Isolation of Liver Mononuclear Cells from a Cholestatic Mice for Single Cell or Single Nuclei Sequencing

Background

Liver is an crucial organ, essential for metabolism and detoxification (Kandilis et al., 2010; Gordillo et al., 2015; Trefts et al., 2017). Underlying its exceptional ability to regenerate in response to injury (Kudira et al., 2016; Sharma et al., 2020), several cellular crosstalk mechanisms work together to initiate the recovery process. Despite significant damage or injury, the liver strives to perform complex functions (Michalopoulos, 2007; Kudira et al., 2016). Due to biliary atresia, primary sclerosing cholangitis, primary biliary cirrhosis, genetic mutations, drugs, immune-mediated, or viral infection, biliary damage is induced and causes obstruction to bile transport, triggering liver fibrosis (Lages et al., 2012; Asai et al., 2015; Taylor et al., 2018; Sharma et al., 2020; Pham et al., 2021). If left untreated, fibrosis progresses to cirrhosis, and ultimately end-stage liver disease, which significantly increases morbidity and mortality. It’s been reported that liver transplantation and clinical follow-up consultation in these patients is an expensive process (Chapman et al., 2010; Bombaci et al., 2019; Lam et al., 2020). To understand the disease mechanisms, and to reduce morbidity and mortality in cholestatic patients, better studies are needed. Cholestasis causes impairment of bile secretion and flow, causing precipitation of cholesterol crystals that form a saturated bile composition. Such precipitated bile acids and cholesterol crystals have significant impact on cell physiology, integrity, and survival (Portincasa et al., 1997; Vavassori et al., 2009; Cipriani et al., 2011; Chen Long et al., 2014; Li et al., 2015; Taylor et al., 2018; Pham et al., 2021). Different studies have demonstrated the modulatory impact of bile acids and their receptors on different immune cells (Matuschek et al., 2019; Kudira et al., 2019; Campbell et al., 2020; Hu et al., 2021). Many well-established models of cholestatic liver disease are used to study liver injury and progression of this disease. Bile duct ligation (BDL) is a surgical model used to induce biliary injury and fibrosis, diet-induced models include chow admixed with xenobiotics, DDC, choline-deficient methionine-supplemented (CDE), and α-naphthyl-isothiocyanate (ANIT) diets, and genetically modified models, like the Mdr2-null mice, an homolog of human multidrug resistance associated protein (MDR3), are used to study cholestatic liver disease (Ravichandra et al., 2021). Chronic feeding of mice with xenobiotic DDC induces hepatobiliary injury and biliary fibrosis, due to precipitation of biliary porphyrin plugs. This model reproduces key histopathological features of PSC disease, such as initiation of ductular reaction by remodeling of biliary compartments, infiltration of inflammatory cells, and periductular fibrosis (Fickert et al., 2007). Delineating the role of specific cells and genes helps in understanding the factors that mediate disease pathogenesis, as previously described by our group (Lages et al., 2012; Kudira et al., 2021c; Pham et al., 2021).Currently, most of the research areas have focused on cellular crosstalk, and gene regulatory network analysis, using single cell RNAseq and snATACseq, or multiome sequencing (MacParland et al., 2018; Dobie et al., 2019; Chen et al., 2020). Current advances in single cell technologies help to better understand the cellular crosstalk that initiates and perpetuates the disease. For all these advanced techniques, the quality of the samples submitted for gene expression/open chromatin analysis plays a significant role in getting high-quality data. Many laboratories have focused on studying highly cholestatic liver diseases, including biliary atresia, primary sclerosing cholangitis disease, and Alagille Syndrome (MacParland et al., 2018; Dobie et al., 2019; Poch et al., 2021). Isolation of good quality hepatic cell fractions (hepatocytes, endothelial cells, cholangiocyte stellate cells, and liver mononuclear cells) and nuclei from cholestatic patients, or from experimental cholestatic mice models, is extremely challenging. We have optimized this protocol for the effective preparation of single cells and single nuclei, for scRNAseq and snATAC-Seq. This protocol will be very useful to the scientific community interested in preparing their cholestatic samples for single cell and single nuclei isolation. Based on an ongoing study on cholestasis, we aimed to publish this protocol to show better methods of cell isolation from an experimental cholestatic mouse model and cholestatic patients. Single cell suspension of liver mononuclear cells isolated from this method is good for flow cytometry, scRNAseq, and snATAC-Seq. This protocol contains two different strategies for preparing hepatic liver cells. We describe in detail the double gradient method for purification of liver mononuclear cells, as this is the best method for cell isolation from DDC-induced cholestatic mice and severe cholestatic patients.