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M. Takaoka, T. Nakamura, H. Suemori, S. Kinoshita; An Investigation of Efficient Cryopreservation for Human Corneal Epithelial Cells by Vitrification. Invest. Ophthalmol. Vis. Sci. 2007;48(13):459.
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An efficient cryopreservation method for human corneal epithelial cells has yet to be clearly confirmed. We examined a useful cryopreservation procedure for human corneal epithelial cells by vitrification, a method which is reportedly effective for human embryos and embryonic stem cells.
Human immortalized corneal epithelial cells transformed with simian virus (SV) 40 (HCE-T) were cultured and stored by vitrification method in vitrifiable media including (1) 40% dimethylsulfoxide (DMSO), (2) 30% DMSO and 10% propane-1,2-diol (PROH), (3) 20% DMSO and 20%PROH, or (4) 10% DMSO and 20% PROH at -196°C for 3 days. After thawing, the cell recovery rate (recovered cells after thawing per cells before freezing) and cell survival rate (trypan blue dye negative cells per recovered cells) were assessed, and then after culturing for several days, colony forming efficiency (CFE) and Bromodeoxyuridine (BrdU) cell proliferation assay were evaluated.
All the cell recovery and survival rates were over 95% and showed no significant difference between the conditions (1-4). CFEs were approximately up to 50% on average and BrdU uptakes were almost the same as those of cells before freezing under all 4 conditions. CFE and BrdU assay showed no significant differences between the conditions.
Vitrification is an efficient method for HCE-T under the several conditions we examined here. Should a standardized cryopreservation method for human corneal epithelial cells be established on the basis of this study, it would greatly contribute to the development of a corneal epithelial stem cell bank in the future.
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