This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Develop a protocol for the proliferation of rhesus ES cells that will improve viability and promote uniformity, consistency and ease of use. The current sub-optimal conditions for rhesus ES cell culture place significant limitations on their use in basic research. Rhesus ES cells are notoriously difficult to grow and maintain in culture. They are considerably more fragile than human ES cells, slower growing and more susceptible to spontaneous differentiation in culture. If they are to be an effective model system for human ES cell culture work, the culture system must be significantly improved. Additionally, the labor required to continuously prepare MEF feeder layers is a major limitation in large- scale production of ES cells. Therefore, elimination of fibroblast feeder cells would greatly improve the efficiency and consistency of ES cell culture. Over the course of this grant period, we have significantly improved rhesus ES cell culture medium by appropriately adjusting physiochemical parameters including pH, osmolarity and gas atmosphere concentrations. Additional we have demonstrated a significant benefit to culture through the addition of specific media additives including bFGF, heparin, and LiCl among others. Integration of all these advances resulted in our most significant breakthrough. By adjusting osmolarity and pH to their most optimal levels, and supplementing the culture medium with specific growth factors, we are now able to reliably culture rhesus ES cells in the absence of serum using conditioned medium. Specifically, addition of high doses of basic FGF to culture medium has, for the first time, allowed the clonal growth of rhesus ES cells in a serum free environment without direct feeder layer contact. Use of conditioned medium is still required at this point. However, current research is focused on the refinement of a feeder-independent, defined medium recently developed for human ES cell culture, and application of that system to obtain feeder-independent rhesus ES cell culture.
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