Development of novel temperature-responsive culture dish modified with bioactive compounds for functional maintenance and proliferation of corneal epithelial cells

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Objectives of this research are development of novel temperature responsive culture dish, which is chemically immobilized with bioactive compounds and to culture and proliferate epithelial stem cells on the novel dishes without using NIH3T3 cells as feeder layer. Exploratory investigation of culture conditions of epithelial stem cells elucidates that a part of bioactive compounds secreted form 3T3 feeder layer functionally play a role of ECM (a scientific paper in preparation). For following exploration of the bioactive compounds, a mouse cell line as positive and negative control was determined from evaluation of behavior of colony formation and multiplayer of corneal epithelial cells, using various kinds of mouse cell lines as feeder layer. The mRNAs isolated from the positive and negative control cells were genetically and quantitatively compared with 30 species of genes derived from ECM, using Taqman PCR method. Likewise, mRNAs isolated from 3T3 feeder layers were also subjected to the Taqman PCR method (a scientific paper in preparation). Consequently, we successfully identified several geneswhich possibly relate to activation of feeder layers. Until now, we are cell-biologically investigating behaviors of the compounds as candidates against corneal epithelial cells, adding gene product or antibody specific for the gene products into the culture medium. Besides addition of the products or the antibody into the medium, we are also trying to modify the surfaces of novel temperature-responsive culture dish with the bioactive compounds via carboxyl groups. We have already found that the novel culture dished modified with bioactive compounds such as RGD and insulin peptides improved adhesion properties and proliferation of endothelial cells. Now, we are promoting investigation of culture conditions of corneal epithelial cells onto the novel culture dished modified with RGD and insulin peptides.