Only a few individual cells within less than 5% of all primary tumors form the cell lines commonly used in cancer research. These growth bottlenecks result in cell lines that are often poor models of primary tumors. Co-culture of primary tumor-derived cells with an irradiated mouse fibroblast feeder layer and ROCK inhibitor, known as the Georgetown Method, offers a way to culture over 80% of tumor-derived cells in vitro to create more representative tumor cell models. In our studies, we optimized the Georgetown Method to culture head and neck cancer cells, including oropharyngeal squamous cell carcinoma, and investigated its mechanism of conditionally immortalizing cells in culture. Differential trypsinization and regular feeder layer replacement were found to significantly improve the efficacy of immortalizing co-cultured cells at both atmospheric and physiological oxygen levels. Medium conditioned by irradiated fibroblasts can also substitute for direct co-culture with a feeder layer. The Georgetown Method was found to maintain low levels of p16 in co-cultured cells, suggesting a potential mechanism by which the Georgetown Method prevents differentiation and senescence. Our ability to culture over 80% of primary tumor-derived cells allows us to test the translational value of tumor-derived cell cultures and xenografts using BH3 profiling. Conditioned medium simplifies maintenance of cell cultures and will also allow us to perform high-throughput screens without the need to separate tumor-derived cells from the fibroblast feeder layer. The Georgetown Method provides opportunities to expand small tissue specimens for future diagnostics, therapeutics, and biobanking.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/14332 |
| Date | 22 January 2016 |
| Creators | Wu, Eric Longhua |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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