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.2007 Nov;2(6):919-27.
doi: 10.2217/17460751.2.6.919.

A xenobiotic-free culture system for human limbal epithelial stem cells

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A xenobiotic-free culture system for human limbal epithelial stem cells

Maria Notara et al. Regen Med.2007 Nov.

Abstract

Aims: Murine 3T3 feeder cells are commonly used for stem cell expansion. Although 'feeder-free' systems are being developed for a variety of stem cells including embryonic, the use of feeder cells currently remains optimal for the expansion of epithelial stem cells. In this study, MRC-5, a human embryonic fibroblast cell line, has been investigated for its potential use as a feeder layer in human limbal epithelial (HLE) cell expansion under serum-free conditions, with the aim of developing a xenobiotic-free culture system for therapeutic corneal regeneration applications.

Materials and methods: MRC-5 feeder cells were compared with J2 3T3 mouse fibroblasts, in both serum-supplemented and serum-free conditions, in terms of their relative ability to support HLE cell metabolic activity, expression of the putative stem cell markers ABCG2 and P63 alpha, cell differentiation using the cornea-specific cytokeratin 3 antibody and colony-forming efficiency.

Results: The proportion of HLE stem cells maintained was determined by functional colony-forming efficiency assays. The metabolic activity results showed that HLE cells cultured on MRC-5 fibroblasts under serum-free conditions proliferated as well as cells cultured on J2 cells under serum-free conditions. Moreover, the HLE cultured on MRC-5 fibroblasts under serum-free conditions expressed high levels of putative stem cell markers ABCG2 and P63 alpha and low levels of the differentiation marker CK3, indicating that they retained poorly differentiated 'stem cell-like' characteristics under those culture conditions. Clonal analysis of HLE cells cultured on growth-arrested feeder layers of J2 and MRC-5 fibroblasts showed that cells expanded on MRC-5 and J2 fibroblasts in serum-free conditions had a colony-forming efficiency of approximately 1.5%, indicating the maintenance of stem cells.

Conclusions: These results demonstrate feasibility of expanding HLE cells for clinical purposes by using a human fibroblast cell line as a feeder layer, avoiding the use of bovine serum, while preserving the proliferative potential and stem cell characteristics of HLE cells.

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