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A comprehensive guide to 
extracellular vesicle counting, 
size determination, and 
characterization
How flow cytometry enables sensitive, 
consistent, and flexible EV analysis
Flow cytometry enables sensitive, consistent, and flexible EV analysis:
• EVs mediate cell-cell communication in the human body. 
Studying them can help researchers understand and 
characterize physiological mechanisms as well as diseases, and 
develop new treatments.
• To thoroughly understand EVs, researchers must be able to 
quantify them with count and concentration, determine their 
size and characterize them.
• Many techniques can be used to analyze EVs, including 
some that can provide a combination of counting, size and 
characterization - but each has drawbacks.
• Nanoscale flow cytometry is emerging as a tool to bring 
depth, efficiency, and quality to EV research, enabling analysis 
of even small EVs.
INTRODUCTION
Accelerating answers
2
Contents
1. Introduction
2. EVs: A goldmine of information
3. How to count and characterize EVs
4. Simplify EV analysis with multi-measurement platforms
5. The growing promise of flow cytometry
6. Nanoscale flow cytometry: Bring clarity to EV analysis
3
01 Introduction
What are EVs and why is it 
important to study EVs? 
Extracellular vesicles, or EVs, are lipid bound vesicles, secreted by cells into the extracellular space. They are 
carriers of biologically active molecules and can travel to different targets to execute specific biological functions. 
The term EV refers to a broad range of carriers that were previously classified into three groups depending on 
their cellular origin — exosomes, microvesicles, and apoptotic bodies. However, there is not yet a consensus on EV 
subtypes, such as exosomes. As a result, nomenclature is being steered towards operational terms that refer to :1
• Their physical characteristics, such as size and density
 - Small EVs: 200 nm
 - Low, medium, or high density EVs
• Biochemical composition, for example CD9+, CD63+, CD81+ EVs, RNA, lipids, etc...
• Descriptions of conditions or cell of origin — podocyte EVs, for instance
EVs are found throughout the human body, including in the brain, eyes, lungs, and reproductive organs, as well 
as in the circulatory, digestive, and urinary systems. Owing to this ubiquity, and their ability to mediate cell-cell 
communication and help maintain homeostasis, EVs offer deep insights to those who study them. Crucially, they 
provide insights about the health and disease profile of their cell and organ of origin, making it easier to diagnose 
and characterize diseases. What’s more, EVs are an ideal candidate for novel drug delivery methods in areas such 
as oncology.
As researchers in a range of fields realize the potential value of better understanding EVs, it is vital they get as 
much information from them as possible — which requires using the most appropriate tools. 
APPLICATION NOTES & CASE STUDIES
• INTO THE WORLD OF EXTRACELLULAR VESICLES (EVs)
Extracellular vesicles (EVs) are non-replicating structures surrounded by a lipid bilayer that are naturally 
released by all cell types. These tiny particles have become a subject of great interest for scientists 
worldwide, who seek to understand their biological roles and explore their potential as therapeutic cargo. 
By unraveling the heterogeneity of EVs, we can enhance our understanding of cell-to-cell communication 
and gain insights into the role they may play in various diseases. This document aims to provide you with 
essential information about EVs and shed light on why they have emerged as crucial entities in the fields 
of biology, immunology, virology, oncology, microbiology, and more.
4
To prepare EVs for analysis, they must be isolated by one of several approaches. Ultracentrifugation is 
the gold standard for EV isolation, as it can reproducibly isolate specific subpopulations of EVs. Additionally, 
researchers can use density modifiers to increase purity, making it a highly effective technique. Size-exclusion 
chromatography (SEC), which separates particles by size, is also a viable option, and is particularly strong at 
removing high-abundance proteins. 
Newer approaches have emerged that simultaneously concentrate and purify the sample. Tangential flow 
filtration, for example, is growing in popularity as a rapid, reproducible, and scalable EV purification approach. 
Another method, dielectrophoresis, can achieve high purity, recovery, and throughput, and is compatible with 
multiple applications including immunochemistry and mass spectrometry. 
Preparation is key
01 Introduction
5
CELL CULTURE
Cultivate productive sources 
of extracellular vesicles with 
automated cell culture, viability 
measurements, and media 
analysis.
SEPARATION 
FROM CELLS, MEDIA
Efficiently refine culture 
supernatant and separate 
desirable microparticles 
from debris and large 
particulates.
PURIFICATION
Flexibly purify the desired 
size and density of extracellular 
vesicle from a heterogeneous sample. 
Customizable protocols can be fit 
to your specific needs.
 VESICLE ANALYSIS
Discover the potential of your 
purified extracellular vesicles: 
perform small particle analyses 
to study physical characteristics, 
and extract RNA to understand
 vesicle cargo.
After purification, EVs are ready for analysis — but this can be challenging for three key reasons:
• Size: Small EVs are difficult to resolve from noise, so often require specialized tools. 
• Heterogeneity: Particles have broad size ranges and variation in cargo, which need accurate 
characterization.
• Lack of standardization: There are limited reference materials or standardized measurement systems for 
EV composition determination, and few standard cell-based platforms to produce EVs. Additionally, the EV 
research community has not yet reached any consensus on standardization, further exacerbating the issue.
The EV research community is taking steps to try to improve EV analysis. The International Society for Extracellular 
Vesicles (ISEV), for example, has tried to overcome a lack of standardization by publishing guidelines for 
Minimal Information for Studies of Extracellular Vesicles (MISEV), which have been created using feedback 
compiled from ISEV expert task forces and more than 1000 researchers.1, 2 However, getting the data needed to 
meet standardization guidelines often requires specialized techniques. Without them, researchers may obtain 
inconclusive and non-reproducible results, which will ultimately stall scientific breakthroughs.
To gain true understanding of EVs, accurate, precise, and reproducible techniques are vital. Thankfully, the field is 
continually and rapidly evolving, and powerful ways to analyze EVs — such as nanoscale flow cytometry — are on 
the horizon. 
The complexities of 
EV analysis
By the end of this eBook, 
you will understand:
• What information about EVs is useful to obtain
• The techniques available to analyze EVs, and their 
strengths and limitations
• How nanoscale flow cytometry is emerging as a useful 
tool for EV analysis
01 Introduction
6
A goldmine of information
EVs contain a wealth of information just waiting to be extracted. Researchers can gather single particle data, 
where they analyze each EV individually for a more detailed, granular picture. However, bulk methods can also 
provide detailed insights into a biological system. 
Ultimately, whether performing bulk 
or single analysis, there are three key 
goals that researchers wants to reach — 
EV concentration, size determination 
and EV characterization. 
