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 (68).”

“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).

“Separation of non-vesicular entities from EVs is not fully achieved by
common EV isolation protocols, including centrifugation protocols or
commercial kits that claim EV or “‘exosome’” purification.”

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.

“Virus” and exosome: can you tell the difference?

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., 2014Arraud 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)

Fig. 1. Towards reproducible measurements of extracellular vesicles. 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 the physical and biochemical heterogeneity of EVs, the complex composition of tissues and biofluids containing the EVs, unidentified variables that affect the presence and function of EVs in a biospecimen, and the lack of both instrument calibration and standardized reporting of methods and results.

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.

Fig. 2. Extracellular vesicles and lipoproteins in human plasma. A transmission electron microscopy image showing five extracellular vesicles (EVs) in the upper right corner. EVs collapse and appear often as cup-shaped structures due to fixation and dehydration. This image illustrates that EVs in human plasma are a small fraction compared to lipoproteins, which appear as white circular particles of various diameter. Scale bar: 200 nm.

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)
“Virus-modified” exosome = the same particles claimed to be “viruses.” In other words, a convenient rescue device.

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.

38 comments

  1. The techniques used for the biochemical and molecular characterization of so-called sub-microscopic particles and the evidence presented for the functions of sub-microscopic particles are just other colossal scams of so-called molecular biology.
    We will not be able to understand anything correct in biology, medicine and all other fields of existence if we judge things in terms of the lie of the existence of atoms and molecules. Chemistry, Bio-Chemistry and Molecular Biology are false sciences, because there are no atoms or molecules. Yes, there is no matter. Everything is “energy” that is structured in countless ways, thus giving rise to what we call being matter.
    No one has ever provided direct and uninterpretable evidence for the existence of atoms and molecules. Atoms and molecules are just theoretical concepts, never proven. The same is true of all the structures purported to exist, although they cannot be seen under a microscope. These are just unproven hypotheses.
    Because there is no “matter” but everything is “energy” (so-called “matter” is just another form of structuring “energy”) … true medicine must be understood only in terms of the manifestations of “energy”. And, of course: no one knows what “energy” is. The structures considered to be “materials” that are observed under the optical microscope, are nothing but forms of manifestation (vibrations) of the fundamental “energy” of which everything is constituted, that is, what is invisible and what is visible but also palpable. True medicine goes beyond the deception of the existence of atoms and molecules of “matter” and explains life in this Underworld by understanding that everything is “vibrating energy.
    We are told that although gel electrophoresis is an indirect and interpretable method, it provides sufficient evidence for the existence of nucleotides that are thought to be the structures that make up the so-called DNA and RNA nucleic acids.
    On the other hand, no one tells us about the direct or indirect methods by which the so-called molecular structures from which the so-called nucleotides are claimed to have formed have been highlighted. I am referring to the so-called nitrogenous bases, the so-called five-atom sugars and the so-called phosphate groups.
    Nucleic acids do not exist. No one actually isolated, purified, or visualized any component of so-called nucleic acids or any DNA fragments. All so-called evidence for the existence of nucleic acids is indirect, circumstantial and subject to interpretation. In fact, no one has actually isolated, purified and visualized any of the hypothetical submicroscopic particles that are the subject of the study of pseudoscience called molecular biology, genetics, virology and immunology. In fact, all the so-called molecular atomistic sciences are just scams, because atoms exist only at the level of theory: Atomist Theory.
    In reality, no one has ever isolated, purified and visualized any submicroscopic particles. All submicroscopic particles are pure inventions. As for the so-called electron microscopy, this is just a laboratory procedure which, like the PCR test, is only useful for claiming all sorts of inventions based on it. The truth is that all so-called atomic-molecular science is deceptive in its purest form and electron microscopy is not true microscopy but just a scam, a deceptive criminal prank.
    ——————————–
    “People don’t realize that molecules themselves are somewhat hypothetical, and that their interactions are more so, and that biological reactions are even more so.”
    – Kary Mullis”
    ————————————-
    All those structures that biologists call cells are highlighted only in decaying tissues due to the fact that by extracting them from living organisms they are disconnected from the energy of life and are deprived of specific nutrients that can only be provided by the body. In addition, dead tissues, which are in the natural process of decomposition, are also severely damaged due to preparatory procedures and microscopy techniques. Even today, the so-called Scientific World does not have the technical capabilities to conduct control experiments on the hypothetical cell structure of the tissues of living organisms, because so-called scientists cannot see live and live what exists and what is it happens in the tissues of a living organism.
    No so-called scientist has so far been able to see a so-called cell in vivo, that is, in the tissues of a living being. Moreover, so far no so-called scientist has been able to see how a so-called cell works in vivo, that is, to see it live, in real time, when it is in the tissues of the living organism. Quite simply, as the doctor and biologist Alain Scohy openly acknowledges, all our supposed knowledge of how the living organism is structured and how it works is done by extrapolating the findings obtained by researching tissue samples extracted from the living organism and which are in the process of decomposition, which in order to be observed under a microscope are also subjected to preparatory laboratory processes that are extremely harmful. In other words, the so-called Cell Biology is the emanation of the consensus of so-called scientists who have decided that observations made on dead, decaying tissue and extremely damaged by laboratory and microscopy procedures can be considered to be exactly what exists in the living organism.
    ————————–
    „Our supposed knowledge, today, of what life is on a microscopic scale, is based only on observations made on dead tissue, which are subject to an astonishingly harmful preparation protocol.”
    – Dr. Alain Scohy
    ————————————-
    Quote:
    Distinction between science and empiricism
    This distinction is very important, but often elided over by lecturers and theoreticians, probably because they don’t like admitting that some of their beliefs may be unsoundly based. Some examples-
    Anaesthetics. I was, personally, astonished when I was told that nobody knows how anaesthetics work. When chemistry improved to the point that pure chemicals which barely exist in nature could be isolated. some of their properties were unpredicted. Ether and chloroform, and nitrous oxide, were found to have anaesthetic effects, which—importantly—were reversible. Today, the mechanical side is far improved: the purity, the quantity delivered, the time measurement, the gas cylinders, the ambulances, are all far more efficient. But the way they operate on the body isn’t known. There are theories, and descriptions of their actions; for example, hypotheses including the cell ‘membrane fluid mosaic’. (These, probably mythical constructions, are what Margaret Thatcher worked on as a chemist). It’s not surprising that occasional anomalies occur.
    Drug testing. If it does what they want, they say it works according to the theory. (‘Beta blockers’.) However, huge numbers of similar molecules are tested; the acid test is always empirical. Any unwanted effect is labelled a ‘side effect’ as though it might go away. The same sort of thing applies with insecticides, which have to be tried out on (for example) caterpillars or species of fly.
    Blood transfusions. The blood types were all found empirically: the important thing was to avoid clotting, rather than work out what made them clot.
    Antibiotics are another example: penicillin was found by accident when a mould, penicillium, was noticed to kill bacterial growths in a petri dish. Lithium, as an anti-manic depressive drug, was found by chance to make lab animals sleepy. Titanium was found by chance to work well in bone replacements: the new musculature adhered well to it. Stainless steel is another example: I doubt anybody really knows how it works. The trick is to alloy metals and test the result. An interesting point here is that many people will claim to know (e.g.) why stainless steel doesn’t rust: that there’s an oxide layer, for example. But if they can’t predict which alloys will work, this arguably is just a form of words restating what they’ve found by trial.
    https://big-lies.org/harold-hillman-biology/index.html?fbclid=IwAR13c5PIMsbYy7de5bLkERnSQupWgnDabAPiwDEt7jmQfx8Ydae_V3MeBcs

