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.
Notara
Maria
M
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
Haddow
David B
DB
MacNeil
Sheila
S
Daniels
Julie T
JT
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