Isolation and Characterization of Trabecular Meshwork Stem Cell Exosomes from the Conditioned Medium

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Exosomes are nanovesicles (30-200nm) whose cargo includes proteins, RNA of different types, DNA, lipids, and small molecules. The exosome-based therapy is gaining significance in modern medicine due to the signal transduction capabilities and non-immunogenic properties of exosomes. In regenerative medicine, stem cellderived exosomes are employed as an alternative in cell therapies. Currently, there are more than 35 ongoing clinical trials worldwide employing exosomes in various disease conditions, including two eye diseases. However, the efficacy of these nanovesicles in glaucoma therapy still remains unexplored. Trabecular meshwork (TM) is a tiny, porous tissue located in the iridocorneal angle of the eye that maintains intraocular pressure (IOP) homeostasis. The adult stem cells are located in the non-filtering region and are drastically reduced in glaucomatous conditions (Sundaresan et al., 2021). Our previous TM stem cells (TMSC) transplantation studies on a human organ culture of anterior segment (HOCAS) model (Iswarya et al., ISSCR 2021) and several other studies on animal models (Yun et al., 2018; Xiong et al., 2021,) promise the application of TMSCs in glaucoma therapy. To evaluate the efficacy of the TMSC exosomes as an alternative to cell-free therapy for glaucoma, we optimized a few in vitro assays, which give insights into the properties of isolated exosomes. This chapter describes protocols to isolate exosomes by ultracentrifugation, characterization by nanoparticle tracking analysis (NTA), Western blot analysis, and transmission electron microscopy (TEM). Further, the functional efficacy of the exosomes was evaluated by in vitro wound healing assay and an anti-oxidant assay.