A standardized approach to Isolate, Culture and Compare Human Conjunctival Epithelial cells from two different sources

Abstract

Dry eye is a disease condition of the ocular surface that is caused by either deficiency of tear production or increased evaporation of tears. The current standard of treatment is purely symptomatic in the form of artificial tears and does not restore the tear function or the eroded conjunctival and corneal epithelium. Establishing an in-vitro culture model of conjunctival epithelial cells is an important tool for testing drug and other cell-based therapies. The current study was to design a culture model to harvest conjunctival cells from a trimmed graft of pterygium surgery or cadaver human conjunctiva. The conjunctival tissue was minced and underwent Dispase II treatment to isolate cells. Different growth media and culture plate coating and supplements were used to test the best cell culture method. The cells were analysed using Immunofluorescence imaging, flow cytometry and Gel Electrophoresis for characterization. The immunofluorescence staining showed CK-19 positive conjunctival epithelial cells. Flow cytometry showed 96% e-cadherin positive cells. Gel Electrophoresis with 1% agarose showed positive for CK-19, CK-15, CK-7 and negative for MUC5AC and MUC4. In conclusion, the conjunctival epithelial cells have been successfully isolated and the cell culture model has been standardized using appropriate media.