Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Canada.
Stem Cells Dev. 2011 Apr;20(4):583-91. doi: 10.1089/scd.2010.0400. Epub 2010 Dec 13.
Conventionally, researchers remove spontaneously differentiated areas in human pluripotent stem cell (hPSC) colonies by using a finely drawn glass pipette or a commercially available syringe needle. However, when extreme differentiation occurs, it is inefficient to purify the remaining undifferentiated cells, as these undifferentiated areas are too small to be isolated completely with the mechanical method. Antibodies can be utilized to purify the rare undifferentiated cells; however, this type of purification cannot be used in xeno-free culture systems. To avoid the loss of valuable hPSCs, we developed a novel method to isolate undifferentiated hPSCs from extremely differentiated colonies that could be easily adapted to xeno-free culture conditions. This protocol involves dissecting away differentiated areas, dissociating the remaining colony into clumps, seeding small clumps into new dishes, and picking undifferentiated colonies for expansion. Using this method, we routinely achieve completely undifferentiated colonies in one passage without the use of antibody-based purification.
传统上，研究人员通过使用精细绘制的玻璃移液管或市售的注射器针去除人多能干细胞 (hPSC) 集落中的自发分化区域。然而，当发生极端分化时，纯化剩余的未分化细胞效率低下，因为这些未分化区域太小，无法通过机械方法完全分离。抗体可用于纯化罕见的未分化细胞；然而，这种类型的纯化不能用于无动物源培养系统。为了避免宝贵的 hPSC 损失，我们开发了一种从极度分化的集落中分离未分化 hPSC 的新方法，该方法易于适应无动物源培养条件。该方案包括解剖分离分化区域，将剩余的集落解离成团块，将小团块播种到新的培养皿中，并挑选未分化的集落进行扩增。使用这种方法，我们通常可以在不使用基于抗体的纯化的情况下，在一个传代中获得完全未分化的集落。