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    1. Simple, how soluble is the filter material with the liquid?
      With fuel, the paper doesn’t swell so it flows through. With water the material swells and stops flow. That’s the first sign of having water in my diesel truck….
      So, what’s your point here?

      Like

      1. Thanks rob. I actually had the Mr Funnel screen filters in mind when I asked the question, that I use for farm equipment. You have any? How do those work then? Is it an electrostatic, hydrophobic deal or what? You can fill the funnel completely with water and nothing will go through.

        Like

  2. You make an important point that most technology comes about via tinkering, not actual science. That’s especially true in the life sciences.

    This means mere technological advancement is never sufficient to validate the science that supposedly underlies it. For example they say GPS uses general relativity, thus “proving the theory,” but this is circular reasoning. There is no need for general relativity in the calculations; they merely decided to use it and probably needed to fudge to make it work.

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    1. Haha , Yeah, I understood that GPS was like any triangulation- you timestamp a signal and measure how long it takes. With 3 different times, you get 3 distances.
      No need for relativity, even if they claim they needed it to “correct” the GPS signal, yeah where’s the proof that the original time/distance calculations needed to be corrected? haha

      You would enjoy these videos that take apart modern scientific “faith”
      debunking qt

      and debunking relativity

      Like

      1. Ah yes, ItsBS has some great videos. Karma Peny, bgaede (Rational Science channel, youstupidrelativist.com), and Miles Mathis round out the package, hitting modern physics from every angle. I think this is important for all scientists to understand the errors in modern physics because physics is the kingmaker, the hard science that sets the tone and standard for the rest, and it has turned to a pile of mush bringing the rest of science down with its horrible example.

        Liked by 1 person

  3. The standards board really took an AK-47 to the whole field there didn’t they. They simply say in a nice way that it’s all junk science so far and we’ll try to do better. By implication, virology is no different as they use the same techniques.

    Virologists find the particles indistinguishable from “viruses” in healthy samples, and it looks like EV research exists simply as a front to patch that gaping hole in virology.

