在 3D 器官型培养中分离和鉴定小鼠和人食管上皮细胞。
Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.
机构信息
Division of Gastroenterology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA.
出版信息
Nat Protoc. 2012 Jan 12;7(2):235-46. doi: 10.1038/nprot.2011.437.
Abstract
This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.
摘要
本方案描述了从小鼠和人食管组织中分离和鉴定食管上皮细胞的方法，以及 3D 器官型培养（organotypic culture，OTC）的应用，后者是组织工程的一种形式。这种模型系统可用于研究上皮-间质相互作用的机制。我们提供了分离和培养多种上皮细胞和成纤维细胞的指南，以及这些细胞类型的遗传操作，作为将其整合到 OTC 之前的准备。该方案包括许多重要的应用，包括组织学、免疫组织化学/免疫荧光、用逆转录病毒和慢病毒载体对上皮细胞和成纤维细胞进行遗传修饰，以过表达基因或 RNA 干扰策略、共聚焦成像、激光捕获显微解剖、单个细胞区室的 RNA 微阵列和基于蛋白质的测定。OTC（3D）培养方案需要 15 天才能完成。
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