A Novel NIH/3T3 Duplex Feeder System to Engineer Corneal Epithelial Sheets With Enhanced Cytokeratin 15-Positive Progenitor Populations

Abstract

Purpose: : To describe a newly devised duplex (two-layered) feeder culture system that reproduces the limbal phenotype from dissociated limbal epithelial cells in vitro.

Methods: : Human limbal epithelial cells were isolated from US eyebank corneas, and expanded by the duplex feeder system consisting of 2 separate layers of 3T3 cells, one in contact with epithelial cells in culture inserts, and a second separate layer covering the bottom of the well. Culture conditions with only one of the feeder layers served as control. To evaluate the effect on growth, cells were fixed at week 2 and stained with Rhodamin B. To evaluate the effect on the quality of cell sheets, confluent cells were air-lift cultured for 1 week, and then immunostained for K3, K12, and K15, or enzymatically dissociated to observe secondary colony formation.

Results: : Proliferation of primary epithelial cells was significantly higher when in direct contact with feeder cells. Duplex feeder cultures also yielded epithelial sheets with small, cuboid basal cells and strong expression of K3, K12 and K15. Furthermore, only duplex feeder layers reproduced the basal K15, suprabasal K12 limbal phenotype. Duplex feeder sheets also produced significantly more secondary colonies compared to cells dissociated from control sheets.

Keywords: cornea: epithelium • proliferation • cell-cell communication