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Abstract
There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5–14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1×104 in a 35-mm dish (9.6 cm2) grew to confluence (about 1.87–2.41×106 cells) in 12–14 days, representing 187–241 fold expansion with over 7–8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.
Highlights
Ocular surface diseases with corneal epithelial stem cell deficiency are sight threatening and often cause blindness [1]
To identify whether human fibroblasts can serve as a feed layer to support ex vivo expansion of human corneal epithelial cells, five commercially available human fibroblast cell lines were tested with mouse 3T3 fibroblasts as controls
Human corneal epithelial cells were isolated from donor corneal limbus, and seeded at 16103 cells/cm2 on these human fibroblast feeder layers with mouse 3T3 cells as controls [22]
Summary
Ocular surface diseases with corneal epithelial stem cell deficiency are sight threatening and often cause blindness [1]. There is great interest in using corneal epithelium generated in vitro to treat these conditions and restore vision [2,3,4,5,6,7] This requires development of culture technique that mimics the in vivo environment. The single-cell clonal growth was first achieved for keratinocytes by co-culturing with mouse 3T3 fibroblasts as a feeder layer [13,14]. This 3T3 fibroblast co-culture system has been proven to be the most powerful system for human epithelial cultivation [7,15,16,17], including human ocular surface epithelia [18,19,20,21], over the past 3 decades. Human corneal epithelium regenerated on 3T3 fibroblast feeder layer preserved more stem/ progenitor cells than explant cultures [20,22,23]
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