Hi,
I trying to establish an ovarian cancer cell line resistant to the drug cisplatin. What is the best method to do so?
I think finding out MIC for cisplatin over cell line will be first step, followed by maintaining the cell lines at MIC for 3-6 passages and then sort live cells through FACS, bring them back to cell culture and cross check if they are still resistant to MIC. MIC (Minimum inhibitory concentration)
Screen toxic and non cytotoxic concentrations of cisplatin. Grow your cells for few passages in less cytotoxic concentrations. Then keep adding increasing concentrations of cisplatin for few more passages. I guess after 8-10 passages you may get resistant cells. Finally use cytotoxic doses, the remaining cells should be again grown with same concentrations.
From
PLoS One. 2013; 8(1): e54193. Published online 2013 Jan 17. doi: 10.1371/journal.pone.0054193PMCID: PMC3547914PMID: 23349823
Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature
Martin P. Barr, 1 , * Steven G. Gray, 1 Andreas C. Hoffmann, 2 Ralf A. Hilger, 2 Juergen Thomale, 3 John D. O’Flaherty, 1 Dean A. Fennell, 4 Derek Richard, 5 John J. O’Leary, 6 and Kenneth J. O’Byrne 1 Pan-Chyr Yang, Editor
"Cisplatin-resistant (CisR) variants of each cell line were derived from each original parental (PT) cell line by continuous exposure to cisplatin (Sigma-Aldrich, UK) following initial dose-response studies of cisplatin (0.1 µM–100 µM) over 72 h from which IC50 values were obtained. Initially, each CisR subline was treated with cisplatin (IC50) for 72 h. The media was removed and cells were allowed to recover for a further 72 h. This development period was carried out for approximately 6 months, after which time IC50 concentrations were re-assessed in each resistant cell line. Cells were then maintained continuously in the presence of cisplatin at these new IC50 concentrations for a further 6 months. While A549 cells were initially treated with IC50 concentrations of cisplatin, cells were sensitive to treatment at this concentration resulting in cell senescence and delayed growth. For this reason, the cisplatin concentration was reduced (IC25) until such time as cells demonstrated sensitivity to cisplatin at the appropriate IC50 concentration. "
Good luck!
rolando
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