Cultivation and characterization of corneal limbal epithelial stem cells on lens capsule in animal material‐free medium

Abstract

AbstractPurpose To develop simple, reproducible and animal‐materials free method for cultivating and differentiating human limbal stem cells (LSCs) into corneal epithelium on human lens capsule (LC) for clinical transplantation.Methods Limbal tissues (2x2x0,25 mm) were harvested from cadavers (44 males and 33 females, age 70,5+9,3 years) within 12 hours of biologic death. All tissue collection complied with the guidelines of the Helsinki Declaration and was approved by the Regional Ethical Committee. Cell viability was measured with the MTT and annexin‐FITC/propidium iodide assays. Characterization of the growing cells was performed by RT‐qPCR and immunfluorescence stainings accordingly.Results Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were tested for positivity for markers of stemness (P63, ABCG2, CK19, ITGA9, VIM), proliferation (Ki‐67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non‐hematopoietic, ‐endothelial and ‐mesenchymal stem cell phenotype, while the cell adhesion molecules, integrins and lectin‐based surface carbohydrate profiling showed a specific pattern on the LESCs.Conclusion We report a method for isolating and expanding cornea LESCs from cadavers or alternatively from autologous donors capable of producing viable cell outgrowth on LC for possible treatment of LESC deficiency.