Purpose: The purpose of this study was to establish a simple, xeno-feeder-free method for cultivating human corneal epithelial cells. Methods: Limbal tissue explants from a cadaver were cultured in Iscove's modified Dulbecco's medium and low-calcium Panserin 801 medium in a 1:1 ratio. The outgrowing cells were characterized by flow cytometry, immunohistochemistry and real-time PCR (rtPCR). Limbal epithelial cells were expanded in a xeno-feeder-free, low-calcium medium and airlifted for 2 weeks each. Results: Migration of fibroblast-like stromal cells initially occurred from the limbal explants, and then epithelial cells migrated and grew on the stromal cells as an autofeeder layer. After airlifting, the cultured epithelium consisted of two to three layers. The cultured cells expressed stem cell-associated markers (ABCG2 and ΔNp63), differentiation markers (CK3 and CK12) and extracellular matrix-associated markers (lumican and decorin). rtPCR showed increased expression of markers for epithelial progenitor cells compared to fresh limbal tissue. Side population cells comprised 0.43 ± 0.04% of the cells (n = 5) in the primary culture. Flow cytometry showed that 49.12, 40.44 and 44.55% of the cells from the explants expressed E-cadherin, ΔNp63 and ABCG2, respectively. Conclusion: This explant culture system using stromal cells as an autofeeder layer was useful in expanding human corneal epithelial cells. This system may offer clinical insight for the expansion of limbal progenitor cells for the reconstruction of the ocular surface.

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