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D.–Q. Li, Z. Chen, X. Wang, S.C. Pflugfelder; Identification of Human Fibroblast Cell Lines as A Feeder Layer Alternative to Mouse 3T3 Fibroblasts in Supporting Human Corneal Epithelial Growth . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2712.
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© ARVO (1962-2015); The Authors (2016-present)
Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for 3 decades. To avoid the use of xeno–components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial growth.
Three primary human fibroblasts (corneal, limbal and conjunctival) were established by explant cultures. Six human fibroblast cell lines, (MRC–5 fetus lung fibroblasts, Detroit 551 fetus skin fibroblasts and 4 newborn foreskin fibroblasts [NSF]) and mouse 3T3 fibroblasts were from ATTC. All these 10 fibroblasts were grown to confluence, treated with mitomycin C, and then seeded at 2x 104/cm2 as feeder layers. Single human limbal epithelial cells isolated from fresh limbal tissue were seeded on these feeders in SHEM media. Cell growth capacity was evaluated on days 5–14. The phenotype of these primary cultures was evaluated by morphology and immunostaining with epithelial markers.
Among 9 strains of human fibroblasts evaluated, three NSF cell lines were identified to support human corneal epithelial growth. These fibroblasts in their 10 passages continually showed a comparative efficiency to the 3T3 feeder layer for colony forming efficiency and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1x104 in a 35–mm dish grew to confluence (about 1.87–2.41x106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on 3T3 or NSF feeders. These limbal cells grew on these human feeders from colonies to epithelial sheets, which expressed keratin 3, keratin 14, connexin 43, p63 and integrin ß1, resembling the phenotype of human corneal epithelium.
These findings demonstrate for the first time that the commercially available human fibroblast cell lines support human corneal epithelial growth. These human feeders may have potential in use for tissue bioengineering and corneal reconstruction to replace mouse 3T3 fibroblasts.
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