EXTRACELLULAR VESICLESin Health and DiseaseExtracellular vesicles (EVs)—exosomes, microvesicles, and apoptotic bodies—are small, membrane-bound vesicles of various sizes. Both prokaryotic and eukaryotic cells release EVs, which are comprised of the origin cell’s plasma membrane and cytoplasmic contents1. EVs carry and transport bioactive cargo, such as proteins, nucleic acids, and lipids, that can participate in intracellular communication. EVs and their cargo can indicate disease, but they are di�cult to characterize due to their small size, variable composition, and heterogeneous refractive indices.

CancerCancer cells can release all three types of EVs

with tumor-speci�c antigens on their surfaces. Inside, they may carry proteins, mRNA, or

miRNA that alter gene expression. Their cargo can stimulate metastasis in healthy recipient

cells, enhance cell migration by degrading the extracellular matrix, and induce angiogenesis

to support a growing tumor7. Depleting cancer EVs to dampen their cancer-enhancing e�ects

is a current area of therapeutic research8.Additionally, analyzing EV cargo from bodily �uid samples (liquid

biopsy) can determine a cancer’s origin. miRNAs are

especially interesting because they are enriched in cancer EVs compared to EVs from normal

cells9.

Brain/Nervous System EVs play a critical role in central nervous system function. They promote neuronal

survival and synapse assembly and plasticity by sending signals between neurons and myelinating glia3. They also contribute to

neurodegenerative diseases like Parkinson’s and Alzheimer’s by carrying plaque-forming

proteins across the blood-brain barrier 4.

Tissue Repair and Wound Healing

While the contents of some EVs may be detrimental, other EVs transport cargo with

bene�cial e�ects. For example, EVs released from stem cells facilitate repair of various body sites,

such as the vasculature and the cornea6,13. EVs can induce in�ammation in the �rst step of tissue repair

and stimulate stem cell di�erentiation and extracellular matrix turnover14. They are especially promising for cancer therapy development, where they can stimulate antitumor responses or deliver

drugs directly to tumor cells15.

Drug Delivery

Because EVs package a variety of biomolecules into cellular membranes, they are natural drug-delivery vehicles. They can cross the blood-brain barrier, target desired cell types, and avoid the

immune response16,17. EVs can carry cytotoxic drugs that directly kill their

targets, or they may hold molecules like siRNAs that knockdown oncogene

expression in tumor cells18.

Immune SystemEVs originating from infected cells and

antigen-presenting cells carry antigens that activate T cells and B cells. They also mediate

cytokines, which stimulate other immune cells and induce in�ammation. Some EVs

circulating in the blood or originating from tumors may induce immunosuppression5.

Pulmonary Disorders EV production is upregulated in many

pulmonary disorders. They contribute to endothelial cell dysfunction, pulmonary

capillary permeability, and increased migration of damage-inducing in�ammatory mediators that contribute to many diseases,

such as COPD, Acute Respiratory Distress Syndrome, and pulmonary hypertension6.

Bodily FluidsEVs have been found in all tested bodily

�uids, including blood, saliva, breast milk, urine, and cerebrospinal �uid1. They travel

to distant body sites via these liquids. Their presence allows for less-invasive testing

methods for certain diseases, such as liquid biopsies for cancer.

Skeletal SystemBone tissues are constantly synthesized

and broken down. This remodeling process requires communication

between many cell types, including osteoclasts, osteoblasts, osteocytes,

and vascular endothelial cells. Signaling molecules and miRNAs found in EVs

can facilitate this communication and induce remodeling and fracture

healing12.

Heart/Cardiovascular SystemThe contents of EVs released by

cardiomyocytes vary based on conditions or stresses. For example, EV release increases in instances of injury or

in�ammation. Damaged heart muscle releases EVs with cargo that promote

repair and healing, such as miRNAs that play a role in cardiac regeneration10.

Liver InjuryLiver injury diagnosis typically occurs through hepatic enzyme detection in blood plasma; however, these enzyme levels do not always re�ect the stage of

liver disease. miRNAs found in EVs released by injured hepatic cells are

promising as biomarkers for liver injury11.

Apoptotic Body

Apoptotic bodies are the largest vesicles, with 1-5 µm diameters. These compartments form during programmed cell death and consist of parts of the dying cell. These vesicles are often eliminated by phagocytes; therefore, they infrequently

participate in cell signaling2.

Microvesicle/ microparticle

These vesicles range in diameter from 100-1,000 nm. They form by directly budding from the plasma

membrane. They are often released after activation of cell surface

markers from platelets, red blood cells, and endothelial cells,

although tumors release them constitutively2.

ExosomeExosomes are the smallest vesicles at 30-120 nm in diameter. They form in multivesicular

bodies (MVBs) through endosomal membrane budding. MVB fusion with the plasma membrane releases the exosomes through exocytosis. They may be released

constitutively or upon induction2.

