A simple dissection method for the isolation of mouse trabecular meshwork cells

Abstract

Purpose The outflow pathway, especially trabecular meshwork (TM), plays an essential role in glaucoma, and the availability of TM cells is crucial for in vitro research. So far, the isolation of TM cells from mice has been anything but manageable due to the small size of the eye. Direct isolation using a stereomicroscope and forceps requires a high grade of dexterity. Indirect isolation is based on the phagocytic properties of TM cells and involves injecting magnetic microspheres into the anterior chamber of live mice followed by isolation. Therefore, a simpler, less expensive, and nonexperimental strategy for isolating mouse TM cells would be desirable. Methods After enucleation, the eyes were cut in half anterior-to-posteriorly. The lens and posterior segment were removed. Iris and the attached ciliary body were gently pulled backward and disconnected from the remaining tissue to expose the TM. By incising through the cornea anteriorly and posteriorly of the TM, the cornea/TM stripe could be isolated. The cornea/TM stripe was cultured with the pigmented side down in a 6-well. The outgrowing pigmented cells were analyzed by immunocytochemistry and mRNA expression for previously described TM cell markers. The phagocytic properties of the cells were additionally confirmed using fluorescent microspheres. Results Pigmented phagocytic cells were the first to grow out of the cornea/TM strips after approximately 4–7 days. Cells were positive for Collagen IV, Fibronectin1, Vimentin, and Actin alpha 2 and could phagocytize fluorescent microbeads. Cross-linked actin networks were visible after 9 days of exposure to TGFB2 (transforming growth factor-beta 2). Additionally, treatment with 500 nM Dexamethasone for one week increased myocilin expression, as previously reported for TM cells. In addition, we proved that this method can also be used in albino mice, which lack pigmentation of the trabecular meshwork. Conclusions The isolated cells show phagocytic properties and specific expression of markers reported in TM cells. Therefore, our dissection-based method is inexpensive and reproducible for isolating TM cells in mice.