Ellerström Catharina, Hyllner Johan, Strehl Raimund
Cellartis AB, Göteborg, Sweden.
Methods Mol Biol. 2010;584:121-34. doi: 10.1007/978-1-60761-369-5_7.
The routine culture and expansion of human embryonic stem (hES) cells has been and is still posing a challenge to researchers wishing to take advantage of the cells' unique potential. In contrast to mouse embryonic stem cells, hES cells usually have to be expanded by tedious mechanical microdissection or by enzymatic dissociation to cell clusters of a very narrow size range.It is essential to use a culture system that allows the robust and reproducible enzymatic dissociation of viable hES cell cultures to single cells to allow the scale-up of hES cell cultures as well as the application of hES cells in various experiments, such as FACS, electroporation, and clonal selection.By the development of enzyme-based protocols, which are less labor intensive and less time consuming, much progress has been made over the recent years with regard to improved culture systems for hES cell. We have developed a culture system that is based on single cell enzymatic dissociation (SCED) in combination with a highly supportive feeder cell layer of human foreskin fibroblasts (hFFs). The culture system allows defined enzymatic propagation while maintaining the hES cell lines in an undifferentiated, pluripotent, and normal state.In this chapter, we will show how hES cells, which have been derived and passaged by traditional mechanical dissection, can be rapidly adjusted to propagation by enzymatic dissociation to single cells. The protocols we describe are widely applicable and should therefore be of general use for the reliable mass cultivation of hES cells for various experiments.
人类胚胎干细胞（hES细胞）的常规培养与扩增一直以来且至今仍然对那些希望利用这些细胞独特潜能的研究人员构成挑战。与小鼠胚胎干细胞不同，hES细胞通常必须通过繁琐的机械显微切割或酶解成非常窄尺寸范围的细胞团来进行扩增。使用一种能够将活的hES细胞培养物可靠且可重复地酶解为单个细胞的培养系统至关重要，这样才能扩大hES细胞培养规模，并将hES细胞应用于各种实验，如荧光激活细胞分选（FACS）、电穿孔和克隆选择。通过开发基于酶的方法，这种方法劳动强度较低且耗时较少，近年来在改进hES细胞培养系统方面取得了很大进展。我们开发了一种基于单细胞酶解（SCED）并结合人包皮成纤维细胞（hFFs）高度支持性饲养层的培养系统。该培养系统允许进行明确的酶促增殖，同时使hES细胞系保持在未分化、多能和正常状态。在本章中，我们将展示如何将通过传统机械切割获得并传代的hES细胞快速调整为通过酶解为单个细胞进行增殖。我们描述的方法广泛适用，因此对于可靠地大规模培养hES细胞用于各种实验应具有普遍用途。
