Exosomes: Fact or Fiction?

In order to know whether the evidence submitted for any invisible entity is credible, one must examine the methods the researchers used in order to obtain their results. Did the researchers follow the scientific method? Were the procedures validated for the intended purposes? Are there technological limitations that need to be addressed? Did the researchers implement the proper controls to factor in all possible confounding variables? Are the methods standardized across all laboratories performing such research? Were the conclusions supported by the evidence and were the results reproducible and replicable?

Often, when trying to find answers to questions such as these, one comes across a litany of excuses for why the current methods employed are incapable of supporting the evidence obtained. In many instances, the promise is made that there will be better technology available in the future and that steps are being made to ensure quality research going forward. However, if the current methods and technology are inadequate and the research lacks standardization leading to a free-for-all where every lab is left to its own individual devices, how can any of the evidence presented be validated? How can scientific fact be determined from scientific fiction?

In the case of exosomes, the evidence submitted for these invisible entities has accumulated at a rapid pace over the last forty years. Research has grown tremendously since these entities were originally found using the same cell culture practices as seen in virology. Since then, the particles assumed to be exosomes have been claimed to have been found in nearly all bodily fluids. They have been given many theoretical functions such as a pivotal role in intercellular communication as well as having a pathological effect identical to “viruses.” In fact, I have shown that exosomes are the very same particles claimed to be “viruses” with the only difference being that exosomes are said to be unable to replicate which is a function never proven for “viruses.”

Just as in the case of virology, the problem encountered with exosome research is that, since the “discovery” of exosomes in the early 1980’s, it has been shown that the methods used to isolate and study these particles are entirely inadequate. The procedures used for purification are not able to separate exosomes from “viruses” as well as other extracellular vesicles and non-EV particles contained within a sample due to similar size, shape, and density. The techniques utilized for the biochemical and molecular characterization are non-specific and remain a difficult challenge. The evidence presented for the functions of these particles has been called into question due to the crude, contaminated, and heterogeneous preparations the exosomes are assumed to be contained within. Much of the characteristics and functions attributed to these fictional entities is unable to be verified experimentally.

The problems encountered by exosome researchers led to the International Society for Extracellular Vesicles to release a series of guidelines in 2014 known as the Minimal Information for Studies of Extracellular Vesicles (MISEV). It was an attempt to provide guidance in standardization of protocols and reporting in the extracellular vesicle field which had been around for 30+ years without any such criteria in place. In the opening statement, the authors admitted to the difficulty in actually isolating the particles being studied in order to characterize them and determine their function. The lack of standardized procedures was generating unreliable results. Thus, they aimed to correct these issues by providing the minimal requirements needed to be addressed for every study:

“Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology.”

“Separation of these non-vesicular entities from EV is not fully achieved by common EV isolation protocols, including centrifugation protocols or commercial kits that claim EV or “exosome” isolation/purification. Also, the composition of recovered EVs vary vastly according to the protocols used (6–8).”

“We recognize that different experimental systems, sources of biological specimens, investigator’s experience and instrumentation used contribute to the heterogeneity of published protocols and the interpretation of results. A framework for providing data and attributing functions to EVs was discussed by the Executive Committee of the International Society for Extracellular Vesicles (ISEV), a group of scientists with collective long-term expertise in the field of EV biology. Here, we propose a series of criteria, based on current best-practice, that represent the minimal characterization of EVs that should be reported by investigators. Adoption of these criteria should aid researchers in planning studies as well as reporting their results. In addition, we suggest appropriate controls that should be included in EV-related functional studies. These controls should support conclusions regarding the functions of EVs and their relationship to physiologic and pathologic mechanisms.”

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4275645/

As admitted by MISEV2014, exosomes can not be purified and isolated from other biological compounds nor characterized properly, thus the validity of any scientific study regarding exosomes over the last 40+ years, including the original study that brought about the exosome concept, should be questioned as to its accuracy. As the same methods are used to “isolate” the identical particles known as “viruses,” the last 70+ years of virology papers should also be in doubt. However, instead of critically examining the previous decades of research, the experts gathered around and set in place an agreed upon criteria and guidance in order for the researchers to “do better” in the future. There is always the promise that the results and conclusions will be verified by advancements in the methods and technology while the problem with the vast amount of accumulated evidence that has been shown to be built upon fraudulent grounds is ignored. This creates an illusion of progress when in fact things stay exactly the same.

Thus, the ISEV set out to re-evaluate and update their guidelines every four years in order to guage any advancements in the field and apply that to their recommendations. They revised MISEV in 2018, with the promise of another update coming this year. The differences they found in four years were minimal. Despite technological advances, exosomes were still unable to be purified and isolated from everything else. In fact, the ISEV admitted that this was an impossibilty. This inability to separate the particles made characterizing them a difficult challenge. The functions ascribed to EVs were unable to be verified experimentally due to the still limited knowledge of their specific molecular machineries of biogenesis and release. Due to the different approaches applied during different studies, MISEV2018 did not propose molecular markers that could characterize specifically each EV subtype. It was also not recommended as a major point of any EV study to try and demonstrate that a function is specific to exosomes as compared with other types of small EVs, due to the issues related to the non-standardized approaches, the insufficient isolation/separation of the particles in order to determine functioning, and the unverifiable results.

Below are highlights from the MISEV 2018 update. The report itself is rather long so I supplied the most important sections:

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

“The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Introduction

In 2014, the ISEV board members published a Position Editorial detailing their recommendations, based on their own established expertise, on the “minimal experimental requirements for definition of extracellular vesicles and their functions” [1]. A list of minimal information for studies of extracellular vesicles (MISEV or MISEV2014) was provided, covering extracellular vesicle (EV) separation/isolation, characterization, and functional studies. The major goal of these recommendations was to sensitize researchers (especially the rapidly growing numbers of scientists newly interested in EVs), as well as journal editors and reviewers, to experimental and reporting requirements specific to the EV field. The ISEV board highlighted the need to consider these issues when making strong conclusions on the involvement of EVs, or specific populations of EVs (exosomes in particular), in any physiological or pathological situation, or when proposing EVs or their molecular cargo as biological markers. By stimulating improved reliability and reproducibility of published EV results, the MISEV2014 authors hoped to further the promise of EVs as biomarkers or for therapeutic applications even in the face of skepticism by some scientists outside the field.

As evidenced by the increasing number of EV publications in high-profile journals, proposing major roles of EVs in numerous physiological pathways from aging to cancer, infectious diseases to obesity, EV science has now clearly achieved widespread interest and enthusiasm well beyond the EV research community. However, the promotion of rigorous EV science is an ongoing process; as EV experts within the ISEV community, we are still concerned to see that major conclusions in some articles are not sufficiently supported by the experiments performed or the information reported. We therefore aim to revise and renew the MISEV recommendations and to continue to work toward their wider acceptance and implementation. In this “MISEV2018” update, a much larger group of ISEV scientists was involved through a community outreach (the MISEV2018 Survey), striving for consensus on what is absolutely necessary, what should be done if possible, and how to cautiously interpret results if all recommendations for controls cannot be followed.

We strongly believe that most of the MISEV2014 recommendations are still valid; however, discoveries and developments within the field during the past four years necessitate certain amendments. This document explains how the 2014 recommendations evolved into MISEV2018 in Tables 1, 2 and 4; provides suggestions for protein markers to validate the presence of EVs (Table 3); and, to highlight the salient points, provides outlines of examplar approaches to address some of the most important experimental issues. Importantly, a 2-page checklist summarizing the major aspects to follow in EV science is provided at the end of this article.”

Note on applicability of MISEV2018: species, cells, sample types, and experimental conditions

“Does MISEV2018 apply to all EV studies, or only to some? EVs appear to be produced by almost all organisms and cell types studied. Yet EV research to date has focused on mammalian EVs, chiefly those of human or mouse origin, and not all cell types or experimental conditions have been closely investigated. In this document, as in MISEV2014, specific examples of molecular markers (such as the markers of EVs in Table 3) are based on studies of specific species, cells, and experimental conditions. Some may be broadly applicable, others less so. Nevertheless, the general principles of MISEV2018 apply to EVs produced by all organisms and all cells. The need to demonstrate presence (or enrichment) of EV markers and absence (or depletion) of putative contaminants, when contents or function of EVs are described, can be generalized to all species, cells, and conditions. We find ourselves at an exciting scientific frontier; where such markers are not yet available, we encourage their development and publication, using the principles of this document as a guide. Additional specific examples may then be incorporated into future MISEV updates.

Nomenclature

ISEV endorses “extracellular vesicle” (EV) as the generic
term for particles naturally released from the cell that are delimited by a lipid bilayer and cannot replicate, i.e. do not contain a functional nucleus. Since consensus has not yet emerged on specific markers of EV subtypes, such as endosome-origin “exosomes” and plasma membrane-derived “ectosomes” (microparticles/microvesicles) [3,4] assigning an EV to a particular biogenesis pathway remains extraordinarily difficult unless, e.g. the EV is caught in the act of release by live imaging techniques. Therefore, unless authors can establish specific markers of subcellular origin that are reliable within their experimental system(s), authors are urged to consider use of operational terms for EV subtypes that refer to a) physical characteristics of EVs, such as size (“small EVs” (sEVs) and “medium/large EVs” (m/lEVs), with ranges defined, for instance, respectively, < 100nm or < 200nm [small], or > 200nm [large and/or medium]) or density (low, middle, high, with each range defined); b) biochemical composition (CD63+/CD81+-EVs, Annexin A5-stained EVs, etc.); or c) descriptions of conditions or cell of origin (podocyte EVs, hypoxic EVs, large oncosomes, apoptotic bodies) in the place of terms such as exosome and microvesicle that are historically burdened by both manifold, contradictory definitions and inaccurate expectations of unique biogenesis. If it is deemed unavoidable to use these or newly coined terms, they should be defined clearly and prominently at the beginning of each publication [5]. If confirmation of EV identity cannot be achieved according to the minimal requirements of this MISEV2018 publication, other terms such as extracellular particle (EP) might be more appropriate.