    Liked by 1 person

    1. Clarifire

      The generous version would be appreciative that the standards board is doing a good job holding the line in a crony capitalist system. This field is obviously in its infancy, and the funding mechanisms of research facilities are made possible by vested interests. Some teams are better than others; as an umbrella organization the standards board is obvious mainly speaking to the latter; Michael S and Jeff presumably look for papers from the former.

      Mike’s blog will stand the test of time as a serious reference site a whole lot better if he leaves his biased and simplistic peanut gallery chatter out of his posts and keeps it to the peanut gallery here, itself.

      Liked by 1 person

      1. @reante

        Did you read the whole of the quoted content from ISEV? It’s long, but if you read it all they’re not just commenting on the lower quality research. They’re commenting on the limits of the field itself, at least heretofore.

        Like

      2. I’ve read most of it – the second half and the first quarter. Sure they’re commenting on the field as a whole. My comment was a patterned one.

        Patterning “junk science” out of characterizations they made such as “difficult’ and “challenging” — however euphemistic they may or may not be being — is facile unless you have some excerpts to back it up.

        And frankly I really don’t get the “hole-patching/rescue device deal. You say it often about microbiology and you said it about dark matter and dark energy. As I’m sure you’re aware from the life experience, sometimes some new shit comes to light, man. And it can’t be ignored. And it messes with some previous shit. That’s life.

        Animist pure Reason holds that something (reality) cannot come from nothing and, therefore, a creator, about which nothing can be known at all, must exist. An extension to this cosmological truth that says reality cannot come from nothing is that holograms also cannot come from nothing; therefore, the Consciousness and Energy symbionts cannot come from nothing. They need sourcing in reality itself otherwise reality is not a self-sufficient dimension and all evidence points to it being so.

        Quantum physics rediscovered the animist understanding that energy is the fundamental medium that Reason otherwise understands is symbiotic with consciousness/spirit. That was no “coincidence,” and my appreciation for the historical human ontological truth coming full circle

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      3. @reante

        Taken as a whole, the position of the ISEV is clear: no reliable science has been done in the field to date. They merely hope that new tech will allow it to be done.

        “New shit comes to light,” yes, but if it invalidates your existing theory you don’t just expand the theory uncritically. Each expansion reduces the chance that the theory is true and increases the chance that you should be erasing the board and starting again. And when there was never a good reason to believe a theory in the first place, and then there have been hundreds of expansions for each new thing that comes to light, we have nothing but a comedy sketch.

        “Energy” is not coherently defined in physics. It’s merely a word they play with. Again, faith that they’re doing *anything* useful in these fields as far as advancing theory (they do gather and summarize a lot of observational data, which can be useful) is misplaced and I’m pretty sure wouldn’t help your endeavors even if it were true.

        Quantum mechanics, like the rest of modern mathemagics since Cantor, is simply a social technology that is very successful at advancing careers, attracting funding, and keeping competitors out. Fascinating from a psychological, linguistic, and sociological perspective but nothing more.

        Like

      4. (hit the send button by accident)

        full circle again is not me making a “naive” connection – it’s just a recognition of how things played out favorably to the truth, (Not that it made any difference to the dominant culture.) And so it appears with dark energy and matter. Their instruments are telling them that these entities are not only most of the universe ‘by volume’ but that they are pervasive and also that they do not interact with each other; pervasiveness and separatism, in ‘darkness,’ are the precise requirements for sourcing ‘light’ holography in ‘light’ reality according to reason because if consciousness and energy are symbiotic in holography then they must of course be apart outside of holography.

        I was only dimly aware of dark matter until you mentioned it. Had never even heard of dark energy that I recall. So thanks.

        I remain open to your physics debunkings if the be from reason.

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      5. To your addition, reante, I would say yes it’s culturally pretty cool that Bohr (and maybe Heisenberg) chose to add a Buddhist/Taoist/Hinduist flavor to his brand of scientism, but that doesn’t mean what you’re apparently thinking.

        He merely was steeped in that culture and the trends of the time of embracing paradox, which in the 1920s was the order of the day in art, math, economics, etc. It was the fashion, and he did it in a more stylish way by looking east. Unlike normal fuzzyheaded incoherent babble dressed up with a mathematics ribbon, he gave us fuzzyheaded incoherent babble with an Eastern flavor and a mathematics ribbon. Maybe it’s even good that it got people thinking more about consciousness and the like, socially speaking, but as far as science it is nothing but nonsense posing as theory. A master move in career advancement and moat building, I’ll grant.

        For a cultural look at modern physics that echos and elaborates on some of what I’m saying here, Miles Mathis’s essay Death by Mathematics has stood the test of time and part of it applies in a different way to virology and the rest:

        http://milesmathis.com/death.html

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      6. No reliable*scientific method*-based characterization has been done to date. And never will be. But holistic science/ systematic knowledge doesn’t limit itself to the false idol known as the ‘method.’ If these experiments existed in a vacuum, then it would be junk science. And in five years it will be junked science lol.

