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Y.–H. Chen, I.–J. Wang, L.–K. Yeh, T.–H. Yang; Behavior of Keratocytes on Chitosan Membrane . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2983.
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© ARVO (1962-2015); The Authors (2016-present)
To observe spheroid–formation behaviors of keratocytes after seeding on chitosan membrane with different conditions of media and to study the charecteristics of keratocyte spheres.
Bovine keratocytes were cultured on culture dish and chitosan membrane under DMEM with 10% FBS, DMEM with 10% horse serum (HS), DMEM with 1%FBS, and DMEM with 1%HS to observe the cell morphology, number of sphere formation .Proliferation rate and cell viability of keratocyte sphere was assessed by MTT assay and calcium–AM tests. Keratocyte spheres were reseeded and cultured on culture dish again to study cell proliferation. Immunocytochemistry was used to check present of vimentin and K3, which are markers of mesenchymal cells and epithelial cells. RT–PCR was used to observe the present of indicators of keratocytes differentiation level, such as alpha–smooth muscle actin (alpha–SMA), keratocan, and ALDH–3.
The numbers of keratocyte spheres were significantly more in 1% FBS and HS groups than in 10% FBS group. However, the size of sphere was larger in 10% FBS and 10% HS groups (130 µm vs 80 µm in diameter). Calceim–AM and reseeding test indicated that cells in the spheres for four groups were alive. MTT assay shows that the sphere proliferated slowly. The results of immunocytochemistry showed that these cells were positive present of vimentin and negative present of K3. The resuts of RT–PCR confirmed the expression of keratocan and alpha–SMA in keratocyte spheres.
The formation of keratocyte spheres on chitosan membrane is a very interesting phenomenon and can be a good model to study the differentiation of keratocytes.
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