Collection and pre-processing: pre-analytical variables

The first step to recover EVs is to harvest an EV-containing matrix, such as fluid from tissue culture or from an organismal compartment. During this pre-analytical phase, an extended constellation of factors, including characteristics of the source, how the source material is manipulated and stored, and experimental conditions, can affect EV recovery. Therefore, it is crucial to plan collection and experimental procedures to maximize the number of known, reportable parameters, and then to report as many preanalytical parameters as are known.

Cell culture conditioned media

For EV isolation/characterization from conditioned media (an ISEV survey found that the majority of responding EV researchers studied conditioned medium [6]), basic characterization of the releasing cells and culture and harvesting conditions must be performed and reported. Some precautions, such as regular confirmation of cellular identity (e.g. by short tandem repeat (STR) profiling or other methods) [7,8] and identification of cell lineage and provenance including mode of immortalization [9], are advisable for all cell studies. Especially important for EV studies is that the percent of dead cells at the time of EV harvest should be indicated, since even a small percentage of cell death could release cell membranes that outnumber true released EVs. Quantifying the percentage of apoptotic and necrotic cells may also be useful. (Note, however, that when cells are treated with high concentrations of EVs, cell-adherent EVs positive for apoptotic markers may skew results [10,11]). Other relevant characteristics of the cells, including state of activation, malignancy, and senescence [12,13], should be reported where applicable.

Culture and harvesting conditions such as passage number (or days in culture for suspension cells), seeding density [14], density/confluence at harvest [14], including any relevant post-confluence characteristics such as development of polarity [15–19] (in that case, were EVs collected globally or separately, from the different parts of polarized cells?), culture volume, culture vessel or bioreactor system
(if used [20,21]), surface coatings, oxygen or other gas tensions (if they differ from standard cell culture) [22,23], stimulation and other treatments [24–30], and frequency and intervals of harvest [14] should be given to allow replication [31,32]. Culture conditions prior to treatment-(s), if any, should also be given. Note that EV recovery depends not only on EV release, but also on re-uptake by cells in culture, which may vary based on culture density and other conditions. Regular checks for contamination with Mycoplasma (and possibly other microbes) are needed, not only because of cellular responses to contamination, but also because contaminating species can release EVs [33–36]. Exact methods of medium collection should be given, as well (e.g. decanting or pipetting from flasks, centrifugation of suspension cell cultures). The suggested parameters are of course non-inclusive, and others may be necessary to report for specific types of cells and experiments, including co-culture systems and organoid cultures [37].

All culture medium composition and preparation details should be provided in methods. This should be customary for cell culture studies, and is doubly important here since supplements like glucose [38–40], antibiotics [41], and growth factors [42] can affect EV production and/or composition. Of special note are medium components that are likely to contain EVs, such as serum. EVs are ideally obtained from culture medium conditioned by cells in the absence of fetal calf serum (FCS or FBS), serum from other species, or other complex products such as platelet lysate, pituitary extract, bile salts, and more, to avoid co-isolation of exogenous EVs. When use of these supplements is unavoidable, experiments should include a non-conditioned medium control to assess the contribution of the medium itself. However, depending on downstream use, it may not be necessary or desirable to deplete EVs [43,44]. In the case of depletion, since nutrient or EV deprivation of cells that are normally cultured in serum- or lysate-containing medium can change cellular behavior and the nature and composition of released EVs [45,46], it is important to specify culture history (how and when the switch to serum-free medium occurred, including acclimatization
steps). Alternatively, cells can be exposed during the EV release period to medium that has been pre-depleted of EVs. Here, too, effects on cells and EVs may be expected [47], and the methods and outcome of depletion vary greatly and should be reported. Several fairly efficient protocols are available, such as 100,000 x g ultracentrifugation of complete medium (or of serum following at least 1:4 dilution) for at least 18 hours [48], centrifugation at enhanced speeds (e.g. 200,000 x g [49]) for shorter periods of time, or tangential flow filtration or other forms of ultrafiltration [50]. Ultracentrifugation at around 100k x g for a few hours or without dilution will not eliminate all EVs or EV-associated RNA [51–53]. Commercial “exosome/EV-depleted” serum and other supplements are available from an increasing number of vendors. Since the method of depletion is usually not indicated, consequences on cell growth and EV release may not be predictable; the exact source, method, and reference of depleted supplements should be given, and the “exosome-free” nature of the product should be checked carefully before use [54]. Additionally, vendors are encouraged to report and benchmark the methods of depletion utilized in their products, while users should report product and lot numbers as well as any pooling of biologicals. Finally, medium preparation details, including heating (heat inactivation) or filtration steps, should be reported. For example, heat inactivation of additives such as serum leads to formation of protein aggregates that may co-precipitate with EVs and thus also change the growth-supporting properties of the serum.

Biological fluids

Since more than 30 types of biofluids exist in mammals, and lavages of numerous compartments add to this number (despite not being true biofluids), MISEV2018 does not provide an exhaustive review of the literature on pre-analytical variables related to all biofluids. Each biological fluid presents specific biophysical and chemical characteristics that makes it different from culture conditioned medium, and this must be taken into account when isolating EVs. For instance, plasma and serum are more viscous than conditioned medium. Plasma and serum contain numerous non-EV lipidic structures (low/very low/high density lipoproteins), milk is replete with fat-containing vesicles, urine with uromodulin (Tamm-Horsfall protein), bronchoalveolar lavage with surfactant, all of which will be co-isolated to various degrees with EVs. In each case, specific precautions to separate EVs from these components may be required. While detailed biofluid-specific reporting guidelines are beyond the scope of this MISEV, we encourage development of such guidelines under the MISEV umbrella.

For EV isolation/characterization from biofluids such as blood plasma, several previous ISEV position papers [55,56] and other publications (for just a few of many examples, see [57–63]) have listed reporting requirements that are important for standardization, and these are still valid today, even if many questions remain about the effects of specific pre-analytical variables on different classes of EVs. Since many of these factors have been covered in these previous publications, we do not review them exhaustively here. To give examples of considerations for blood derivatives such as plasma: donor age, biological sex, current or previous pregnancy, menopause, pre/postprandial status (fasting/non-fasting), time of day of collection (Circadian variations), exercise level and time of last exercise, diet, body mass index, specific infectious and non-infectious diseases, medications, and other factors may affect circulating EVs [64,65]. Similarly, technical factors including fluid collection volume, first-tube discard, type of container(s), time to processing, choice of anticoagulant (for blood plasma) [66–68], mixing or agitation, temperature (storage and processing), description of transport (if any), whether tube remained upright before processing, exact centrifugation or filtration procedures, degree of hemolysis, possible confirmation of platelet and lipoprotein depletion prior to storage [69–73], and other parameters should be clearly indicated. Overall, except some that are specific of plasma/serum (such as platelet removal and coagulation), the above listed technical details of collection condition apply to all biofluids and must be reported. Of course, it may be that not all variables have been
recorded for archived samples, and this should be acknowledged where applicable.

Tissue

As a special case of pre-analytical issues, a rapidly increasing number of groups have reported isolation of tissue EVs. These studies may involve short-term culture of tissue explants [74] such as ex vivo tumors [75], or placenta [76,77], or extraction from whole tissues [78–84]. Many of the same considerations that apply for cellular and biofluids studies also apply here, including confirmation of provenance and condition. Especially for EV extraction from tissue, it is challenging to ensure that recovered vesicles are truly from the extracellular space, rather than being intracellular vesicles or artefactual particles released from cells broken during tissue harvest, processing (e.g. mechanical disruption), or storage (including freezing). This may be especially challenging in a tissue like brain, where similar procedures are used to collect synaptosomes [85]. Even apparently pure tissue-derived EVs can contain endosome components, which could correspond to components of intracellular vesicles including unreleased intraluminal vesicles of late endosomes/multivesicular bodies (MVBs) that are released artifactually during tissue processing. The recent awareness of these challenges has led researchers to perform gentle tissue disruption (i.e. with the goal of separating EVs from cells and extracellular matrix, but not disrupting cells) and several steps of further separation (including density gradients), followed by strict characterization of multiple negative markers, leading to more convincing tissue-derived EV preparations [79]. Use of genetically modified models to trace EV release from specific cells [83] is also a useful approach. More research is clearly needed and encouraged into the isolation, characterization, and function of tissue EVs, as compared with intracellular vesicles and/or non-vesicular extracellular particles (EPs).

