It takes a lot of work to pull meaningful insights from tiny vesicles residing in complex, heterogenous biofluids – particularly when variation can come from just about everywhere. Physiological status, storage conditions, and methods of analysis are all sources of variation – and isolation methods are no exception. The need for reproducibility in the extracellular vesicle (EV) field is so widely recognised that a Rigor and Standardization subcommittee formed, encompassing task forces spanning a range of specialised subgroups addressing different biofluids and specific challenges.
The ability to reduce variation in extracellular vesicle (EV) isolation is a key contributor to size exclusion chromatography-based qEV columns rising rapidly in popularity for the isolation of exosomes and other EVs for a wide range of biofluids and cell culture media. Now, research groups across fundamental research, diagnostics, and therapeutic applications continue to reap the benefits of qEV isolation.
qEV columns are highly effective at removing soluble protein from EV-containing samples. This is of particular importance as the co-isolation of non-EV components can impact the accuracy of downstream analysis and subsequently skew the results. To address this, qEV columns have been optimised to maximise the separation of EVs from soluble protein, for a wide range of column sizes. Gen 2 qEV columns are built with a proprietary, high-performing resin which enables this high-resolution separation (Figure 1).
Figure 1. Comparison of total protein elution levels and concentration of extracellular vesicles and similarly sized particles >60 nm between qEV10/35 nm Gen 2 and qEV10/70 nm Gen 2 columns with 10 mL of human plasma loaded, normalised for buffer volume. EV concentration was measured using an Exoid and protein levels by bicinchoninic acid (BCA) assay. *Nb: the default buffer volume values differ for qEV10/35 nm Gen 2 (23.2 mL) and qEV10/70 nm Gen 2 (22.9 mL) columns. **Volumes are labelled as the highest volume in that sample i.e., label “5” refers to the volume from 0.0-5.0 mL after the buffer volume, label “10” refers to the volume from 5.0-10.0 mL after the buffer volume and so on.
The use of qEV columns removes a significant amount of variation associated with human error. As optimised and standardised columns, qEV columns improve reproducibility by enabling the same isolation approach to be used within and between studies. In contrast, many EV isolation approaches rely extensively on manual methods (e.g., ultracentrifugation (UC), density gradient centrifugation, and homemade SEC columns), and the irreproducibility of these methods have been widely documented. For example, quantitative differences in EV yield are thought to be due to both equipment and operator-dependent technical variability of UC1, while density-based isolation methods rely on UC itself and therefore share the same sources of variation. Meanwhile, going the ‘DIY’ route of column creation introduces unnecessary potential for error, with efficient and standardised column production requiring a high degree of optimisation (and subsequently, time), resources and skill.
Whereas UC subjects EVs to intense gravitational force, making EVs prone to degradation, aggregation and fusion2,3, qEV columns provide a gentle approach. SEC-based qEV columns enable the isolation of intact, functional EVs.
The convenient and quick nature of qEV columns has enabled qEV isolation to become one of the most popular EV isolation methods. While qEV columns are an effective and efficient tool for EV isolation, they are just one part of the puzzle. Further efficiency and reproducibility can be achieved when qEV columns are used alongside the Automatic Fraction Collector (AFC), which guides you through the isolation process and introduces the valuable element of automated volume measurement and collection. The AFC allows you to get on with other tasks while isolation is occurring, allowing you to scale and streamline your methods and save precious time in your day.
Figure 2. Three Automatic Fraction Collectors (V2) with Gen 2 qEVoriginal / 35 nm columns loaded.
Overall, the purity, reproducibility, and gentle nature of qEV columns, alongside the efficiency and scalability offered by the AFC, have made qEV isolation a widely used method for EV isolation. Designed and manufactured under a quality system certified to ISO 13485:2016, SEC-based qEV columns are built to the highest standard, allowing you to spend more time focusing on your research questions.
qEV columns are available in a wide range of sizes to suit sample loading volumes from 150 µL to 100 mL, and can therefore be used to isolate EVs from cell culture media and a comprehensive range of biofluids. This makes qEV columns not only a convenient addition to your EV isolation workflow, but an essential one for the maximisation of reproducibility, efficiency, and isolate purity.
Want to learn more about the qEV Isolation platform? Head over to the Izon Science YouTube channel where there is a range of qEV videos, from How qEV and Size Exclusion Chromatography (SEC) Works For Exosome Isolation to Customise Exosome and Extracellular Vesicle Isolation With qEV and the AFC.
qEV columns have been used in over 650 publications from 2015 to 20214. To keep up to date with the high-quality research enabled by qEV isolation, subscribe to our newsletter for a quarterly summary of the latest qEV publications, and check out the qEV Publication Watch from 2022:
Figure 3. Number of articles published from 2014 to 2021. Data from app.dimensions.ai; search included qEV columns + 'Izon'.
To find out which columns are right for you, follow the two-step process outlined here.