Lin Sabrina, Talbot Prue
Department of Cell Biology & Neuroscience and Stem Cell Center, University of California Riverside, Riverside, CA, USA.
Methods Mol Biol. 2011;690:31-56. doi: 10.1007/978-1-60761-962-8_2.
Mouse embryonic stem cells (mESCs) were first derived and cultured almost 30 years ago and ever since have been valuable tools for creating knockout mice and for studying early mammalian development. More recently (1998), human embryonic stem cells (hESCs) have been derived from blastocysts, and numerous methods have evolved to culture hESCs in vitro in both complex and defined media. hESCs are especially important at this time as they could potentially be used to treat degenerative diseases and to access the toxicity of new drugs and environmental chemicals. For both human and mouse ESCs, fibroblast feeder layers are often used at some phase in the culturing protocol. The feeders - often mouse embryonic fibroblasts (mEFs) - provide a substrate that increases plating efficiency, helps maintain pluripotency, and facilitates survival and growth of the stem cells. Various protocols for culturing embryonic stem cells from both species are available with newer trends moving toward feeder-free and serum-free culture. The purpose of this chapter is to provide basic protocol information on the isolation of mouse embryonic fibroblasts and establishment of feeder layers, the culture of mESCs on both mEFs and on gelatin in serum-containing medium, and the culture of hESCs in defined media on both mEFs (hESC culture medium) and Matrigel (mTeSR). These basic protocols are intended for researchers wanting to develop stem cell research in their labs. These protocols have been tested in our laboratory and work well. They can be modified and adapted for any relevant user's particular purpose.
小鼠胚胎干细胞（mESCs）最早于近30年前获得并培养，自那时起一直是创建基因敲除小鼠以及研究早期哺乳动物发育的宝贵工具。最近（1998年），人类胚胎干细胞（hESCs）已从囊胚中获得，并且已经发展出多种方法在复杂和限定培养基中体外培养hESCs。此时hESCs尤为重要，因为它们有可能用于治疗退行性疾病以及评估新药和环境化学物质的毒性。对于人类和小鼠胚胎干细胞，在培养方案的某些阶段通常都会使用成纤维细胞饲养层。饲养细胞——通常是小鼠胚胎成纤维细胞（mEFs）——提供一种能提高接种效率、有助于维持多能性并促进干细胞存活和生长的底物。有各种培养这两种物种胚胎干细胞的方案，最新趋势是朝着无饲养层和无血清培养发展。本章的目的是提供有关分离小鼠胚胎成纤维细胞和建立饲养层、在含血清培养基中在mEFs和明胶上培养mESCs以及在限定培养基中在mEFs（hESC培养基）和基质胶（mTeSR）上培养hESCs的基本方案信息。这些基本方案适用于希望在其实验室开展干细胞研究的研究人员。这些方案已在我们实验室进行测试且效果良好。它们可以根据任何相关用户的特定目的进行修改和调整。
