Parris Alyson, Williams Mark R
School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR47TJ, UK.
Methods Mol Biol. 2015;1212:141-61. doi: 10.1007/7651_2015_197.
The intestinal epithelium is one of the most rapidly renewing tissues in the human body and fulfils vital physiological roles such as barrier function and transport of nutrients and fluid. Investigation of gut epithelial physiology in health and disease has been hampered by the lack of ex vivo models of the native human intestinal epithelium. Recently, remarkable progress has been made in defining intestinal stem cells and in generating intestinal organoid cultures. In parallel, we have developed a 3D culture system of the native human colonic epithelium that recapitulates the topological hierarchy of stem cell-driven tissue renewal and permits the physiological study of native polarized epithelial cells. Here we describe methods to establish 3D cultures of intact human colonic crypts and conduct real-time imaging of intestinal tissue renewal, cellular signalling, and physiological function, in conjunction with manipulation of gene expression by lentiviral or adenoviral transduction. Visualization of mRNA- and protein-expression patterns in cultured human colonic crypts, and cross-validation with crypts derived from fixed mucosal biopsies, is also described. Alongside studies using intestinal organoids, the near-native human colonic crypt culture model will help to bridge the gap that exists between investigation of colon cancer cell lines and/or animal (tissue) studies, and progression to clinical trials. To this end, the near native human colonic crypt model provides a platform to aid the development of novel strategies for the prevention of inflammatory bowel disease and cancer.
肠道上皮是人体中更新速度最快的组织之一，具有屏障功能以及营养物质和液体运输等重要生理功能。由于缺乏天然人类肠道上皮的体外模型，健康和疾病状态下肠道上皮生理学的研究受到了阻碍。最近，在确定肠道干细胞和生成肠道类器官培养物方面取得了显著进展。与此同时，我们开发了一种天然人类结肠上皮的三维培养系统，该系统重现了干细胞驱动的组织更新的拓扑层次结构，并允许对天然极化上皮细胞进行生理学研究。在此，我们描述了建立完整人类结肠隐窝三维培养物的方法，并结合慢病毒或腺病毒转导对基因表达的操纵，对肠道组织更新、细胞信号传导和生理功能进行实时成像。还描述了培养的人类结肠隐窝中mRNA和蛋白质表达模式的可视化，以及与来自固定黏膜活检的隐窝进行交叉验证。除了使用肠道类器官的研究外，近乎天然的人类结肠隐窝培养模型将有助于弥合结肠癌细胞系和/或动物（组织）研究与向临床试验推进之间存在的差距。为此，近乎天然的人类结肠隐窝模型提供了一个平台，有助于开发预防炎症性肠病和癌症的新策略。
