In extracellular vesicle (EV) research, there is a tale as old as time (or at least as old as the late-2000s, maybe early 2010s):
Researcher tests biofluid/conditioned culture media and finds that it impacts cells or model animals – yay!
Researcher then isolates EVs from the biofluid/conditioned culture media and still sees the impact – double yay!
But is that really enough to suggest that the EVs themselves are having the effect? Those writing the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines1 certainly don’t think so, and we’re inclined to agree with them. Especially if you’re planning on turning your discovery into a therapeutic. You’ll want to be sure that the active ingredient is in your EVs before you push forward with production.
The anatomy of an extracellular vesicle isolate
Here’s the thing, the uncomfortable truth: No isolation technique will isolate only EVs. There will always be some contamination from one thing or another. Mostly this contamination comes in the form of soluble protein. When isolating EVs using our qEV Gen 2 columns, your EV isolate should be depleted of around 99% of the protein from the original sample. Even then, 1% remains, and this could skew your results. It’s unlikely to, but it could. Especially if your sample is highly enriched for a particularly potent protein.
So, what do you do? If the EV isolate recapitulating the impact of the whole starting sample isn’t enough, where do you turn next?
Luckily, the path forward is a relatively easy one to walk. You already have what you need. Especially if you use qEV columns to isolate your EVs. Simply collect both the EV-rich volume (which we call the pooled collection volume, or PCV) and the post-PCV volume which is rich in proteins. Testing both alongside each other will answer the question of where the active ingredient lies. It should go one of three ways:
- You see an impact from the EV-rich volume and either none or less from the protein-rich volume? Congratulations, you have yourself an EV-associated active factor.
- You see no or little impact from the EV-rich volume but do see an impact from the protein-rich volume? Also congratulations! You have yourself an active ingredient in the protein-rich portion. It could be anything though, sorry about that.
- Both have the same level of impact. Maybe there’s an active factor in both fractions.
In theory, this tactic should work. But how does it play out in practice?
The EV-rich vs protein-rich tactic
The EV-rich vs protein-rich tactic was employed by Yang et al. (2023).2 They wanted to know which component of their mesenchymal stem cell (MSCs) conditioned media was having a beneficial effect on cartilage tissue regeneration. Articular cartilage is that smooth white cartilage covering the ends of bones in a joint. This tissue is avascular and so is ill-adapted to self-repair after being damaged. Since MSCs likely don’t survive transplantation to any significant degree,3 perhaps it is their secretome that has positive impacts on tissue regeneration?
The first thing that Yang et al. did was to identify where EVs and proteins were eluting. This is a great first step, as it helps you determine where to take your PCV and any post-PCV samples. As you can see from Figure 1, there was a clean and clear separation between EVs and proteins in the qEV fractions. This allowed them to take clean EV-rich and protein-rich samples. We’ll call these MSC EVs and MSC proteins, respectively.
How do EV-rich vs protein-rich fractions compare in vitro?
Next the authors turned to the real business of the project, comparing the MSC EVs and MSC proteins with MSC conditioned media. They also did this comparison with and without hypoxia as they had found that MSCs exposed to hypoxia produced conditioned media which was more beneficial for joint repair. Could the beneficial component/s produced in response to hypoxia be within EVs? They set out to find out.
For all treatments, normoxic treatments had no effect, so we will focus on the hypoxic treatments.
Chondrocytes are important to joint regeneration. Their proliferation (Figure 2A) was improved by hypoxic MSC conditioned media (yay!) and by hypoxic MSC EVs (double yay!), but less so by hypoxic MSC proteins (triple yay!). A similar pattern was seen in undesirable inflammatory-induced senescence (i.e., that state in which cells continue to exist but shut down their proliferative capabilities, among other pathways) in chondrocytes (Figure 2B). So what does this mean? Well, going by our three-outcome system above, it means that at least some of the beneficial components of MSC conditioned media on chondrocyte biology are within EVs.
* = p < 0.05 vs (A) serum-free media or (B-E) inflammatory stimuli unless otherwise indicated. The difference between the inflammatory stimuli (Inflam. stimuli) and the lack of inflammatory stimuli (No. inflam. stimuli), or the impact of serum-free media is shown by the shaded area.
Another important factor in cartilage regeneration is the extracellular matrix (ECM; Figure 3). Inflammatory stimuli (in this case, IL-1β) damages the ECM in part by increasing breakdown by ADAMTS54 and altering the collagen makeup to favour non-articular cartilage collages such as collagen I over the usual collagen II. Can MSC EV treatments reverse this?
Hypoxic MSC conditioned media reversed the impact of IL-1β, moving towards a normalisation of the extracellular matrix. Hypoxic MSC EVs did the same, almost perfectly replicating the degree of normalisation seen with conditioned media. However, hypoxic MSC soluble proteins were decidedly lacklustre, not really doing anything except mildly improving collagen II secretion.
Clearly then, the beneficial factor(s) for the extracellular matrix is EV-associated. But what does this all mean for actual joint regeneration? For this, the authors turned to a rat model.
The regenerative potential of mesenchymal stem cell extracellular vesicles
When it comes to in vivo studies, Yang et al. did not include the soluble protein control. Which is a shame, because whilst EVs were found to be more potent in vitro, that’s not necessarily true in vivo. We understand that this might reduce the number of animals needed for experiments, but at what cost in the long run? If the therapeutic fails, then those animals were used in vain anyway. It’s better to do it properly.
Instead, Yang et al. went with the tactic of comparing MSC conditioned media with a lower and higher concentration of EVs, all of which they introduced by intra-articular injections. A drill-induced injury in the trochlear groove of the femur was introduced prior to treatment. Could MSC EVs help in tissue regeneration?
As in vitro, normoxic treatments were of no help. However, the regeneration score of the joint was drastically improved by hypoxic MSCs conditioned media and EVs. The synovitis score (i.e., inflammation of the synovial membrane) was lowered by hypoxic MSC conditioned media, as compared to the control treatment of serum-free media. However, whilst there was a trend towards a reduction synovitis with hypoxic MSC EVs, this did not reach statistical significance.
The power of appropriate comparisons
When it comes down to it, Yang et al., managed to show that that it was most likely the EV component of their isolate having a beneficial effect, at least in vitro. Isolating your EVs using qEV columns allows you to effectively separate the EV-enriched and protein-enriched portions of your sample. Comparing them to the starting sample puts you in the best position to produce an effective therapeutic, whatever format that might take. It should be considered the gold standard when assessing potency.
If EV therapeutics development is your thing, then we can help. We have a proven track record in EV isolation for therapeutics development, and our scientists can help to design a customised sample processing and EV isolation workflow suited to your therapeutic and scale of production. Which means that if you are at the start of your EV therapeutic journey or well on your way, we can help propel you towards success. Take a look at what we can do to help.