        But it’s not now junk science; it’s immature, experimental science based in reasonable industrial object modeling. And it’s struggling. That’s as it should be.

        And it’s all totally unnecessary and wasteful. We don’t need to know or even explore any of it… note to self.

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      7. Merely rejecting their conclusory explanations, reante, doesn’t mean there’s nothing to explore in their experiments or observational data, but since they are doing experiments that are far removed from the experiments one with the right paradigm would be doing, the possibility of learning much of use is not high. Either way, that’s beyond the scope of the claims made by me or this blog.

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      8. What’s the paradox?

        Can’t abide by eastern religions myself. Worse than western religions in some ways. Just about controlling minds.

        Like

      9. Particle and a wave at the same time. Double slit experiment. Various paradoxes in modern physics that you’ve probably heard of.

        Like

      10. Why would watching light bend in the night sky with telescopes, and deciding based on all your best patterned prior observations, that it must be a distributed, dark gravitational force permeating reality, be a wrong paradigm according to reason and minus the shitty ethics that made the telescopes possible?

        What’s the right paradigm? Just enjoying the stars and the milky way before turning in for the night?

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      11. I don’t know what the right theory of physics is, reante, but the mainstream model is ridiculous. Dark matter was simply hypothesized due to holes in their theories. It’s not based on just patterned observations, but on assumptions.

        I think our physics discussion might be outlasting its welcome in this small comment section unless we can tie it back to virology.

        Liked by 2 people

      12. Well this physics stuff is all part of the same conversation that I’ve been having, anyway. As above so below: the parallels are clear; the first primordial earth soup was patterned on the cosmic soup. It’s turtles all the way down baby. The point of disagreement also mirrors that of the exosome topic. You can’t put intelligence in a box. It simply can’t be done.

        Regarding the ridiculous paradoxes of quantum mechanics — and I haven’t spent any significant time looking at the field — my feeling is that animism can probably take the paradox out of them as is necessitated by Reason since paradoxes do not exist in *holographic* reality.

        Previously I’ve always said that “paradoxes don’t exist in reality” — minus the “holographic” modifier — but given this recent and still-provisional development whereby dark matter and dark energy exist as two separatisms within the realm of Reality that lies outside of the five-sense holography, we must now include the modifier.
        The Home Cosmology previously stated that source consciousness (the C in ‘god’) and source energy (the A (for application) in ‘god’) exist separately outside of Reality and, colloquially in ‘god’/Creator. But I realize now that that is paradoxical. Reality by definition must be whole unto itself. There is no basis in Reason for Source ‘materals’ to be ‘wormholing’ into symbiosis holographic reality from elsewhere. They are here: without the dark matter fabric underlayment permeating our intelligent body holograms we have no pulling power against entropy; we have no biochemical bonding, no hydrostatic ability. The physicists would do well to consider, if they haven’t already, that gravity, too, is nested. Nesting circumvents paradox. The ridiculous wave-particle duality, given more thought, can probably be seen to be representative of the event horizon at which the two darks and the light holography are all participating in the trangulation of further holographic genesis, and as such not paradoxical because the ‘paradox’ is only so when looking at it from the limited light holography perspective without accounting for the unitary event horizon.

        The collapse of the wave function is metaconsciousness looking at holography. Hence all the object modeling that humans in surplus societies are wont to do.

        Dark energy is how we grow. It is the push. Life grows outwards: take some commodity cropland in most of north America (excluding the South), and implement a kickass grazing program for 5 years until you have the bluegrass-white clover dominant ‘god’ pasture, the peak (fertility) pasture of civilization between about 35 and 55 degrees latitude. You don’t need to seed it, it just comes in as soil fertility increases. If you nurture that ‘god pasture’ you will maximize your milk and meat production and the pasture itself will steadily growing higher and higher above sea level, because holography grows outwards as a fractal of the dark, radiating push of the dark energy fabric underlayment that suffused all holography. When we look at the groundheave surrounding shallow rooted species (conifers) of large trees we are looking at sea level drop relative to that heave. Species like oak don’t visibly cause ground heave because the heave, the soil growth, is much more distributed.

        Forbes apparently has some of the better mainstream journalism around because this is the second deep layperson’s article I’ve posted of theirs, the other being on exactly the whats and whys of the purported synthetic mRNA structure in the vaxxxes.

        The early part of the article, Clarifire, addresses the hole-patching:

        https://www.forbes.com/sites/startswithabang/2020/11/10/why-arent-scientists-more-skeptical-of-dark-matter/?sh=3947e2a35e3a

        Haven’t read Mathis yet. Will do so.

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      13. Reante, what you are calling “dark matter” and “dark energy” could very well be valid and important concepts, but they have no necessary relation to what the modern physicists are talking about.

