Extracellular vesicles or EVs are particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate.
EVs are complex signalling vehicles that can target recipient cells thanks to the EV surface molecules.
Once recognised or internalised, EVs are able to modify the physiological state of the recipient cell.
We know now that all living cells have the capacity to release EVs,
that means eukaryotes including mammalian cells, fungi, plants, protozoa,
and prokaryotes like bacteria.
As seen here,
each eukaryotic cell releases a heterogenous population of EVs,
with three broad categories of EVs depending on their biogenesis mechanism,
historically named exosomes, microvesicles, and apoptotic bodies.
The most studied EV type is exosomes.
Exosomes have a defined biogenesis mechanism: they form by inward budding of the limiting membrane of early endosomes,
which mature into multivesicular bodies and are secreted when these compartments fuse with the plasma membrane and contents get released into the extracellular space.
Exosomes are typically less than 160 nm in diameter and small as 40 nm.
Because of the defined biogenesis mechanism,
exosomes are enriched in endosome-derived markers that can be used to validate the presence of this type of EVs in a sample.
Despite having this unified origin,
the exosome population released from a single cell can be very heterogeneous and will adapt depending to the stimulus the cells are exposed to.
In this example we can see that there are exosome subtypes that can have more specific sizes ranges and molecular composition.
It is well established that exosome roles are very diverse,
not only in pathological conditions but in normal cellular activities.
Exosomes are associated with immune responses, wound healing, pregnancy, viral pathogenicity, cardiovascular diseases,
central nervous system–related diseases,
and cancer progression to name a few.
Another important and second-most studied EV type are microvesicles.
Microvesicles are formed by budding of the plasma membrane,
a releasing process that is driven by calcium-dependent signalling,
activity of several enzymes, cytoskeleton remodelling,
and externalization of specific phospholipids.
Microvesicles are shed under normal conditions and their release increases with various stimuli and pathological settings.
Microvesicles fall in a diverse range of sizes,
from 100 to 1,000 nm in diameter and they may also be called ectosomes or microparticles.
Microvesicles can package an array of distinct biomolecules,
like lipids, genetic material and proteins such as transcription factors, cytokines, and growth factors.
Markers for microvesicles include surface molecules from parental cells and integrins or CD40 among others.
Apoptotic bodies are EVs specifically generated through apoptotic cell disassembly.
Apoptotic bodies are large EVs, typically 100 nm to 5 μm in diameter.
Apoptotic bodies can harbour DNA,
RNA and proteins too,
therefore can mediate intercellular communication through the transport of these biomolecules and may contribute to the development of various disease states.
There are some markers that can help identify apoptotic bodies, like histones,
annexin V and the presence of organelles.
Other types of EV originated from significantly different cells, are prokaryotic EVs.
Prokaryotes or bacterial cells differ from eukaryotic cells,
as they have a faster growth pace and don’t have organelles.
Therefore, all molecules, machinery and processes occur free in cytoplasm.
Bacteria are smaller than eukaryotic cells,
ranging from 0.5 to 2 or 3 um and have complex and different cell wall compositions.
These unique growth and structural characteristics have resulted that EVs can be generated by different mechanisms,
depending on their cell wall composition or exposure to environmental stimulus as shown in the diagram.
Bacterial EVs have been described with sizes between 50 and 300 nm and may contain a diverse cocktail of molecular cargo,
like DNA, RNA, proteins, lipids and metabolites.
EVs can be found nearly everywhere.
Some of the most studied EV samples are listed here.
Biofluids for example, are any biological fluids obtained from the body of an animal.
In the human body,
every different cell type can release EVs into their surrounding space,
forming a complex mixture of EVs with different origins and composition in biofluids like plasma, urine and milk.
The study of biofluid EVs has significant potential for diagnostic purposes as circulating Evs reflect pathological states from originating cells.
Another well used EV sample type is that produced by lab cellular culture.
In this aspect, many different types of cells can be cultured in vitro, like mammalian cells, bacteria, and fungi.
An advantage of Evs produced by cultured cells is that production process can be controlled and optimised to meet different down-stream outcomes.
Here is an example of the complexity of human biofluid samples regarding the presence of non-EV structures that overlap in the size range of Evs,
leading to potential contamination of EV samples,
depending on the EV isolation method.
