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Hongyu Ying, Sanja Turturro, Xiang Shen, Beatrice Yue; 3-DIMENSIONAL (3D) CULTURES OF HUMAN TRABECULAR MESHWORK CELLS. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5676.
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© ARVO (1962-2015); The Authors (2016-present)
To establish in vitro 3-dimensional (3D) culture systems to replicate more closely the native tissue environment for human trabecular meshwork (TM) cells.
Human TM tissues dissected from corneo-scleral rings of 10- and 31-year old donors (Illinois Eye Bank) were cultured on Falcon Primaria flasks (referred to as 2D cultures) in complete medium. Cells from 3rd or 4th passage of 2D cultures were used for the 3D culture system. Commercially available 3D hydrogel platforms such as 3DLife (Cellendes), Puramatrix (BD Biosciences), QGel (Qgelbio), Extracel (Glysan Biosystems) and cell culture inserts including 3D Insert (3D Biotek), Preset VECELL (Nacalai USA), and Alvetex (Reinnervate) were tested. Cell morphology was assessed by light microscopy and hematoxylin and eosin staining. Cell proliferation was examined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay. The expression of genes including myocilin, matrix GLA protein (MGP), angiopoietin-related protein 7 (CDT6/ANGPTL7), fibronectin and collagen type I was assessed by RT-qPCR using Taqman gene expression assays. The protein levels of myocilin, fibronectin, and collagen type I were examined by immunofluorescence staining.
Human TM cells cultured on QGel and Alvetex platforms demonstrated high cell viability, growth, and migration. Elongation of cells was observed in QGel by day 2 along with formation of tubular networks. TM cells cultured on Alvetex were distributed in multilayers. Their proliferative activity was substantially reduced by day 3 post seeding. Contrasting the 2D culture system, the gene expression of myocilin and MGP in TM cells on the Alvetex and QGel 3D platforms was high and that of CDT6/ANGPTL7, fibronectin and collagen type I was low. Such gene expression patterns resembled those observed in TM tissues.
The cell morphology, proliferative activity, and gene expression profile of TM cells cultured in QGel and Alvetex 3D scaffolds mimicked those in TM tissue. These 3D cultures may serve as effective in vitro models, allowing insights into the in vivo activities, characteristics, and stress responses of TM cells as well as signaling events that take place in native tissue.
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