        Paradoxes and contradictions exist only in diction, that is, only in language. Never in reality in any way. Neither does probability exist in reality; probability is just a subjective state of given observer’s knowledge/ignorance. (Yes by saying “reality” I am using physicalist language, a sloppier phrasing than subjective epistemology, but that is what the level of analysis we call physics calls for to speak in a non-unwieldy manner. If we break the phrasing down to the more precise first-person epistemological level, we can speak nearly without baggage or errror but at much greater length and with a lot more effort in crafting the sentences or diagrams.)

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      14. Right on Clarifire. Once again I think with physics we’re dealing with a situation where the ridiculous conclusions or framings or parlance of and by the field is essentially the result of a cultural problem of clueless urbanity born of limited organic life experience. Obviously there’s a lot of crazy ass noise in the field. The key as always is to focus by filtering.

        That they don’t know what to do with ‘the darks,’ and despite themselves, is fitting. In the articles the word “mystery” keeps coming up; mystery is the alternate translation of “spirit” in the ubiquitous animist conception of “the great spirit in the sky.”

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      15. Clarifire

        I moved on to mathis’ “explaining the ellipse” article. I didn’t realize he has a great mind, I only was aware of his valid antisemitic ‘drumpf not trump’ stuff. He is right about the need to triangulate ellipses in order to explain them. Just as is the need to triangulate holistic reality with the two darks and the the light (holography). Mathis triangulating the ellipse IS him triangulating the two darks and the light. The “E/M field” he is referring to is dark energy exactly, the pushing force (repulsive in the context of holographic body ellipses).

        The “compound” or “differential” effect that requires is exactly the nested gravity I referred to previously, that resolves mainstream paradox. The nested gravity results in a continuously variable ‘net gravity’ that is the creative tension between the two dark fields in separation acting on the two celestial bodies which themselves are the two dark fields in symbiosis.

        http://milesmathis.com/ellip.html

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      16. Reante, that is interesting. I actually haven’t read all Mathis’s old papers, including that one. He’s a true polymath, a rare beast these days. There is a great deal to learn from his science site, whether his theories are correct or not. That he is also an accomplished studio artist is no coincidence; he brings an easy familiarity with visual modeling to physics where it was so sorely needed. It’s a field drowning in words and equations with only token images and animations to satisfy the lay audience who would otherwise be tempted to write them off.

        I will set that article for later reading and consider what you’ve said.

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  4. I think science left the building for good in the 1980’s after reading Robert Kennedy’s “The Real Anthony Fauci” whereby the predictions failed to come true, the testing failed, the drugs failed to save lives and the entire riddle of AIDS/HIV has yet to be solved. This fantasy is not supported by the scientific method and exacting virology, but by the medical mafia disinformation and propaganda machine.

    The pretend science of virology is the basis for the execution of the strategy that says a well and healthy patient is revenue lost. A sick and diseased patient is a revenue gold mine. Not even the “expert” doctors question their mindless march towards satisfying the AMA and big pharma. They put their faith in the master con artist of all time…fauci.

    Liked by 1 person

    1. Tom, did you notice that the first chapter is RFK Jr’s book is titled “A Mismanaged Pandemic”? Do you think we are experiencing a “pandemic”? Or that the number of “COVID deaths” stated in that chapter is based upon facts? Does the book present science which is any better than Fauci’s?

      Like

    2. Coronaviruses are all dressed up with no place to go.
      “Thin section electron micrograph of a cell infected with the coronavirus infectious bronchitis virus showing virus particles assembling in a Golgi region. These virus particles will eventually be exported by vesicles to the cell surface where they are released.”
      https://www.hopkinsmedicine.org/news/articles/the-weird-way-coronaviruses-assemble-their-offspring
      . . .”Let us assume that Hillman is correct about the endoplasmic reticulum and Golgi body being artefacts of histological preparation and electron microscopy (not existing in life). Considering that coronaviruses are said to be assembled at the endoplasmic reticulum-Golgi interface[9], what does this mean for our understanding of virus assembly? . . .
      . . . Let us assume Hillman is correct and macromolecular cell receptors don’t exist. Considering that viruses are said to interact with host cell receptors as the preliminary step to penetration[10], what does this mean regarding our understanding of how viruses penetrate cells, if they do at all?”
      https://www.newbraveworld.org/modern-medicine-is-currently-in-dire-straights/

      Liked by 1 person

  5. Dear Mr Stone (or others),

    I’m firmly on your side of the aisle here, but one question has been plaguing me. We all know about the Rosenau experiment where, despite best efforts, they were unable to transmit the ‘spanish flu’ to uninfected people. That’s pretty convincing evidence that the ‘spanish flu’… wasn’t infectious.

    However, have they never took a dog infected with ‘doggie flu’, put him in a room with 1000 other dogs, and watched the ‘flu’ spread like wildfire in a more or less predictable way?

    I realize that they can’t do this with humans because of ‘ethics’, but no such bounds exist with animals.

    If they haven’t done this then virologists go from being incompetent… to malicious.

    Liked by 1 person

      1. I came across a link an article a while , somehow, did not attach the original article and cannot find the original article.

        Anyway will post it .