 
Key takeaways
To get deeper insights from EVs, researchers must:
• Count: 
 Ascertain how many EVs there are per sample
• Determine their size: 
 Calculate individual EV size and distribution 
• Characterize: 
 Determine cargo composition of each EV02 EVs: A goldmine of information
7
In EV counting, researchers determine the number of EVs per sample. Counting is highly useful for biomarker 
discovery and disease diagnosis, as it can be used to determine physiological activity in the cellular and 
organ systems where the EVs originated. 
The measurement can also help researchers monitor disease progression and therapeutic efficacy, too. 
For example, increased EV levels may show disease progression, whereas a decrease may indicate a positive 
response to therapy.
Determine count and 
concentration
02 EVs: A goldmine of information 
Size determination
APPLICATION NOTES & CASE STUDIES
• History of EV Research & Methods
THE DISCOVERY OF EXTRACELLULAR VESICLES (EVs):
TRACING THE JOURNEY FROM INCEPTION TO PRESENT DAY
 
Researchers also need to characterize the EV itself and identify protein cargo. Analyzing EVs in this way helps 
researchers achieve three important goals:
• Understand EV biological functions: Decipher specific messages/signals that EVs carry between cells.
• Diagnose diseases: Uncover disease-specific molecules (DNA, RNA, microRNAs etc.) in cargo. 
• Investigate therapeutic potentials: Discover EV composition and ensure they provide precise/consis-
tent dosing when used therapeutically.
Characterization
8
When working with EVs, researchers usually deal with polydisperse samples. By measuring EV size, it is possible 
to differentiate between different subtypes, helping better understand their origin/function. It can also help them 
determine size distribution, which is vital to understand physiological or pathological conditions. And finally, 
measuring EV size is pivotal to standardization and quality control of EV preparations, which is particularly useful 
for therapeutic applications.
03 How to count and characterize EVs 
Size determination
Characterization
9
ELISA: Analyze cargo and determine heterogeneity
Strengths
 
✔ Compatible with a range of samples
✔ High resolution
✔ High specificity 
✔ Quantitative
✔ Reproducible
✔ Simple to perform
Count
Characterization
Many approaches to count and characterize EVs and determine their sizes exist, each having its own strengths 
and limitations. In this chapter, we explore the different methods available to researchers to either count or 
characterize EVs as described in Chapter 2.
Spectrophotometry: Quantifying EVs
Strengths
 
✔ High resolution
✔ High sensitivity
✔ Simple to perform
✔ Requires no other reagents 
Limitations
x Affected by impurities
x Indirect (measures protein content in 
μg/mL, but not EV concentration)
x Low throughput
EVs
Limitations
x Limited dynamic range
x Sample preparation can alter protein 
structure, affecting accuracy 
x Target-dependent
Mass spectrometry-based proteomics: Analyze cargo in bulk
Strengths 
✔ Can identify protein aggregates 
 and contaminants
✔ High resolution
✔ High specificity and sensitivity 
✔ High throughput
Immunoblotting/SDS-PAGE: Identify cargo in bulk
Strengths 
✔ Compatible with small volumes
✔ Cost-effective
✔ Multiplexing capabilities
✔ High specificity and sensitivity
✔ Rapid turnaround time
✔ Simple to perform
03 How to count and characterize EVs 
Characterization
EVs
10
EV array and bead capture: Use for protein profiling/phenotyping
Strengths
 
✔ Easy to perform
✔ High throughput
✔ Multiplexing capabilities
✔ High sensitivity
Limitations
x Biased by dependency on tetraspa-
nin or other singular affinity site
x Difficult to standardize
x Doesn’t define true EV heterogenei-
ty (array consists of one marker per 
bead)
x Limited to known targets
Limitations
x Complex sample preparation
x Limited quantifiability
x Potential for antibody cross-reactivity
x Prone to variability
Limitations
x Complex data analysis
x Difficult to detect/quantify low ab-
undance components in complex 
samples
x Expensive
x Long run time per sample
x Requires larger sample volume
Analyzing one aspect of 
EVs at a time: effective but 
complicated
Many techniques are available to count or characterize EVs, each with its own strengths. However, they also have 
their own limitations, which need to be carefully assessed and balanced in line with research aims. What’s more, as 
each technique only provides one type of measurement, researchers will need to combine multiple approaches to 
get the EV data they need — which can be complex, time-consuming, and costly. There are, however, techniques 
that provide multiple measurements, simplifying EV analysis, which are explored in Chapter 4.
 
Key takeaways
• Researchers can use spectrophotometry to count EVs 
indirectly, but it is affected by impurities
• Many techniques are available to characterize EVs, but 
each has its own drawbacks that need to be considered 
• Methods that obtain multiple measurements can 
 simplify EV analysis
Mass spectrometry-based proteomics: Analyze cargo in bulk
Strengths 
✔ Can identify protein aggregates 
 and contaminants
✔ High resolution
✔ High specificity and sensitivity 
✔ High throughput
Immunoblotting/SDS-PAGE: Identify cargo in bulk
Strengths 
✔ Compatible with small volumes
✔ Cost-effective
✔ Multiplexing capabilities
✔ High specificity and sensitivity
✔ Rapid turnaround time
✔ Simple to perform
03 How to count and characterize EVs
11
EV array and bead capture: Use for protein profiling/phenotyping
Strengths
 
✔ Easy to perform
✔ High throughput
✔ Multiplexing capabilities
✔ High sensitivity
Analytical ultracentrifugation (AUC): 
Isolate EVs, quantify them, and measure size, heterogeneity, and shape
Strengths
 
✔ Can be analyzed during the same step
✔ Good for broad size ranges
✔ High resolution
✔ Wide range of data obtained
Resistive pulse sensing: 
Quantify and determine size profile of single EV particles
Strengths
 
✔ Compatible with small volumes
✔ Cost-effective
✔ Multiplexing capabilities
✔ High specificity and sensitivity
✔ Rapid turnaround time
✔ Simple to perform
04 
Rather than needing to perform different techniques for the analyses needed, some methods allow you to obtain 
multiple measurements at once. For instance, many systems can be used to both quantify EVs and determine their size. 
12
Limitations
✘ Difficult to analyze highly heterogeneous samples
✘ Relatively high upfront cost
Limitations
✘ Complex sample preparation
✘ Limited quantifiability
✘ Potential for antibody cross-reactivity
✘ Prone to variability
Quantification and size measurements
Transition electron microscopy (TEM): 
Quantify EV and measure their size. Individual EVs can be counted in micrographs to determine concentration.
Strengths
 
✔ Can elucidate specific EV features (e.g., surface proteins) 
 when used with immunolabelling
✔ Direct visualization
✔ High resolution (can see detailed morphology of individual EVs)
✔ High sensitivity
* The sample section analyzed may not represent the whole EV population.