We can see different sub-populations of Evs but also structures with similar size range are Very-low-density lipoproteins (VLDL) or chylomicrons also known as ultra-low-density lipoproteins,
or different types of viruses.
Regarding the conditioned culture media with mammalian cells,
Classic Tissue culture uses flasks to grow adherent cells as a monolayer in a 2D fashion.
Cell culture depends on the cell line,
culture media and incubation parameters,
however it is also very common to add Fetal Bovine Serum to cell culture media,
which contains significant amounts of bovine EVs.
To avoid contamination,
it is best to use lab-made or commercial vesicle-depleted serum,
serum replacements,
or culture cells without serum when possible.
EV production depends on some important parameters like cell density, incubation conditions, and oxygen levels.
Another option for EV production is to grow cells in Bioreactors or high-density systems.
This bioreactor, for example has two chambers:
a top chamber for nutrient media separated from a bottom chamber for cells to grow on a support membrane.
The separation between chambers is done using a 10 kDa membrane that allows nutrients to pass through but not EVs,
so cell released EVs are retained in this compartment.
There are lots of advantages of using bioreactors: there is no subculturing cells every 2-4 days,
therefore it is cheaper if plasticware and labour work are considered.
Also with bioreactors,
released EVs are already concentrated into a very small volume,
and high-density systems often do not require FBS.
Finally, the use of a 3D cell growth structure better mimics cancer cells in vivo.
Culture of bacteria for EV production has historically been done with cells growing in suspension in very large vessels,
however biofilm is the most common growth mode in bacteria and is now starting to get attention in EV research.
Suspension bacterial cultures can reach very high cell densities in 8 or 10 hours,
which is an advantage for fast and efficient EV production.
For successful removal of bacterial cells which may have sizes as small as 0.5 um,
centrifugation and filtration is preferred.
Attention must be paid to detailed recording of culture conditions,
as quick bacterial growth enables a rapid response to environmental changes through modulation of released EVs.
How can EVs be isolated from any complex EV sample? There are few different EV separation methods with strengths and limitations regarding the duration and ease of the method,
the extent of EV loss during the protocol and specificity of resulting EV preparation.
With this in mind, Size exclusion chromatography, or SEC,
has become very popular among EV researchers as an EV isolation technique as it provides favourable outcomes in all aspects mentioned before.
MISEV, a guideline developed by expert EV researchers,
has classified EV isolation methods based on their EV recovery and specificity properties,
positioning SEC as a high specificity method for EVs.
In addition, MISEV recommends minimal characterisation of purified EV preparations to demonstrate the presence of true EVs
and not co-isolated contaminants as well as presence of the specific EV subtype that researchers aim to isolate
and the absence of non-EV structures or other EV subtypes that are not of interest.
With all the methods and technologies available to study EVs, each field is facing specific challenges.
Academic researchers often are pioneers in discoveries, often diving into unknowns.
Most EV research focuses on characterising the biophysical and functional heterogeneity of EVs from different type of samples and determining biological roles in cellular activities or diseases.
EV research provides the knowledge foundation to any potential medical applications.
In Diagnostics,
the challenge is using the smallest amount and least invasive biofluid to accurately identify a disease with sufficient sensitivity and specificity,
within the complex and natural variability among all individuals or disease states.
Luckily, EVs are present in all biofluids and contain a cocktail of molecules which have potential to be used as biomarkers.
The field of EV-based therapeutics has quickly expanded,
as EVs have the potential to be used for numerous medical benefits.
Administration of EVs could restore the lack or deficiency of a particular molecule or be used to intervene in pathways and regulate physiological processes as treatment.
Attractiveness of therapeutic application of EVs relies on several EV properties,
including efficient cell entry and cargo delivery,
low immunogenicity,
low cytotoxicity,
high biocompatibility,
and tissue tropism.
The field also requires mass production of EVs,
where precise and reproducible high standard and safe EV preparations are mandatory.
Izon Science supports the growing areas of EV diagnostics,
therapeutics and research by providing a rapid and reproducible solution for EV isolation.
This is a critical part of reducing the measurement error in analysis downstream.
SEC-based qEV isolation columns are available to support a range of sample loading volumes.
Alongside this,
Izon also offers instruments for tunable resistive pulse sensing analysis to enable precise physical characterisation,
an important component of characterising EVs for research and dose measurement.
You can learn more by visiting Izon’s website at www.izon.com