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E.A. Sander, J.C. Downs, C.F. Burgoyne, E.A. Nauman; Effects of Limited O2 and Nutrient Availability on Cell Number and Viability in Cultured Scleral Fibroblasts . Invest. Ophthalmol. Vis. Sci. 2004;45(13):2183.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In glaucoma, optic nerve head neural and connective tissue damage may be an astrocyte mediated response to decreased nutrient and oxygen levels. To model this form of connective tissue damage, we investigated scleral fibroblast response to nutrient and oxygen limited culture conditions. Methods: Scleral fibroblasts were harvested from New Zealand White rabbit eyes, grown to confluency, and plated into 24–well plates. Treatment groups were exposed to 5 or 21% O2, and 2 or 10% fetal bovine serum (FBS). In Group A, medium was replaced every 2–3 days. In Group B, medium was never replenished. Cell viability was determined with live/dead staining and total cell number (TCN) with LDH assays conducted at weeks 1, 2, 3, and 4. Extracellular matrix quality was assessed with Fourier transform infrared spectroscopy (FTIR) and staining. Results: Group A– For all conditions, except 10% FBS & 5% O2, TCN increased through week 2. Cells cultured with 10% FBS & 5% O2 quickly became oxygen limited and decreased to a stable population. Cells cultured with 2% FBS & 5% O2 increased to 235 million cells per well by week 4 and showed few signs of cell death. Cells grown with 10% FBS & 21% O2 maintained a TCN near 300 million through week 4, and nearly all cells were viable. Group B– Viability decreased substantially by week 4 for cells cultured in 5% O2 regardless of serum levels. Compared to Group A, TCN was lower in groups cultured in 21% O2, but live/dead revealed a largely viable cell population that was visually similar to those in Group A. FTIR analysis of both groups show spectral signatures typical of spectra obtained from rabbit sclera. Conclusions: Cells cultured with 10% FBS proliferate more rapidly than cells cultured with 2% FBS. Proliferation and cell viability is dependent on cell density and oxygen levels. When cell density is low, high oxygen levels can impede proliferation but augment cell viability when glucose levels are low. High cell density with low oxygen levels result in cell death. In vitro models of glaucomatous damage should adjust for cell density, oxygen, and nutrient levels.
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