EV separation and concentration: how MISEV2014 evolves in 2018

Absolute purification, or complete isolation of EVs from other entities, is an unrealistic goal (as for many biological products). For this reason, and since the various combinations of EVs and media are colloids [98], here we use the terms separation and concentration. Separation (colloquially referred to as purification or isolation) of 1) EVs from other non-EV components of the matrix (conditioned medium, biofluid, tissue) and 2) the different types of EVs from each other, are achieved to various degrees by the different techniques available. Concentration is a means to increase numbers of EVs per unit volume, with or without separation. The term “enrichment” can refer to increasing concentration, i.e. EV counts relative to volume, or to increasing EV counts/markers relative to another component. The extent of separation or concentration can be assessed by characterization, which will be detailed in the next section.

How pure should an EV preparation be? The answer depends on the experimental question and EV end use, and often segregates by basic and clinical research. Highly purified EVs are needed to attribute a function or a biomarker to vesicles as compared with other particles. Less pure EVs may be required in other cases, such as when a biomarker is useful without pre-enrichment of EVs, or in certain therapeutic situations where function is paramount, not the definitive association of function with EVs. Of note, some presumed contaminants may co-isolate with EVs and may even contribute to EV function. Therefore, the choice of separation and concentration method must be informed by factors that may vary between studies such that there is no one-size-fits-all approach. More details on this issue (function and co-isolated factors) are given in section 5c-d (p.24).

At the end of 2015, according to a worldwide ISEV survey [6], differential ultracentrifugation was the most commonly used primary EV separation and concentration technique, with various other techniques, such as density gradients, precipitation, filtration, size exclusion chromatography, and immunoisolation, used by 5–20% of respondents each. Relative success of these different methods in terms of recovery and specificity to EVs (as compared to non-vesicular components), or to EV subtypes, has been addressed in a previous ISEV
Position Paper (see Figure 1 of [56]), and is summarized in Table 1 below. To achieve better specificity of EV or EV subtype separation, most researchers use one or more additional techniques following the primary step, such as washing in EV-free buffer, ultrafiltration, application of density gradients (velocity or flotation), or chromatography [6,99–102].

A variety of additional techniques or combinations of techniques have been or are currently being developed, some of which may become more prominent in the coming years if they achieve better recovery or specificity than legacy methods (and this must be demonstrated as in, e.g. [103]). Such methods include tangential flow filtration and variations thereon [21,104–110], field-flow fractionation (FFF) [111], asymmetric flow field-flow fractionation (AFFF, A4F, or AF4) [112–114], field-free viscoelastic flow [115], alternating current electrophoretics [116,117], acoustics [118], variations on size exclusion chromatography (SEC) [100,119–121], ion exchange chromatography [122–124], microfiltration [125], fluorescence-activated sorting [126,127] (especially for larger EVs including large apoptotic bodies [128] and large oncosomes [129]), deterministic lateral displacement (DLD) arrays [130], novel immunoisolation or other affinity isolation technologies [131–138], including lipid affinity [139], novel precipitation/combination techniques [140–142], hydrostatic filtration dialysis [143], highthroughput/high-pressure methods such as fast protein/high perfomance liquid chromatography (FPLC/ HPLC) that involve some form of chromatography
[144] and a wide variety of microfluidics devices that
harness one or more principles, including some of those mentioned above [145–153]. Of course, combinations of methods will continue to be used and may outperform single-method approaches.

Table 1 summarizes the instructions given in MISEV2014 for EV isolation (left column), and their updates in MISEV2018 (right).

EV characterization: how MISEV2014 evolves in 2018

EV characterization by multiple, complementary techniques is important to assess the results of separation methods and to establish the likelihood that biomarkers or functions are associated with EVs and not other co-isolated materials. The need for guidelines for characterization was emphasized by a consortium study led by Hendrix and colleagues [161]. These authors found that only about half of EV-related articles published within a five-year time period included positive markers of EVs, and only a small minority complemented positive with negative markers to track co-isolated non-EV components. ISEV recommends that each preparation of EVs be 1) defined by quantitative measures of the source of EVs (e.g. number of secreting cells, volume of biofluid, mass of tissue); 2) characterized to the extent possible to determine abundance of EVs (total particle number and/or protein or lipid content); 3) tested for presence of components associated with EV subtypes or EVs generically, depending on the specificity one wishes to achieve; and 4) tested for the presence of non-vesicular, co-isolated components.

Table 2 summarizes the instructions given in MISEV2014 for EV characterization, and their updates in MISEV2018. These recommendations apply to EVs
from all sources, including non-mammalian and non-eukaryotic cells and organisms.

Quantification of EVs

Since quantifying EVs themselves remains difficult (see below), as minimal information, the total starting volume of biofluid, or, for conditioned medium, number of cells or mass of tissue at the time of collecting, should be indicated for each experimental use. If the latter is not possible, for instance due to culture conditions (such as periodic collection in continuous bioreactor-based cultures [162]), number of cells at initiation of culture, expected doubling time, and frequency of collection must be indicated. For some biological fluids, like urine, the volume depends strongly on pre-analytical conditions (especially intake of liquid by the donor), thus additional means of normalization should be considered, such as urinary creatinine, as routinely done in the clinic for albumin [163].

EVs have a particulate structure and contain proteins, lipids, nucleic acids, and other biomolecules. Quantification of each of these components can be used as a proxy for quantification of EVs, but none of these values is necessarily perfectly correlated with EV number.

Particle number can be measured by light scattering technologies, such as nanoparticle tracking analysis (NTA); by standard flow cytometry for larger EVs [164–167] or high resolution flow cytometry for smaller EVs [127,168–176]; by resistive pulse sensing (RPS) for a wide range of sizes, depending on pore size [177]; by cryo-EM [174]; by a platform combining surface plasmon resonance (SPR) with AFM [178]; or by other techniques with similar capabilities. Accurate quantitation may be possible only within a certain concentration and size range that varies by platform; where possible, this range (or the minimum and maximum diameter measured) should be reported along with concentration. The method of volume determination in flow cytometry should be reported and potential swarming/coincidence artefacts controlled for [179]; a more detailed guideline article on specifics of flow cytometry analysis of EVs is in preparation by members of ISEV, ISCT and ISAC. Some devices for particle
quantification have the advantage of providing accurate sizing information amongst a complex mixture of particles (see Table 2-c: single vesicle analysis). This is not the case for dynamic light scattering (DLS), which is accurate only for monodisperse particle populations [180]. Particle counting by light scatter, RPS, and similar techniques typically results in overestimation of EV counts since the techniques are not specific to EVs and also register co-isolated particles including lipoproteins and protein aggregates. Possibly, ongoing development of fluorescence capabilities of NTA devices may ultimately allow EV-specific measurement [181], although assay sensitivity and the tendency of labeling antibodies and lipid dyes to form particles pose substantial hurdles to such applications [127,182]. Additionally, particle counting technologies may be biased towards certain particle size ranges (especially 50–150 nm [183,184]) because of pore sizes (RPS), size of calibrator used, sensitivity (for example, smaller particles scatter less light), and ability to cope with multidispersity (DLS versus NTA) [185]. Finally, proprietary
software used for analysis of data from each device may apply unknown selection and other processing of data, resulting in differences in absolute values obtained by different software or different versions of the same
software (see example in [183]).

Total protein amount can be measured by various colorimetric assays [Bradford or micro-bicinchonic acid (BCA)] or fluorimetric assays, or by global protein stain on SDS-PAGE. The EV sample concentration must be
within the linear range of the reference curve. However,
protein quantification can result in overestimation due to co-isolated protein contaminants (such as albumin from culture medium or plasma/serum), especially when the less specific methods of EV separation are used, or conversely can prove not sensitive enough if highly specific methods yield pure EVs. In addition, results may vary depending on the use or not of detergent to disrupt EVs and expose the entire protein content prior to performing the assay; nature and concentration of the detergent must be indicated.

Quantification of total lipids can be achieved, e.g. by sulfophosphovanilin assay [186], by measuring fluorescence of phospholipid dyes that fluoresce only when incorporated into lipid bilayers, such as DiR [187], or by total reflection Fourier-transform infrared spectroscopy [188]. However, the latter requires specialized equipment, and the former two types of assays may be insufficiently sensitive for small amount of EVs. In addition, whether these techniques equally detect all EVs independent of their specific lipid composition must still be established.

Quantification of total RNA can be performed by global RNA assays including profiles obtained by capillary electrophoresis instruments (see recommendations in Table 1 of [56]). Such measurements are difficult to recommend at this time for EV quantification or purity assays, though, since exRNAs associate in abundance with other circulating and potentially co-separating entities: chiefly ribonucleoproteins [189,190], but also a range of particles including exomeres [112] and lipoproteins [191]. RNA measurements remain, however, an important parameter to report in studies of extracellular RNA.

Quantification of specific molecules. Other methods of EV quantification, like ELISA [192] bead-based flow cytometry [193,194], aptamer- and carbon nanotube-based colorimetric assays [195], and SPR on surfaces such as antibody-coated nanorods [178,196,197], can be used to quantify the amount of one or more specific molecules in the EV preparation. These are generally proteins (usually the tetraspanins CD9, CD63 and/or CD81, but sometimes tumor-specific proteins or other molecules such as lipids [139]) and can be used to estimate the amount of EVs containing this particular component, rather than total EVs. These methods provide additional information to the above methods and are in line with characterization recommended in part 4b (p.16).

