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Nataliia Paone, Maria E Sousa, Sangita P Patel; Effects of oxygen concentration on viability of human corneal endothelial cells (HCEnCs). Invest. Ophthalmol. Vis. Sci. 2020;61(7):1454.
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As treatment of corneal endothelial diseases advances towards HCEnC injection therapies, optimizing in vitro culture conditions can maximize cell viability and minimize cell stress. The purpose of our study was to investigate the role of O2 concentration in primary HCEnC culture. In the human eye anterior chamber, [O2] is 2-3% while HCEnC are typically cultured at ambient O2 ([O2]A; O2 in air is 21%). Increased [O2] can contribute to cellular oxidative stress. We tested the hypothesis that HCEnC cultured at physiologic O2 ([O2]2.5%) would have greater cell viability (CV) compared to HCEnC cultured at [O2]A.
Protocols were approved by the University at Buffalo and VA IRBs. Corneas were used within 18 hours of death for HCEnC cultures (10 corneas from 8 donors; mean 79 years; range 59-92 years; 3 female). Dissociated cells from each cornea were distributed in 2 culture plates. One plate was placed in a standard 5% CO2/air tissue culture incubator ([O2]A). The other was placed in an enviromental chamber (Billups Rothenberg, San Diego, CA) at 2.5% O2, 5% CO2, 92.5% N2, 37°C. Cells were expanded for 1 week in growth medium and then matured for 1-2 weeks in minimal medium. The mature HCEnCs were subject to reversing culture conditions ([O2]A↔[O2]2.5%) to test their ability to adapt to altered [O2]. CV was measured with the RealTime-Glo Cell Viability Assay (Promega, Madison, WI). Metabolically active cells reduce the proprietary substrate to generate a quantifiable luminescent signal. The assay was performed at multiple time points (Fig. A). Raw data were normalized with log2 transformation and means were compared using paired, two-tailed Student’s t-test with significance as p < 0.05.
CV was significantly increased in HCEnC cultured under [O2]2.5% compared to [O2]A (Table 1, G1). Regardless of initial culture condition, when [O2] was reversed, cells at [O2]2.5% showed significantly higher CV than those at [O2]A (Fig. A and Table 1, M1-M3). No significant differences were found between males and females.
Our results support our hypothesis that HCEnCs cultured at [O2]2.5% show greater viability compared to those grown at [O2]A and adapt rapidly to changes in [O2]. Further studies of HCEnCs cultured at physiologic [O2] may impact our techniques for HCEnC expansion and understanding of oxidative stress in corneal endothelial disease such as Fuchs endothelial corneal dystrophy.
This is a 2020 ARVO Annual Meeting abstract.
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