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B. Kim, C. J. Roberts, D. M. Grzybowski, P. Weber, Y. Zhao; Development of in vitro 3D Model of Human Trabecular Meshwork Using Polymer Microfibers. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4853.
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© ARVO (1962-2015); The Authors (2016-present)
To develop a 3D in vitro model to resemble the in vivo trabecular meshwork (TM) and to address one of the limitations of currently employed monolayers in studying the hydraulic resistance of TM structures and the impact on primary open-angle glaucoma (POAG).
Polyetherurethane urea (PEUU), and polycaprolactone (PCL) microfibers were prepared by electrospinning. Polyethylene terephthalate (PET) was obtained from a commercial vendor. The substrates were sterilized by 70% ethanol overnight, kept in DPBS for 3 hours, and reserved in culture medium until cell seeding. The primary cultured TM cells were seeded on the microfibers at the same concentration. The seeded microfibers were replaced in new plates after 8 hours and cell proliferation reagent (Alamar blue®) was then added to cell culture media at the ratio 1:10. Cell proliferation rates were measured every 24 hours during the entire culture (5 days). In order to examine the morphological change and production of extracellular matrix (ECM) materials, laser confocal immunofluorescence microscopy and scanning electron microscopy were performed. The cells cultured on planar cover slips were used as experimental control for investigating the effects of microfibers.
Proliferation tests show the microfibers support 3D cell proliferation. The cell morphology is vastly different from that in the control group. In particular, cells in PCL and PEUU fibers develop laminated cell clusters, while cells in PET grow on the fiber surfaces. Interestingly, the cells in PCL and PEUU develop extensive ECM materials, which accumulates within the fiber matrix and form collagen networks. Such structures are believed to play an important role in regulating the hydraulic resistance.
Cell morphology and expression of ECM materials imply that polymer microfibers provide a viable solution for development of in vitro models which outperform currently used monolayers in resembling natural TM structures. This model provides a promising starting point for systematic investigation of the development and evolution of POAG.
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