Propagation of Embryonic Stem Cells in Stirred Suspension Without Serum
Propagation of Embryonic Stem Cells in Stirred Suspension Without Serum
复制标题
DOI：
10.1002/btpr.57
复制
发表时间：
2008-11-01
影响因子：
2.9
通讯作者：
Tzanakakis, Emmanuel S.
中科院分区：
文献类型：
--
作者：
Kehoe, Daniel E.;Lock, Lye T.;Tzanakakis, Emmanuel S.
胚胎干细胞（ESCs）具有无限的自我更新能力和沿多细胞系分化的能力，是生物技术应用和细胞治疗的极好起始材料。然而，要发挥ESCs的潜力，就需要发展可扩展的系统，以便以有管制的方式大量生产它们。在这里，我们描述了一种在搅拌悬浮生物反应器和无血清培养基中将小鼠ESCs （mESCs）扩增为多能聚集体的方法。最初，不依赖饲养细胞的mESCs在培养皿中培养适应无血清条件。此外，我们还探讨了配备三角形叶轮和挡板的旋转烧瓶是否支持mESC聚集体的培养。在这些血管中无血清培养4天后，活mESC浓度增加了近20倍，而细胞活力没有明显下降。即使在连续传代后，mESCs仍保持了多能性标记物Oct3/4、Rex1和SSEA-1的高表达。更重要的是，当诱导分化时，这些细胞采用了所有三种胚层的命运，即神经外胚层和心脏中胚层。和最终的内胚层。这些发现表明，干细胞可以在无血清条件下在可扩展的搅拌悬浮培养中繁殖，而不会丧失其多能性。
Embryonic stem cells (ESCs) with their unlimited capacity for self-renewal and ability to differentiate along multiple cell lineages are a superb starting material for biotechnology applications and cellular therapies. However, realization of the potential of ESCs requires the development of scalable systems for their production in large quantities and in a regulated manner. Here, we describe a methodology for the expansion of mouse ESCs (mESCs) as pluripotent aggregates in a stirred suspension bioreactor and in medium without serum. Initially, the culture of feeder cell-independent mESCs in dishes was adapted to serum-free conditions. Also, we explored whether spinner flasks equipped with a triangle-shaped impeller and baffles support the culture of mESC aggregates. Serum-free culture in these vessels resulted in an almost 20-fold increase in the live mESC concentration over 4 days without significant loss of cell viability. Even after consecutive passages, mESCs retained high expression of pluripotency markers Oct3/4, Rex1 and SSEA-1. More importantly, when differentiation was induced these cells adopted fates of all three germ layers namely neuroectoderm, cardiac mesoderm. and definitive endoderm. These findings demonstrate that stem cells can be propagated under serum-free conditions in a scalable stirred-suspension culture without loss of their pluripotency.