Objective: To investigate the effects of repeating-70℃freeze thawing on human choroidal melanoma cell line OCM-1. Methods: OCM-1 cells were frozen by repeating -70℃ freeze thawing with various durations and frequencies. Then the inhibit rate of cells was examined by MTT essay. The cell viability was measured by monoclonal formation assay. We also used the HE staining, immunohistochemistry staining and the laser-scanning confocal microscopy (LSCM) to investigate the morphological changes of the cells. Results: The growth of OCM-1 cells was inhibited by repeating -70℃ freeze thawing in time-dependent and frequency-dependent manners (P < 0.01). Different morphous including necrosis and apoptosis of the cells could be observed after -70℃ freeze thawing by the LSCM. Conclusion: Repeating -70℃ freeze thawing can not only kill cells directly and induce considerable cells to apoptosis, but also inhibit the growth of the survivals. The kill and wound ratio of the cells disposal with different times and frequencies present variance. And the distinction when treated with different frequencies during the same time is much more significant than different times with the same frequency, which guide clinical workers to choose repeating cryotherapy with short term method instead of single cryotherapy with long term in choroidal melanoma treatment.
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