Single and multiple measures and implications for purity. Quantification methods are the most informative for EVs recovered by separation methods with the highest expected specificity (Table 1a-category 3), and for these preparations, one quantification method may suffice; in contrast, more than one quantification should be used for EVs recovered from low-specificity methods. Importantly, ratios of the different quantification methods may provide useful measures of purity. For example, protein:particle ratio [198,199], protein: lipid ratio [186,188,200] and RNA:particle [201] have been proposed as possible purity metrics, although their applicability across protein, lipid, RNA and particle quantification methods remains to be established. Techniques that measure multiple parameters at once, such as colloidal nanoplasmonic assays or infrared (IR) spectroscopy [188,199] may be good optional methods, despite the need for specific sensors or other equipment.

Absolute EV sizing and counting methods are currently imperfect and will require further improvement, aided by appropriate EV reference standards that are now in development [202]. Nevertheless, current methods can provide a reasonable indication of particles per volume and particle size distributions that are best interpreted when combined with general (Table 2b) and single-particle (Table 2c) characterization.

Characterization of EVs by their protein composition

Selection of proteins for use as EV markers. Since MISEV2014, the growing recognition of the existence of many different types of EVs, of different sizes and cellular origins, has led to publication of several studies comparing the protein composition of at least two subtypes of EVs isolated from the same secreting cells. Some studies compared EVs recovered by medium speed centrifugation (called large oncosomes [203], ectosomes [204], microvesicles [205], cell debris [206], or large [207] or medium [208] EVs), with those recovered by 100,000 x g ultracentrifugation (called exosomes in the first four studies, small EVs in the last two), and several of these applied additional separation in density gradients. Another study used differential filtration to separate large microvesicles retained by 0.65 micron filters, and small “exosomes” passing through 0.1 micron filters [209]. Others further separated the high speed pellet to identify subpopulations of small EVs bearing different surface markers such as A33 antigen (GPA33) vs EPCAM [19], lipid moieties binding Cholera Toxin, Annexin-V or Shiga Toxin [139], or tetraspanins CD63, CD9, and/or CD81 [208]. EVs were also separated by floating at different densities within a sucrose gradient (defined as high density “HD-exo” vs low density “LD-exo”) [210] or eluting at different time points in asymmetric flow field-flow fractionation (AF4) (small “exo-S” vs large “exo-L”) [112]. These studies together provide a rich source of potential EV subtype-specific markers. However, since they were performed with different separation approaches and with different cellular sources of EVs, it is still not possible to propose specific and universal markers of one or the other type of EVs, let alone of MVB-derived “exosomes” as compared with other small EVs.

Consequently, MISEV2018 does not propose molecular markers that could characterize specifically each EV subtype. Of note, although the ISEV board tried in MISEV2014 to propose general rules applying to all EVs, some suggestions of MISEV2014 were still biased by an “exosome–oriented” view of EVs. Specifically, Table 1 of MISEV2014 listed, as primary components to analyze in EVs, 2 categories of proteins present or enriched in EVs/exosomes (membrane bound and cytosolic proteins), plus another global category of proteins « not expected in EVs/exosomes » (such as mitochondria, Golgi, or nuclear proteins), and a last category of « contaminants ». In this updated version, MISEV2018, reference to exosomes and the proteins expected or not in them (the previously called “negative controls” of “exosome” preparations) have been deleted, reflecting an evolving understanding of the subtypes of EVs and their associations with other entities.”

New recommendation: determine the topology of EV-associated components

“Importantly, the luminal versus surface topology of various EV-associated components, including nucleic acids, proteins, glycans, etc, is not entirely strictly determined. Theoretically, components localized in the cytosol of EV-secreting cells should be inside EVs, and hence protected from mild degradation by proteases or nucleases. While this protection is usually observed, some studies have unexpectedly found proteins [266], RNAs [267], and DNA [41] on the EV surface and sensitive to digestion. It is not yet clear whether this unexpected topology is due to debris from dead or dying cells, or is instead the outcome of as-yet unknown mechanisms of transport of intracellular compartments across membranes that could occur in some physio- or pathological conditions. Certainly, even a small degree of contamination with intracellular material (with the reverse topology to EVs) would complicate interpretation.”

Functional studies: how MISEV2014 evolves in 2018

“Table 4 summarizes the previous and updated recommendations on functional analysis of EVs. More detailed justification for these recommendations and proposed protocols follows the Table. The goals of these recommendations are to avoid over-interpretations or classical artefacts when analyzing functions of EVs. It is important to consider several issues when attributing a functional activity to EVs in general, or an EV subtype in particular. We describe here the controls and processes that should be included in all functional studies, unless limited amounts make it impossible to perform them. For clinical applications, for instance, after a first step of pre-clinical validation following these recommendations, systematic analysis may not be possible (see previous Position Paper on clinical applications) [95].

Determine the specific versus common functions of different types of EVs

An important point to keep in mind is that, when analyzing exclusively the function of a single type of EV (for instance either small EVs or large EVs that have been called ectosomes, microvesicles or microparticles in different studies), one may miss the most active EV subtype for the particular function studied. Even if a function is found in the concentrated small EV preparation, it could also be present, and even possibly more concentrated, in other EV subtypes that had been eliminated during the small EV isolation process: keeping large EVs (e.g. “microvesicles”) and
comparing their activity to that of small EVs should be a first step in all functional studies. In addition, when a function found in EVs may be due to soluble molecules that may or may not associate specifically with EVs, one must consider the possibility that the EV-associated function is only a minor fraction of the non-EV-bound soluble protein. Comparing quantitatively the effects of EV fraction(s), EV-depleted fraction(s), and also the unfractionated initial fluid, will identify the relative contributions of each to total activity.

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.”

Demonstrate that the activity is predominantly associated with EVs rather than with soluble mediators

“Typically, an EV-associated activity is explored by 1) separation and concentration of EVs from a biofluid or cell culture media, 2) application of EVs to a recipient cell or organism, and 3) observation of a readout phenotype. However, to convincingly argue that a detected readout/function is EV-borne, it must be determined that the activity is specifically enriched in EVs (possibly with non-EV components), and not instead due to low amounts of a highly active soluble molecule remaining non-specifically in the EV preparation. This point is
particularly important when the proposed or suspected active molecule on EVs is a cytokine/growth factor/metabolite usually described as secreted in a soluble form. For this step, one must compare quantitatively the activity present in/on the EVs versus in the remaining EV-depleted biofluid, using the same amounts of
materials in terms of initial volume of biofluid. When evaluating the relative importance of EVs and soluble mediators, it may be worth remembering that EVs and
soluble mediators may have combinatorial (e.g. synergistic) effects on cells [275,276].

Demonstrate the specific association of the activity with EVs rather than with co-isolated components

Especially when dealing with concentrated preparations enriched in small EVs, one must keep in mind that such preparations potentially contain non-EV components (ribonucleoprotein aggregates, lipoproteins, exomeres, etc.). The proportion of such co-isolated components differs tremendously with the type of protocol used to separate EVs, with some (like polymer-based concentration) displaying particularly abundant contaminants, and also remnants of the precipitating agent that can affect cell function [277,278]. In the case of cells infected experimentally or unintentionally (e.g. mycoplasma) with microbes, functional microbial factors may also be co-isolated with EVs. Therefore, the functional activity of an EV preparation may be borne by EVs, or by the additional components, or by a combination of both. One must determine which of these three possibilities is the case. If small amounts of working materials do not make it possible to perform these additional investigations, the authors can explain this situation and interpret their data as activity present in EV-enriched preparations, rather than EV-specific activity.”

Determine whether a function is specific to exosomes, as compared with other small EVs

“As highlighted here, it is now clear that different types of EVs can present functional activities that are as important to explore as those elicited by late endosome-derived exosomes. However, in the last decade, many studies have focused exclusively on demonstrating association of a given function with exosomes. This section explains the technical limitations of such studies, and why they are not sufficient to conclude, as is generally done, that exosomes have specific functions compared with other EVs.”

“These cell treatment approaches have great potential and deserve more development; however, it is important to recognize several caveats.

(1) Small EV–containing fractions potentially contain EVs originating from late endosomes (“exosomes”) and others originating from the cell surface (plasma membrane), with both classes sharing common molecular players, including the ESCRT components TSG101, VPS4, and/or Alix [308–310]. Therefore consequences of decreasing or increasing global secretion of heterogeneous populations of small EVs should not be interpreted in terms of functional effects of exosomes, but rather of small EVs in general.
(2) Tools described until now to block or enhance exosome secretion have not been well evaluated for their possible effect on secretion of other EVs. For instance, ionophores, such as ionomycin, are also potent inducers of large EV and microparticle secretion [207,311]. Conversely, in one study, inhibition of neutral sphingomyelinases was shown to enhance secretion of larger plasma membrane-derived EVs while decreasing that of small EVs [312]. Another example is monensin, often used to stimulate EV secretion, being an inhibitor of apoptotic body formation [167]. Therefore, it is likely that putative exosome modulators will have different consequences in different cells and under different conditions, and it is important to carefully quantify the toxicity of each treatment in each experimental system, to exclude artefactual effects on EV recovery due to increased cell death.
(3) Some EV release modulators affect other major intracellular pathways that might indirectly affect EV secretion and modify cell functions in general (like general intracellular trafficking, secretory, or autophagy pathways). Consequently, not only EV amount, but also EV composition may be changed, together with changes in protein expression and physiology of the secreting cells. As an example, Rab27a inhibition also decreased secretion of some non-EV-bound soluble factors [313,314]. Another caveat to consider is that disrupting the secretion of one EV type may disrupt the production of other EV types, such that the functional EV type may be masked by the over-production of an antagonistic one, leading to an erroneous conclusion that the disrupted EV type is the functional EV. Therefore, demonstrating that only late endosome-derived exosomes bear an analyzed function remains challenging. Some previous studies managed to rescue an observed effect by re-introducing purified exosomes (or rather small EV pellets) into the functional in vitro or in vivo assays [292,313,314]. This approach is indeed recommended, with careful interpretation taking into account the degree of rescue and the required amount of EVs.

