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

Figures & data

Table 1. Considerations for EV separation/enrichment.

Major recommendations of MISEV2014.	Validity and/or Update in 2018
a) There is no single optimal separation method, so choose based on the downstream applications and scientific question. Separation of non-vesicular entities from EVs is not fully achieved by common EV isolation protocols, including centrifugation protocols or commercial kits that claim EV or “‘exosome’” purification.	Still valid. Any newly developed or applied technique for EV isolation must indicate to which of the 4 recovery/specificity options below it aims, and provide characterization information (see Table 2) to show the extent of success. Different methods may be positioned on a recovery vs specificity grid, ranging from low to high in each dimension. Note that the degree of specificity of a particular method might vary depending on the type of biofluid from which EVs are separated. 1) high recovery, low specificity: methods that recover the highest amount of extracellular material, whatever its vesicular or non-vesicular nature, i.e. whole or near-whole concentrated secretome. Examples of protocols include but are not limited to: precipitation kits/polymer (PEG or others), low molecular weight cutoff centrifugal filters with no further separation step, and lengthy or very high speed ultracentrifugation without previous, lower-speed steps. 2) intermediate recovery, intermediate specificity: methods that recover mixed EVs along with some amount of free proteins, ribonucleoproteins, and lipoproteins, depending on the matrix. Examples of protocols: size-exclusion chromatography [154,155], high molecular weight centrifugal filters [102], differential ultracentrifugation using intermediate time/speed with or without wash, tangential flow filtration, and membrane-affinity columns [155,156]. 3) low recovery, high specificity: methods that recover a subtype (or a few subtypes) of EVs with as few non-vesicular components as possible. Subtypes of EVs can be separated by their size (e.g. by filtration, which must be combined with another method such as SEC to eliminate non EV components), their density upon either flotation or pelleting in a density gradient, their surface protein, sugar, or lipid composition (immuno- or other affinity isolation including flow cytometry for large particles), or other biophysical properties such as surface charge. Note that the designation of “low recovery” is relative to total EVs, and that high recovery of specific subtypes may be possible using these techniques. 4) high recovery and high specificity, which may not be achievable as of this writing.
b) Report all details of the method(s) for reproducibility	Still valid. Methods reporting is now facilitated by the EV-TRACK knowledgebase [161] (see also checklist, p.42). ISEV strongly recommends that authors deposit experimental details with EV-TRACK. Examples for classical techniques: centrifugation (g-force, rotor, ultracentrifuge, adjusted k-factor, tube type, adaptor if relevant, time, temperature, and brake)^a; gradients (materials, densities, volumes, and whether top-down (pelleting) or bottom-up (flotation) gradients were done, further processing); chromatography (matrix nature, pore size, volume; volume of loaded sample; volume and number of fractions, type of the elution buffer; further processing of fractions); immunoaffinity (antibody reference and amount per volume of fluid, particle number, or protein amount, incubation time and temperature, matrix, recovery). Necessary technical detail reporting may have to be established for newly developed techniques.^b-d
c)	MISEV2018 additional recommendation: Some protocols, including those associated with many commercial kits, may result in EV populations bound to or mixed with introduced components such as antibodies, beads, polymers, and more. These materials may affect downstream profiling or functional studies and may also render the EVs unusable for therapeutic applications. Particular care in performing functional experiments must be taken (see Table 4), with controls including procedural controls and possibly with further separation of EVs.^e,f

^aFor ultracentrifugation, the k factor can be determined from the rotor type, tube/adapter, and centrifuge speed: the k factor represents the relative pelleting efficiency of a given centrifuge rotor at maximum rotation speed; for runs with a rotational speed lower than the maximum rotor-speed, the k factor has to be adjusted: k_adj = k x (maximum rotor speed/actual rotor speed)²; we recommend that all possible parameters be reported [157,201]. See also a web calculator based on a theoretical model of centrifugation and meant for conversion of protocol parameters between rotors [158].

^bFor filtration techniques, one must take care to remove cells and other large membranous structures prior to ultrafiltration; otherwise, large structures may disintegrate and re-form as small vesicles after passing through the filter [159]. Reference numbers of all filters should be specified, as filter type has been found to influence recovery profoundly [102].

^cSEC: the pore size of the matrix should be taken into account. For example, if the pore size excludes EVs > 70 nm in diameter, a population of vesicles may be excluded.

^dBoth size exclusion chromatography (SEC) and density gradients may co-isolate EVs and certain lipoproteins. Sequential techniques may be needed to achieve separation [160,213].

^eNote, however, continuing concerns about the specificity and effects on vesicles of certain precipitation techniques [103,278].

^fThis Table does not address several important considerations that are beyond the scope of these guidelines, such as ease of use, cost, and potential for Good Manufacturing Practice (GMP) production.

