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Promote stem cell growth and inhibit differentiation new medium
In order to keep hESCs alive and pluripotent, researchers usually culture them on trophoblast cells (that is, a layer of mouse embryonic fibroblasts), or in clusters on microcarriers. But culturing stem cells on these substrates is quite expensive, and the culture cannot remain undifferentiated for a long time, even though all the necessary growth factors are present.
The research team is studying the differentiation of hESC clusters into neural cells, and they have been using Invitrogen's Neurobasal medium. This medium can maintain the growth and normal phenotype of nerve cells for a long time without a trophoblast. During the study, the researchers noticed that not all cells have differentiated. They suspected that the choice of culture medium caused this phenomenon, so they began to concentrate on studying how the culture medium promotes stem cell growth and inhibits differentiation.
In order to verify their theory, the research team customized the culture medium. They added a serum substitute to Neurobasal medium and added proteins needed for stem cell growth, including extracellular matrix components, neurotrophic factors, fibroblast growth factor 2 and activin A. The researchers cultivated hESCs in conventional serum-free media and customized media. After three weeks, there were more undifferentiated hESC cells in the customized medium.
With this new medium, the research team tested three different hESC lines. They analyzed the culture using fluorescence activated cell sorting (FACS) after 7 and 20 weeks and found that 90% of the cells still maintained pluripotency. This breakthrough created the possibility of large-scale expansion of embryonic stem cells in a computerized automated system. In addition, by changing the culture conditions, it is possible to further instruct stem cells to grow into specific cell types for research or patient transplantation.
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