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Betsabe Hernández-Sedas, Maria Dolores Montalvo Parra, Cesar E. Calzada-Rodriguez, Judith Zavala, Jorge E Valdez; 3D epiflourescence analysis of a bioengineered CEC scaffold. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1202.
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© ARVO (1962-2015); The Authors (2016-present)
The corneal endothelium (CE) engineering approaches usually reports cell morphology and biomarkers expression, however, in order to determine the similarity with the healthy human CE, further in vitro analysis are needed. The purpose of this project is to identify the monolayer arrangement through a 3D analysis of a bioengineered corneal endothelium.
Corneal endothelial cells (CEC) proliferated with a two-phase system over a collagen vitrigel (CV) membrane were incubated for 30 min with 10mM Calcein-AM and washed with PBS. Construct was mounted on a glass coverslip and observed with a white field epifluorescence microscope using an Apotome background removing system. Apical and basal cell morphology were assessed using contrast microscopy: scaffold without cells and construct in the cellular side were compared.
Cells over the construct showed hexagonal morphology in the apical side and a finger-like processes expanding toward the CV membrane in the basal side, resembling healthy human corneal endothelial monolayer.
CECs harvested with a two-phase culture approach over a collagen vitrigel membrane are able to form a monolayer with healthy CE arrangement. This results taken together with the previous reported gene and protein expression, demonstrate the potential of this tissue engineered CE replacement.
This is a 2020 ARVO Annual Meeting abstract.
a) Cells over CV membrane showing polygonal morphology in light microscopy. b) 3D structure showing finger-likeprocesses in the basal side (arrow). (10x)
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