Table 2. Steps of EV characterization.

Major recommendations of MISEV2014.	Validity and/or Update in 2018
a) No recommendation on quantification	New in MISEV2018: As a rule, both the source of EVs and the EV preparation must be described quantitatively. Source: Number of cultured cells (possibly an estimate, if adherent, as well as intervals of harvest, where applicable), total starting volume of biofluid, or weight/volume/size of tissue at the time of collection must be indicated for each experimental use. The appropriate parameters will vary by source, however; e.g. for urine, volume alone may not be meaningful, and other parameters, such as creatinine level, might be reported. For the EV preparation, global quantification of EVs should be provided. There is no single perfect quantification method. The most commonly used are total protein amount and total particle number. Total lipid quantification could be also considered. None of these components are exclusively associated to EVs, though: proteins are also soluble, particles can be protein aggregates, and lipids are also present in lipoproteins. Thus, ratios of proteins:particles, lipids:particles or lipids:proteins should be reported along with global quantification estimates as a measure of purity and thus reliability of the quantity measure.
b) General characterization. Show: i. At least three positive protein markers of EVs, including at least one -transmembrane/lipid-bound protein -cytosolic protein ii. At least one negative protein marker	Still valid but has evolved with increasing knowledge of the existence of different EV types. Table 3 gives categories of proteins to consider for characterization and some examples. At least one protein of each category 1 to 3 must be evaluated in any EV preparation (at least each time pre-analytical and/or EV isolation conditions are modified). Analysis of proteins of categories 4–5 is recommended for studies that focus on one or more EV subtypes (e.g. small EVs < 200 nm, vs larger EVs: category 4), or that have identified a functional soluble factor in EVs (category 5). 1. All EVs bear proteins associated with the membrane or outer membrane (prokaryotic cells), or with plasma membrane and/or endosomes (eukaryotic cells). To demonstrate the presence of a lipid bilayer in the material analysed, at least one transmembrane or GPI-anchored extracellular protein must be shown. Examples include mammalian proteins expressed (nearly) ubiquitously in all cell types (1a), and proteins specifically expressed in some cells (1b). 2. In all EVs, the lipid bilayer encloses cytosolic material (eukaryotic cells, Gram-positive bacteria) or periplasmic material (Gram-negative bacteria) from the secreting cell. To demonstrate that the material analysed contains more than open cell fragments, at least one cytosolic/periplasmic protein with lipid or membrane protein-binding ability must be shown (2a). Other cytosolic proteins are more promiscuously associated with EVs and other structures and thus should be only optionally used as EV markers (2b). 3. Purity controls include proteins found in most common co-isolated contaminants of EV preparations: depending on the source of EVs, expected contaminants from category 3a (lipoproteins and serum-derived materials), or 3b (urine), should be evaluated. 4. Proteins present in subcellular compartments other than the plasma membrane and endosomes, which may be present in certain EV subtypes (eukaryotic cells). 5. Soluble extracellular proteins with functional activities (cytokines, growth factors, extracellular matrix) may be detected in EVs: their mode of association to EVs (via a specific or promiscuous receptor? Or internal?) should be determined. (See Table 2 part d), below.
c) Characterization of single vesicles: use two different but complementary techniques, for example: i. electron or atomic force microscopy (and show both close-up and wide-field) ii. single particle analyzers (not electron microscope-based)	Still valid, but has evolved with a rapidly increasing number of techniques used to analyze EVs. i. Techniques providing images of single EVs at high resolution, such as electron microscopy and related techniques, scanning-probe microscopy (SPM) including atomic-Force microscopy (AFM), or super-resolution microscopy: these techniques are not interchangeable in the information they provide. When reporting results, both close-up and wide-field images must be provided. ii. Single particle analysis techniques that estimate biophysical features of EVs from other techniques than high-resolution images: size measured by resistive pulse sensing (electric field displacement), or light scattering properties [nanoparticle tracking analysis (NTA), high resolution flow cytometry, multi-angle light scattering coupled to asymmetric flow field-flow fractionation (AF4)]; or fluorescence properties [fluorescence correlation spectroscopy (FCS), high-resolution flow cytometry]. Chemical composition measured by Raman spectroscopy. Other techniques are being developed that may combine these two categories but have not yet been widely used (see 4c p.20). Whatever technique is used, all experimental details for both acquisition and analysis must be reported. Note that not all techniques are equally adapted to all EVs: large EVs (> 400 nm) and very small EVs (< 50 nm) are not well quantified by all NTA; small EVs are not easy to detect by most common flow cytometers. Some large EVs (and aggregates of small EVs) can be imaged by light/fluorescence microscopy. EVs smaller than the refraction limit or resolution of a microscope can still be detected by fluorescence, but no structural information can be obtained, and a single EV cannot be distinguished from a small EV cluster purely based on structural details.
d)	MISEV2018 additional characterization. We now recommend that the topology of EV-associated components be assessed, that is, whether a component is luminal or on/at the surface of EVs, at least for those required for a given EV-associated function. Topology may be particularly important for certain classes of biomolecules. Protease and nuclease digestions, detergent permeabilization, and antibodies to outer epitopes (should bind) or inner epitopes (should not bind) can be used to probe topology.

