Hi, my name is Michele Trott and in this video,
I will be sharing some considerations for the storage of EV samples derived from milk,
urine, and cell culture media.
As mentioned in the previous video,
considering EV storage is an important part of obtaining results that reflect the original physiological state of EVs in the sample.
Researchers need to be confident that their methods have not altered the recovery,
physical properties or biological functions of the EVs.
This includes all steps prior to EV analysis,
and pre-analytical processes such as sample collection,
handling, processing and EV isolation.
As there are unique complexities associated with each sample type,
ideally there should be standardised,
sample-specific protocols for sample collection,
handling and EV isolation that maintain EV function and integrity.
It is often impractical to isolate and analyse EVs immediately after sample collection,
therefore it may be necessary to introduce the additional variable of short or long-term sample storage.
Storage may be required pre or post EV isolation,
and this has started to gain attention among researchers as a variable worth considering for all sample types.
Milk-derived EVs have been studied in many species and milk EVs from farmed animals have received particular attention for their therapeutic potential.
Like other biofluids, milk has its own complexities and challenges for EV isolation.
It has an abundance of other nanostructures with a similar size as EVs such as fat globules,
casein micelles and aggregated proteins.
Also, milk contains host and bacterial cells, therefore by definition is not a sterile biofluid.
One of the first recommendations for milk samples is to remove cells and the cream layer before storage,
otherwise stress-induced EVs may be released during apoptosis.
Also, caution must be taken when removing or separating the fat layer before continuing for processing or storage,
as the fat layer is positioned in the upper phase after centrifugation.
EV isolation in high-casein milk samples is improved by casein-removing treatments,
including treatment options with EDTA,
acetic acid or hydrochloric acid.
Urine is a convenient and popular source of EVs that can be sourced repeatedly,
non-invasively, and in large quantities.
Urine is not a sterile biofluid,
therefore minimising the influence of bacterial contamination is a significant factor for urinary EV analysis.
Also, urine contains significant amounts of aggregating glycoprotein uromodulin or Tamm Horsfall, which can entrap EVs.
In general, studies have shown that protease inhibitors prevent the significant degradation of membrane-bound proteins and small EVs,
when added to fresh unprocessed urine immediately after collection.
Fresh urine should be processed at room temperature or 37°C,
as cold temperatures can promote uromodulin aggregation and reduce EV recovery.
Otherwise, thawed frozen urine samples should be followed with processing at 4°C.
Researchers have used different approaches to eliminate uromodulin.
For example, as shown in the figure here,
the addition of DTT to cell and debris free urine results in the significant removal of filament-looking uromodulin aggregates.
Other methods include the addition of Zinc Sulfate to cell-free and debris-free urine with consecutive steps to remove uromodulin.
Meanwhile, others may remove uromodulin by adding detergents like CHAPS to the final EV preparation.
A single freeze-thaw cycle in cell-free urine has been shown to significantly decrease EV concentration,
therefore freeze-thaw cycles should be minimised.
Urine should be stored at −80°C as storage at higher temperatures, even -20C, can increase EV loss.
EVs released by cultured cells is the most effective way of producing significant amounts of EVs for downstream analyses,
as systems can be easily scaled and optimised as needed.
In this way, the composition of cell culture derived EVs can be heavily determined by cell culture parameters.
Therefore, it is recommended to keep a thorough record of the EV production phase.
Also, it’s important to be aware that the common cell culture supplement,
fetal bovine serum,
is itself a source of EVs.
Serum is an important component for sustaining cell growth,
however you may choose to use EV-depleted fetal bovine serum,
and this may be lab-made or commercially available.
It is a good idea to make assessments of cell death in cell culture; as if this occurs,
the sample will become contaminated with EVs derived from stress-induced and/or dead cells.
Although most cases of microbial contamination might quickly become apparent,
some microbes such as Mycoplasma have dormant stages that enable them to remain unnoticed in cell culture for days or weeks.
Therefore, appropriate and frequent checks for microbial contamination should be performed.
As with the majority of the other EV sample types,
it is recommended to remove cells and large debris before storage,
to avoid the in vitro generation of cell-derived EVs or EV degradation.
It is good practice to characterise fresh EV preparations, so that you can assess potential storage effects.
For storage of post-EV isolation samples and all types of purified EV preparations,
rapid freezing is often preferred over slow freezing.
Freezing at −80°C is a standard method of storage in buffer-resuspended EVs,
and storage at higher temperatures has been shown to alter many different EV properties.
Another thing to be aware of is that storage protocols can also affect EV zeta potential,
which is a measure of the effective charge of a particle in a certain medium.
Zeta potential can affect interactions between an EV and a cell, and it may alter EV stability or aggregation.
This figure shows how storage can affect zeta potential.
After storing EVs at −80°C for six months, EVs shifted to having a positive charge.
A multitude of physiological roles has been attributed to the EV RNA cargo,
leading to significant interest in the diagnostic and biomarker potential of this biomolecule.
Therefore, protecting EV-RNA cargo during storage is incredibly important.
Storage at -80°C has been reported to stabilise RNA cargo in small EVs,
whereas storage at -20°C or 4°C has shown significant RNA reduction.
This is in line with the knowledge that RNA is highly sensitive to temperature.
In addition, we can see in this figure that long-term storage over 2 years leads to significant RNA degradation in small EVs,
as shown by the reduction in peak size.
Because of this,
long-term storage is not recommended and this also highlights the need for studies aimed at identifying RNA-protecting strategies.
Thanks for watching and I hope this was helpful.
To learn more about storing EVs, and other sample types, be sure to download the app note on EV storage linked below.