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Chun-Yen Liu, Eddie Wang, Nikki Tjahjono, Chun-hong Xia, Xiaohua Gong; Investigation of differentiation and cell junctions in 3D Spherical Micro-Chambered Lens Epithelial Cells In vitro. Invest. Ophthalmol. Vis. Sci. 2018;59(9):892. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the mechanisms of various growth factors including FGF, IGF and vitreous extracts in lens epithelial cell (LEC) regulation, proliferation and differentiation in 3D micro-chambered beads.
Primary cultured LECs from transgenic mice with membrane-tdTomato and nuclear GFP labeling in C57BL/6 background were harvested and mixed with gelatin. The mixture were poured into lecithin/paraffin solution for microbeads formation. Cell-embedded microbeads were washed and mixed with Matrigel and GelMA/photoinitiator solutions, and then irradiated by UV light for gel formation. Gels with cell beads were moved to dishes filled with culture mediums with and without addition of growth factors and proteins and then incubated. Proliferation and differentiation results were collected by confocal laser scanning microscope observation.
2D culture reveals that expressions of gap junction Cx50 and Cx46 are regulated by FGF in different manners such as stimulation or inhibition. 3D culture system shows LECs sank to the beads bottom after gel formation. Then some cells migrated across the chamber equator to the top, and then completed by day 3-5. Without addition of growth factors treatment, 3D cells remained thin layers covered around the beads. With bFGF, LECs proliferate and differentiate in part to become thicker cell layers but unable to fill in the lumen of 3D cell balls. Interestingly, the vitreous extracts can induce the LECs to proliferate and differentiation to fill in the lumen of 3D cell ball.
The synthesis of artificial lens can be achieved in vitro easily by our novel method. Cells remained their viability and proliferation ability to cover the inner area of the microbeads. Our results showed FGF can induce fiber cell differentiation but inadequate to fill in the lumen. Besides, FGF will affect the expression of Cx50 and Cx46 in different manners. By using this in vitro model, we can easily study the effect of addition of proteins to LEC inside the beads by measuring the changes of thickness of cell layer. Immunostaining will also be conducted to evaluate the LEC differentiation in 3D beads under various conditions.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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