Table 3. Protein content-based EV characterization. At least one protein of categories 1a or 1b, 2a (optionally 2b), 3a or 3b must be analysed to demonstrate the EV nature and the degree of purity of an EV preparation. Analysis of proteins of category 4 is required when claiming specific analysis of small EVs, and of category 5 to document functional activities. Examples of proteins commonly found in mammalian cell-derived EVs are provided, but other proteins that fall into the provided categories can be used, particularly for analysis of EVs from prokaryotic (bacteria) or non-mammalian eukaryotic sources (including parasites and plants). XX = human gene names. XX* or XX** used for families of multiple proteins, for example for integrins: ITGA* indicates any integrin alpha chain.

Category	1- Transmembrane or GPI-anchored proteins associated to plasma membrane and/or endosomes	2- Cytosolic proteins recovered in EVs	3- Major components of non-EV co-isolated structures	4- Transmembrane, lipid-bound and soluble proteins associated to other intracellular compartments than PM/endosomes	5- Secreted proteins recovered with EVs
Use for	All EVs	All EVs	All EVs as purity control	Subtypes of EVs (e.g. large oncosomes, large EVs) and/or pathologic/atypical state	Functional component of EVs: need to determine the mode of association with EVs
	1a: non-tissue specific. Tetraspanins (CD63, CD81, CD82); other multi-pass membrane proteins (CD47; heterotrimeric G proteins GNA*) MHC class I (HLA-A/B/C, H2-K/D/Q), Integrins (ITGA*/ITGB*), transferrin receptor (TFR2); LAMP1/2; heparan sulfate proteoglycans including syndecans (SDC*); EMMPRIN (BSG); ADAM10; GPI-anchored 5ʹnucleotidase CD73 (NT5E), complement-binding proteins CD55 and CD59; sonic hedgehog (SHH)	2a: with lipid or membrane protein-binding ability. ESCRT-I/II/III (TSG101, CHMP*) and accessory proteins: ALIX (PDCD6IP), VPS4A/B; ARRDC1; Flotillins-1 and 2 (FLOT1/2); caveolins (CAV*); EHD*; RHOA; annexins (ANXA*); Heat shock proteins HSC70 (HSPA8), and HSP84 (HSP90AB1) note that both are abundant also in exomeres; ARF6; syntenin (SDCBP); microtubule-associated Tau (MAPT, neurons)	3a: lipoproteins (produced by liver, abundant in plasma, serum). ApolipoproteinsA1/2 and B APOA1/2, APOB; APOB100; albumin (ALB)	4a: nucleus. Histones (HIST1H**); Lamin A/C (LMNA)	5a: Cytokines and growth factors. e.g. TGFB1/2; IFNG, VEGFA, FGF1/2, PDGF*, EGF, interleukins (IL*)…
	1b: cell/tissue specific. Some TSPANs: TSPAN8 (epithelial cell), CD37 and CD53 (leukocytes), CD9 (absent from NK, B and some MSC); PECAM1 (endothelial cells); ERBB2 (breast cancer); EPCAM (epithelial); CD90 (THY1) (MSCs); CD45 (PTPRC) (immune cells), CD41 (ITGA2B) or CD42a (GP9) (platelets); Glycophorin A (GYPA) (RBC); CD14 (monocytes), MHC class II (HLA-DR/DP/DQ, H2-A*); CD3* (T cells); Acetylcholinesterase/AChE-S (neurons), AChE-E (erythrocytes); amyloid beta A4/APP (neurons); multidrug resistance-associated protein (ABCC1)	2b: promiscuous incorporation in EVs (and possibly exomeres). Heat shock protein HSP70 (HSPA1A), cytoskeleton: actin (ACT*), tubulin (TUB*); enzymes (GAPDH)	3b: protein and protein/nucleic acid aggregates. Tamm-Horsfall protein (Uromodulin/UMOD) (urine); ribosomal proteins	4b: mitochondria IMMT, cytochrome C (CYC1); TOMM20	5b: adhesion and extracellular matrix proteins. Fibronectin (FN1), Collagen (COL**), MFGE8; galectin3-binding protein (LGALS3BP), CD5L; fetuin-A (AHSG)
				4c: secretory pathway (endoplasmic reticulum, Golgi apparatus) calnexin (CANX); Grp94 (HSP90B1); BIP (HSPA5), GM130 (GOLGA2)
				4d: others (autophagosomes, cytoskeleton…). ATG9A, Actinin1/4 (ACTN1/4), cytokeratin 18 (KRT18)

Table 4. EV-associated and EV-excluded biological activities.

