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Monika Valtink, Patricia Donath, Lilla Knels, Richard Funk, Katrin Engelmann; Effects of different culture media and deswelling agents on human corneal endothelial and epithelial cell survival. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2026.
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The choice of culture medium influences the quality and viability of donor corneal tissue. We developed a screening system to monitor the effects of different media and media supplements on the viability of human corneal endothelial and epithelial cells.
The human corneal endothelial cell line HCEC-12 (Bednarz et al., Acta Ophthalmol Scand 2000) and the human corneal epithel cell line HCEp (Araki-Sasaki et al., IOVS 1995) were cultured in four different corneal organ cultivation media systems (serum-supplemented: MEM + 2% FCS, CorneaMax®/ CorneaJet®, serum-free: Human Endothelial-SFM, Stemalpha 2 and 3) with and without two different deswelling agents (6% w/v dextran T500, 7% w/v HES 130/0.4). Standard growth media F99HCEC and DMEM/F12 served as controls. After 5 days in the test media, cell viability was assessed by flow cytometric analyses of apoptotic cell death (fragmentation of nuclei by sub-G1 DNA content), by vital staining with YO-PRO®-1 and propidium iodide to quantifiy the number of apoptotic and necrotic cells, and quantification of intracellular reactive oxygen species (ROS). As negative controls, cells were incubated with 0.5 μmol/l staurosporin for 4 hours or 200 μmol/l hydrogen peroxide for 3.5 hours prior to analyses.
It was observed that all MEM-based media were not suitable to support HCEC and HCEp survival under stress conditions, resulting in significantly increased numbers of apoptotic and necrotic cells after staurosporin or hydrogen peroxide application. HCEC survival was markedly improved when cultured in SFM-based media even in the presence of staurosporine or hydrogen peroxide. Likewise, HCEp survival was supported in SFM w/o deswelling agent or SFM + dextran, but not in SFM + HES. The media CorneaJet®, CorneaMax® and CorneaMax® + HES supported HCEC survival better than MEM-based media, but not as good as SFM-based media. HCEp viability was supported by CorneaJet® medium, but not by CorneaMax® with or without HES. Stemalpha-based media were not suitable to support viability of the used cell lines in the applied cell culture system.
The screening system yields reproducible data and can be used to evaluate the suitability of culture media and supplements to support corneal cell viability, and can easily be implemented into routine lab procedures. The choice of medium for corneal organ cultivation should be reconsidered.
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