Cellular redox state predicts in vitro corneal endothelial cell proliferation capacity.
Cellular redox state using the non-invasive mitochondrial autofluorescence technique of redox fluorometry was evaluated as a predictor for corneal endothelial proliferative capacity in vitro. Human corneal endothelial cells (HCEC) harvested from eye bank corneas were cultured in plates with two different coating substrates; type I collagen and poly-D-lysine. Cellular autofluorescence was measured with both DAPI (excitation: G365, emission: bandpass 445/50) and FITC (excitation: bandpass 450-490, emission bandpass 515-565) filter sets on days 3, 5, 7, and 14. The redox fluorometric ratio was calculated as net "DAPI" signal intensity divided by net "FITC" signal intensity. Normalized redox ratio was calculated as redox ratio divided by individual cell size. Cellular proliferation was analyzed by live cell count on days 2, 7, and 14. Mitochondrial staining was performed on days 4 and 14. The poly-d-lysine substrate decreased the proliferation capacity of HCEC in comparison to type I collagen out to 2 weeks (p=0.045). The cellular redox fluorometric ratio decreased significantly as the cells proliferated (p<0.001). The cells cultured on type I collagen coated plates exhibited significantly lower redox fluorometric ratios than cells cultured on poly-D-lysine coated plates at day 7 (p=0.015). Normalized redox ratio showed significantly lower value in type I collagen coated plates at days 7 (p=0.015) and 14 (p=0.039). Correlated cell proliferation capacity was significantly higher on type I collagen coating at days 7 and 14 (p=0.045 and p=0.049 respectively). HCECs showed different growth potential in vitro on different culture surface coating agents. This difference was well correlated with cellular redox ratios determined using redox fluorometry. Cellular redox ratio can be a potential predictor of cellular proliferation capacity.
Park
Choul Yong
CY
Department of Ophthalmology, Wilmer Ophthalmological Institute, Johns Hopkins University, 255 Woods Building, 600 North Wolfe Street, Baltimore, MD 21287, USA.
Zhu
Zejin
Z
Zhang
Cheng
C
Moon
Christina S
CS
Chuck
Roy S
RS
D018929:Cell Culture Techniques / Q000379:methods; D049109:Cell Proliferation; D048429:Cell Size; D024042:Collagen Type I; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005125:Eye Banks; D005470:Fluorometry; D006801:Humans; D008239:Lysine; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D010084:Oxidation-Reduction
D024042:Collagen Type I; D008239:Lysine