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K.A. Kobuch, B. Wild, P. Eberl, C. Framme, A. Opolka, E. Wild, A. Goepferich, V.–P. Gabel; New Strategies for Vitreous Substitution: Towards a Cellbased Regenerative Therapie of the Vitreous . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1466.
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© ARVO (1962-2015); The Authors (2016-present)
With the final aim of a cell–based reconstruction of the vitreous and the vitreoretinal interface for therapy of vitreoretinal diseases, we determined basic requirements for a biologic implant with respect to cell–source, proliferation and redifferentiation of hyalocytes in tissue culture and performed a structural analysis of the vitreous cortex.
Hyalocytes were isolated from the vitreous or from the ciliary body of porcine or human eyes. After mechanical and enzymatic dissociation, the cells were proliferated for 3 passages in culture flasks, than grown to redifferentiate in 12 well dishes. Basic culture conditions for proliferation, redifferentiation and expression of extracellular matrix were determined. Hyalocytes from different sources and in different stages were characterized by immunohistochemistry (S100, CD68, Coll.I, Coll.II, Coll.IX) and by light– and electron microscopy (TEM, SEM). Expression of Coll.I and Coll.II was determined by PCR. The structural integration of hyalocytes to extracellular matrix was evaluated in native vitreous or in hyalocyte–cultures by SEM. Intravitreal Coll.IX was examined in a Coll.IX–knock–out mouse model.
A sufficient number of vital hyalocytes could be isolated and proliferated from porcine and human vitreous. Cells from the vitreous as well as from the nonpigmented epithelium of the ciliary body showed typical positive markation for S100 and negative for CD68. Under modified culture–conditions in 12–well plates, the cells redifferentiated as concluded from reduced proliferation, increased number of intracellular, collagen–containing vacuoles, and production of extracellular matrix. After 2–3 weeks, extracellular matrix formed network–like, Coll.II–positive structures. Coll.II–expression was demonstrated immunohistochemically by light– and electron microscopy and by PCR after 3 to 8 weeks of culture. By light microscopy and immunostaining for Coll.IX, the vitreous of Coll.IX–deficient mice showed no structural difference to the wild type.
Culture–conditions for isolation, proliferation and redifferentiation of hyalocytes from porcine and human eyes could be established. Cells from the ciliary body showed evidence to be vitreal precursor cells. These results may represent the first steps on the way to a cell–based regenerative therapy of the vitreous.
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