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.
journal_name
Regen Medjournal_title
Regenerative medicineauthors
Notara M,Haddow DB,MacNeil S,Daniels JTdoi
10.2217/17460751.2.6.919subject
Has Abstractpub_date
2007-11-01 00:00:00pages
919-27issue
6eissn
1746-0751issn
1746-076Xjournal_volume
2pub_type
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journal_title:Regenerative medicine
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pub_type: 杂志文章,随机对照试验
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更新日期:2014-05-01 00:00:00
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pub_type: 杂志文章,meta分析,评审
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更新日期:2018-12-01 00:00:00
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journal_title:Regenerative medicine
pub_type: 杂志文章
doi:10.2217/rme-2017-0063
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更新日期:2012-09-01 00:00:00
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journal_title:Regenerative medicine
pub_type: 杂志文章
doi:10.2217/17460751.1.1.59
更新日期:2006-01-01 00:00:00
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journal_title:Regenerative medicine
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更新日期:2009-01-01 00:00:00
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journal_title:Regenerative medicine
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更新日期:2012-01-01 00:00:00
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更新日期:2008-03-01 00:00:00