Limitations
✘ Access limitations 
✘ High running costs
✘ Laborious sample preparation, making it low 
throughput and challenging to standardize
✘ Potential bias*
Simplify EV analysis with 
multi-measurement platforms
04
13
Simplify EV analysis with 
multi-measurement platforms
Simplify EV analysis with 
multi-measurement platforms
Super-resolution microscopy (SRM): 
Determine EV size and enables detection and quantification of single proteins and nucleic acids at the 
sub-vesicular level.
Strengths
 
✔ Accurate
✔ High resolution
✔ Precisely images/analyzes EVs in cells or solution
✔ Uses low sample volume
* The sample section analyzed may not represent the whole EV population.
As well as quantifying and determining the size of EVs, some techniques can characterize them, too.
Combined quantification, size profiling, 
and characterization
Nanoparticle tracking analysis (NTA): 
Measure EV size and concentration. Enables phenotyping with limitations.
Strengths
 
✔ Allows real-time visualization
✔ High sensitivity✔ Rapid turnaround time
✔ Relatively wide dynamic range
✔ Simple to perform
* The right concentration is essential for accurate results. † Larger particles more strongly influence the profile obtained. ‡ NTA uses Brownian motion and cannot see all
Limitations
✘ Laborious sample preparation* 
✘ Less reproducible owing to time/test and operator 
variation
✘ Poor results for polydisperse suspensions†
✘ Requires high concentration
✘ Uses estimates‡
Limitations
✘ Complex sample preparation
✘ Manual process-heavy, limiting speed and throughput
✘ No size distribution analysis
✘ Potential bias*
Dynamic Light Scattering (DLS): 
Determine EVs size and size distribution. 
Strengths
 
✔ Low hands-on time
✔ Low running costs
✔ Low sample volume required
✔ Measures a wide range of particle sizes
✔ Rapid turnaround
✔ Simple to perform
* The sample section analyzed may not represent the whole EV population.
Limitations
✘ Poor results for polydisperse suspensions*
✘ Low resolution
Simpler EV analysis 
requires a trade-off
14
04 
Simplify EV analysis with 
multi-measurement platforms
Researchers can potentially reduce instrumentation and reagent costs, save time, and simplify their EV analysis 
by determining size and quantity using the same instrument. Both SRM and DLS can characterize, count and 
determine the size of EVs, making them highly useful techniques. 
However, these methods also have drawbacks. Not only is SRM low throughput, for example, but it is time-
consuming — despite the many types of data it can obtain. DLS has a quicker turnaround time than SRM, but the 
resolution is poorer, and it isn’t as effective when there are a range of particle sizes.
So, is there a more optimal technique available, where researchers don’t have to make a trade-off between 
throughput and resolution? Flow cytometry may be the answer, providing higher resolution and higher throughput 
— even for polydisperse samples. 
 Key takeaways
• Multi-measurement platforms can simplify EV analysis, 
combining multiple approaches in one system
• Both DLS and SRM can count, quantify, and 
 characterize EVs
• DLS is simple-to-use and rapid, but is lower resolution 
and only suitable for monodisperse samples
• SRM offers higher resolution, but has limited 
 throughput and risks potential bias
While flow cytometry is a well-established technique for analyzing cells and particles, it is currently underused for 
EV analysis, yet it can count, determine the size of, and characterize EVs. So how does it work?
Flow cytometry detects and measures the physical and chemical characteristics of a particle by measuring 
fluorescence intensity produced by fluorescently labeled antibodies specific to proteins on or in the EVs. 
Accordingly, researchers can obtain detailed characterization by identifying specific subsets and assessing cargo.
Flow cytometry brings 
three benefits to EV 
research: depth, 
efficiency, and 
quality.
Confidential - Company Proprietary
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05 How to count and characterize EVs
15
Depth
• Compatible with 
 limited sample sizes
• High sensitivity
• Single-particle 
 resolution
Efficiency
• High throughput*
• Rapid turnaround 
(hours)
• Simultaneous 
 multi-parametric data 
collection
Quality
• High dynamic 
range and precision, 
to characterize hete-
rogeneous and poly-
disperse samples
• Quantitative data 
• Reproducible
*Flow cytometry can measure thousands of events per second. 
Despite all its strengths, flow cytometry has one significant drawback: it does not provide precise size 
measurements for EVs lower than 100 nm. It is also important to note that when using positive or negative 
pressure, the flow cytometer needs a hall sensor to accommodate a counting estimate.
“While traditional flow cytometry has many strengths, 
it does not provide precise size measurements 
for EVs lower than 100 nm”
Flow Cytometry 
Key takeaways
• Flow cytometry brings depth, efficiency, and quality 
 to EV research
• The technique doesn’t provide precise measurements 
for smaller EVs 
• Nanoscale flow cytometry is emerging as a tool 
 to help researchers analyze smaller EV subpopulations
Nanoscale flow cytometry is emerging as an alternative to traditional flow cytometry for EV analysis. While 
it is built on similar principles to flow cytometry, as the name implies, nanoscale flow cytometry works on 
the nanoscale — making it ideal for analyzing small EVs. With nanoscale flow cytometry, researchers could 
reproducibly: 
• Fully characterize diverse EVs
• Accurately quantify, measure, and identify EV subpopulations
• Obtain robust and high-quality data, even for small EVs
So far, this promising technology is not yet widely adopted. However, new advances are continually happening, 
and highly sensitive, consistent, and flexible nanoscale flow cytometers will soon be within reach. To get the most 
from nanoscale flow cytometry, researchers will need a system that can simultaneously count, measure, 
and identify EV subpopulations below 100 nm.
“New advances are happening all the time, and highly sensitive, 
consistent, and flexible nanoscale flow cytometers 
will soon be within reach”
Characterization
16
05 How to count and characterize EVs 
EVs play a vital role across the human body, and their precise, sensitive, and reproducible analysis is key to better 
understanding and characterizing diseases and developing innovative therapies. Many techniques can be used 
to analyze EVs: from single parameter characterization using ELISA, to multi-measurement platforms such as DLS 
and SRM. However, most existing technologies require researchers to make a trade-off, whether it is throughput, 
accuracy, or resolution. 
With flow cytometry, researchers can quickly, accurately, and reproducibly count, determine the size of, and 
characterize EVs at high throughput. And soon, nanoscale flow cytometry will redefine what it is 
possible to achieve, allowing characterization of even small EVs. Armed with this technology, 
researchers will be able to unlock new insights into biological systems, such as the detection and characterization 
of biomarker EVs in liquid biopsy samples — making it quicker and simpler to advance understanding in areas such 
as health and disease than ever before. 
Interested in learning how to advance 
your EV research? Whether you have 
questions or are interested in a demo, 
we’re here to help.
