Hi, my name is Michele Trott and I am here to share considerations for storing EV-containing samples prior to EV isolation.
Specifically, those derived from blood, semen and saliva.
I will also be sharing general tips about the storage of purified EV preparations after EV isolation.
The most critical steps in EV research are EV isolation and analysis,
with both steps including many variables that directly impact results.
As it is typically impractical to perform EV isolation and analysis on the same day as sample collection,
sample storage is often necessary.
Researchers want to be confident that their results reflect the original physiological state of EVs.
They want to know that all pre-analytical steps such as sample collection, handling
or EV isolation are consistent in terms of their impact on recovery,
and that physical properties or biological functions of the EVs have not been significantly affected.
It’s also important to note that EVs are heterogenous,
so some EV subpopulations may be differentially impacted by pre-analytical factors.
Each sample type comes with its own complexities.
So ideally, for each sample type and stage of processing – like sample collection,
handling and EV isolation - there would be standardised protocols that maintain EV function and integrity.
In reality, there are still significant knowledge gaps around the impact that these procedures have on EVs.
And for some less common sample types, this remains unexplored.
In general,
sample storage may be required before or after EV isolation and this has started to gain attention among researchers as an important source of variability.
Blood-derived EVs are commonly used in biomarker discovery,
as the composition of circulating EVs can vary with disease.
Blood is also one of the most complex biofluids and is typically analysed in the form of serum or plasma – samples which differ significantly in EV composition.
Plasma is the liquid portion of whole blood collected after cells have been removed from a blood sample treated with an anticoagulant agent.
In contrast, serum is the liquid fraction of whole blood that is collected after the blood has been allowed to clot.
As coagulation is driven by platelets,
platelet-derived EVs are present in serum samples,
which may or may not be of interest to you.
In general, there are two major pre-analytical variables that can affect EV composition in blood samples.
Firstly, the physiological factors of the individual from which EVs were collected.
And technical factors can have an impact,
such as the vessel used to collect blood,
the processing duration,
and storage protocols used.
When collecting many blood samples, it can be impractical to remove cells immediately.
This is not ideal, as blood cells can continue to release EVs in vitro.
When dealing with unprocessed blood samples,
it is therefore best to: centrifuge samples to remove cells within an hour of collection,
leave blood samples at room temperature,
and avoid agitation.
Removing platelets is a critical step if you don’t want platelet-derived EVs included in your analysis,
since activated platelets can release significant amounts of EVs into the sample,
overtaking the EV population collected originally from blood.
As shown in this figure,
there are protocols available for reducing or removing the platelet load,
that harness size differences between platelets and blood cells.
It is important to highlight that studies have revealed different responses of microvesicles or exosome populations to different protocols,
for example to the storage of unprocessed blood.
Therefore, more studies are needed to fully unravel the impacts on these EV subtypes.
Once plasma or serum is obtained, EV samples are a lot more stable and tolerant of wider temperature ranges.
and researchers continue to investigate the effects of storage temperatures and duration on EV samples.
For example, a reduction of EV concentration and increase in protein
has been demonstrated in plasma stored at negative eighty degrees for 6 months, compared to 4 weeks
suggesting potential EV damage – or aggregation over time.
Repeated freeze-thaw cycles of plasma or serum can decrease EV functionality too,
therefore it is recommended to prepare aliquots for different downstream applications.
Moving to a less commonly used diagnostic fluid, seminal EVs are also gaining relevance.
For example,
anti-HIV-1 activity has been demonstrated in EVs,
highlighting their potential as therapeutics or cancer biomarkers.
Understanding the storage effects on seminal EVs could enable the use of samples from large sperm biobanks for research.
In general, seminal EVs may be derived from a range of cellular origins,
for example epididymosomes released by the epididymal epithelium,
and prostasomes,
released by prostate epithelial cells.
Similar to other EV samples,
the analysis of fresh samples is preferable where possible,
but again, this is often impractical.
It is recommended that fresh semen samples are left to liquefy at room temperature immediately after collection.
Spermatozoa and large pieces of undissolved seminal gel are pelleted by centrifugation allowing the storage of seminal plasma contained in supernatant at -80°C until EV isolation.
Visible light can affect sperm motility,
therefore all semen samples are typically maintained in the dark prior to analysis,
as a precaution.
Saliva-derived EVs have also caught the attention of researchers due to their biomarker potential.
Saliva is not only a complex biological fluid containing antimicrobial compounds,
antibodies and glycoproteins,
but also is a non-sterile biofluid with a significant contribution of EV release from microbial cells.
Many factors can impact the composition of saliva EVs,
and these should be recorded,
including details like food and drink intake,
smoking and physical activity,
the presence of oral disease,
and teeth brushing given its potential for blood contamination.
Due to the high viscosity of saliva,
a 1 to 1 dilution of saliva with PBS or Tris-buffered saline is recommended before continuing with processing.
Cell removal by low-speed centrifugation is recommended before sample storage at -80°C.
Regardless of the origin or type of EV sample,
purified EV preparations obtained after EV isolation are often considered to be fairly stable.
However, the choice of EV suspension buffers,
storage time, and temperature can still have a significant effect on the EVs.
Best practice suggests that for every developed procedure involving collection,
processing and EV isolation from a specific sample type,
the resulting purified EV preparation should initially be fully characterised fresh after isolation and prior to storage,
ruling out microbial contamination during the EV isolation process and storage.
Overall, there are many unknowns regarding the storage of purified EV preparations.
Phosphate-buffered saline (PBS) is mostly used for EV resuspension and storage,
However, other buffers such as Hank’s Balanced Salt Solution or Tris-buffered saline are also used.
Freeze drying and subsequent storage at -80°C has been studied and compared to storing small EVs at the same temperature,
without freeze-drying first.
While EV bioactivity was unaltered,
small EV concentration and integrity was not assessed,
therefore more comprehensive studies are required.
The addition of cryoprotectants to EV storage buffers in purified EV preparations may prove beneficial.
In this figure we can see that EVs stored with trehalose show greater EV bioactivity against macrophages in comparison to EVs stored with PBS.
Well that’s all for part 1 of this video series – be sure to keep an eye out for part 2,
with more tips about the storage of EV samples from milk,
urine and cell culture conditioned media and some more general considerations about storing purified EV preparations after EV isolation.
In the meantime, you can learn more by downloading our app note about EV storage, which is linked below.
Thanks for watching!.