Checklist for Standardization of Extracellular Vesicle Characterization by Flow CytometryThe following experimental conditions and results should be reported in standard units19

Source, sample collection, isolation, and storage variables

Staining method and reagent descriptions according to MIFlowCyt guidelines20

All washing and dilution steps

Settings used for bu�er-only controls and samples. Data should be recorded for a set period of time rather than a set event count

Detergent type, concentration, and lysis results, if applicable

Trigger channels and thresholds usedfor detection

Fluorescence calibration: materials and methods, catalog and lot numbers, reference units for standards, type of regression used, and plot showing arbitrary versus standard data for reference particles. Fluorescence parameters in units of ERF, MESF, or ABC beads

Light-scatter calibration details needed to repeat the model. Use standardizedunits of nm2

Flow cytometry calculation of EV diameter, surface area, and/or volume

Approximation of EV refractive index

Link to data in a public repository

Refractive Index in Flow Cytometry

For more information visit beckman.com/nanoscale

Nanoscale �ow cytometry is often used to analyze EV samples. Fluorescence from stained samples and forward and side light scatter are detected to gather data, such as size and granularity, that can be used to di�erentiate particles. The scatter measurements depend on several variables, including the refractive indices of the sample and the suspension medium (sheath �uid) in the �ow cytometer.

The refractive index (RI) contributes signi�cantly to a medium’s light scattering ability. Refraction—the bending of light—occurs as a beam of light passes from one medium (such as sheath �uid in a �ow cytometer) to another with a di�erent refractive index (such as a cell or EV). As light bends at the interface between the media, it may interact with light scatter detectors in the �ow cytometer. Light passing through two media with similar RI does not bend greatly, while there is greater light refraction as the RI of a material increases compared to the initial medium.

Light beam

Medium 1

Medium 2

Refraction at the interface of the

two media

Air (vacuum) 1

Water 1.33

Saline 1.35

Ethanol 1.36

Cells 1.35-1.40

Bacteria 1.42

Silica beads 1.44

Polystyrene beads 1.59

Refractive Index @ 589 nm

1)Y. Yuana et al., “Extracellular vesicles in physiological and pathological conditions,” Blood Rev, 27:31-9, 2013. 2)B. György et al., “Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles,” Cell Mol Life Sci, 68:2667-88, 2011.3)V. Budnik et al., “Extracellular vesicles round o� communication in the nervous system,” Nat Rev Neurosci, 17:160-72, 2016. 4)B.M. Coleman, A.F. Hill, “Extracellular vesicles--their role in the packaging and spread of misfolded proteins associated with neurodegenerative diseases," Semin Cell Dev Biol, 40:89-96, 2015. 5)P.D. Robbins, A.E. Morelli, “Regulation of immune responses by extracellular vesicles,” Nat Rev Immunol, 14:195-208, 2014.6)E. Letsiou, N. Bauer, “Chapter six - Endothelial extracellular vesicles in pulmonary function and disease,” in Current Topics in Membranes, P Belvitch, S. Dudek, eds., Academic Press, 2018, pp. 197-256. 7)P. Lampropoulos et al., “TGF-beta signalling in colon carcinogenesis,” Cancer Lett, 314:1-7, 2012. 8)A.M. Marleau et al., “Exosome removal as a therapeutic adjuvant in cancer," J Transl Med, 10:134, 2012.9)L. O’Driscoll, “Expanding on exosomes and ectosomes in cancer,” N Engl J Med, 372:2359-62, 2015. 10)D.A. Chistiakov et al., “Cardiac extracellular vesicles in normal and infarcted heart," Int J Mol Sci, 17:63, 2016.11)L. Morán, F.J. Cubero, “Extracellular vesicles in liver disease and beyond,” World J Gastroenterol, 24:4519-26 2018.12)S.-C. Tao, S.-C. Guo, “Extracellular vesicles in bone: “dogrobbers” in the “eternal battle �eld”,” Cell Commun Signal, 17:6, 2019.13)J.D. Zieske et al., “Extracellular vesicles and cell-cell communication in the cornea,” Anat Rec (Hoboken), 2019. 14)A.M. Silva et al., “Extracellular vesicles: immunomodulatory messengers in the context of tissue repair/regeneration,” Eur J Pharm Sci, 98:86-95, 2017.15)B. You et al., “Engineering exosomes: a new direction for anticancer treatment,” Am J Cancer Res, 8:1332-42, 2018.16)D.M. Sun et al., “A novel nanoparticle drug delivery system: the anti-in�ammatory activity of curcumin is enhanced when encapsulated in exosomes,” Mol Ther, 18:1606-14, 2010.17)T.Z. Yang et al., “Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio,” Pharm Res, 32:2003-14, 2015.18)F.N. Faruqu et al., “Preparation of exosomes for siRNA delivery to cancer cells,” J Vis Exp, 2018. 19)“MIFlowCyt-EV (draft),” Extracellular Vesicles Flow Cytometry Working Group, 2017, www.ev�owcytometry.org/links/. 20)“Minimum information about a �ow cytometry experiment (MIFlowCyt) checklist (Numbered in accordance with MIFlowCyt 1.0 document),” Cytometry Part A,77A: 813, 2010.

2019 Beckman Coulter Life Sciences. Research use only. Not for use in diagnostic procedures. FLOW-6078PST10.19