Until we achieve unambiguous identification of specific, unique biogenesis machineries affecting only a given subtype of EVs, we are left with trying to isolate EV subtypes after they have left the cell. For example, if multi-tetraspanin-bearing EVs are true exosomes in a particular cell system, an EV preparation could be depleted of such EVs and the activity quantified in comparison with that of an irrelevant IgG- or mock-depleted population.

How to attribute particular effects mediated by EVs to specific EV components

Many publications include knock-out or knock-down of a certain bioactive protein or RNA in the EV donor cell, after which the effects of the modified EV on target cells are compared with the effects of non-modified EVs. If the native effect of EVs is lost, the authors conclude that EV activity was due to the specifically targeted protein or RNA. However, such a conclusion may or may not be valid in the absence of an extensive characterization of EVs released by the cells depleted for the targeted molecule. Indeed, deletion of the protein/RNA of interest may also lead to major alterations of the secreting cell, resulting in additional changes to the quantity or molecular contents of EVs, which could also explain the changes in EV-induced effects on target cells. While a complete omics analyses of the modified EV population may be beyond the scope of many studies, there should be an awareness that other EV components may have changed as well. At a minimum, a small-scale analysis of EV number or common EV-associated proteins in the modified and WT conditions must be performed. Finally, Direct EVs engineering (e.g. to deplete the particular putative active molecule) may overcome the issue of alterations in the secreting cells. However, possible loss/alteration of EV cargo due to EV manipulation may also occur.

Consider whether an EV-dependent function is specific to a given EV source

Finally, in all cases, one must be careful in claiming a specific function of EVs from a particular source: it is one thing to claim that the EV fraction from Cell X is potent (versus other fractions), another to claim that Cell X EVs are potent versus those from other cells. For example, do my mesenchymal stromal cell (MSC) EVs do something special, or do milk EVs, urine EVs, cancer cell EVs do the same? Of course, it will not be possible to compare EVs from all different sources, thus the final message must reflect this uncertainty.”

Exceptions to compliance with MISEV guidelines

Some situations may arise in which strict adherence to the MISEV guidelines is difficult. Not all biofluids, for example lacrimal fluid, are available in sufficient volume to separate EVs and perform multiple tests with each sample; also, only limited numbers of EVs may be harvested from small numbers of patient-derived cells, small organoids, and more. In such cases, multiple samples might be pooled to establish the reliability of the separation method(s) and characterize EVs before further characterization or functional studies are performed with individuals samples. If even this solution is impractical, authors should indicate the limit of detection of each applied EV characterization technology and demonstrate that the available material falls below this limit. However, applying this “escape clause” means that EVs cannot be rigorously demonstrated, requiring that authors mention (and reviewers insist on) the caveats of alternative interpretations, i.e. that EVs may contribute, but not necessarily exclusively, to an observed phenomenon or molecular signature.”

doi: 10.1080/20013078.2018.1535750.

The technical challenges related to the purification and isolation of exosomes has led to a great amount of data being generated that can not be experimentally verified. The technological advances that were supposed to establish higher quality results have not yet come to fruition. The standardized approaches that were supposed to establish verifiable and reproducible results have not been widely implemented. This has led to a reproducibility crisis in exosome research that is still ongoing today. The problem of inaccurate data, inconsistent reporting, and lack of replication was addressed in this 2021 review of the EV literature, including the issue of having insufficient means of isolating exosomes and differentiating them from microvesicles. In fact, it is stated that there is no possible way to clearly differentiate “microvesicles” from “exosomes” by either morphology, size, or function and that it is recommended that all EVs should be considered as a single entity:

Critical Review of the Evolution of Extracellular Vesicles’ Knowledge: From 1946 to Today

“In order to systematically review the impact of the 2013 ISEV criteria for the characterization of EVs in scientific production, the EV-TRACK knowledgebase consortium was
convened to record experimental parameters of EV-related studies [5]. A review of a checklist of 115 parameters based on the MISEV guidelines, related to sample type, preanalytical variables, isolation protocol, and characterization method in 1742 experiments published in 2010–2015, revealed widespread heterogeneity in EV isolation methods and inconsistent reporting of important experimental parameters [1–3,5]. Moreover, it is important to underline that only 18% of experiments include both qualitative and quantitative analysis, and 50% did not achieve more than 20% of EV–METRIC (a checklist to assess the completeness of reporting of generic and method-specific information necessary to interpret and reproduce the experiment, according to Reference [5]). These analyses revealed that a large number of publications on EVs contained insufficient information for unambiguous interpretation or replication of experiments [5].”

“It was amply apparent and worrisome to experts within the ISEV community that the continuous large increase in EV publications often reported major conclusions that were not sufficiently supported by the experiments performed or the information reported. These circumstances prompted a revision and renewal of the MISEV recommendations that brought to date the new knowledge in the area, along with a commitment by the ISEV to continue to work toward their wider acceptance and implementation.”

“Because exosomes are mostly analyzed as bulk isolates, their heterogeneity in composition and population is often overlooked. It still remains a challenge to isolate specific populations of exosomes, and technological advances are urgently required to address this problem.

Most of the recent basic research on EVs is focused on MVB-derived exosomes, with little or no attempts in investigating the membrane-derived 100 to 1000 nm EVs (“microvesicles”). It seems at present there is no possible way to clearly differentiate “microvesicles” from “exosomes” by either morphology, size, or function; thus, it is recommended that all EVs should be considered as a single entity. However, the fact that we cannot differentiate them once they are released from the cell does not necessarily mean that they are all the same. They ought to be different, but we cannot tell them apart.”

https://www.google.com/url?sa=t&source=web&rct=j&url=https://www.mdpi.com/1422-0067/22/12/6417/pdf%3Fversion%3D1623888852&ved=2ahUKEwj8iJ_oqeX4AhW3JjQIHT5gArEQFnoECBEQAQ&usg=AOvVaw0XLQh9NS0ZATrsLUKy_V7j

In June 2022, a review of the reproducibility of exosome research was published. In this review, we can find many reasons for the inabilty to reproduce exosome research. It is stated that the heterogeneity (the quality or state of being diverse in character or content) of the EVs affects the ability to isolate and detect them. It is admitted that there are no distinguishable populations of small and large EVs. In fact, the size of the EVs contained in a preparation depends entirely on the chosen method(s) employed for the separation or enrichment of the sample. The method used for purification/isolation provides different EV preparations even from the very same starting material, thus showing that it is the purification method itself creating the size of the particles seen as they are broken apart into smaller pieces.

It is also shown that EV preparations contain many non-EV components which makes separation and isolation a challenge. Even though it is known that there will be other substances in the samples, insufficient attention is paid to critical confounders which have not yet been entirely recognized. Due to these factors, detecting, distinguishing, and isolating EVs by optical means is a challenge and the presence of non-EV particles may interfere with separation and characterization of EVs. This has led to the general rule that when EVs are isolated by one method, (i.e. a method isolating EVs based on size, charge, density, or biochemical composition,) the isolated EVs are likely to be impure and contain confounders. The paper concludes that EV research is working towards a reproducible standard by focusing on infrastructure and instrument calibration which offers the promise of reproducibility, thus showing that to date, this inability to verify experimental results by reproduction and replication has not been solved:

Reproducibility of extracellular vesicle research

“Cells release membrane-delimited particles into the environment. These particles are called “extracellular vesicles” (EVs), and EVs are present in fluids contacting cells, including body fluids and conditioned culture media. Because EVs change and contribute to health and disease, EVs have become a hot topic. From the thousands of papers now published on EVs annually, one easily gets the impression that EVs provide biomarkers for all diseases, and that EVs are carriers of all relevant biomolecules and are omnipotent therapeutics. At the same time, EVs are heterogeneous, elusive and difficult to study due to their physical properties and the complex composition of their environment.

This overview addresses the current challenges encountered when working with EVs, and how we envision that most of these challenges will be overcome in the near future. Right now, an infrastructure is being developed to improve the reproducibility of EV measurement results. This infrastructure comprises expert task forces of the International Society of Extracellular Vesicles (ISEV) developing guidelines and recommendations, instrument calibration, standardized and transparent reporting, and education. Altogether, these developments will support the credibility of EV research by introducing robust reproducibility, which is a prerequisite for understanding their biological significance and biomarker potential.”

1. Introduction

Extracellular vesicles (EVs) is an umbrella term for different types of vesicles that are released by cells, including the endosome-origin exosomes and the plasma membrane-origin ectosomes or microvesicles of living cells, and the apoptotic bodies of apoptotic cells (Yáñez-Mó et al., 2015). The term “extracellular vesicles” was introduced by the International Society for Extracellular Vesicles (ISEV) because the different types of EVs are often indistinguishable (Théry et al., 2018). Studying EVs is challenging for several reasons, which will be briefly outlined in Part 2: Challenges. These challenges will be illustrated by explaining relevant examples, which include the physical properties of EVs underlying their heterogeneity and how this heterogeneity affects isolation and detection, the complexity of blood to illustrate the difficulties encountered when studying EVs in a body fluid, and flow cytometry as an EV detection method that likely will produce reproducible measurement results in the near future.