 TALK TO AN EXPERT 
 
06
17
Characterization
Nanoscale flow cytometry: 
Bring clarity to EV analysis
beckman.com
2024-GBL-EN-104736-v1
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nano flow cytometer. With the ability to count, size, and characterize 
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Breaking the boundries of EV detection
SHIFT PERSPECTIVE. 
ACHIEVE MORE.
For Research Use Only. Not for use in diagnostic procedures.
© 2024 Beckman Coulter, Inc. All rights reserved. Beckman Coulter, the stylized logo, and the Beckman Coulter 
product and service marks mentioned herein are trademarks or registered trademarks of Beckman Coulter, 
Inc. in the United States and other countries. 
1 Théry C, Witwer KW, AikawaE, et al. Minimal information for studies 
of extracellular vesicles 2018 (MISEV2018): A position statement of 
the International Society for extracellular vesicles and update of the 
MISEV2014 guidelines. Journal of Extracellular Vesicles. 2018;7(1). doi:1
0.1080/20013078.2018.1535750
2 Welsh JA, Goberdhan DC, O’Driscoll L. Minimal information for 
studies of extracellular vesicles (MISEV2023): From basic to advanced 
approaches. Journal of Extracellular Vesicles. 2024 Feb;13(2):e12404. 
doi: 10.1002/jev2.12404.
https://www.beckman.com/flow-cytometry/research-flow-cytometers/cytoflex-analyzer-platform/cytoflex-nano-2024
18
THE DISCOVERY OF 
EXTRACELLULAR VESICLES (EVs): 
TRACING THE JOURNEY 
FROM INCEPTION TO 
PRESENT DAY
1. UNVEILING THE 
SECRETS OF BLOOD 
CLOTTING: THE EARLY 
DAYS OF EV RESEARCH
Chargaff: During his work to establish a 
centrifugation protocol for separating clotting 
factors from cells, Chargaff observed that 
“the addition of the high-speed sediment to 
the supernatant plasma brought about a very 
considerable shortening of the clotting time.”
1945
Wolf: Wolf’s work involved the identification of 
platelet dusts, which he described as a “material 
in minute particulate form, sedimentable by 
high-speed centrifugation and originating 
from platelets but distinguishable from intact 
platelets.” This study also provided the first 
electron microscopy images of EVs.
1967
Nunez: Nunez’s study focused on the presence 
of small extracellular vesicles in the bat thyroid 
gland during arousal from hibernation. The authors 
observed multivesicular bodies (MVBs) near the 
apical membrane and proposed that the fusion of 
the MVB’s outer membrane with the apical plasma 
membrane could lead to the release of the vesicles 
into the luminal space.
1974
1946
Chargaff and West: In their study, Chargaff 
and West identified a particulate fraction that 
“sedimented at 31,000 x g and had high clotting 
potential.” They suggested that this fraction 
likely included “a variety of minute breakdown 
products of blood corpuscles” in addition to the 
thromboplastic agent.
Aaronson and others: Studies conducted by 
Aaronson and colleagues on various organisms 
indicated that vesicular structures extruded 
from cells were not unique to mammals. For 
example, they observed vesicles budding from 
cells and isolated them through centrifugation in 
Ochromonas danica, a flagellated alga. EVs were 
also shown to be released by Candida tropicalis, 
Corynebacterium, Acinetobacter, Escherichia coli, 
and other species.
1971–1979
Crawford: Crawford’s research demonstrated 
that EVs contain lipids and carry cargo such as 
ATP and contractile proteins.
1971
2. UNRAVELING THE EV 
ENIGMA: ACCUMULATING 
EVIDENCE, UNCERTAIN ROLE
Multiple Researchers: During this period, 
researchers actively searched for “virus-like 
particles” that could potentially cause diseases, 
including infections and cancer. However, it 
was noted that labeling structures with the 
morphological characteristics of naturally 
occurring vesicles from multivesicular bodies and 
microvesicles associated with epithelial cells as 
“virus-like” was unwarranted.
1956–1975
1986
Gawrisch: Gawrisch’s study revealed that the 
lateral diffusion of lipids and proteins in vesicle 
membranes differed between extracellular vesicles 
(EVs) and red blood cells (RBCs). The diffusion 
was higher in EVs, likely due to their lower protein 
content and random lipid composition compared 
to RBC membranes.
Smalley: Smalley’s research highlighted 
the production of extracellular vesicles by 
Porphyromonas gingivalis and showed their 
interaction with human polymorphonuclear 
leukocytes.
1990
Vidal & Stahl: Their work led to a better 
understanding of vesicular trafficking, 
including the components of EVs.
1993
Escola: Escola’s study confirmed the presence 
of tetraspanins as components of EVs.1998
1983
Johnstone and Stahl: Their work demonstrated 
the release of intraluminal vesicles from cells, using 
reticulocyte maturation as a model. They defined 
these vesicles as exosomes, which were released 
from the lumen of multivesicular bodies upon fusion 
with the plasma membrane. This discovery unveiled 
the exosome secretion pathway.
Johnstone: Based on the internalization and 
shedding of EV components at different times, 
it was suggested that exosomes served as a 
major route for externalizing obsolete membrane 
proteins. This finding challenged the perception of 
EV release as solely a waste disposal mechanism.
1991
Raposo: Raposo’s study revealed that EVs derived 
from immune cells had the ability to present 
antigens. This expanded the understanding of the 
utility of EVs, particularly in the development of 
therapeutic strategies, such as in cancer treatment 
(as demonstrated by Zitvogel in 1998).
1996
Johnstone: This study demonstrated that 
exosomes released from reticulocytes 
retained enzymatic activity.
1989
19
Figure 1: Regulatory Functions of EVs. The central EVs express specific markers on their surface and interact with various immune cells, 
including T cells, Natural Killer cells (NK), and dendritic cells (DC). Regulatory T cells (Treg), cytotoxic T lymphocytes (CTL), adenosine 
triphosphate (ATP), and adenosine monophosphate (AMP) are involved in these interactions. Please note that the size of the extracellular 
vesicle in the figure has been intentionally exaggerated for illustrative purposes.
EV or not EV
Currently, there is a faction of scientists within the scientific 
community who use the term “exosome,” while originally 
these structures were referred to as “microparticles.” 
This discrepancy in nomenclature can lead to confusion, 
particularly when arbitrary size ranges are assigned to 
different types of vesicles. To address this issue, it has 
been suggested that the umbrella term “extracellular 
vesicles” (EVs) should be used to describe non-
replicating structures that are bounded by a lipid bilayer. 
