Joplin Ruth, Kachilele Stivelia
Liver Research Laboratories, Institute of Biomedical Research, University of Birmingham Medical School, Birmingham, UK.
Methods Mol Biol. 2009;481:193-206. doi: 10.1007/978-1-59745-201-4_16.
The human intrahepatic biliary epithelium is composed of a morphologically heterogeneous population of epithelial cells. During liver cirrhosis, new biliary ductular structures develop at the portal margins that express markers of immaturity such as CD56 and Bcl-2. These markers are also expressed transiently on immature biliary duct precursors during embryological development; thus their reappearance during cirrhosis suggests a recapitulation of ontogenesis during some liver conditions. Here we describe methods, based on the differential expression of membrane markers, for separating immature biliary epithelial cells from those associated with mature ducts. We also describe two- and three-dimensional culture models for the maintenance of mature and immature populations in vitro. Both populations readily establish colonies in monolayer culture but only cells from mature ducts can be maintained in medium-term culture as serially proliferating, passageable cultures; immature cells deteriorate and detach within 2-3 weeks of isolation. In three-dimensional collagen gel culture, both mature and immature populations form duct-like structures with clearly definable lumena that persist for up to 6 weeks.
人肝内胆管上皮由形态学上异质性的上皮细胞群体组成。在肝硬化过程中，新的胆小管结构在门脉边缘形成，这些结构表达不成熟标志物，如CD56和Bcl-2。这些标志物在胚胎发育过程中也在未成熟胆管前体上短暂表达；因此它们在肝硬化期间的再次出现表明在某些肝脏疾病状态下个体发生过程的重现。在此我们描述基于膜标志物差异表达从与成熟胆管相关的细胞中分离未成熟胆管上皮细胞的方法。我们还描述用于在体外维持成熟和未成熟细胞群体的二维和三维培养模型。这两种细胞群体在单层培养中都易于形成集落，但只有来自成熟胆管的细胞能够在中期培养中作为连续增殖、可传代培养物维持；未成熟细胞在分离后2 - 3周内会退化并脱落。在三维胶原凝胶培养中，成熟和未成熟细胞群体都形成具有清晰可定义管腔的管状结构，这些结构可持续长达6周。
