Methodology matters. Changing one thing in your workflow can completely change your results. This is as true with extracellular vesicle (EV) isolation as it is for any other technique, but what does it mean for your research?
To discuss this, we turn to Kronstadt et al (2022)1 as a case study, delving into the detail of their results and talking about how and why contamination may be present in your EV isolates.
Hint: using qEV Gen 2 columns can significantly reduce contamination compared with other techniques!
It’s all about size. Or is it?
How can you be sure that the bioactive activities of an EV isolate are down to the EVs themselves?
To investigate this, Kronstadt et al (2022) employed the use of the trusty HEK293T cells. EVs were then isolated using ultracentrifugation (UC), tangential flow filtration (TFF) or size exclusion chromatography using qEVoriginal 35 nm Series Gen 2 columns (qEV Gen 2). They found that EV size was statistically unchanged, regardless of isolation technique (Figure 1). So far, so good.
Until, that is, the ability to inhibit lipopolysaccharide (LPS)-induced inflammation in mouse macrophages was investigated. Here, it is important to note that HEK293T cells are not thought to release anti-inflammatory EVs.
To test whether erroneous bioactivity could be detected, authors looked at the secretion of three cytokines/chemokines and undertook various experiments to understand where any inhibitory properties were coming from. ELISAs were used to measure cytokine/chemokine secretion.
Active EV component or FBS contaminant?
When applied to macrophages, EVs from isolates obtained from all three techniques resulted in the inhibition of LPS-provoked IL-6 secretion. Thus, the inhibitory factor was observed in samples isolated by all techniques, suggesting the inhibitory factor may indeed be EV-based. As fresh media was equally capable of inhibiting IL-6 as was HEK293T conditioned media, the inhibitory factor likely comes from the media rather than the cells (Figure 2). Specifically, it comes from the fetal bovine serum (FBS).
Is it all in the FBS?
When investigating LPS-provoked RANTES secretion (a chemokine), only UC isolation (and, in other experiments, TFF) resulted in inhibition. As UC is associated with a greater degree of protein contamination than qEV isolations, it is possible that the inhibitory factor in this context was a soluble protein. As with IL-6 inhibition, this inhibitory factor appears to come from FBS in the media rather than from cells themselves (Figure 3).
Non-EV components from cells can also contaminate EV isolates
Finally, for TNF-α, only TFF (and in, other experiments, UC) isolated the inhibitory factor. This factor appears to be present in the fresh media and, to a greater extent, the HEK293T conditioned media (Figure 4). This suggests that likely the inhibition of TNF-α secretion is a due to a non-EV contaminant secreted by HEK293T cells, which may also be present in media.
The bottom line
Overall, it is clear that isolating EVs with the qEV Gen 2 columns gives the purest EV isolate, which is freer of contamination from non-EV sources than either UC or TFF. The purer the isolation method, the lower the chance that something will be present in your isolation which is not an EV.
However, the authors also highlighted an important methodological consideration when using FBS. FBS should be added to media and then depleted for EVs using UC, rather than being depleted before being added to media. No matter how good an isolation method is, it cannot differentiate between the EVs you want to be in your sample, and the EVs you don’t want to be in your sample.
What does that mean for your EV isolation?
So, you may have just changed your EV isolation (or you might be contemplating doing so) and are seeing different results than you were before. Whilst this is super frustrating, it’s important to understand the reason for this change. With the qEV Gen 2 range, we have improved the purity of EV isolates more than ever before. As you can see in Figure 5, the Gen 2 range has a significantly higher EV-to-protein ratio, which is generally used in the EV field as a measure of purity. With such high purity and a fast and reproducible isolation, there are so many reasons to choose qEV Gen 2 columns for your EV workflow.
This increased purity may also change what you thought were the properties of your EVs, either compared to Legacy columns or other methods. This might mean changing a few hypotheses and that’s great! Sure, changing your hypothesis is tedious, but ultimately it’s a good thing. It means that you are more right now than you were before. Our ideas of what is ‘true’ in science always change as methods improve. Be at the forefront of the accuracy revolution and make the switch to improved EV isolation with the qEV Gen 2 columns today. Gen 2 columns are now available to suit sample sizes from 150 µL to 100 mL, and you can even buy in bulk for a discount!
To discuss how the qEV Gen 2 column range can help you achieve greater purity in your EV isolations, or to enquire about bulk discounts, contact our team.
