Wednesday, 20 June 2018

Exosome isolation using ultracentrifugation

An interview with Dr Karina Serban, Assistant Professor of Medicine, National Jewish Health, conducted by April Cashin-Garbutt, MA (Cantab)

How important is exosome isolation in your research?

In the outpatient clinic and in the laboratory our current research studies aim at understanding the mechanisms of release and clearance and the biological functions of exosomes and membrane particles in the plasma of individuals who develop emphysema, a form of chronic obstructive pulmonary disease characterized by enlargement of airspaces and loss of alveoli.
Soluble components within the cigarette smoke (CS) initially inhaled and then absorbed into the circulation are the most common injury that leads to emphysema and COPD. We have showed that acute exposure to CS is sufficient to stimulate the release of exosomes and membrane particles from lung endothelial cells with highly pro-inflammatory potential based on their cargo analysis: pro-coagulant and pro-inflammatory surface membrane molecules, bioactive lipids, and miRNAs.
The exosomes and membrane particles are extremely small, nanometer in size, circulating particles released within the bloodstream by structural, endothelial cells or circulating cells, like leukocytes or platelets. We have isolated exosomes and membrane particles using differential ultracentrifugation. This method allowed us to pellet the exosomes and membrane particles based on their density, to achieve complete separation from the fluid phase, and to resuspend the pellet in various buffer required for downstream quantitative (flow cytometry counting), qualitative (membrane proteins, lipids, miRNAs), and functional studies (uptake by macrophages).
Since not all smokers develop emphysema we believe that exosomes abundance in the blood of active smokers will allow us to identify the individuals at risk for lung function decline, frequent infectious exacerbations, and frequent systemic comorbidities associated with cigarette smoking and COPD (e.g. arterial and venous thrombotic events, cachexia, or lung cancer).
Besides their role in the diagnosis of smokers at risk, exosomes may account for the paracrine and epigenetic signalling associated with the cargo they may carry, e.g. bioactive lipids (e.g. ceramides), surface membrane receptors and signalling molecules (e.g. phosphatidylserine), or miRNAs (e.g. -125a, -126, and -191).

Traditionally, what methods have been used for the isolation of exosomes and other extracellular vesicles (EVs)?

Over the last fifteen years methods to enrich for a pure extracellular vesicles population have been under careful scrutiny in an attempt to standardize the nomenclature, the isolation and analysis methods.
Following the International Society for Extracellular Vesicles position statement we use the following nomenclature of EVs populations we isolated in our laboratory from human samples (e.g. blood, BAL fluid) or cell culture supernatants (primary human epithelial and endothelial cells, human macrophages). Exosomes were defined as particles ranging from 30-150nm, the remaining of the EVs were membrane particles (200nm – 1μm) and apoptotic bodies (3-5μm).
Several methods including filtration and immunoaffinity isolation have been used since 1970s. The ultracentrifugation method for separation of small size particles has been introduced in the mid 20th century when it was used to isolate enveloped viruses.
Larger EVs (microparticles and apoptotic bodies) are pelleted at low-speed centrifugation forces in the 10,000 – 20,000 X g range. Smaller EVs, including exosomes require high-speed forces (100,000 – 200,000 X g).
Due to concerns of EVs aggregation and protein- or lipid-aggregates “contamination” during high-speed ultracentrifugation several protocols recommend PBS washing step and sucrose density gradients for efficient separation of the exosomes from the protein- or lipid-aggregates.
A newer isolation method, polymeric precipitation using ExoQuick from System Bioscience is less laborious than the previous methods, however there are concerns about the purity of the EVs in the precipitate and the limited use in further analysis methods (e.g. electron microscopy, flow cytometry, western blot).
Our laboratory has used differential ultracentrifugation followed by flow cytometry and optical single particle tracking (Nanosight NS300) techniques for isolation and quantification of EVs from endothelial cell culture supernatants. Nanosight uses light scattering and Brownian motion in order to obtain the size distribution and concentration measurement of EVs in liquid suspension. Both techniques produced similar results allowing us to isolate and count a heterogeneous EVs population, enriched for exosomes, that also contains membrane particles, but no apoptotic bodies.