        DOES VACCINATION MAKE SENSE? – Horses. and Dogs Source : deepL translate , 2001 pdf

        My experience on the subject of inoculation for horses.
        In 1975, as an apprentice in the equine profession on a large stud and farm (120 horses, 500 cattle, 1000 pigs) with a test stable and a farm veterinarian, the new foals were inoculated with vitamins and tonics as a preventive measure against the so- called folic lameness. Many of them died within a short time, with the symptoms of Folic Paralysis – fever – inflamed joints – refusal to feed – unsteady gait – lying down etc. In my 25 years of breeding, during which I never vaccinated the foals, I never experienced a single case of Folic Paralysis. About 15 years ago, influenza vaccination was recommended for show horses and I had a show horse vaccinated. It coughed in a weakened form for 4 months, which again made me against vaccination.
        When the German Equestrian Federation (FN) decided in 2000 to make vaccination compulsory, horse owners in my stable also had them vaccinated. One gelding, who had just recovered from pneumonia and was nevertheless found fit for vaccination by the vet, suffered severe colic one week after vaccination and died.
        A second gelding, in perfect health, suffered a short severe colic 1 day after the 2nd vaccination, i.e. 4-6 weeks after the first vaccination, which he survived. In 2001, again after the first vaccination of 4 young horses, two of them started coughing, then the whole herd of young horses. 6 weeks later, one eye of a vaccinated 6 year old became cloudy and slightly inflamed for no apparent reason.
        Shaken up by a severe vaccination damage of a child (now weak-sensed a foster case) from the neighbouring village, I started reading books by vaccination critics (well- known doctors). I was appalled and it seemed unbelievable to me that many scientific studies and statistics never come out in the open, in which the harmfulness of vaccination is proven beyond doubt. Through a website of the association Wissenschaft, Medizin und Menschenrechte e. V. (Science, Medicine and Human Rights), I was made aware of Dr Lanka and K. Krafeld. After I attended a lecture evening with the two gentlemen and then demanded proof from the offices, authorities and the FN, against what vaccination was recommended or made compulsory, and I received no, or only false, so-called scientific proof in response, it is clear to me that I will never again vaccinate a living being, whether human or animal. I attribute many illnesses of humans and horses from my circle of acquaintances to vaccination. I cannot understand why, despite the known high side effects and the unprovable theory of which pathogens need to be vaccinated against, more and more people are

        advocating or forcing vaccination. Unless it is to support the vaccine manufacturers and the professions that live off them. I sincerely hope that many people who read this letter will also inform themselves more thoroughly and then have an objective insight into the subject of vaccination. Diana Herrmann
        At the last meeting of the Reiterring Oberrhein, one of the topics discussed was the compulsory vaccination of show horses, which was suggested by one of our member clubs. It turned out that this member club, despite several requests to the FN, did not receive any answer to his question about the reason for compulsory vaccination, which astonished the other clubs.
        Reiterring Oberrhein e.V. Joachim Georgii, 1st Chairman ————-
        A study on vaccinations for dogs
        Animal lovers insist that animals are more sensitive than humans. Animals react faster and more directly to things that are bad for them. Animals react more promptly to vaccinations. Horse owners react extremely upset after the experience of two years of compulsory vaccinations for show horses. This is because horses are getting diseases that were known to be caused by poisoning before the introduction of compulsory vaccination. The stud owners are in a dilemma. Because an unvaccinated horse is not admitted to the tournaments and a vaccinated one cannot participate in a tournament because of the vaccination problems.
        What is obviously not possible with humans has been done with animals, especially with dogs.
        For her book: “Canine Health Census Vaccine Survey” Catherine Ó Driscoll researched vaccination problems in dogs. The study began in October 1996 and a questionnaire was developed with the assistance of Christopher Day, Jean Dodds DVM, and Dr. Viera Scheibner. The questionnaire was advertised in Dog World Magazine and people were asked to complete it. At the time of publication, 607 vaccination problems were reported among nearly 2678 dogs.
        This represents more than one fifth of the population. The main interest of the study was to determine if there was a temporal relationship between the illnesses and the previous vaccination. The rationale was that if no correlation between vaccination and disease could be established, the dogs’ illnesses would have to be evenly distributed in the period between two vaccinations.
        The percentage of illnesses should then not exceed 25 %. In fact, the evaluation of the

