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G.C. W. Nareyeck, G. Prescher, M. Zeschnigk, S. Mrzyk, D. von der Haar, N. Bornfeld, H. Schilling, G. Anastassiou; Results of Long Term Culture of Uveal Melanomas and Characterization of a Cell Line with Monosomy 3 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1195.
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Purpose:Several established cell lines derived from uveal melanomas exist. However, based on the published data none of the available cell lines has a monosomy of chromosome 3. Since recent data suggest that uveal melanomas with or without monosomy 3 represent two distinct tumor entities with different metastatic potential it would be of great experimental importance to obtain a cell line with monosomy 3. Methods:Tumor material from 23 uveal melanomas was isolated for cultivation immediate after surgery. The tumor was cut into small pieces followed by trypsin treatment for several minutes. Singled tumor cells were plated out on untreated culture flasks or on culture flasks containing irradiated feeder layer cells. Hams F12 medium with 10% FCS was used. Several additives (insulin, EGF, transferrin) were tested. Choleratoxin and G418 were used occasionally. Estimation of growth pattern, cell morphology and immunocytochemical characterization were performed regularly. The chromosome 3 status of the primary tumor was assessed by microsatellite analysis. Conventional cytogenetic analysis and proliferation assays were performed in a single case of a well proliferating cell line. Results:In 14 out of 23 tumors (61%) cultivation failed (1–2 passages in a mean culture time of 290 days). In 8 tumors (35%) a slow and irregular growth pattern was seen (3–5 passages in a mean culture time of 241 days). There was no association between the chromosome 3 status of the primary tumor and the growth potential of the tumors in culture. Only cells derived from tumors with monosomy 3 formed structures similar to "vascular patterns" in culture but this had no impact upon cell growth. The addition of insulin, EGF and transferrin had no positive effect on cell growth. One tumor (4%) showed a continuous proliferation after an initial phase of slow growth (300 days). The doubling time in the last 10 passages was approximately 300 hours. Immunocytochemistry for HMB45 and S100 was positive. Cytogenetic analysis showed a hypodiploid karyotype with monosomy 3 and other chromosomal aberrations. Conclusions:The difficulties in establishment of a cell line were similar in both groups with monosomy 3 or disomy 3 though some morphological details differed during cultivation. The establishment of a uveal melanoma cell line with monosomy 3 opens new perspectives for experiments exploring the mechanisms involved in dissemination of uveal melanoma.
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