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Tor P. Utheim, Øygunn A. Utheim, Jon Roger Eidet, Maria de La Paz, Edward Messelt, Borghild Roald, Darlene A. Dartt, Torstein Lyberg; Transportation Simulations Of Cultured Human Limbal Epithelial Cells Subjected To Eye-bank Storage. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1839. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The aim of the present study is to investigate the effects of transportation simulations on the morphology, phenotype and viability of cultured human limbal epithelial cells (HLEC) subjected to four days of storage at 23°C. Reliable methods for cell transportation are becoming increasingly more important with the centralization of production units, due in part to strict regulatory demands imposed on tissue laboratories.
HLEC cultured for 3 weeks were transferred to closed glass containers. The cultures were divided into five experimental groups, and all were subjected to four days of storage at 23°C in HEPES buffered Minimal Essential Medium (MEM) supplemented with antibiotics. Storage bottles containing cultured HLEC were completely filled with medium for groups 1-3 and 5, but for group 4 they were only ¾ full. Pluronic F-68, a water-soluble triblock copolymer with shear protecting properties, was added to the medium in group 5. Transportation simulations were initiated after 3 hours of storage. In group 1 (control group) HLEC cultures were stored but were not subjected to transportation simulations. In group 2 cultures were stored and agitated for 6 h at 200 rotations per minute (rpm) using an orbital shaker. HLEC cultures in groups 3, 4 and 5 were stored and subjected to orbital shaking at 200 rpm for 36 h. Cell morphology and phenotype were analysed by light microscopy and immunohistochemistry, respectively. A calcein-acetoxymethyl ester/ethidium homodimer-1 assay was used to assess cell viability.
HLEC morphology appeared unchanged in all but group 4, where the number of desmosomes and hemidesmosomes was significantly lower than the other groups. The number of cell layers (2.8 - 3.5), cell viability (96.4% - 97.5%), and cell phenotype (C/EBPβ, ABCG2, deltaNp63α, p63, Bmi-1, Caspase-3, Ck3, Ki67 and PCNA expression), did not demonstrate any significant differences between the five experimental groups.
HLEC subjected to four days of storage at 23°C in HEPES-MEM are able to withstand long-term transportation simulations especially well when cell storage containers are completely filled with medium. We hypothesise that the lack of air above the medium in the container reduces mechanical stress, i.e. shearing forces exerted on cells.
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