearance and drug metabolism.[1]
liver sinusoidal endothelial cells (LSEC) cultured for 12 hours upon isolation were rinsed twice with pre‐ warmed Dulbecco's phosphate‐buffered saline (DPBS), incubated with 5 μg/mL Alexa Fluor 488 acetylated low‐density lipoprotein (Ac‐LDL) (L23380, Invitrogen) and 1 μmol L−1 Hoechst diluted in medium A without phenol red for 30 minutes at 37°C protected from light
Isolation of non‐parenchymal cells (NPC) resulted in similar yields of LSEC in all groups, but as expected, the yield of hepatic stellate cells (HSC) isolated from Ch livers was significantly greater than Ct
Summary
The liver is the largest internal organ in humans being the main site for macromolecule synthesis and storage, blood clearance and drug metabolism.[1]. LSEC (19% of the liver mass) are highly specialized endothelial cells characterized by the presence of transcellular pores called fenestrae and the lack of basal membrane.[5,6] This unique cell type lines the physical barrier between blood and hepatocytes but is involved in regulating sinusoidal blood flow, tissue homeostasis, immune response and macromolecular waste clearance.[7,8] HMΦ (10%) represent the resident tissue macrophages, which upon liver damage synthesize and secrete immune modulators.[9,10] HSC. Isolated cells obtained with the presented protocol were tested for purity, yield and functionality
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