胃肠道干细胞在球状体培养中的体外扩增与基因修饰
In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.
作者信息
Miyoshi Hiroyuki, Stappenbeck Thaddeus S
机构信息
Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA.
出版信息
Nat Protoc. 2013 Dec;8(12):2471-82. doi: 10.1038/nprot.2013.153. Epub 2013 Nov 14.
Abstract
It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.
摘要
能够在体外培养富集的干细胞群体是很有用的。将干细胞培养成组织球体是允许成年上皮细胞进行可持续培养的关键方法。胃肠道干细胞可以通过使用来自支持性细胞系（L-WRN）的条件培养基来增殖。本方案描述了如何制备条件培养基以及如何从小鼠胃肠道培养富含干细胞的上皮球体。这些球体也适合用重组慢病毒进行基因改造。该系统使许多已用永生化细胞系进行的细胞生物学检测能够应用于球体。从小鼠分离上皮细胞单位最多需要2小时，3天内可获得富含干细胞的胃肠道球体。用慢病毒进行基因改造的球体可在2周内获得。
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