        reporting forms shows that 55% of the diseases occurred in the first three months after vaccination. This means that the incidence of disease was more than twice as high in the first quarter of the year after vaccination
        . In detail, the following breakdown emerged from the study:
        Cancer- 31% within 3 months after vaccination.
        Cramps- 63% within 3 months after vaccination
        Meningitis- 75% within 3 months after vaccination
        Heart disease- 26.8% within 3 months after vaccination
        Kidney damage- 40.5% within 3 months after vaccination
        Paralysis- 52% within 3 months after vaccination
        Abdominal paralysis- 64.7% within 3 months after vaccination
        Liver damage- 47% within 3 months after vaccination
        Impaired concentration of the dog – 68.4% within 3 months after vaccination Autoimmune disease – 54.8% within 3 months of vaccination. If vaccinations had no effect on the reported diseases of dogs, the disease rates would have to be around 25%. This is the case for heart disease, which is the only disease reported.
        Dogs that contracted the diseases they were vaccinated against: Hepatitis- 63.6% within 3 months of vaccination.
        Parainfluenza- 50% within 3 months of vaccination
        Parvovirosis – 68.2% within 3 months after vaccination distemper – 55.6% within 3 months after vaccination Leptospirosis – 100% within the first 3 months after vaccination.
        Here, the baseline value of the disease should be set at 0 %, since the
        vaccination should protect against the disease
        The vaccination was supposed to protect against the disease that it triggered in the reported dogs within three months.
        It is doubtful whether this study can be directly applied to humans. The study certainly shows that vaccinations should not be seen without a connection to the subsequent diseases. Certainly, the time period after vaccinations in humans must be extended. And therein lies the problem with the surveys. Who thinks about the connection with a vaccination after several years. Veronika Widmer

        Liked by 1 person

  6. The diagnosis is a hoax. There are no diseases. There are only automatic processes for adjusting the intensity of energy flows, adjusting the structure of tissues, and adjusting the efficiency of functions (including sensory functions and tissue regeneration functions) in order to cope with all harmful existential factors:
    – negative feelings (fears, sorrows, envy, hatred)
    – toxicity from chemicals (including medicinal chemicals), toxicity from some of the concentrated metals and minerals, toxicity from artificial radiation
    – physical and intellectual overload
    – quantitative and qualitative malnutrition
    – weather exposure
    – iatrogenic
    All the adaptive changes that our being constantly operates in order to be able to cope with the harmfulness of the factors listed above, have as their only objective: survival. That is why the fight against the constant changes that take place in the body and the fight against the unpleasant symptoms we face as our body adapts to cope with the harmfulness of the factors listed above, means even fighting the automatic processes that our being rebalances its energies and regenerates its tissues.
    There are no viruses, pathogenicity of microorganisms and genes. Which is why there is no infectious-contagious or genetic disease. There is also no autoimmune disease. Symptoms attributed to so-called autoimmune diseases are created by the body to adapt to harmful psycho-emotional factors and / or toxic chemical factors (chemical synthesis substances in industry, agriculture, animal husbandry, poultry and fish, from the food industry, from the pharmaceutical industry, from cleaning and hygiene products, from construction and landscaping materials, etc.) to metals and minerals that become toxic by concentration, as well as to some biological products obtained by degradation or concentration organic matter. Also, the so-called nosocomial (hospital) infections are the consequence of drug poisoning, not of any microorganisms. As for the various changes in body tissues that are declared to be “malignant cancers”, they are created by the body itself in order to enhance its various functions, in accordance with the needs imposed by various harmful factors. And when harmful factors no longer act as intensely and for a long time on the being, tissue proliferations become unnecessary, which is why the body breaks them down and eliminates them.
    P. S. There are no diseases of unknown cause. The harmful factors that disrupt our energies and damage our tissues, forcing our being to enter the state of self-healing called disease, are known because they are part of our lives:
    – negative feelings
    – chemical toxins (industrial, agricultural, food, pharmaceutical, etc.)
    – some of the concentrated metals and minerals
    – some of the concentrated biological products
    – artificially generated radiation
    – physical and / or intellectual overload
    – Insufficient night’s sleep
    – quantitative and / or qualitative malnutrition
    – weather exposure
    – iatrogenic
    The extremes of life exhaust our being because it forces us to make adaptive changes in energy, tissue, function and sensory.
    Our being adapts by over-stimulating the intensity of vital energy flows in order to cope with cold, fear, malnutrition, sleep deprivation, physical and intellectual overload. If exposure to the harmful factors listed above is not eliminated or limited, increasing the intensity of vital energy flows above normal unbalances them and depletes them long enough to lead to sudden deaths.
    Our being adapts by under-stimulating vital energy flows to cope with heat, sadness, overeating, excessive sleep, sedentary lifestyle. If exposure to these harmful factors is not eliminated or reduced, decreasing the intensity of vital energy flows below normal will decrease the efficiency of tissue regeneration processes, accelerating tissue degradation, which will further disrupt vital energy flows, accelerating tissue degeneration and this vicious circle will lead to death.
    In addition (and unfortunately) there are also concentrated substances in nature (metals, minerals, organics), there are also artificial substances (chemical, pharmaceutical) and there is also energy radiation (artificial) to which our body is forced to adapt either by over-stimulating or under-stimulating vital energy flows, as in the case of the harmful factors listed above.
    Therefore, the path to a healthier and longer life lies in a philosophy of life that will lead man to understand the above. More directly: Nothing without God.
    Our being is a living and reactive structure, made up of substantiated (materialized) energies and unsubstantiated energies that work together to adapt in the most efficient way possible to all existential factors in order to survive as long as possible.
    The efficiency of our being’s mechanisms to adapt to existential factors is influenced by:
    – the physical and energetic characteristics inherited from the parents;
    – living conditions;
    – working conditions;
    – lifestyle;
    – the feelings of the soul;
    – toxicity;
    – iatrogenic;

    There are two types of cancer: those that increase tissue function and those that increase the rate of tissue regeneration.
    Cancers are nothing but natural processes of automatic self-healing mechanisms in order to survive.

