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Jennifer Ramos, Ana Fernandez, Laura Garcia-Posadas, Antonio Lopez-Garcia, Yolanda Diebold, Marta Lopez, Pilar Puente, Juan Jose Vazquez, Javier Iglesias; Porcine Conjunctival Epithelial Cells: Isolation and Characterization. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3982.
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To isolate, culture and characterize porcine conjunctival epithelial cells obtained from four different anatomical areas (bulbar, fornix, palpebral, and mucocutaneous junction) by combined enzymatic digestion.
Porcine eyes (n=15) were obtained from a local slaughterhouse. Conjunctival porcine biopsies were digested in two-step enzymatic solutions (collagenase type I solution for 15-19 hours and trypsin/EDTA solution for 2 hours). Obtained cells were expanded in vitro under epithelial growth conditions trying to minimize stromal contamination. Cell proliferation rate was estimated through a growth curve. Immunocytochemistry against cytokeratins (CK) 7 and 19, vimentin, CD90, Fibroblast Surface Protein 1 (FSP-1), MUC5AC and HPA lectin was done in order to characterize cell phenotype. To determine whether basic and/or acidic mucopolysaccharides were secreted by cultured cells, alcian blue-periodic acid Schiff (PAS) staining was used. Cells from passage 2 were used for proliferation assays and cells from passages 1, 5 and 12-15 were used for characterization studies. Student’s t-test was used for analyses.
Cells isolated from conjunctival enzymatic digestions were predominantly epithelial-like cells. The highest cell numbers were obtained from the fornix (p<0.05). Mucocutaneous junction-cells proliferation rate was significantly lower than those of the other anatomical areas (p<0.05). We found CK7 and CK19 (epithelial markers) positive cells, and positivity for HPA lectin antigens, confirming the presence of mucin secretory granules. Additionally, alcian blue-PAS reagent revealed the presence of acidic mucopolysaccharides, but not the basic ones.
Depending on the area from which the cells were isolated, the cell numbers obtained and their proliferation were different, although Goblet cell-like morphology was observed in each sample. We have obtained epithelial cells from porcine conjunctiva with secretory properties and have subculture them up to 15 times. Therefore, this makes possible to develop an in vitro model for further conjunctival studies.
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