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S. Raeder, T. Paaske Utheim, O. Olstad, M. de la Paz, T. Lyberg; Genome-Wide Transcriptional Analysis of Cultured Human Limbal Epithelial Cells Following Hypothermic Storage in Optisol-GS. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1773.
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© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated the feasibility of eye bank storage of cultured human limbal epithelial cells (HLEC) in serum based medium. However, storage in serum free media would be advantageous to reduce the risk of transmitting prions or animal viruses. In the present study we therefore investigated the transcriptional pattern in HLEC cultures following 2, 4, and 7 days of conventional hypothermic corneal storage in Optisol-GS (Bausch & Lomb, Irvine, CA).
3-week HLEC cultures were transferred to polypropylene containers and stored in Optisol-GS for 2, 4, and 7 days at 4°C. 100 ng RNA was extracted from 5-mm biopsy punch samples using RNeasy Micro Kit (Qiagen, Hilden, Germany) and subjected to the GeneChip HT One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA) and GeneChip HT IVT labeling kit (Affymetrix). Labeled and fragmented single stranded DNAs were hybridized to the GeneChip Human Gene 1.0 ST Array (28869 genes, Affymetrix). Gene expression of stored HLEC was compared with 3-week HLEC cultures using the non-paired Student’s t-test and a nominal significance level of 0.001.
No genes were significantly increased in HLEC cultures following 2-days storage, whereas histone cluster 1 H4d (HIST1H4D), histone cluster 1 H3f (HIST1H3F), histone cluster 1 H4b (HIST1H4B), histone cluster 1 H4k (HIST1H4K), and histone cluster 1 H4j (HIST1H4J), which are involved in internucleosomal linker DNA organization, were significantly increased after four days of storage (> 2 fold). Following seven days of hypothemic storage, HIST1H4D, HIST1H4B, RNA, HIST1H4K, histone cluster 1 H4c (HIST1H4C), histone cluster 1 H4j (HIST1H4J), histone cluster 1 H2bb (HIST1H2BB), RNA U5E small nuclear (RNU5E), and small nucleolar RNA C/D box 3B-1 (SNORD3B-1) were significantly increased in HLEC cultures (> 2 fold).
Our data indicate that gene expression of linker histones, previously reported to be associated with apoptotic DNA fragmentation, is upregulated during hypothermic storage of HLEC cultures in Optisol-GS.
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