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Updated: May 22, 2026
A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons
Published on: October 6, 2019
Protocol for vital dye staining of corneal endothelial cells
Sunju Park1, Alan G Fong, Hyung Cho
1Department of Ophthalmology and Visual Sciences, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA.
Purpose:
To describe a step-by-step methodology to establish a reproducible staining protocol for the evaluation of human corneal endothelial cells.
Methods:
Four procedures were performed to determine the best protocol. (1) To determine the optimal trypan blue staining method, goat corneas were stained with 4 dilutions of trypan blue (0.4%, 0.2%, 0.1%, and 0.05%) and 1% alizarin red. (2) To determine the optimal alizarin red staining method, goat corneas were stained with 2 dilutions of alizarin red (1% and 0.5%) and 0.2% trypan blue. (3) To ensure that trypan blue truly stains damaged cells, goat corneas were exposed to either 3% hydrogen peroxide or to balanced salt solution, and then stained with 0.2% trypan blue and 0.5% alizarin red. (4) Finally, fresh human corneal buttons were examined; 1 group was stained with 0.2% trypan blue and another group with 0.4% trypan blue.
Results:
For the 4 procedures performed, the results are as follows: (1) trypan blue staining was not observed in any of the normal corneal samples; (2) 0.5% alizarin red demonstrated sharper cell borders than 1% alizarin red; (3) positive trypan blue staining was observed in the hydrogen peroxide exposed tissue in damaged areas; (4) 0.4% trypan blue showed more distinct positive staining than 0.2% trypan blue.
Conclusions:
We were able to determine the optimal vital dye staining conditions for human corneal endothelial cells using 0.4% trypan blue and 0.5% alizarin red.
