Abstract
Purpose :
The short supply of donor corneas is exacerbated by the unsuitability of donors with insufficient endothelial cell density (ECD). Few studies have investigated promoting corneal endothelial cell (CEC) proliferation to increase the ECD. We hypothesize that pre-transplantation treatment of proliferative tissue-cultivated corneas may increase corneal ECD.
Methods :
Human or rabbit corneas were excised and cultured for 7 days in standard corneal organ culture medium DMEM+10% FBS. Corneas were treated by PBS or lysophosphatidic acid (LPA) from Day 2 on. Cell density and central corneal thickness were measured. Samples were then fixed, paraffin-embedded and examined morphologically by HE-staining and by imunofluorescent staining of YAP-1, ZO-1, Na-K-ATPase, SMA and BrdU labeling. Tissue lysates, cell extracts, and condition medium were subjected to Western blot, cDNA microarray, and ELISA respectively Functional assay was assessed by creating a rabbit model of damaged corneal endothelium, followed by transplanting the cultured corneal buttons. Data were compared using the Student's unpaired t-test.
Results :
In this tissue culture system, we observed that the airlift cultures were superior to immersion cultures with respect to both transparency and thickness, and that LPA increased the rabbit corneal ECD, number of BrdU-positive cells, and improve wound healing. We also observed an indirect effect of LPA on CEC proliferation mediated by the stimulation of interleukin-1β (IL-1β) secretion from stromal cells. Human corneal tissues treated with LPA or IL-1β contained significantly more Ki-67-positive cells than the untreated group. The LPA- or IL-1β-treated cultured tissue remained hexagon-shaped, with ZO-1 expression and no evidence of endothelial-mesenchymal transition.
Conclusions :
Our novel protocol of tissue culture may be applicable for eye banks to optimize corneal grafting.
This is a 2020 ARVO Annual Meeting abstract.