3. UNLOCKING THE FUNCTIONAL SECRETS OF EVs: 
DECIPHERING THEIR ROLE IN CELLULAR PROCESSES
4. NAVIGATING THE GAPS IN THE FIELD OF EVs
The beginning of the 21st century marked a turning 
point in understanding the role of extracellular vesicles 
(EVs) in both physiological processes and diseases. 
Scientists began to investigate the content of EVs using 
different approaches, including proteomics, lipidomics, 
genomics, and biochemistry. These studies shed light on 
the potential roles of EVs in different contexts.
This research focused on immune cells, inspired 
researchers in the field of immune therapies against 
cancer, as demonstrated by the work of Chaput 
(2003) and Zitvogel (2005). Ratajczak (2006) made 
a breakthrough when demonstrating that cell-derived 
microvesicles could reprogram other cells through the 
horizontal transfer of mRNA and protein delivery, a 
discovery highlighted the significant impact EVs could 
have on cellular behavior.
Furthermore, it became evident that EVs could play 
crucial roles in supporting healing processes, such as in 
the case of infections (Colino, 2007) or trauma (Bruno, 
2009). For example, compelling evidence suggests 
that EVs have multiple roles in regulating the immune 
response (Figure 1).
The establishment of the International Society for 
Extracellular Vesicles (ISEV) in 2011 has played a crucial 
role in fostering consensus on this terminology. The 
definition of EVs as non-replicating structures delimited 
by a lipid bilayer has been formalized in the current 
recommendations outlined in the Minimal Information 
for Studies of Extracellular Vesicles (MISEV) guidelines 
(2018). The official journal of the society, the Journal 
of Extracellular Vesicles (JEV), further reinforces the 
accepted terminology and provides a platform for 
research in the field.
20
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2024-GBL-EN-104970-V2
Ensuring reproducibility
A major topic of debate in the field of extracellular 
vesicles (EVs) revolves around the selection of methods 
for their isolation and enrichment. Various techniques 
are available, including ultracentrifugation, ultrafiltration, 
size-exclusion chromatography, precipitation methods, 
immunoaffinity-based assays, magneto-immunocapture, 
microfluidic immunochips, and lipid nanoprobes. The 
choice of method depends largely on the specific 
objectives of the study.
In terms of EV characterization, several biophysical 
approaches have been developed, such as nanoparticle 
tracking analysis and reverse pulse sensing, which enable 
the counting and sizing of EVs. Further characterization 
involves methodologies like proteomics, genomics, 
lipidomics, and more. While these techniques provide 
insights into EV populations as a whole, flow cytometry 
allows for the individual characterization of EVs. Single-
EV information is particularly valuable as it unravels the 
heterogeneity within EV populations. However, ensuring 
the accuracy and reproducibility of studies is crucial. To 
address this, various working groups came together in 
2011 to establish the International Society for Extracellular 
Vesicles (www.isev.org), which has played a pivotal role 
in advancing the EV field.
In 2018, the society published updated guidelines for EV 
analysis known as MISEV (Minimal Information for Studies 
of Extracellular Vesicles). These guidelines aim to address 
controversies and questions surrounding EV research. 
As stated by Ramirez et al., “a persistent concern in 
flow cytometry is the reliable distinction between EVs 
carrying specific protein markers and those that do 
not, in order to accurately measure the proportion of 
EVs of a particular type.” Quantitative and qualitative 
analyses of EV heterogeneity within samples are crucial 
for comprehensive understanding.
5. CONCLUSIONS
The research on extracellular vesicles (EVs) has evolved 
into a distinct field, complete with its own dedicated 
society and scientific conferences. The discovery of 
EVs has illuminated a previously unknown realm that 
plays a crucial role in regulating various physiological 
processes. Further studies in this field hold the potential 
to unravel the heterogeneity of EVs and elucidate 
their diverse functions. Moreover, they could pave the 
way for identifying novel biomarkers of diseases. By 
monitoring cell-specific EVs, researchers can search for 
specific biomarkers that are present on or within EVs. 
This approach offers a non-invasive means of defining 
disease biomarkers, as it only requires a simple blood 
draw. Promising results have already been demonstrated 
in the context of Alzheimer’s disease.
Furthermore, EVs are currently the subject of intense 
investigation for their potential use as carriers to 
transport therapeutic compounds to target cells or 
organs. By engineering EVs loaded with drugs that 
can specifically target certain cells, such as tumors, 
researchers hope to overcome challenges associated 
with conventional therapies. This targeted delivery 
approach could improve the efficacy of treatments 
and minimize unwanted side effects. This has important 
implications, as current therapies are often systemically 
adminisstered, resulting in suboptimal functionality and 
potential to cause harm to health tissues
References
Asleh et al. Extracellular vesicle-based liquid biopsy biomarkers and their application in precision immuno-oncology. Biomark Res 11, 99, 2023. 
Ramirez et al. Technical challenges of working with extracellular vesicles. Nanoscale 10, 881-906, 2018.
Théry et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular 
Vesicles and update of the MISEV2014 guidelines. J. Extracell Vesicles. 7(1):1535750, 2018.
Couch et al. A brief history of nearly EV-erything – The rise and rise of extracellular vesicles. J. Extracell Vesicles. 10(14):e12144, 2021.
Tao-Ran et al. Extracellular vesicles as an emerging tool for the early detection of Alzheimer’s disease. Mech Aging and Dev. 184:111175, 2019.
21
22
exposure to radioactivity contributed to her developing 
leukemia, serving as a costly lesson learned by mistake. 
These anecdotes highlight the incredible journey of 
scientific curiosity.
The story of extracellular vesicles (EVs) is akin to the 
narrative of scientific curiosity crossing various disciplines 
such as hematology, cell biology, virology, and more. 
These diverse fields eventually converged to uncover the 
existence of these small biologically active vesicles. The 
knowledge gained from each domain played a crucial 
role in unveiling the significance of EVs as an essential 
component of our physiology.
EVs have gained significant interest in the field of 
biodiscovery, despite being a relatively recent discovery. 
THE DISCOVERY OF EVs
Science and medicine have been at the forefront of 
some of the most fascinating discoveries in history. Take, 
for example, the charming story of penicillium mold 
accidentally falling into an agar plate filled with bacteria, 
leading to the serendipitous discovery of antibiotics. This 
discovery was a result of learning by chance. Another 
remarkable story is that of Barry Marshall, who bravely 
self-administered Helicobacter pylori to demonstrate its 
effect on peptic ulcers. This bold experiment ultimately 
earned him a Nobel Prize, showcasing the power of 
learning through experimentation.
We mustn’t forget the extraordinary Marie Curie, who 
used to carry radium tubes in her pocket. Sadly, her 
INTO THE WORLD OF 
EXTRACELLULAR 
VESICLES (EVs)
Extracellular 
Vesicles (EVs)
23
exposure to radioactivity contributed to her developing 
leukemia, serving as a costly lesson learned by mistake. 
