Abstract
Small extracellular vesicles (sEVs) are membrane bound packets 30-200 nm in diameter which contain DNA, RNA, lipids, and proteins that are used for signaling and transporting cargo between cells. It is known that cancerous cells alter the molecular composition of sEVs. 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 (SEC) is more cost effective and allows for faster exosome isolation compared to the ultracentrifugation (UC) predominately used in research settings. The isolation of sEVs by UF and a multi-step SEC from the SKOV3 cell line, a high-grade serous ovarian carcinoma (HGSOC) as well as sEVs derived from fetal bovine serum (FBS) were performed to determine the conditions necessary for high purity, high yield, and size specific EVs for cancer detection. Exosomes extracted from FBS have high levels of lipoproteins, which are also prevalent in human blood samples, and was used to further elucidate the capability of high-resolution SEC to purify samples with tighter vesicle size distribution without sacrificing EV yield. Issues and solutions will be discussed associated with the SEC extraction process in regard to EV purity and yield with emphasis on early cancer detection. The sEVs were extracted on both a manual system and an automated AKTA 25L Fast Protein Liquid Chromatography System. The extracted EVs were characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), field emission scanning electron microscopy (FE-SEM), BCA protein analysis, and flow cytometry to determine size, morphology, protein levels, and positive EV identification, respectively. This work has developed this methodology for extracting malignant EVs of high purity, size separation in size range of 100-200 nm and yield per sample volume for eventual integration into a SERS-based diagnostic system for early cancer detection in the future.