2. Challenges

2.1. Heterogeneity of extracellular vesicles

The first size distributions of EVs that were published for human plasma and urine in 2014, showed that EVs range in diameter from less than < 100 nm to 1 µm or larger (van der Pol et al., 2014; Arraud et al., 2014). These publications were important for several reasons. Firstly, the size distributions showed that there are no distinct peaks of “small EVs” and “large EVs” (as was once assumed of exosomes and microvesicles), but rather that EVs can be of almost any size or diameter in a continuum. In other words, based on size, there are no distinguishable populations of small and large EVs. Thus, the size of EVs contained in a preparation will depend on the physical basis of the chosen method(s) employed for separation or enrichment of the sample, and different methods will provide different EV preparations starting from the very same material (Veerman et al., 2021).”

“Apart from their heterogeneity in size, the density of EVs also causes challenges, especially when isolating EVs. For example in the case of blood plasma and serum, the density of EVs hardly differs from the density of their environment, and this low “density contrast” makes it difficult to isolate EVs by centrifugation (Rikkert et al., 2020). Importantly, fluids like blood plasma and serum also contain non-EV particles such as high-density lipoprotein particles (Yuana et al., 2014) and platelets (Rikkert et al., 2020) which overlap in density with EVs. Thus, isolation of blood plasma EVs by density gradient centrifugation is complex (Zhang et al., 2020).

Regarding the biochemical composition, EVs contain lipids, nucleic acids, metabolites and proteins (Yáñez-Mó et al., 2015). Thus far, the compositional knowledge has been obtained using techniques that analyze the bulk composition of multiple EVs, and more, often insufficient attention has been paid to critical confounders as they are not yet all recognized. Some techniques provide information on the global biochemical composition at the level of single EVs, such as Raman spectroscopy (Enciso-Martinez et al., 2020), but these techniques are in their infancy regarding EV analyses, and more research is needed to determine the real combination and stoichiometry of the molecules forming a vesicle. Also, the current techniques poorly enable analysis of time-dependent changes in the EV populations.

Taken together, it is clear that the physical properties of EVs cause challenges for (optical) detection and isolation, but we have come to a point where technological innovations in detection and isolation of EVs, combined with standardization efforts and robust reporting, will improve reproducibility to such an extent that multicenter-studies will soon become possible (Nieuwland et al., 2020). 

2.2. Complexity of fluids containing extracellular vesicles

Often, EVs are present in complex (body) fluids with high concentrations of cells, non-EV particles, and soluble proteins. Commonly, EVs are separated from cells by differential centrifugation. There is at least one challenge, and that is the separation of EVs from platelets in blood plasma. Because platelets are small cells (2–4 µm), lack a nucleus and have a density close to EVs, it is difficult to separate platelets from EVs by centrifugation (Rikkert et al., 2018). Consequently, “platelet-free” plasma will still contain platelets (Rikkert et al., 2021). Soluble proteins present less of a problem, because the bulk of proteins can be separated from EVs with differential ultracentrifugation combined with washes or by size exclusion chromatography (Böing et al., 2014). However, the presence of non-EV particles, including lipoproteins (plasma), protein aggregates and even viruses, cause more problems since they may overlap in size and density with EVs (Zhang et al., 2020). This is illustrated in Fig. 2, which shows the presence of a few EVs in a multitude of lipoproteins. Similarly, conditioned culture medium often contains EVs, lipoproteins (including chylomicrons) and soluble proteins from the serum used to culture cells (Zhang et al., 2020), and milk contains not only EVs but also casein particles, milk fat globules, and possibly lipoproteins that co-isolate with EVs (Hu et al., 2021). Thus, the presence of non-EV particles may interfere with separation and characterization of EVs.

2.4. Isolation, detection, analysis and data reporting

After the collection of EV-containing fluids, most downstream methods require isolation of EVs prior to analysis, for example to perform proteomics or lipidomics. The choice of an isolation method depends on the EV-containing (body) fluid studied, the downstream assay, whether or not the presence of particular confounders or reagents (e.g. anticoagulants added to blood to prevent clotting) may interfere with the downstream assay results, and the final application, which can range from basic research assays to routine diagnostics. As explained in the previous sections, the physical properties of EVs and the complexity of the EV-containing fluids present challenges to EV isolation. In principle, the currently available methods used to isolate EVs separate particles essentially based on either size, charge, density, or biochemical composition. As a rule of thumb, one can state that when EVs are isolated by one method, i.e. a method isolating EVs based on size, charge, density, or biochemical composition, the isolated EVs are likely to be impure and contain confounders. Therefore, combinations of separation methods are now being explored. For example, plasma EVs can be purified by separation based on size followed by separation based on density. In the first step, EVs are separated from soluble proteins and small lipoproteins such as HDL by size-exclusion chromatography, and in the second step EVs are separated from chylomicrons and LDL (larger lipoprotein particles) by density gradient centrifugation (Karimi et al., 2018).”

“As explained, there are multiple known and probably even more unknown variables that may affect EV measurement results. As long as we do not know all variables and have limited tools to assess and quantitate the effects of such variables in a reproducible manner, detailed reporting of characteristics of the collected biospecimen and the applied pre-analytical procedures remain important.”

3.5. Towards standardization

“Metrology is the science of measurements and their applications, and comprises traceable accuracy, precision and repeatability of a measurement, often with a help of a “standard”, to help data interpretation and comparison between different measuring systems. Although biological systems are difficult if not impossible to standardize, principles of metrology are now being explored in the EV field to pave way to reproducibility.”

4. Summary

“In the first part we summarized the challenges of studying EVs, and in the following part how the field is actively moving towards traceable and reproducible measurements via a community-built infrastructure. Instrument calibration is expected to pave the way towards monitoring possible variations caused by the biospecimen and pre-analytical procedures, screening the efficacy of isolation procedures, performing multi-center studies, and finally, establishing reference ranges of cell-type specific EVs in body fluids for clinical use. Furthermore, by initiating the Rigor and Standardization Subcommittee and promoting task force activities, education and transparent reporting, there is an already proven and growing awareness amongst EV researchers about the relevance of producing and reporting traceable and reproducible results (Nieuwland et al., 2020). Importantly, the developed infrastructure may place EV research at a pole position in the field of (bio)medical research by producing robust and reproducible data, which in turn may contribute to the overcoming of reproducibility problems in science.”

https://www.sciencedirect.com/science/article/pii/S0171933522000292?via%3Dihub

In Summary:

• Specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly
• An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation
• Claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given the still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs.
• In 2014, the ISEV board members published a Position Editorial detailing their recommendations, based on their own established expertise, on the “minimal experimental requirements for definition of extracellular vesicles and their functions”
• The ISEV board highlighted the need to consider these issues when making strong conclusions on the involvement of EVs, or specific populations of EVs (exosomes in particular), in any physiological or pathological situation, or when proposing EVs or their molecular cargo as biological markers
• The ISEV were still concerned to see that major conclusions in some articles are not sufficiently supported by the experiments performed or the information reported
• In this “MISEV2018” update, a much larger group of ISEV scientists was involved through a community outreach (theMISEV2018 Survey), striving for consensus on what is absolutely necessary, what should be done if possible, and how to cautiously interpret results if all recommendations for controls cannot be followed
• EVs appear to be produced by almost all organisms and cell types studied
• EV research to date has focused on mammalian EVs, chiefly those of human or mouse origin, and not all cell types or experimental conditions have been closely investigated
• The general principles of MISEV2018 apply to EVs produced by all organisms and all cells
• The need to demonstrate presence (or enrichment) of EV markers and absence (or depletion) of putative contaminants, when contents or function of EVs are described, can be generalized to all species, cells, and conditions
• In other words, if one is going to claim something is an exosome and proceed to describe its contents and functions, there must be an absence of contaminants (i.e. it must be purified and isolated)
• Since consensus has not yet emerged on specific markers of EV subtypes, such as endosome-origin “exosomes” and plasma membrane-derived “ectosomes” (microparticles/microvesicles) assigning an EV to a particular biogenesis pathway remains extraordinarily difficult unless, e.g. the EV is caught in the act of release by live imaging techniques
• Authors are urged to consider use of operational terms for EV subtypes that refer to physical and biochemical characteristics of EVs in the place of terms such as exosome and microvesicle that are historically burdened by both manifold, contradictory definitions and inaccurate expectations of unique biogenesis
• The first step to recover EVs is to harvest an EV-containing matrix, such as fluid from tissue culture or from an organismal compartment
• An extended constellation of factors can affect EV recovery including:
  1. Characteristics of the source
  2. How the source material is manipulated and stored
  3. Experimental conditions
• An ISEV survey found that the majority of responding EV researchers studied conditioned medium
• Especially important for EV studies is that the percent of dead cells at the time of EV harvest should be indicated, since even a small percentage of cell death could release cell membranes that outnumber true released EVs
• Quantifying the percentage of apoptotic and necrotic cells may also be useful
• When cells are treated with high concentrations of EVs, cell-adherent EVs positive for apoptotic markers may skew results
• In other words, it is important to look out for apoptotic and necrotic cells in a sample as cell death can release numerous membranes including EV’s with apoptotic “markers”…but let’s just ignore this and continue to pretend the particles are all different…
• Note that EV recovery depends not only on EV release, but also on re-uptake by cells in culture, which may vary based on culture density and other conditions
• Regular checks for contamination with Mycoplasma (and possibly other microbes) are needed, not only because of cellular responses to contamination, but also because contaminating species can release EVs
• All culture medium composition and preparation details should be provided in methods
• This should be customary for cell culture studies, and is doubly important here since supplements like glucose, antibiotics, and growth factors can affect EV production and/or composition
• Of special note are medium components that are likely to contain EVs, such as fetal bovine and other serums
• EVs are ideally obtained from culture medium conditioned by cells in the absence of fetal calf serum (FCS or FBS), serum from other species, or other complex products such as platelet lysate, pituitary extract, bile salts, and more, to avoid co-isolation of exogenous EVs
• Keep this co-isolation of exogenous (i.e. outside the body) in mind also for virology studies using FBS
• In the case of depletion, nutrient or EV deprivation of cells that are normally cultured in serum- or lysate-containing medium can change cellular behavior and the nature and composition of released EVs
• Alternatively, cells can be exposed during the EV release period to medium that has been pre-depleted of EVs and the effects on cells and EVs may be expected and the methods and outcome of depletion vary greatly and should be reported
• Heat inactivation of additives such as serum leads to formation of protein aggregates that may co-precipitate with EVs and thus also change the growth-supporting properties of the serum
• Each biological fluid presents specific biophysical and chemical characteristics that makes it different from culture conditioned medium, and this must be taken into account when isolating EVs
• For instance, plasma and serum are more viscous than conditioned medium and contain numerous non-EV lipidic structures (low/very low/high density lipoproteins), milk is replete with fat-containing vesicles, urine has uromodulin (Tamm-Horsfall protein), bronchoalveolar lavage with surfactant, all of which will be co-isolated to various degrees with EVs
• In each case, specific precautions to separate EVs from these components may be required (why would it not be required?)
• While detailed biofluid-specific reporting guidelines are beyond the scope of this MISEV, they encouraged development of such guidelines under the MISEV umbrella
• To give examples of considerations for blood derivatives such as plasma which may affect circulating EVs:
  1. Donor age
  2. Beiological sex
  3. Current or previous pregnancy
  4. Menopause
  5. Pre/postprandial status (fasting/non-fasting)
  6. Time of day of collection (Circadian variations)
  7. Exercise level and time of last exercise
  8. Diet
  9. Body mass index
  10. Specific infectious and non-infectious diseases
  11. Medications
  12. Other factors
• Other than all of that, nothing effects obtaining EV’s from plasma…
• Especially for EV extraction from tissue, it is challenging to ensure that recovered vesicles are truly from the extracellular space, rather than being intracellular vesicles or artefactual particles released from cells broken during tissue harvest, processing (e.g. mechanical disruption), or storage (including freezing)
• Even apparently pure tissue-derived EVs can contain endosome components, which could correspond to components of intracellular vesicles including unreleased intraluminal vesicles of late endosomes/multivesicular bodies (MVBs) that are released artifactually during tissue processing
• The recent awareness of these challenges has led researchers to perform gentle tissue disruption (i.e. with the goal of separating EVs from cells and extracellular matrix, but not disrupting cells) and several steps of further separation (including density gradients), followed by strict characterization of multiple negative markers, leading to more convincing tissue-derived EV preparations
• More research is clearly needed and encouraged into the isolation, characterization, and function of tissue EVs
• Absolute purification, or complete isolation of EVs from other entities, is an unrealistic goal
• Separation (colloquially referred to as purification or isolation) of 1) EVs from other non-EV components of the matrix (conditioned medium, biofluid, tissue) and 2) the different types of EVs from each other, are achieved to various degrees by the different techniques available.
• Concentration is a means to increase numbers of EVs per unit volume, with or without separation
• The answer for how pure an EV preparation should be depends on the experimental question and EV end use, and often segregates by basic and clinical research
• Highly purified EVs are needed to attribute a function or a biomarker to vesicles as compared with other particles
• Less pure EVs may be required in other cases, such as when a biomarker is useful without pre-enrichment of EVs, or in certain therapeutic situations where function is paramount, not the definitive association of function with EVs
• Of note, some presumed contaminants may co-isolate with EVs and may even contribute to EV function
• At the end of 2015, according to a worldwide ISEV survey [6], differential ultracentrifugation was the most commonly used primary EV separation and concentration technique
• A variety of additional techniques or combinations of techniques have been or are currently being developed, some of which may become more prominent in the coming years if they achieve better recovery or specificity than legacy methods
• Hendrix and colleagues found that only about half of EV-related articles published within a five-year time period included positive markers of EVs, and only a small minority complemented positive with negative markers to track co-isolated non-EV components
• Quantifying EVs themselves remains difficult
• EVs are said to have a particulate structure and contain:
  • Proteins
  • Lipids
  • Nucleic acids
  • Other biomolecules
• Quantification of each of these components can be used as a proxy (i.e. stand-in) for quantification of EVs, but none of these values is necessarily perfectly correlated with EV number
• Particle number can be measured by light scattering technologies, however, accurate quantitation may be possible only within a certain concentration and size range that varies by platform
• Particle counting by light scatter, RPS, and similar techniques typically results in overestimation of EV counts since the techniques are not specific to EVs and also register co-isolated particles including lipoproteins and protein aggregates
• Possibly, ongoing development of fluorescence capabilities of NTA devices may ultimately allow EV-specific measurement, although assay sensitivity and the tendency of labeling antibodies and lipid dyes to form particles pose substantial hurdles to such applications
• Proprietary software used for analysis of data from each device may apply unknown selection and other processing of data, resulting in differences in absolute values obtained by different software or different versions of the same software
• Protein quantification can result in overestimation due to co-isolated protein contaminants (such as albumin from culture medium or plasma/serum), especially when the less specific methods of EV separation are used, or conversely can prove not sensitive enough if highly specific methods yield pure EVs
• Results may vary depending on the use or not of detergent to disrupt EVs and expose the entire protein content prior to performing the assay
• Quantification of total lipids requires specialized equipment, and the former two types of assays may be insufficiently sensitive for small amount of EVs
• In addition, whether these techniques equally detect all EVs independent of their specific lipid composition must still be established
• Quantification of total RNA is difficult to recommend at this time for EV quantification or purity assays since exRNAs associate in abundance with other circulating and potentially co-separating entities: chiefly ribonucleoproteins but also a range of particles including exomeres and lipoproteins
• Ratios of the different quantification methods may provide useful measures of purity
• For example, protein: particle ratio, protein: lipid ratio and RNA: particle have been proposed as possible purity metrics, although their applicability across protein, lipid, RNA and particle quantification methods remains to be established
• Absolute EV sizing and counting methods are currently imperfect and will require further improvement
• Since studies on the selection of proteins for use as EV markers were performed with different separation approaches and with different cellular sources of EVs, it is still not possible to propose specific and universal markers of one or the other type of EVs, let alone of MVB-derived “exosomes” as compared with other small EVs
• MISEV2018 does not propose molecular markers that could characterize specifically each EV subtype
• In this updated version, MISEV2018, reference to exosomes and the proteins expected or not in them (the previously called “negative controls” of “exosome” preparations) have been deleted, reflecting an evolving understanding of the subtypes of EVs and their associations with other entities
• When determining the topology of EV-associated components, the luminal versus surface topology of various EV-associated components, including nucleic acids, proteins, glycans, etc, is not entirely strictly determined
• Theoretically, components localized in the cytosol of EV-secreting cells should be inside EVs, and hence protectedfrom mild degradation by proteases or nucleases
• While this protection is usually observed, some studies have unexpectedly found proteins, RNAs, and DNA on the EV surface and sensitive to digestion
• It is not yet clear whether this unexpected topology is due to debris from dead or dying cells, or is instead the outcome of as-yet unknown mechanisms of transport of intracellular compartments across membranes that could occur in some physio- or pathological conditions
• Even a small degree of contamination with intracellular material (with the reverse topology to EVs) would complicate interpretation
• The goals of these 2018 recommendations for studying the functions of EVs are to avoid over-interpretations or classical artefacts when analyzing functions of EVs
• Demonstration that a function is specific to exosomes (EVs of endosomal origin), as compared with other types of small EVs, is not recommended as a major point of any EV study
• 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
• It must be demonstrated that the activity is predominantly associated with EVs rather than with soluble mediators
  • Typically, an EV-associated activity is explored by:
    1. Separation and concentration of EVs from a biofluid or cell culture media
    2. Application of EVs to a recipient cell or organism
    3. Observation of a readout phenotype
  • However, to convincingly argue that a detected readout/function is EV-borne, it must be determined that the activity is specifically enriched in EVs (possibly with non-EV components), and not instead due to low amounts of a highly active soluble molecule remaining non-specifically in the EV preparation
• It must be demonstrated that the specific association of the activity is with EVs rather than with co-isolated components:
  • Especially when dealing with concentrated preparations enriched in small EVs, one must keep in mind that such preparations potentially contain non-EV components (ribonucleoprotein aggregates, lipoproteins, exomeres, etc.)
  • The proportion of such co-isolated components differs tremendously with the type of protocol used to separate EVs, with some (like polymer-based concentration) displaying particularly abundant contaminants, and also remnants of the precipitating agent that can affect cell function
• It must be determined whether a function is specific to exosomes, as compared with other small EVs
  • In the last decade, many studies have focused exclusively on demonstrating association of a given function with exosomes yet there are technical limitations of such studies and they are not sufficient to conclude, as is generally done, that exosomes have specific functions compared with other EVs
• Small EV–containing fractions potentially contain EVs originating from late endosomes (“exosomes”) and others originating from the cell surface (plasma membrane), with both classes sharing common molecular players
• Tools described until now to block or enhance exosome secretion have not been well evaluated for their possible effect on secretion of other EVs
• Therefore, it is likely that putative exosome modulators will have different consequences in different cells and under different conditions, and it is important to carefully quantify the toxicity of each treatment in each experimental system, to exclude artefactual effects on EV recovery due to increased cell death
• Some EV release modulators affect other major intracellular pathways that might indirectly affect EV secretion and modify cell functions in general
• Another caveat to consider is that disrupting the secretion of one EV type may disrupt the production of other EV types, such that the functional EV type may be masked by the over-production of an antagonistic one, leading to an erroneous conclusion that the disrupted EV type is the functional EV
• Demonstrating that only late endosome-derived exosomes bear an analyzed function remains challenging
• Until unambiguous identification of specific, unique biogenesis machineries affecting only a given subtype of EVs is achieved, researchers are left with trying to isolate EV subtypes after they have left the cell
• When figuring out how to attribute particular effects mediated by EVs to specific EV components, many publications include knock-out or knock-down of a certain bioactive protein or RNA in the EV donor cell, after which the effects of the modified EV on target cells are compared with the effects of non-modified EVs
• If the native effect of EVs is lost, the authors conclude that EV activity was due to the specifically targeted protein or RNA
• However, such a conclusion may or may not be valid in the absence of an extensive characterization of EVs released by the cells depleted for the targeted molecule
• Deletion of the protein/RNA of interest may also lead to major alterations of the secreting cell, resulting in additional changes to the quantity or molecular contents of EVs, which could also explain the changes in EV-induced effects on target cells
• It must be considered whether an EV-dependent function is specific to a given EV source
• In all cases, one must be careful in claiming a specific function of EVs from a particular source
• It will not be possible to compare EVs from all different sources, thus the final message must reflect this uncertainty
• The guidelines allow for applying an “escape clause” if strict adherence can not be achieved, which means that EVs cannot be rigorously demonstrated, requiring that authors mention (and reviewers insist on) the caveats of alternative interpretations, i.e. that EVs may contribute, but not necessarily exclusively, to an observed phenomenon or molecular signature

