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Carlos-Alberto Rodriguez-Barrientos, Judith Zavala, Victor Treviño, Raul Aguirre, Jorge E Valdez, ; Gene expression profile analysis of cultured corneal endothelial cells in Mitogenic/Resting culture system for applications in tissue engineering.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5187.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the benefits of include a resting culture medium after a mitogenic culture medium to maintenance the specific gene expression markers for corneal endothelial cells.
4 young New Zealand white males rabbits were used. Corneal endothelial cells (CEC) were isolated. After five days in culture with mitogenic culture medium (mM), the first subculture was carried out and CEC was split into two populations, one of these were used to test the effect of the resting culture medium (rM) and the other one continued in mM. After cell culture in P1 with mM and rM reached confluence (80-90%), cells were subculture (P2) and continued with the same scheme of culture medium. For RNA extraction, culture cells in P2 were used only when cultures exhibit growth and morphologic characteristic similar to those of early passage cells. RNA was isolated and microarray analysis of gene expression was performed pooling 4 samples of CEC cultured in mM and 4 samples cultured in rM. An analysis of the difference in the gene expression pattern between cells in mM and rM was made.
Cells cultured in mM showed fibroblastic morphology while cells in rM were polymorphic with more tendencies to hexagonality. A top of highly different expressed genes based on their p value between cells cultured in mM and rM was obtained from microarray data. Genes highly different expressed and more significant in cells cultured in pM are related to: Catalytic activity (p value=0.000062), Collagen degradation (0.0099), Metalloendopeptidase (0.036), filopodium assembly (0.017), Cell motion (0.0055), G1/S transition of mitotic cell cycle (0.027), and regulation of apoptosis (.0044). In contrast, cell culture in rM express terms for collagen (6.2E-09), Collagen type IV (4.5E-06), TIMP metallopeptidase inhibitor 2 (0.074), cell cycle arrest (0.0018), p53 signaling pathway (.052), Wnt receptor signaling (.033), actin filament bundle (0.023), morphogenesis (.00065), and focal adhesion (1.6E-06).
Our results support the use of a Mitogenic/Resting culture system for CEC. And describe for the first time using microarray technology the benefits of resting culture medium. This step had the capacity to induce the expression of specific gene that reflects as closely as possible the in vivo conditions of normal corneal endothelial cells.
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