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Jamie Floss, Christian F Mueller, Maria Sousa, Sangita P Patel; Growth characteristics of primary human corneal endothelial cells (HCEnC) at ambient and 2.5% oxygen. Invest. Ophthalmol. Vis. Sci. 2021;62(8):813.
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The corneal endothelium in vivo is exposed to approximately 2.5% O2 ([O2]2.5) while standard culture conditions with ambient air expose HCEnC to ~18% O2 ([O2]A). Oxidative stress is known to contribute to corneal endothelial disease. The purpose of this study was to evaluate the effects of [O2]A and [O2]2.5 on the growth of primary HCEnC cultures. We hypothesized the physiologic condition of [O2]2.5 would be more favorable to growth of HCEnC than [O2]A.
Protocols were approved by the Univ at Buffalo and VA IRBs. Human donor eyes were enucleated within 16 hrs of death and used immediately for cultures. Dissociated HCEnCs from each cornea were distributed into 96-well format culture plates; one plate at [O2]A and another in an environmental chamber at [O2]2.5 (2.5% O2, 5% CO2, balance N2; Billups-Rothenberg, Inc., Del Mar, CA). Following 10-14 days of growth in complete medium, cells were matured for 10-14 days in basal medium. Nuclei were stained with DAPI and digital fluorescence images acquired for cell count analysis (ImageJ, NIH) with conversion to cells/mm2 (endothelial cell density; ECD) based upon growth area. Positive cell growth for each well was considered ECD>50 cells/mm2. We compared mean ECD under each condition with two tailed Student’s t-tests and compared percentage of wells with growth under each analysis condition with two tailed Fisher exact tests with statistical significance at p<0.05.
We analyzed 34 corneas from 20 donors (mean age 72 yrs; range 36-98 yrs; 16 female corneas; 18 pseudophakic corneas). Percentage of wells with growth were greater at [O2]2.5 vs [O2]A overall and notably in corneas from pseudophakic eyes but did not reach statistical significance (Table). Of the wells with growth, there were no significant differences in ECD between [O2]A and [O2]2.5 overall or when compared by age or lens status.
Our results suggest that [O2]2.5 is more favorable for initiating HCEnC growth than [O2]A but does not result in increased ECD. The favorable effects of [O2]2.5 on growth were most apparent with pseudophakic donors which may reflect increased cell stress in these donor corneas, relieved by culture under physiologic O2 conditions. These findings contribute to our understanding of culture conditions to optimize HCEnC expansion for in vitro study or transplantation.
This is a 2021 ARVO Annual Meeting abstract.
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