A Protocol for Primary Culture of Non-Pigmented Ciliary Epithelium Cells

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Here, we provide a methodology for routine primary culture of porcine nonpigmented ciliary epithelium (NPE), a cell type that plays a critical role in AH formation. Using fresh eyes from adult pigs, the dissection protocol allows us to obtain the entire ring of NPE as a ruffled sheet of cells attached to the vitreous. Dispersal of the cells for initial culture is made possible by treatment with collagenase and hyaluronidase. We describe the seeding density, time to confluence, and trypsinization protocol for NPE cells established in primary culture. Comparison of the freshly isolated and cultured NPE cells includes analysis of several proteins known to be characteristic of the NPE. This includes measuring the expression of xenobiotic transporters (PgP, BCRP, MRP4, MRP 2, MRP1), connexins (Cx-43 and Cx-50), sodium-hydrogen exchangers, and carbonic anhydrases. The diffusion barrier property of cultured NPE monolayers is also examined. Prior to the development of this technique in 2007, experts in the field did not believe it was feasible to culture and propagate NPE. Our success hinged on being able to seed freshly isolated NPE cells at a sufficiently high density. This, in turn, hinged upon being able to isolate a relatively large quantity of NPE cells, first by isolating the entire ciliary ring from each eye, then by dispersion of cells by enzyme treatment of the cell-to-vitreous body attachment.