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T. P. Utheim, S. Raeder, J. Eidet, C. Stormo, M. de la Paz, O. A. Utheim, B. Roald, T. Lyberg; Storage of Cultured Human Limbal Epithelial Cells in Optisol-GS at 23°C versus 5°C. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1778.
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Successful storage of cultured human limbal epithelial cells (HLEC) in a serum-based medium for one week at ambient temperature (23°C) has been reported. However, the feasibility of storage in serum-free media has never previously been demonstrated. In the present study, we compared the effect of Optisol-GS storage at 23°C versus conventional hypothermic storage (5°C) on cultured HLEC.
Three-week HLEC cultures were transferred to polypropylene containers and stored in Optisol-GS for 4 days at 23°C and 5°C. Morphology and phenotype were analysed by light microscopy and immunohistochemistry, respectively. A calcein-acetoxymethyl ester/ethidium homodimer 1 assay was used to assess viability, and the Mann-Whitney U test was applied for statistical evaluation.
Storage of cultured HLEC for 4 days at 5°C led to extensive detachment of basal cells from the amniotic membranes in sharp contrast to storage for 4 days at 23°C, where the cells attached well to the amniotic membranes in all the cultures. The phenotype, assessed by p63, ΔNp63, ABCG2, K19, K3, Cx43, Ki67, and PCNA, was maintained after storage at 23°C. Basal layer viability of cultured HLEC was 93.1% ± 6.1% and 55,0% ± 27.2% after 4 days of storage at 23°C and 5°C, respectively, compared with 98.9% ± 0.7% prior to storage (P=0.12 and P<0.001, respectively).
The present study demonstrates the feasibility of serum-free storage of cultured HLEC. The temperature during storage in Optisol-GS strongly affected the morphology and viability of cultured HLEC, favoring storage at 23°C. Conventional hypothermic use of Optisol-GS for storage of cultured HLEC is clearly unsatisfactory.
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