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


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.
UCL Institute of Ophthalmology, Cells for Sight Transplantation and Research Programme, Ocular Repair and Regeneration Biology Unit, 11-43 Bath Street, London, EC1V 9EL, UK. m.notara@ucl.ac.uk
Maria
M
Notara
David B
DB
Haddow
Sheila
S
MacNeil
Julie T
JT
Daniels
D016475:3T3 Cells; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D016895:Culture Media, Serum-Free / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D010641:Phenotype; D013234:Stem Cells / Q000166:cytology*; D015262:Xenobiotics / Q000737:chemistry*
D016895:Culture Media, Serum-Free; D015262:Xenobiotics