Isolation and characterization of corneal epithelial extracellular vesicles. Corneal epithelial extracellular vesicles (CE-EVs) were isolated by treating a semi-confluent layer with a starvation medium (Opti-MEM) for 48h to trigger EV production. After the starvation treatment with Opti-MEM, one third of the treated flasks were analyzed for the live/dead cell ratio using trypan blue staining, which showed 97-99% cell viability. Additionally, cell counts after treatment revealed a nearly fourfold increase (378%) compared to initial seeding, indicating proliferation (in normal growth media) and robust survival (in the Opti-MEM) during the treatment. The medium was then collected and purified by tangential flow filtration (TFF) followed by concentration of the sample with an Amicon® Ultra Centrifugal Filter. TFF was used for EV-purification instead of the more commonly employed ultracentrifugation (UC) due to the reported higher yield in TFF purification. Purified CE-EVs were stored in the fridge and used within 1-2 weeks or otherwise frozen at -20°C in freezing media containing PBS with HEPES, Albumin, and Trehalose as described elsewhere. Nanoparticle tracking analysis (NTA) was then performed to determine the concentration and size of the CE-EVs. NTA was measured of samples before, during and after purification to see how the size, distribution, and number of particles were affected (Figure ). In Figure , the size distribution of the purified CE-EVs (from 9 batches) is presented and the main peak for the samples were found to be in the range of 110 ±12 nm which correlates to the size range of EVs. The size and morphology of CE-EVs were also studied by transmission electron microscopy (TEM) (Figure ). The size was found to be slightly smaller (around 60-70 nm) compared to NTA and aggregation could be observed. . For all batches (n=8) the most common surface markers were the EV-specific markers CD63 (most expressed, set to 100%), CD9 (59.4±24.4%), and CD81 (62.5±10.2%). Aside from the EV-specific markers, the most expressed surface protein was CD29 (integrin β1) with 32.8±13.8%. CD29 is part of many cell surface receptors which are responsible for the interaction of cells with ECM proteins . Moreover, in most of the batches CD49E (integrin α5) was also highly expressed (average 32.6±23.6%) which together with CD29 constitutes the fibronectin receptor (integrin α5β1). CD29 can constitute receptors with multiple other integrin α subunits that are not included in the MACSPlex EV kit, such as CD49C (integrin α3). Another highly expressed protein was the CD44 (17.9±8.6%); a transmembrane cell surface receptor responsible for both cell-cell adhesion and cell-matrix interaction, which is usually upregulated after tissue injury. Further abundantly expressed surface markers were CD142 (17.6±4.5%), CD40 (16.3±3.9%), CD105 (14.1±12.5%), CD146 (12.1±12.6%), HLA-ABC (11.0±6.5%), CD326 (7.4±4.8%), MSCP (5.5±5.1%) and HLA DRDPRDQ (4.8±4.5%). These proteins are known to have crucial roles in cell-adhesion and immunomodulation (Figure ).