• From the thousands of papers now published on EVs annually, one easily gets the impression that EVs provide biomarkers for all diseases, and that EVs are carriers of all relevant biomolecules and are omnipotent therapeutic
• At the same time, EVs are heterogeneous, elusive and difficult to study due to their physical properties and the complex composition of their environment
• This overview addressed the current challenges encountered when working with EVs, and how the researchers envisioned that most of these challenges will be overcome in the near future
• Right now, an infrastructure is being developed (i.e. not in place currently) to improve the reproducibility of EV measurement results
• Altogether, these developments will support the credibility of EV research by introducing robust reproducibility, which is a prerequisite for understanding their biological significance and biomarker potential
• The term “extracellular vesicles” was introduced by the International Society for Extracellular Vesicles (ISEV) because the different types (including the endosome-origin exosomes and the plasma membrane-origin ectosomes or microvesicles of living cells, and the apoptotic bodies of apoptotic cells) of EVs are often indistinguishable
• The challenges in studying EVs include the physical properties of EVs underlying their heterogeneity and how this heterogeneity affects isolation and detection and the complexity of blood to illustrate the difficulties encountered when studying EVs in a body fluid
• Size distributions of EVs showed that there are no distinct peaks of “small EVs” and “large EVs” (as was once assumed of exosomes and microvesicles), but rather that EVs can be of almost any size or diameter in a continuum
• In other words, based on size, there are no distinguishable populations of small and large EVs
• Thus, the size of EVs contained in a preparation will depend on the physical basis of the chosen method(s) employed for separation or enrichment of the sample, and different methods will provide different EV preparations starting from the very same material
• The density of EVs also causes challenges, especially when isolating EVs
• In the case of blood plasma and serum, the density of EVs hardly differs from the density of their environment, and this low “density contrast” makes it difficult to isolate EVs by centrifugation
• Importantly, fluids like blood plasma and serum also contain non-EV particles such as high-density lipoprotein particles and platelets which overlap in density with EVs
• The compositional knowledge of EVs has been obtained using techniques that analyze the bulk composition of multiple EVs, and more, often insufficient attention has been paid to critical confounders as they are not yet all recognized
• The current techniques poorly enable analysis of time-dependent changes in the EV populations
• It is clear that the physical properties of EVs cause challenges for (optical) detection and isolation
• In other words, after 40 years, they are still unable to accurately distinguish between EVs by sight (as in EM images)
• The current lack of reproducibility in extracellular vesicle (EV) research hinders progress in understanding their biological role and theranostic applicability
• Isolation and analyses are hampered by:
  1. The physical and biochemical heterogeneity (diverse in character and content) of EVs
  2. The complex composition of tissues and biofluids containing the EVs
  3. Unidentified variables that affect the presence and function of EVs in a biospecimen
  4. The lack of both instrument calibration and standardized reporting of methods and results
• Often, EVs are present in complex (body) fluids with high concentrations of cells, non-EV particles, and soluble proteins
• Because platelets are small cells (2–4 µm), lack a nucleus and have a density close to EVs, it is difficult to separate platelets from EVs by centrifugation
• The presence of non-EV particles, including lipoproteins (plasma), protein aggregates and even “viruses,” cause more problems since they may overlap in size and density with EVs
• Similarly, conditioned culture medium often contains EVs, lipoproteins (including chylomicrons) and soluble proteins from the serum used to culture cells and milk contains not only EVs but also casein particles, milk fat globules, and possibly lipoproteins that co-isolate with EVs
• Thus, the presence of non-EV particles may interfere with separation and characterization of EVs
• EVs collapse and appear often as cup-shaped structures due to fixation and dehydration during election microscopy preparation
• The physical properties of EVs and the complexity of the EV-containing fluids present challenges to EV isolation
• In principle, the currently available methods used to isolate EVs separate particles essentially based on either:
  1. Size
  2. Charge
  3. Density
  4. Biochemical composition
• As a rule of thumb, one can state that when EVs are isolated by one method, i.e. a method isolating EVs based on size, charge, density, or biochemical composition, the isolated EVs are likely to be impure and contain confounders
• There are multiple known and probably even more unknown variables that may affect EV measurement results
• They do not know all variables and have limited tools to assess and quantitate the effects of such variables in a reproducible manner
• Although biological systems are difficult if not impossible to standardize, principles of metrology are now being explored in the EV field to pave way to reproducibility (i.e. there is no reproducibility currently)
• The field is actively moving towards traceable and reproducible measurements via a community-built infrastructure (i.e. no current reproducible measurements)
• Instrument calibration is expected to pave the way towards:
  • Monitoring possible variations caused by the biospecimen and pre-analytical procedures
  • Screening the efficacy (the ability to produce a desired or intended result) of isolation procedures
  • Performing multi-center studies
  • Establishing reference ranges of cell-type specific EVs in body fluids for clinical use
• The developed infrastructure may place EV research at a pole position in the field of (bio)medical research by producing robust and reproducible data, which in turn may contribute to the overcoming of reproducibility problems in science (i.e. once again, there is no current reproducibility as they are trying to work towards it)

It should be apparent that in order to trust the validity of any scientific claims, especially regarding invisible entities, the methods utilized to study them must be verified. The technology used must be shown to be effective for the task assigned to it. The results that are obtained must be able to be reproduced and replicated independently. The practices between labs and researchers should be standardized in order to ensure that these issues are taken care of from the very start. However, this is rarely, if ever, the case.

Using the example of exosome research, we can see that for the vast majority of the 40+ years of rapidly expanding research, no such quality controls existed. Researchers were left to their own devices in order to generate whatever experimental data that suited their needs and purposes. This data was collected yet never validated. An entity was “discovered” yet the methods needed for the complete separation of the assumed exosome particles from everything else do not exist. Due to this inability to separate exosomes from “viruses” and other extracellular vesicles of the same size, shape, and density, the characterization and functioning of these entities remains unverifiable. The “specific” proteins and molecular markers claimed to be able to be used to differentiate these particles from each other have never been validated nor shown to come from the entity assumed to be within the sample. As they look the same as the other non-EV and “viral” particles, exosomes can not be distinguished based on optical means through electron microscopy imaging. Just like their “viral” counterparts, the entities known as exosomes can not be isolated, characterized, nor differentiated from the other components within the sample.

Knowing this, how can any of the papers purporting to show these entities and describe their biological functions be taken seriously? How can any of the information be trusted? The fact that exosome research is marred in a reproducibility crisis should tell you everything you need to know about the validity of exosome research. Reproducibility is admitted to be a prerequisite for understanding their biological significance and biomarker potential. Yet, in order to attain this required reproducibility, we must await future technology and the implementation of standardized methods. As this does not exist, we are left with a big heaping pile of creative writing in the guise of exosome research that has accumulated over the last four decades. If you find yourself bored, look for exosome research next to the virology papers in the science fiction section at your local library.