These anecdotes highlight the incredible journey of 
scientific curiosity.
The story of extracellular vesicles (EVs) is akin to the 
narrative of scientific curiosity crossing various disciplines 
such as hematology, cell biology, virology, and more. 
These diverse fields eventually converged to uncover the 
existence of these small biologically active vesicles. The 
knowledge gained from each domain played a crucial 
role in unveiling the significance of EVs as an essential 
component of our physiology.
EVs have gained significant interest in the field of 
biodiscovery, despite being a relatively recent discovery. 
THE DISCOVERY OF EVs
Science and medicine have been at the forefront of 
some of the most fascinating discoveries in history. Take, 
for example, the charming story of penicillium mold 
accidentally falling into an agar plate filled with bacteria, 
leading to the serendipitous discovery of antibiotics. This 
discovery was a result of learning by chance. Another 
remarkable story is that of Barry Marshall, who bravely 
self-administered Helicobacter pylori to demonstrate its 
effect on peptic ulcers. This bold experiment ultimately 
earned him a Nobel Prize, showcasing the power of 
learning through experimentation.
We mustn’t forget the extraordinary Marie Curie, who 
used to carry radium tubes in her pocket. Sadly, her 
INTO THE WORLD OF 
EXTRACELLULAR 
VESICLES (EVs)
Extracellular 
Vesicles (EVs)
Many scientists are now focused on understanding the 
diversity of EVs, their role in physiological functions, and 
their potential involvement in various diseases.
In the 1930s, a biochemist named Chargaff was working on 
developing a centrifugation protocol to separate clotting 
factors from blood. During this process, he observed that 
adding the high-speed sediment to the supernatant plasmasignificantly shortened the clotting time.1 This observation 
brought to light the existence of EVs, a distinct family of 
biomolecules present in blood. With the assistance of 
West,2 they further identified a “particulate fraction” that 
sedimented at 31,000 x g and exhibited high clotting 
potential and thromboplastic properties. This fraction 
included EVs. Initially, it was hypothesized that these isolated 
fractions were composed of various minute breakdown 
products of blood corpuscles. However, it took nearly two 
more decades of research to identify and differentiate 
the material in minute particulate form, sedimentable by 
high-speed centrifugation, and originating from platelets 
but distinct from intact platelets. These findings align 
closely with the characteristics of EVs.3 The first electron 
microscopy images of this “platelet dust” provided 
scientists with visual insights into the true appearance of 
EVs (Figure 1). 
• Extracellular vesicles (EVs) possess a phospholipid 
membrane that typically has a thickness of 
approximately 5 nm. 
• These vesicles exhibit a wide range of sizes, with 
most fluids containing EVs of varying sizes. 
• The minimum size of EVs is dependent on their 
composition and ranges from 30-50 nm in human 
blood plasma. 
• Generally, the size distribution of EVs has a peak 
below 200 nm.
• In human blood, spherical EVs smaller than 500 nm 
constitute around 95% of the total population.
EVs AT A GLANCE
Figure 1. Urinary EVs (uEVs) characterization by transmission electron 
microscopy (TEM). Representative picture of TEM characterization 
of uEVs isolated with ultracentrifugation. Bertolone et al. Front. 
Endocrinol. 2023 14:1096441. Published under a Creative Commons 
Attribution 4.0 International License: http://creativecommons.org/
licenses/by/4.0/  
The subsequent work by Crawford played a crucial role 
in identifying the presence of lipids and proteins within 
the membrane of EVs, shedding light on their functional 
properties.4 In a fascinating study on bat hibernation, 
scientists initially observed how EVs are generated from 
cells through the fusion of multivesicular bodies with the 
plasma membrane before being released.5 This discovery led 
to the realization that EVs are not exclusive to mammalian 
cells but are produced by various cell types.6 During the 
1980s, researchers focused on unraveling the intricacies of 
membrane trafficking and the assembly of EVs, specifically 
examining how proteins are included or excluded.7
Initially referred to as exosomes (small microvesicles), the 
term “extracellular vesicles” was officially adopted in 2011 to 
define these non-replicating structures enclosed by a lipid 
bilayer. The same year witnessed the establishment of the 
International Society of Extracellular Vesicles (ISEV), which 
developed guidelines (MISEV) to standardize the analysis 
and reporting of EV research.8 Since then, numerous topics 
have emerged, including the study of cargo trafficking, 
biomarkers, cancer, cardiovascular diseases, infectious 
diseases, drug delivery, and many more. These areas of 
research highlight the tremendous importance of EVs in 
various fields of study.
THE IMPORTANCE OF EVs 
IN SCIENCE AND MEDICINE
The discovery of the world of extracellular vesicles (EVs) 
has brought to light previously unknown aspects of how 
the body functions, how cancer cells spread, and how 
pathogens disseminate. However, there is still much 
investigation needed to fully decipher the secrets of EVs. 
Scientists now recognize that EVs play a central role not 
only in physiological processes but also in pathological 
conditions. This recognition has led to a significant surge in 
research over the past decade, with thousands of scientific 
papers published in 2022 alone (Figure 2).
0
1000
2000
3000
4000
5000
6000
7000
1950 1960 1970 1980 1990 2000 2010 2020 2030
Figure 2. The number of publications related to extracellular vesicles 
(EVs). This figure represents the number of publications retrieved 
from 1950 to the present, using the keyword “extracellular vesicles” 
(source: Pubmed.com).
24
EVs are produced by all cell types across different life 
kingdoms. Their biogenesis can be categorized into two 
types of release: (i) budding from the plasma membrane, 
resulting in larger microvesicles and apoptotic bodies, and 
(ii) release of vesicles derived from endosomes. 9 EVs are 
small in size (ranging from 30 to 500 nm) and possess 
an intraluminal region. Due to their size and ability to 
carry proteins and nucleic acids, scientists are focused on 
understanding their functions and exploring their potential 
as biomarkers.
Research has revealed that EVs play a role in various 
physiological processes, including angiogenesis, cellular 
migration, and cell-to-cell signaling. Their abundance, 
and their ability to easily enter target cells suggest their 
involvement in numerous physiological processes, both in 
peripheral circulation and tissues (Figure 3). In fact, EVs have 
the capacity to cross the blood-brain barrier, indicating a 
potential role in neurodegenerative diseases.10 Additionally, 
EVs can serve as biomarkers for peripheral screening tools 
in diseases like Alzheimer’s (among other applications).11
There is growing interest in the therapeutic potential 
of EVs. Clinical trials are investigating the use of EVs as 
cargo vehicles to deliver compounds to target cells. By 
engineering EVs to express specific markers, they can be 
targeted to specific sites in the body and deliver cargo 
inside the cells. This targeted delivery offers advantages 
in terms of specificity compared to conventional drug 
delivery methods. Transmembrane proteins like tetraspanins 
(e.g., CD9, CD63, CD81), often used as markers of EVs, 
• EVs are derived from 
the endosomal system, 
leading to the formation 
of multivesicular bodies.
