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Catherine Jackson, Peder Aabel, Jon Roger Eidet, Edward B Messelt, Torstein Lyberg, Tor Paaske Utheim; The effect of temperature on the viability, morphology and phenotype of epidermal cell sheets after 14 days of storage.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5179. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate the effects of temperature on the viability, morphology and phenotype of cultured human epidermal keratinocytes following two weeks of xenobiotic free storage. Epidermal cell sheets may be used in the treatment of limbal stem cell deficiency (LSCD) as demonstrated in animal models. With adequate preservation of key characteristics of cultured epidermal cells, storage of such sheets in sealed containers may pave the way for a novel option for treating LSCD with considerable ease in logistics, transportation and quality control using autologous cells.
Human epidermal keratinocytes were cultured until confluence and subsequently stored at nine different temperatures (4°C, 8°C, 12°C, 16°C, 20°C, 24°C, 28°C, 32°C and 37°C) for 14 days in a buffered MEM/Hepes medium. Cells that were not stored served as a control. Cell viability and dead cells were measured using calcein-AM (live)/ethidium homodimer-1 (dead). Cell metabolism indicators were measured by a blood gas analyzer. Morphology was investigated by light microscopy and scanning electron microscopy. Phenotype analysis was performed using a number of immunocytochemistry markers associated with proliferation, progenitor or differentiated cell character: ABCG2, PCNA, P63, C/ebpδ, Bmi-1, deltaNp63α, Occludin, and CK14. In addition, flow cytometry analysis of JC1 was carried out.
Viability was reduced after storage at all temperatures, except 24°C, where viability compared to control was 95.2 ± 10.4%. Morphology was also best maintained at 24°C. Cell-cell contact was discontinuous between 4°C and 16°C, whereas cells lost normal epithelial sheet morphology and formed clumps between 28°C and 37°C. Lactate and glucose levels increased and decreased respectively in association with increased storage temperature. The pO2 in the storage medium decreased steadily from 4°C to 37°C. All phenotype markers appeared to demonstrate variation in expression pattern within the range of 4-37°C. The expression of PCNA was highest at 20°C and 24°C and dropped at both lower and higher temperatures. ABCG2 expression was equivalent to control at 24°C.
The optimum temperature when comparing overall viability, morphology and phenotype for xenobiotic free storage of cultured epidermal cell sheets for 14 days is 24°C.
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