A novel method for establishment and characterization of extrahepatic bile duct epithelial cells from mice

Abstract

Culture of extrahepatic bile duct epithelial cells is a useful model to investigate physiology of extrahepatic bile duct epithelia and hepatobiliary disease mechanisms. The aim of this work was to establish and characterize a primary murine extrahepatic bile duct epithelial cell culture. Epithelial cells were isolated from extrahepatic bile ducts of BALB/c mice that were intraperitoneally injected with newborn bovine serum to induce the proliferation of extrahepatic bile ducts' epithelial cells and cultured on rat tail type I collagen-coated plastic culture flask containing DMEM/HamF12 with 10% FBS and 10 ng/ml epidermal growth factor at 37°C in an incubator with 5% humidified CO(2). The cells showed typical morphologic characteristics of epithelial phenotypes with cobblestone appearance in monolayer within 5-6 d after culture; they were positive against anticytokeratin-19 immunostaining. Transmission electron microscopy showed typical bile duct epithelia with microvilli on the cytomembrane, Golgi complex, massive mitochondria, and rough endoplasmic reticulum in the cytoplasmic. The growth curve of the epithelial cells was determined by a MTT assay which showed a normal sigmoidal growth curve. This culture technique might be a reliable method for isolation, purification, and primary culture of extrahepatic bile duct epithelial cells that can serve as a model for in vitro studies on the pathophysiology of hepatobiliary diseases as well as pharmacological and toxicological targets relevant to hepatobiliary diseases.

Development and functional characterization of extrahepatic cholangiocyte lines from normal rats

“…Ultrastructural analysis shows that extrahepatic bile ducts are lined by polarized cholangiocytes and have morphological phenotypes similar to that of intrahepatic cholangiocytes [42]. Intact tight junctions and microvilli (characteristic of intra- and extra-hepatic biliary epithelia) [27, 43] were present in EBDs and ECCs. Similar to EBDs, ECCs express CK-19, secretin, SR, SSTR 2 , CFTR and AE2 and respond to secretin and somatostatin with changes in cAMP levels.…”

“…Secretin-induced choleresis is associated with increased cAMP levels in extrahepatic bile duct tissue of humans and baboons [15]. While several in vitro culture models are available for evaluating the function of rodent and human intrahepatic cholangiocytes [20–26], limited in vitro tools exist for evaluating the pathophysiology of the extrahepatic common bile duct [27–29]. Therefore, the aim of the study was to develop a cell line (ECCs) from normal extrahepatic bile ducts.…”

HMGB1-Promoted and TLR2/4-Dependent NK Cell Maturation and Activation Take Part in Rotavirus-Induced Murine Biliary Atresia

“…As described in our previous study [43], extrahepatic cholangiocytes were isolated, cultured and identified. The purified cholangiocytes were >95% pure [43].…”

“…The purified cholangiocytes were >95% pure [43]. The method of macrophage culture was described in Supplemental Protocol S1 .…”

Microvesicles secreted from human multiple myeloma cells promote angiogenesis

“…Transmission electron microscopy (TEM) and fluorescence microscopy (FM) TEM was performed as previously reported [25] . For FM, the purified MM-MVs were stained using the PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich, St Louis, MO, USA) according to the kit's instructions, and the MM-MVs were observed by FM (Olympus, Tokyo, Japan).…”