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Carl Sheridan, Laura Currie, Natalie Hill, Sharon Mason, Ian Grierson; Decellularisation and repopulation of the bovine trabecular meshwork (BTM) with primary BTM cells and BTM-derived spheres.. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4676.
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© ARVO (1962-2015); The Authors (2016-present)
Progenitor cells have been found in the transition zone between TM and corneal endothelium and can be cultured to generate progenitor cell colonies as cell populated spheres. These cells have a tendency to differentiate into a specific cell type and have the potential to replace differentiated cells which are lost and thus improve outcome for damaged and diseased tissue. The loss of TM cells with age and in POAG is well known and we evaluated the potential of cells to repopulate decellularised TM.
TM dissected and placed in TC treated flasks for explant culture in DMEM 30% FCS and cells detatched with trypsin/EDTA for routine population expansion, maintained in media containing 10% FCS. Cells were detached with Accutase to optimise cell viability for sphere culture. 2,500 cells/cm were seeded onto 35mm dishes either tissue culture treated (control) or non-tissue culture treated (spheres). Cells were maintained with serum free DMEM/F12 containing L-glutamine and B27 (1X, bFGF 10ng/ml, EGF 20ng/ml). The decellularisation process involved incubating TM in 1. Hypotonic Solution to lyse cells for 16hours at 4C 2. A detergent to remove cellular fragments (24 hours at 25C) and 3. Isotonic solution (3x 30 min at 25C).
Quantification of decellularised BTM reseeded with primary cells demonstrated BTM positively stained with Alexa Fluor 488 Phalloidin and Propidium Iodide (PI) at 7, 10, and 20 days post seeding. Control decellularised BTM was negative for both Phalloidin and PI. The nestin positive spheres appeared to settle in the TM and single cells (nestin negative) appeared to have migrated out from the spheres into the TM tissue. PI counts showed 30% of TM appeared to be repopulated by both TM cells and sphere derived cells by day 20.
BTM successfully recellularised, (after successful decellularisation), with BTM primary cells and with BTM-derived whole spheres.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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