Lightning Talk & Poster Australasian Extracellular Vesicles Conference 2018

Two step isolation procedure for exosome purification from blood   (#57)

Candice de Boer 1 , Dee Blackhurst 1 , David Marais 1 , Neil Davies 1
  1. University of Cape Town, Observatory, CAPE TOWN, South Africa

Exosomes are secreted nano-sized vesicles (30-100 nm) found in body fluids such as blood, urine, saliva and cerebrospinal fluid with potential as biomarkers and therapeutic agents. It is important to understand the physiological functions of the exosomes without the presence of co-isolated lipoproteins that could obscure interpretation. For example, we found that low density-lipoprotein (LDL) contamination can influence exosome uptake studies employing fluorescent nucleic acid and lipid labels. The aim of this study was to purify human serum exosomes away from lipoprotein contamination through density gradient ultracentrifugation followed by size exclusion chromatography (SEC). When exosomes were isolated by SEC alone, transmission electron microscopy and western blotting showed the majority of exosomes were present in fractions 8-9. ELISA indicated a significant presence of ApoB containing lipoproteins (very low (VLDL); intermediate (IDL), LDL and chylomicrons) in these fractions (up to 15 µg/ml). SEC alone successfully removed high-density lipoprotein (HDL) contamination with ApoA1 only detected from fractions 12 onwards. When density gradient ultracentrifugation was introduced upstream from SEC, no ApoB lipoproteins or HDL were detected in fractions 8 and 9. Exosomal markers (CD9 and TSG101) and vesicles (30-100 nm) were present albeit at lower concentration. This study demonstrates that SEC is required to remove HDL from serum exosomes according to size and density gradient ultracentrifugation to remove VLDL, IDL and LDL according to density. Combining the two isolation techniques isolates purer serum exosomes from blood that are more suitable for biomarker discovery, molecular composition determination and their biological function analysis.