    All tissue proliferations are adaptive processes generated by the body to improve tissue functions. Self-healing is also a function of the body’s tissues. All manifestations of the body, no matter what they consist of and no matter how dramatic, have as their sole objective survival.

    The explanation given by the New German Medicine for the reason why tissue mass losses occur during the acute conflict generated by strong feelings of sadness, is wrong. The idea that in the acute phase of strong feelings of sadness the body generates erosions of the blood vessels, mammary ducts or bile ducts in order to improve blood flow, milk flow or bile flow is completely illogical, because the body permanently makes these things only by dilating or contracting blood vessels or mammary ducts or bile ducts. The reason for the loss of tissue mass during the active conflict created by strong feelings of sadness is related to the imbalance and depletion of vital energy flows in those tissues, which is why tissue regeneration becomes so slow that it can not fully recover degeneration.

    Like

    1. I think both orthodox and a lot alternative medicine are resisting universal biology as both would have to retrain , there is huge money in both out of selling indulgences/ false promises.

      I have been reading alternative medical sites for over 10 years eg. functional medicine and others and was on the fence but know that most is wrong as based on false concepts of biology .ie disproven cellular biology, disproven molecular genetics and there is no immune system as told.
      I noticed that in some ‘freedom’ circles they jump from one bad science to another one as it is based on reductionist approach to health. They are stuck in a material explanation of life and illness.
      The body-soul medicine is a much better explanation
      That is a reason why many people are not open to accept that there is no virus k etc. as they need an answer. If it is not a virus what is it?

      “ Biological life is about maintaining and increasing the flow of energy.
      All biological structures, perception and behaviour serve this goal.

      The basis of life is a substance that emerges from water.
      This substance is referred to by physics as ice form X and by its discoverer as 
      dense water.

      This viscous substance is one of four states that water can assume.

      This substance, which is energy and at the same time the building and information substance of life, 
      connects all matter, organisms and functions.”

      source- w-plus.
      ——-

      The essence of five Biological laws.- called laws as applicable tieach case.

      1. First Law
      All diseases start with a conflict shock that affects one equally on a psychic, cerebral and organ level.
      It’s cause is not the psychic , the brain or the organ. The cause is the shock that triggers something in the soul and starts problem on all three levels.

      2 Second Law
      Every disease goes through 2 stages active and healing ie. if the conflict gets resolved it evolves into a linear form.
      But that seldom happens in people .
      Because of the symptoms they cause themselves new conflicts or because of oxygen or energy the body cannot-get over the healing phase , they relapse and the cycle keeps repeating itself .
      This depends on many factors eg. the overall health of the person, our emotions, fear , they way we interpret our symptoms.
      Many factors can influence how the disease will progress.
      Eg if have symptoms and go to the doctor, a diagnosis creates extra conflict and can lead to a never ending cycle , the worst conflict can be from a diagnosis.

      3 Third Law
      The changes happen on a tissue level and can be explained on an embryological level.
      Depending on the embryological layer ( 4 layers, ectoderm, 2 types of mesoderm, endoderm) from which the organ stems we can identify what type of conflict occurred

      4 Forth Law .
      The microbes optimise the healing .
      Bacteria, fungi are never our enemy, they are our servants, they are working for us, they are our cellular garbage cleaners.
      It is true that once the person has been in a stage of conflict a long time , a cellular mass builds up that needs to be decomposed in the healing phase., then you need a large number of microbes. They will decompose that large mass triggering inflammation, pain , pus, bleeding ie with symptoms. But that is not the fault of the microbes but the fault if the person that stayed in that conflict for a long time

      5 Fifth Law.
      All diseases are a reality.
      The quintessence- there is a special biological problem with meaning , ie.the basis of this biological process is to keep us alive,

      Like

      1. 1 Thessalonians 5:21
        New International Version
        21 but test them all; hold on to what is good,

        Everyone has to do their own critical research, including on the New German Medicine, which 99, (9)% of its followers idolize as perfect. Even if the New German Medicine promotes more truths than other schools of understanding the state of health, the state of injury and the state of self-healing, for several reasons which I will not detail, because we would waste time unnecessarily, even the New German Medicine, by its complicated way of being and by its errors, is also a dead end that blocks the progress in understanding that comes when we manage to understand things so as to summarize them until they become simple and clear enough that to be understood by any man.

        Liked by 1 person

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