• These EVs carry cell-
derived proteins on their 
surface.
• Subsequently, they are 
transported to the cell 
membrane and fuse 
with it.
• Once fused, they are 
released from the cell into 
the extracellular space.
• EVs can then bind to 
target cells that express 
the corresponding 
receptor that they 
themselves express.
• Once inside the target 
cell, they have the ability 
to release their cargo.
Figure 3. Biogenesis of vesicles from cells and their anchoring into target cells. EVs and their cargos are enclosed within microvesicular 
bodies (MVB) that migrate to the plasma membrane for release into the extracellular space.
Secreting Cell
RNAs
Proteins
MVB
Cytosol
Cytosol
Microvesicles
RNAs
Recipient Cell
Extracellular 
Vesicles
also play a role in this process.12 Once inside the cells, the 
cargo interacts with intracellular components, influencing 
signaling pathways and ultimately leading to desired 
outcomes such as activation, suppression, or modulation 
of cellular functions.
This constant physiological process, where EVs modulate 
cellular functions from within, impacts the interaction of 
target cells with their microenvironment, particularly in 
tissues. It is through the release of EVs that some cancer 
cells can spread to other tissues, as these vesicles have 
the ability to modulate the tissue microenvironment in 
favor of cancer cell dissemination.13 This area of research is 
highly active, as finding ways to suppress this action could 
potentially reduce the spread of cancer cells throughout 
the body.14
CENTRIFUGATION AT THE 
GENESIS OF EV DISCOVERY
The study of extracellular vesicles (EVs) and the 
development of the EV field have heavily relied on 
centrifugation protocols since Chargaff’s initial discovery. 
Over the years, scientists have refined protocols to enrich, 
isolate, and characterize EVs. The ideal separation protocol 
would offer high yield, heterogeneity, and efficiency 
while minimizing time and costs. Centrifugation, being a 
well-established technique in laboratories, has been the 
method of choice that has propelled the field toits current 
state. There are various methods for isolating EVs, and the 
How EVs Are Generated
25
specific downstream applications dictate the appropriate 
protocol to employ, each with its own advantages and 
disadvantages.15 Body fluids such as plasma, serum, urine, 
cerebrospinal fluid, and milk are commonly used as starting 
materials for EV separation.
Ultracentrifugation, including differential centrifugation, 
is a widely employed isolation method for EVs. While a 
combination of techniques may be utilized, centrifugation 
is almost always included in the isolation protocols (Figure 
4). Density gradient centrifugation, filtration, and size-
exclusion chromatography have gained interest among 
researchers. However, subsequent purification methods still 
involve ultracentrifugation, density gradient centrifugation, 
or chromatography techniques. Magnetic bead separation, 
although offering a targeted approach, may compromise 
the true heterogeneity of EVs. The isolation of EVs entails 
the removal of contaminants and the separation of EVs 
from other particles, such as chylomicrons and lipids, 
present in biological fluids or cell culture media (Figure 
4). Ultracentrifugation utilizes centrifugal force to separate 
components based on size and/or density. Differential 
ultracentrifugation techniques involve multiple spins at 
progressively increasing speeds to eliminate contaminants 
and pellet EVs. Density gradient ultracentrifugation is a high-
resolution purification technique that separates particles 
based on physical properties (size, shape, mass, and/or 
density) by employing a medium with graded densities, 
such as sucrose or iodixanol. While density cushion 
ultracentrifugation strikes a balance between throughput 
and purity, density gradient ultracentrifugation provides the 
highest purity, and differential ultracentrifugation allows for 
the highest throughput. Density gradient and differential 
ultracentrifugation yield the cleanest samples and preserve 
the structural integrity of EVs compared to other methods 
like size exclusion chromatography or precipitation. 
The question of scalability, especially in the context of 
manufacturing and EV therapy, becomes important when 
deciding which isolation method to employ.16-19
MOVING FORWARD WITH EV RESEARCH
From the initial discovery of EVs to the present day, several 
decades have passed. The recent excitement surrounding 
EVs stems from the potential applications that could arise 
from a deeper understanding of their generation, regulation, 
and manipulation for therapeutic purposes. Since EVs are 
produced by all types of living cells and have the ability 
to regulate numerous processes, the possibilities for their 
applications are limitless. However, this also emphasizes 
the need for further research to unravel the secrets of 
EVs. The scientific community is eager to standardize 
Figure 4. Isolating EVs involves the removal of contaminants and the separation of EVs from other particles, such as chylomicrons and 
lipids, present in biological fluids or cell culture media. Ultracentrifugation utilizes centrifugal force to separate components based on 
their size and/or density. Differential ultracentrifugation techniques employ multiple spins at progressively increasing speeds to first 
eliminate contaminants and then pellet EVs. Density gradient ultracentrifugation is a high-resolution purification technique that relies on 
a medium with graded densities, such as sucrose or iodixanol, to separate particles based on their physical properties such as size, shape, 
mass, and density.
26
the isolation, characterization, and use of EVs, as this will 
enhance reproducibility and facilitate the translation of 
findings into clinical use.
There are still important characteristics of EVs that need 
to be clarified. Firstly, their heterogeneity is a significant 
aspect to explore. EVs are not a single entity, as their 
composition and size can vary even within the same cell. 
The generation and composition of EVs are likely to be 
continuously regulated processes influenced by the cell’s 
status and received signals (Figure 5). Characterizing EVs 
individually, which can be achieved using technologies like 
flow cytometry, will contribute to a better understanding 
of their heterogeneity. Secondly, it is crucial to develop 
scalable methods for generating EVs in vitro for delivery 
to patients. Defining standard workflows will be essential 
in this regard, and centrifugation plays a crucial role in the 
manufacturing process. Lastly, mapping markers associated 
with different classes of EVs (e.g., cells of origin, healthy 
vs. cancer cells, activation status, disease biomarkers) is 
necessary to expedite ongoing therapeutic efforts.
MORE INFORMATION
To learn more, please visit these resources on the Beckman 
Coulter Life Sciences website.
Figure 5. Representation of the size and heterogeneity of EVs. High-density lipoproteins (HDLs), low-density lipoproteins (LDLs), and 
very low-density lipoproteins (vLDLs) are included for comparison. The average sizes of antibodies, viruses, and bacteria are also shown.
