Journal Article

Cushioned-Density Gradient Ultracentrifugation (C-DGUC) improves the isolation efficiency of extracellular vesicles

Raffai, Robert L., Chung, Allen, Duong, Phat, Bouchareychas, Laura

2019

Overview

Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Agglomeration / Animals / Biology and Life Sciences / Cell culture / Cell Fractionation - methods / Cell Line / Cell membranes / Centrifugation, Density Gradient - methods / Centrifuges / Chromatography / Concentration gradient / Conditioning / Contaminants / Contrast media / Culture media / Culture Media, Conditioned / Data recovery / Density / Density gradients / Deoxyribonucleic acid / DNA / EDTA / Engineering and Technology / Extracellular vesicles / Extracellular Vesicles - metabolism / Extracellular Vesicles - ultrastructure / Glycols (Class of compounds) / Impact analysis / Lipoproteins / Macrophages / Macrophages - metabolism / Macrophages - ultrastructure / Methods / Mice / Microscopy, Electron, Transmission / Nanoparticles / Nanoparticles - metabolism / Nanoparticles - ultrastructure / Penicillin / Physical Sciences / Polyethylene glycol / Polyethylene Glycols / Polyols / Protein folding / Proteins / Proteins - metabolism / Purity / Research and Analysis Methods / RNA - metabolism / Sedimentation / Sedimentation & deposition / Triiodobenzoic Acids / Ultracentrifugation / Ultrafiltration / Vascular surgery / Vesicles / Yield