2026-04-14T17:45:31Zhttps://uvadoc.uva.es/oai/request

oai:uvadoc.uva.es:10324/509492021-12-15T21:46:03Zcom_10324_1138com_10324_931com_10324_894col_10324_1226

García Posadas, Laura Maldonado López, Miguel José Diebold Luque, María Yolanda 2021-12-15T09:28:58Z 2021-12-15T09:28:58Z 2022 Experimental Eye Research, 2022, vol. 214, 108878 0014-4835 https://uvadoc.uva.es/handle/10324/50949 10.1016/j.exer.2021.108878 Human corneal epithelial cells are needed to study corneal pathophysiology in vitro. Due to the limitations of cell lines, the use of primary cells is highly desirable, but the scarcity of human tissues, along with ethical issues, make it difficult to accomplish all required experiments. In advanced surface ablation (ASA), the central corneal epithelium is removed and discarded. We hypothesized that ASA samples could be used to perform in vitro assays. In this study, 29 samples from patients undergoing ASA were recovered in supplemented DMEM/F12 culture medium, RIPA buffer, or RLT lysis buffer. The first aim was to determine whether cells could be maintained in culture. Although with the explant technique, tissue pieces did not attach to the culture surface, after disaggregation, cells showed high viability (90.0 ± 6.0%), attached to plates, and remained viable for up to 14 days. The second aim was to elucidate if ASA samples could be used to study protein or gene expression. Cytokeratin-3, ZO-1, Ki67, and E-cadherin protein expression were confirmed by immunofluorescence. Total protein (485.8 ± 115.8 μg) was isolated from cells in RIPA buffer, and GAPDH was detected by Western blotting, indicating that samples are adequate for protein studies. RNA (9.0 ± 3.6 μg) was isolated from samples in RLT lysis buffer, and GAPDH gene expression was studied by PCR, confirming that samples were also suitable for gene expression studies. These results suggest that samples obtained from corneal surface ablation procedures may constitute a valuable source of human cells to accomplish in vitro studies. eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/4.0/ © 2021 The Authors Attribution-NonCommercial-NoDerivatives 4.0 Internacional Epithelial cells removed in advanced surface ablation (ASA) surgery can be used as a source of corneal samples to perform in vitro studies info:eu-repo/semantics/article