Abstract
Purpose :
During refractive surgery using the advanced surface ablation technique (ASA), an epithelial cell sheet is removed from the central cornea and discarded. We hypothesized that these cells could be useful to develop in vitro models. The purpose of this study is to characterize this corneal epithelial sheet and to optimize a protocol to culture these cells.
Methods :
This study was approved by the ethics committee of the University of Valladolid. Corneal epithelial cells were obtained from 26 patients who underwent ASA. Central cornea was exposed to 20% ethanol for 30 s to debride the epithelium. Initial cell viability was assessed using LIVE/DEAD Assay. Samples were also embedded in OCT Compound or in paraffin, sectioned, and stained. The expression of corneal (CK3, CK12), cell barrier (E-cadherin and ZO-1) and proliferative (Ki67) cell markers were assessed by immunofluorescence. For cell culture the tissue was cut in half, disaggregated with trypsin/EDTA for 10 and 15 min and cells were seeded with supplemented DMEM/F12 medium. Cell proliferation was measured with AlamarBlue assay in 3 different conditions: 1) fetal bovine serum (FBS) vs human serum (HS); 2) addition of 1µM hydrocortisone; 3) 10 mg/ml type I collagen surface coating (Col1). Data are shown as mean ± standard deviation. A Student’s t-test was used to analyze differences.
Results :
A 60.97±7% of the cells were alive in the epithelial tissue. Despite showing edema in different layers, regions of well-preserved cell-cell junctions, expressing E-cadherin and ZO-1 were observed. All cells expressed CK3 and CK12, and some of them were also positive for Ki67. Trypsinization for 15 min allowed to obtain 233,667±34,249 cells with a viability of 47.7±8,5%, whereas 10 min provided with 139,600±20,243 cells with 20.5±9% viability. Cells showed higher metabolic activity when cultured with FBS than with HS (2.7-fold increase, p=0.001). Cell proliferation was significantly increased with hydrocortisone (1.7-fold increase, p<0.001) and Col1 coating (4.0-fold increase, p<0.001).
Conclusions :
Corneal epithelial tissue obtained from ASA refractive surgery retained viability and key marker expression, being therefore a valuable source of cells. Culture conditions can be optimized with the addition of specific compounds, facilitating the development of in vitro models.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.