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R. Takagi, M. Yamato, A. Kushida, K. Nishida, J. Yang, M. Utsumi, C. Kohno, Y. Tano, T. Okano; Profiling of Extracellular Matrix Molecules Expressed by Mouse Feeder Layer Cells Screened by Limbal Epithelial Cell Colony Forming Assay . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2711.
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© ARVO (1962-2015); The Authors (2016-present)
Mouse 3T3 feeder layer has been utilized for epidermal and corneal epithelial cell culture to promote tissue–like cell stratification. However, the molecular mechanism underlying the epithelial–feeder layer interactions remains poorly understood. Here, we examined feeder layer activity of six different mouse cell lines in terms of colony forming efficiency of primary limbal epithelial cells including corneal epithelial stem/progenitor cells.
Six mouse fibroblasts lines were examined as feeder layers, and colony forming assay with primary rabbit limbal epithelial cells was performed. Expression of mRNA for twenty nine extracellular matrix molecule genes was quantified by TaqMan PCR after reverse transcription for two highest and two lowest cell lines in colony forming efficiency.
When epithelial cells and feeder layers were separated by culture inserts, colony forming efficiency was significantly lower than that when both cells were cultured on the same dish surfaces, implying that direct contacts between these cells and/or pericellular extracellular matrix deposition by feeder layers have an important role in feeder layer activity. With TaqMan PCR assay, significant difference in expression correlated with colony forming efficiency was observed for some extracellular matrix molecules including tenascin C, type XII collagen alpha1 chain, and type XVII collagen alpha1 chain.
Decreased colony forming efficiency under the condition where epithelial cells and feeder layers were separated by culture inserts suggests that direct contacts and/or efficient extracellular matrix deposition is essential for the feeder layer activity. Correspondingly, mouse cell lines showed varied colony forming efficiency for limbal epithelial cells, and significantly different profiles of extracellular matrix proteins.
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