Abstract
Purpose :
Extracellular Vesicles (EVs) are a heterogeneous group of lipid bilayer membrane structures released by all cells. EVs contain specific sets of lipids, proteins, DNA and RNA and are being recognized as key players in cell signaling, homeostasis, and the pathology of various diseases. Most EVs studies have been accomplished by using cell supernatants or different body fluids, but the number of studies on tissue derived EVs is still limited. To characterize and gain insight on the role of EVs derived from the retina and RPE tissue, it is important to establish practical and efficient protocols for their isolation. Here we describe a step-by-step procedure of a novel, optimized and efficient method to isolate EVs directly from both human retina and RPE tissues.
Methods :
Our protocol includes enzymatic treatment of dissociated postmortem retina and RPE tissues followed by differential ultracentrifugation and density separation. The isolated EVs were characterized by transmission electron microscopy (TEM), Nanosight (NTA) and Exoview analysis. In addition, western blot against well-known EV specific markers and non-EV markers was used to determine the quality and purity of EV preparations. Furthermore, we analyzed the proteomic cargo of retina- and RPE tissue-derived EVs and validated the proteins found by western.
Results :
EVs trapped within the retina and the RPE were successfully isolated while maintaining the integrity of the vesicles and their cargo. The presence of EVs during each isolation step was confirmed via NTA with additional Exoview analysis after final recovery. Characteristic clear rounded membrane vesicles within a size range of 30–170 nm was observed by TEM. The physical characteristics of the vesicle preparations (ultrastructural morphology and size), and their biochemical composition (presence of Syntenin, CD63, and absence of GM130) confirmed that they fulfill the criteria for EVs. Furthermore, proteomic profiling of tissue-derived EV cargo revealed that EVs were enriched in proteins involved in biological processes associated with retina physiology.
Conclusions :
This study represents the first human retina and RPE EV isolation protocol to date. This method will enable researchers to gain insight on EV populations in the eye, explore their role in cell-to-cell communication and, provide a novel approach for elucidating mechanisms behind ocular pathogenic conditions.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.