Isolation and fractionation of blood plasma extracellular vesicle (EV) subpopulations using size exclusion chromatography

Abstract

Extracellular Vesicles (EVs) are membrane enclosed structures that are shed from cells into extracellular space carrying cargo from their parent cells. The main areas of current EV research include fundamental biology studies focused on EV roles in cell-to-cell communication and cell biogenesis in health and disease, and EV potential for clinical diagnostics and drug delivery. However, it is difficult to isolate EVs at high purity. The current gold standard isolation method based on ultracentrifugation and differential centrifugation is complicated, expensive, and can take several days. There is a need for straightforward, low-cost, robust, quick, and reproducible techniques that can be applied to processing of complex physiological fluids in clinical and biological labs. In this thesis, I propose a method using various size exclusion chromatography stationary phases to isolate EVs from proteins and lipoprotein particles in blood plasma samples, and to fractionate EVs into different size subpopulations. Several implementations of size-exclusion chromatography were developed and evaluated for this purpose. In addition, a fluorescence-based detection method was developed for characterization of EV abundance. This detection method was then applied to assess the performance of two EV fractionation columns that demonstrated the potential for separating EV subpopulations. Using Sephacryl S-1000 and Sephacryl S-500 in addition to the commonly used Sepharose CL-2B, EVs can be isolated from these contaminants, and the initial results of fractionation into EV subpopulations appear promising.