Abstract：
Organoids are novel in vitro models to study intercellular cross talk between the different types of cells in disease pathophysiology. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), scaffold-free multicellular three-dimensional cholangiocyte organoids (3D-CHOs) were developed using primary liver cells derived from normal subjects and patients with PSC. Human liver samples from healthy donors and patients with PSC were used to isolate primary cholangiocytes [epithelial cell adhesion molecule (EpCam)+/ cytokeratin-19+], liver endothelial cells (CD31+), and hepatic stellate cells (HSCs; CD31-/CD68-/desmin+/vitamin A+). 3D-CHOs were formed using cholangiocytes, HSCs, and liver endothelial cells, and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence, quantitative RT-PCR, and transmission electron microscopy. Transcription profiles for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, and SCTR), fibrosis (ACTA2, COL1A1, DESMIN, and TGFβ1), angiogenesis (PECAM, VEGF, CDH5, and vWF), and inflammation (IL-6 and TNF-α) confirmed PSC phenotypes of 3D-CHOs. Because cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal immunofluorescence was used to demonstrate localization of the neurokinin-1 receptor within cytokeratin-19+ cholangiocytes and desmin+ HSCs. Moreover, 3D-CHOs from patients with PSC confirmed PSC phenotypes with up-regulated neurokinin-1 receptor, tachykinin precursor 1, and down-regulated membrane metalloendopeptidase. Scaffold-free multicellular 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared with two-dimensional cell culture, which can be used in PSC disease-related research.
摘要：
类器官是新的体外模型，用于研究疾病病理生理学中不同类型细胞之间的细胞间串扰。为了更好地了解驱动原发性硬化性胆管炎（PSC）进展的潜在机制，我们使用来自正常和PSC患者的“原代”肝细胞开发了无支架的多细胞3D胆管细胞类器官（3D-CHO）。来自健康供体和PSC患者的人类肝脏样本用于分离“原代”胆管细胞（EPCAM/CK-19），肝内皮细胞（LECs，CD31+),和肝星状细胞（HSC，CD31-/CD68-/Desmin+/维生素A+)。3D-CHOs是用胆管细胞形成的,HSC,和LEC,并保持存活长达1个月。通过免疫荧光进一步表征分离的原代细胞系和3D-CHOs，qRT-PCR,和透射电子显微镜。胆管细胞的转录谱（SOX9，CFTR，EpCAM,AE,SCT,SCTR),纤维化(ACTA2，COL1A1，DESMIN，TGFβ1),血管生成（PECAM，VEGF,CDH5,vWF),炎症（IL-6，TNF-α）证实了3D-CHOs的PSC表型。由于胆管细胞发展神经内分泌表型并表达神经调质，共聚焦免疫荧光表明，神经激肽-1受体（NK-1R）位于CK-19胆管细胞和结蛋白-HSC内。此外，PSC患者的3D-CHOs证实了NK-1R上调的PSC表型，速激肽前体1和下调的膜金属内肽酶。与2D细胞培养相比，无支架多细胞3D-CHOs在模拟PSC体内表型方面显示出作为体外模型的优越性。可用于PSC疾病相关研究。
