title: A novel NIH/3T3 duplex feeder system to engineer corneal epithelial sheets with enhanced cytokeratin 15-positive progenitor populations.
abstract: Corneal epithelial cell sheets co-cultivated with feeder cells are used to reconstruct the ocular surface in stem cell-depleted eyes. The present study was conducted to investigate the optimal method of using feeder cells in the interest of preserving progenitor cells in cultivated sheets. We compared the phenotype and secondary colony forming efficiency (CFE) of cell sheets that were engineered using 3T3 feeder cells as a separate layer or as a contact layer. We also devised a novel "duplex feeder" system that consists of two separate layers of feeder cells. After cells reached confluence, cells were cultured at the air-liquid interface to allow full stratification. Stratified sheets were then analyzed using immunohistochemistry and secondary colony formation. Contact feeder cultures and duplex feeder cultures yielded epithelial sheets with small, cuboid basal cells with strong expression of keratin (K)3, K12, and K 15. Furthermore, only duplex feeder layers reproduced the basal K 15, suprabasal K12 limbal phenotype where epithelial stem cells reside. A similar effect was observed when cornea stroma-derived progenitor cells were used as feeder cells. Duplex feeder sheets also produced significantly more secondary colonies than cells dissociated from single layer sheets, suggesting that the duplex feeder system produces transplantable sheets with a higher yield of progenitor cells.
authors.affiliation: Department of Ophthalmology, Keio University School of Medicine, Tokyo, Japan.
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Miyashita
authors.forename: Shigeto
authors.initials: S
authors.lastname: Shimmura
authors.forename: Kazunari
authors.initials: K
authors.lastname: Higa
authors.forename: Satoru
authors.initials: S
authors.lastname: Yoshida
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Kawakita
authors.forename: Jun
authors.initials: J
authors.lastname: Shimazaki
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tsubota
mesh_terms: D000818:Animals; D018920:Coculture Techniques; D003114:Colony-Forming Units Assay; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D007633:Keratins / Q000096:biosynthesis; D051379:Mice; D041681:NIH 3T3 Cells / Q000166:cytology* / Q000378:metabolism; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism
chemical_list: D007633:Keratins