Kowalski Michael P, Yoder Amy, Liu Li, Pajak Laura
Beckman Coulter, Inc., Indianapolis, IN 46268, USA.
J Biomol Screen. 2012 Oct;17(9):1171-9. doi: 10.1177/1087057112452783. Epub 2012 Aug 15.
Despite significant use in basic research, embryonic stem cells have just begun to be used in the drug discovery process. Barriers to the adoption of embryonic stem cells in drug discovery include the difficulty in growing cells and inconsistent differentiation to the desired cellular phenotype. Embryonic stem cell cultures require consistent and frequent handling to maintain the cells in a pluripotent state. In addition, the preferred hanging drop method of embryoid body (EB) differentiation is not amenable to high-throughput methods, and suspension cultures of EBs show a high degree of variability. Murine embryonic stem cells passaged on an automated platform maintained ≥ 90% viability and pluripotency. We also developed a method of EB formation using 384-well microplates that form a single EB per well, with excellent uniformity across EBs. This format facilitated high-throughput differentiation and enabled screens to optimize directed differentiation into a desired cell type. Using this approach, we identified conditions that enhanced cardiomyocyte differentiation sevenfold. This optimized differentiation method showed excellent consistency for such a complex biological process. This automated approach to embryonic stem cell handling and differentiation can provide the high and consistent yields of differentiated cell types required for basic research, compound screens, and toxicity studies.
尽管胚胎干细胞在基础研究中得到了广泛应用，但它们刚刚开始被用于药物发现过程。在药物发现中采用胚胎干细胞的障碍包括细胞培养困难以及向所需细胞表型的分化不一致。胚胎干细胞培养需要持续且频繁的操作，以将细胞维持在多能状态。此外，胚状体（EB）分化的首选悬滴法不适用于高通量方法，并且EB的悬浮培养显示出高度的变异性。在自动化平台上传代的小鼠胚胎干细胞保持了≥90%的活力和多能性。我们还开发了一种使用384孔微孔板形成EB的方法，每个孔形成单个EB，EB之间具有出色的均匀性。这种形式便于高通量分化，并能够进行筛选以优化定向分化为所需的细胞类型。使用这种方法，我们确定了将心肌细胞分化提高七倍的条件。这种优化的分化方法对于如此复杂的生物学过程显示出出色的一致性。这种胚胎干细胞处理和分化的自动化方法可以为基础研究、化合物筛选和毒性研究提供所需的高产量且一致的分化细胞类型。
