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Maria Dolores Montalvo, Judith Zavala; Evaluation of cultured corneal endothelial cells morphology with an easy and free tool. Invest. Ophthalmol. Vis. Sci. 2018;59(9):1376.
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© ARVO (1962-2015); The Authors (2016-present)
Hexagonal shape of corneal endothelial cells (CECs) is a parameter of health and function. Measuring CECs shape in patients is achieved with specular biomicroscopes but these are not applicable for cell cultures. Since polygonal shapes resemble to circular ones as the number of equal sides increases, we propose to obtain circularity (C), roundness (R) and aspect ratio (AR) indexes with a free image processing software to measure hexagonal shape conservation of CECs cultured with a two-phase system.
Rabbit CECs were cultured with a two-phase approach: whole corneal endothelium was cultured in mitotic media until confluence, passaged and cultured in resting media until confluence. CECs were photo documented in each phase: 3 photographs of rabbit CECs acquired after cornea obtention (uncultured), 5 of mitotic media phase, 5 of resting phase. NIH Image J software was used to analyze the morphology. Scale was set on each photograph and, ~25 cells were delimited with free shape ROI. C, AR and R were obtained using Image J, Analysis menu, Measure tool. Also, 3 specular biomicroscopy images of normal human CECs were analyzed. Total cells for each group were: 21 for uncultured rabbit, 68 for human, 128 for mitotic media and, 120 for resting media. Statistical analysis was performed with one-way ANOVA, all pairwise multiple comparison procedure by Holm-Sidak method (P= 0.001). C (4π area/ perimeter2) and R (4 area/π major axis2) 1 = perfect circle; 0 = elongated shape. AR (major axis/minor axis) = 1, axes are equal, there is radial symmetry as in perfect hexagons.
Human and uncultured rabbit CECs showed to be alike in terms of R (human 0.83, SD= 0.09; uncultured rabbit 0.78, SD=0.11; P=0.26) and AR (human 1.22, SD=1.14; uncultured rabbit 1.30, SD=0.18; P=0.75); C values resulted different (Human 0.76, SD=0.05; uncultured rabbit 0.77, SD=0.6). As expected, all other comparisons made among groups showed cells are statistically different in terms of C, R and AR.
Our preliminary data indicates Image J Measure tool provides three useful shape descriptos to evaluate changes occurred during the two-phase transition of CECs cultured with this system. R and AR show human and uncultured rabbit CECs can be used as shape control. Further analysis using different culture approaches will set the best parameters to be measured in order to obtain the greatest reproducibility.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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