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Eddie Wang, Dong Wang, Byron Mui, Cindy Lam, Xiaohua Gong; Development of 2D and 3D Culture Systems for Lens Epithelial Expansion and Differentiation. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2626.
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© ARVO (1962-2015); The Authors (2016-present)
We currently have few techniques for expanding and maintaining the phenotype of primary lens epithelial cells (LECs) and for controlling their differentiation into 3D lens-like tissues in vitro. These limitations greatly inhibit our ability to perform basic biological studies on the lens. Therefore, the goal of this study is to determine factors that will allow for primary lens epithelial cell expansion in the absence of native matrices and to begin creating a 3D culture system that mimics the environment of the developing lens.
2D Culture: We isolated lens epithelial cells (LECs) from the lens capsules of mice using dispase and TrypLE. The LECs were then cultured in the presence or absence of small molecules such as a TGF-beta pathway inhibitor and a Wnt pathway agonist. Cell morphology and the expression of LEC markers were then examined by immunolabeling.<br /> <br /> 3D Culture: GFP positive primary LECs were grown in suspension in the presence of electrospray synthesized alginate microspheres. After 24 hours spheres with attached cells were embedded in Matrigel and visualized over time by fluorescent microscopy.
2D Culture: LECs in the presence of the Wnt agonist behaved similarly to those in control media. The presence of the TGF-beta pathway inhibitor prevented the majority of cells from adopting a mesenchymal morphology. These cells also retained more epithelial cell markers such as E-cadherin.<br /> <br /> 3D Culture: We determined electrospray conditions that allowed for synthesis of 200 micron diameter alginate spheres. Poly ornithine coated spheres were able to promote LEC attachment. Once embedded in Matrigel the cells underwent minimal proliferation but did appear to survive.
A TGF-beta pathway inhibitor is sufficient to improve the maintenance of primary LEC phenotype in the absence of native matrix. We are seeking further optimization of culture conditions to improve the long term proliferation of these cells. Furthermore, the primary LECs are able to attach to alginate microspheres and survive being embedded in Matrigel. By dissolving the alginate spheres we hope to create a similar 3D environment to that of the lens vesicle.
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