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Weiming Mao, Yang Liu, Robert Wordinger, Abbot Clark; A magnetic bead-based method for mouse trabecular meshwork cell isolation. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3527. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Glaucoma is a leading cause of visual impairment worldwide. The most important risk factor of glaucoma is elevated intraocular pressure (IOP). In primary open angle glaucoma (POAG) patients, damage to the trabecular meshwork (TM) causes ocular hypertension. The mouse model has been widely used for glaucoma research. However, due to the extreme small size of the mouse eye, it is very difficult to dissect pure mouse TM (MTM) tissues and establish MTM cell strains. To circumvent these problems, we took the advantage of the phagocytic property of TM cells, and developed a novel magnetic bead-based method that enables us to isolate pure MTM cells.
After anesthesia, 1 μl magnetic beads (2.0-2.9 μm in diameter) were intracamerally injected into one of the BLAB/c mouse eyes. To study the localization of the beads, mice were sacrificed 1-7 days post-injection, and eyes were enucleated for transmission electron microscopy (TEM). To isolate MTM cells, anterior segments were dissected from 10-15 sterilized mouse eyes 7 day post-injection. The tissues were digested with collagenase A for 2 to 4 hours. MTM cells were purified with a strong magnetic field as well as repeated washings. Purified MTM cells were cultured serially from 96-well plates to 25 cm flasks, and were further characterized.
TEM studies showed that the magnetic beads located specifically in the mouse TM, but not in corneal or scleral fibroblast cells. Cultured MTM cells were spindle-like and they did not tend to grow into clusters that are usually seen in fibroblast cell cultures. These TM cells expressed TM markers including collagen IV, laminin, and α-smooth muscle actin, as shown by immunocytochemistry studies. Similar to human TM cells, MTM cells treated with 100 nM dexamethasone also showed the formation of cross-linked actin networks (about 2-fold increase, n=5 or 6, p<0.001) and induction of myocilin, as revealed by immunofluorescent staining and western immunoblotting, respectively.
The magnetic bead-based method is effective in the isolation of pure MTM cells with minimal requirement of micro-dissection techniques. This will be a useful tool for TM cell isolation from small animals for glaucoma research.
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