10 nm 30 nm 50 nm 120 nm 200 nm 500 nm 1000 nm
Small EVs Large EVs
BacteriaVirus
HDLs LDLs vLDLs
Antibody
Extracellular Vesicles Overview
Flow Cytometry Overview
CytoFLEX nano for EV Research
27
1. Chargaff, E. J. (1945). Cell structure and the problem of blood 
coagulation. 160(1), 351–359
2. Chargaff, E., & West, R. (1946). The biological significance of the 
thromboplastic protein of blood. Journal of Biological Chemistry, 
166(1), 189–197
3. Wolf, P. (1967). The nature and significance of platelet products in 
human plasma. British Journal of Haematology, 13(3), 269–288
4. Crawford, N. (1971). The presence of contractile proteins in platelet 
microparticles isolated from human and animal platelet-free 
plasma. British Journal of Haematology, 21(1), 53–69
5. Nunez, E. A., Wallis, J., & Gershon, M. D. (1974). Secretory 
processes in follicular cells of the bat thyroid. 3. The occurrence 
of extracellular vesicles and colloid droplets during arousal from 
hibernation. American Journal of Anatomy, 141(2), 179–201
6. Couch Y, Buzàs EI, Di Vizio D, Gho YS, Harrison P, Hill AF, Lötvall 
J, Raposo G, Stahl PD, Théry C, Witwer KW, Carter DRF. A brief 
history of nearly EV-erything - The rise and rise of extracellular 
vesicles. J Extracell Vesicles. 2021 Dec;10(14):e12144
7. Harding, C., Heuser, J., & Stahl, P. (1983). Receptor-mediated 
endocytosis of transferrin and recycling of the transferrin receptor 
in rat reticulocytes. Journal of Cell Biology, 97(2), 329–339
8. Théry, C., Witwer, K. W., Aikawa, E., Alcaraz, M. J., Anderson, J. 
D., Andriantsitohaina, R., Antoniou, A., Arab, T., Archer, F., Atkin-
Smith, G. K., Ayre, D. C., Bach, J.-M., Bachurski, D., Baharvand, H., 
Balaj, L., Baldacchino, S., Bauer, N. N., Baxter, A. A., Bebawy, M., 
& Zuba-Surma, E. K. (2018). Minimal information for studies of 
extracellular vesicles 2018 (MISEV2018): A position statement of 
the International Society for Extracellular Vesicles and update of 
the MISEV2014 guidelines. Journal of Extracellular Vesicles, 7(1), 
1535750
9. Welsh JA, Arkesteijn GJA, Bremer M, Cimorelli M, Dignat-George 
F, Giebel B, Görgens A, Hendrix A, Kuiper M, Lacroix R, Lannigan 
J, van Leeuwen TG, Lozano-Andrés E, Rao S, Robert S, de Rond 
L, Tang VA, Tertel T, Yan X, Wauben MHM, Nolan JP, Jones JC, 
Nieuwland R, van der Pol E. A compendium of single extracellular 
vesicle flow cytometry. J Extracell Vesicles. 2023 Feb;12(2):e12299. 
doi: 10.1002/jev2.12299. PMID: 36759917; PMCID: PMC9911638
10. Jeyaraman M, Rajendran RL, Muthu S, Jeyaraman N, Sharma 
S, Jha SK, Muthukanagaraj P, Hong CM, Furtado da Fonseca 
L, Santos Duarte Lana JF, Ahn BC, Gangadaran P. An update 
on stem cell and stem cell-derived extracellular vesicle-based 
therapy in the management of Alzheimer’s disease. Heliyon. 2023 
Jun 29;9(7):e17808. doi: 10.1016/j.heliyon.2023.e17808.PMID: 
37449130; PMCID: PMC10336689
11. Cheng, L., Doecke, J., Sharples, R. et al. Prognostic serum 
miRNA biomarkers associated with Alzheimer’s disease shows 
concordance with neuropsychological and neuroimaging 
assessment. Mol Psychiatry 20, 1188–1196 (2015)
12. Ginini L, Billan S, Fridman E, Gil Z. Insight into Extracellular Vesicle-
Cell Communication: From Cell Recognition to Intracellular Fate. 
Cells. 2022 Apr 19;11(9):1375. doi: 10.3390/cells11091375. PMID: 
35563681; PMCID: PMC9101098
13. Kosaka N, Yoshioka Y, Fujita Y, Ochiya T. Versatile Roles of 
Extracellular Vesicles in Cancer. J Clin Invest (2016) 126:1163–72
14. Xu Y, Yao Y, Yu L, Zhang X, Mao X, Tey SK, Wong SWK, Yeung CLS, 
Ng TH, Wong MYM, Che CM, Lee TKW, Gao Y, Cui Y, Yam JWP. 
Clathrin light chain A-enriched small extracellular vesicles remodel 
microvascular niche to induce hepatocellular carcinoma metastasis. 
J Extracell Vesicles. 2023 Aug;12(8):12359
15. Gardiner, C., Vizio, D. D., Sahoo, S., Théry, C., Witwer, K. W., 
Wauben, M., & Hill, A. F. (2016). Techniques used for the isolation 
and characterization of extracellular vesicles: Results of a 
worldwide survey. Journal of Extracellular Vesicles, 5, 32945
16. Dong X, Dong JF, Zhang J. Roles and therapeutic potential of 
different extracellular vesicle subtypes on traumatic brain injury. 
Cell Commun Signal. 2023 Aug 18;21(1):211
17. García-Fernández J, Fuente Freire M. Exosome-like systems: 
Nanotechnology to overcome challenges for targeted cancer 
therapies. Cancer Lett. 2023 May 1;561:216151. doi: 10.1016/j.
canlet.2023.216151
18. Li G, Chen T, Dahlman J, Eniola-Adefeso L, Ghiran IC, Kurre P, Lam 
WA, Lang JK, Marbán E, Martín P, Momma S, Moos M, Nelson DJ, 
Raffai RL, Ren X, Sluijter JPG, Stott SL, Vunjak-Novakovic G, Walker 
ND, Wang Z, Witwer KW, Yang PC, Lundberg MS, Ochocinska MJ, 
Wong R, Zhou G, Chan SY, Das S, Sundd P. Current challenges 
and future directions for engineering extracellular vesicles for 
heart, lung, blood and sleep diseases. J Extracell Vesicles. 2023 
Feb;12(2):e12305. doi: 10.1002/jev2.12305. Erratum in: J Extracell 
Vesicles. 2023 Mar;12(3):e12314. PMID: 36775986
19. Estes S, Konstantinov K, Young JD. Manufactured extracellular 
vesicles as human therapeutics: challenges, advances, and 
opportunities. Curr Opin Biotechnol. 2022 Oct;77:10277
References
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