High Resolution Size Exclusion Chromatography (SEC) Purification and Isolation of Small Vesicles in Ovarian Cancer Cell Lines

Small vesicles are spherical membrane bound packets 30-150 nm in diameter which contain DNA, RNA, lipids, and proteins that are used for signaling and transport of cargo between cells. It is known that cancerous cells alter the molecular composition of small vesicles. Thus, detection of different types of cancer can be performed by molecularly comparing secreted small vesicles of malignant cells to those of healthy cells through various detection methods such as Polymerase Chain Reaction (PCR) and Surface Enhanced Raman Scattering (SERS). Currently, there are multiple methods being investigated to extract vesicles. Ultrafiltration (UF) in combination with size exclusion chromatography are more readily transferred to a clinical lab setting due to lower cost and faster exosome isolation compared to the predominately used method of ultracentrifugation (UC) currently used in research settings. The isolation and purification of small vesicles were extracted from the SKOV3 cell line, a high-grade serous ovarian carcinoma (HGSOC) using ultrafiltration (UF) and high-resolution size exclusion chromatography (SEC). The vesicles were extracted on an AKTA 25 protein purification SEC system using a 1 cm diameter by 30 cm long high resolution Tricon column which was packed with Sepharose CL-6B resin. High-resolution SEC coupled with UF allows for lower levels of contaminating proteins and other biomolecules in vesicle containing media and tighter vesicle size distribution than low resolution SEC that is predominately used for SEC vesicle isolation. The isolated SKOV3 exosomes were characterized by dynamic light scattering (DLS), field emission scanning electron microscopy (FE-SEM), Protein analysis, and Bead-based flow cytometry to determine size, morphology, protein levels, and semi-quantitative vesicle concentration, respectively. Bead based flow cytometry was used to identify proteins on the surface of the exosomes with emphasis on the tetraspanins, molecular scaffolds which are known to be overexpressed in small vesicles of cancerous cells. Exosomes extracted from fetal Bovine Serum (FBS), which have high levels of lipids and lipoproteins which are also prevalent in human blood and serum samples was used to further elucidate the capability of high-resolution SEC to purify samples with tighter vesicle size distribution. The SKOV3 cell line exosomes were harvested for eventual integration on a novel photonic array design currently in development for SERS detection of malignant exosomes.