Preparation of a Single Cell Suspension from the Murine Iridocorneal Angle

Background

Glaucoma is a progressive neurodegenerative disease with no cure, affecting over 60 million individuals worldwide and leaving 8 million blind (Quigley and Broman, 2006). Current therapy is focused on reduction of intraocular pressure (IOP), the only treatable risk factor for disease progression. IOP is determined by the ratio between aqueous humor secretion by the ciliary body and drainage through tissues in the iridocorneal angle, including the trabecular meshwork and Schlemm’s canal, which comprise the conventional outflow pathway. Patients with glaucoma, including primary congenital glaucoma, exhibit defects in tissues of the conventional outflow pathway leading to decreased aqueous humor outflow and elevated IOP (Tektas and Lütjen-Drecoll, 2009; Braunger et al., 2015; Zagora et al., 2015). The importance of these tissues to glaucoma pathogenesis, and consequently as therapeutic targets, has focused intense research interest in understanding their physiology, molecular phenotypes, and the cell-cell interactions governing their function. However, due to the small sizes of the tissues involved and their location within the iridocorneal angle, relatively little is known about the transcriptomes or overall phenotypes of Schlemm’s canal or drainage channel endothelial cells, potential differences between inner and outer wall endothelial cells of Schlemm’s canal or cells of the trabecular meshwork.

Recently, single cell RNA sequencing has been used to identify cell types in the iridocorneal angle region of mice, humans, and other model species (van Zyl et al., 2020; Patel et al., 2020; Thomson et al., 2021). These studies have provided exciting hints about the cells making up the angle and identified potential new therapeutic targets, but the extreme rarity of Schlemm’s canal endothelial cells within published datasets [van Zyl et al. (2020) identified just 25 Schlemm’s canal endothelial cells after sequencing 13,833 cells (0.18%), while our group has identified 109 in a dataset of 19,232 (0.57%)] has emphasized the need for sequencing of more cells, in healthy and disease conditions, to better understand the full complexity of this important tissue.

In the present protocol, we optimized a straightforward method for dissecting and dissociating the murine iridocorneal angle to obtain a single cell suspension of healthy cells to be used for downstream applications such as single cell RNA sequencing or flow cytometry. Previous studies utilizing the full anterior segment have reported that corneal and ocular surface epithelial cells represent over 50% of total cells in the dataset (van Zyl et al., 2020). While glaucoma-relevant iridocorneal angle cell populations remain rare, by removing the majority of the cornea during dissection, our method substantially increases their yield, resulting in a ~200% increase in proportion of Schlemm’s canal endothelial cells and ~80% increase in trabecular meshwork cells within the sample. Depending on the tissue(s) of interest, this protocol could be combined with flow cytometry or antibody-conjugated bead-based methods to enrich the sample in relevant cell types prior to library preparation. We have also found that this procedure can be readily adapted to dissociate cells of the cornea or posterior eye with similar results and cell viability.