Major recommendations of MISEV2014.	Validity and/or Update in 2018
a) Dose–response studies	Still valid In addition: quantitative comparison of the activity of conditioned medium or biofluid 1) before, 2) after elimination of EVs, and 3) the EVs themselves, keeping in mind that the EV fraction may include co-isolated/contaminating materials. Additional suggested control: quantitative comparison of the activity of the targeted versus the “discarded” EV subtypes (see part 1 below)
b) Negative or background controls. For conditioned medium, negative control = complete medium that has not been conditioned by cells, but still processed in the same way as conditioned medium	Still valid. For biofluids, negative controls of disease-associated functions = fluids from healthy, untreated or otherwise matched donors
c) Controls to assess influence of soluble or non-EV macromolecular components c-i.) Density gradients or other rigorous separation method to show activity is intrinsic to EVs, not just associated or c-ii.) EV depletion to remove activity or c-iii.) EV/cell labelling (e.g. fluorescent labelling, with careful interpretation)	Still valid. Increasingly, it is possible to separate EVs from non-EV components by multiple methods, e.g. density gradients and size exclusion chromatography. It is recommended that functional assays be performed after rigorous separations, comparing EV and non-EV fractions to identify what proportion of activity is associated with each fraction (in case it is not EVs). If the activity is primarily associated with EVs, depleting the EVs should also deplete the activity. The refined separation must be performed at least for a set of biological replicates, but not necessarily systematically afterwards.

Table

Text Box 1. Example of a change from MISEV2014 as a paradigm for considering negative markers.

Argonaute proteins, chiefly AGO2, the most abundant family member in mammals, have been previously proposed as negative markers of EVs. In blood plasma and perhaps other fluids, most extracellular AGO is indeed found outside of EVs [189,190,216]. However, the canonical view of biogenesis of microRNAs (miRNAs) and their subsequent protection from degradation depends on AGO proteins. According to this reasoning, if a mature miRNA is detected, whether in cells, in EVs, or elsewhere, it is most likely protected by AGO (although other associations have been reported [56]). Limits of detection of proteins versus amplified nucleic acids may have contributed to the interpretation that miRNAs are present in EVs without AGO protein. It is in any case now established that AGO can be found in mammalian [217,218] and non-mammalian [219] EVs, not just outside EVs.

Table

Text Box 2. Example of a possible change from MISEV2018 in the future as markers of non-EV co-isolated components.

The Lyden group recently reported that lipidic structures called “exomeres” contain lipids and a limited set of membrane-bound proteins, but do not feature a lipid bilayer and thus do not qualify as EVs. Exomeres were found to co-isolate with small EVs recovered from many cultured cell lines [112] but could be separated from EVs by AF4 (which unfortunately is not implementable in some laboratories). It may be necessary to evaluate this potential contaminant in all EV preparations. Proteomic analyses of exomeres identified various proteins in Table 3-category 2, above: heat shock proteins (HSPA8, HSPA1A, HSP90AB1), actin (ACT*), tubulin (TUB*), and GAPDH. Such proteins thus probably do not qualify as EV-specific components. In addition, a few proteins were identified exclusively in exomeres: hemoglobin (HBA1/A2), IDH1, MAT1A, transmembrane FAT4 (protocadherin, transmembrane plasma membrane), and EXT1/2 (exostosin, transmembrane, Golgi). Although this exhaustive study was performed with several cell lines, suggesting that the listed proteins are good candidates as specific markers of exomeres co-isolated with EVs, these results still have to be confirmed by other groups to reach validation and inclusion in the next update of the MISEV guidelines.

Table

Text Box 3.

As an aside, although we do not go into great detail on this point, many functional studies presume or investigate EV uptake. Time-courses and environmental determinants of EV uptake have been studied for some time [272-274], but challenges exist [275]. Detection within the cell of signal from an EV-labeling dye or other entity does not necessarily mean that the EV or its cargo has been internalized. Some labeling substances are very long-lived, can exist separate from the presumably labeled entity, and can form EV-mimicking particles that are difficult to separate from EVs. Another potential artifact is that labeling EVs with lipophilic or surface-coating fluorophores may modify physicochemical characteristics of EVs, thus altering detection mode and/or uptake by target cells. Although we cannot yet make firm recommendations, we urge researchers to be aware of these issues and to consider that each specific EV-donor/EV-recipient pair may behave in a different manner.

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