When getting down to the size of nanoparticles and the expected billions of identical particles at that level, it would be logical to assume that completely separating the exact particle a virologist is looking for from everything else in the sample is downright impossible. Thus, asking virologists to completely purify and isolate the suspected “viral” particles from the unaltered sample from a sick patient may seem like a Herculean task and an unfair demand.
However, this is the corner Virology and Germ Theory has backed itself into. In order to claim a particular particle is a “virus” and can cause the symptoms of disease associated with it, logic dictates that it must be completely separated from all other potential variables/factors in order to prove that particular particle is indeed the cause of disease. This is the only logical way to show that no other particles in the sample could have been the cause of disease and in the case of genomics, that the DNA/RNA sequences belongs to only that particular particle which is believed to be a “virus.”
We can find out if complete purification/isolation is possible by looking at exosome research and the methods used. “Viruses” are considered exosomes in every sense of the word as they are identical in size, shape, and appearance. The methods used to purify/isolate exosomes are the same ones which are supposed to be used for “viruses” but which are never done, especially in the absence of toxic cell culture processes. These methods are considered the best available purification/isolation methods today.
Let’s look at a few of them briefly:
Recent advances and challenges in the recovery and purification of cellular exosomes
“Although different approaches for the separation, purification, and analysis of exosomes have been explored, so far, there is no methodology providing enough robustness regarding purification yield, selectivity, and reproducibility. In fact, because of the inherent biochemical properties of these vesicles and the enormous differences between them that depend on the matrix from which they are obtained, usually a combination of techniques needs to be tailored to achieve the desired purification outcomes. In this sense, different physical and/or chemical methodologies have been studied and proposed to achieve a good exosome purification yield. This work aims to establish the current state of the art in exosome isolation strategies highlighting the advantages and disadvantages of each of the most commonly used techniques while presenting a perspective of the future of this important topic.”
“Furthermore, many challenges arise when isolating these naturally‐occurring particles. For example, exosomes belong to a large domain of extracellular vesicles, some of which present overlapping physicochemical properties 55. Moreover, exosomes themselves exhibit high heterogeneity in size, cargo, and surface markers.”
“The current golden standard for exosome isolation is ultracentrifugation 58. As known, this technique exploits the particle movement principle due to gravitational acceleration in an inertial field 59. Differential and density gradient ultracentrifugation are among the most commonly used ultracentrifugation methods for exosome isolation 60.
Differential ultracentrifugation is commonly known as the pelleting method, because it allows to obtain pellets containing exosomes. It is also referred to as the simple ultracentrifugation method since it only requires several ultracentrifugation steps 61. To date, this is the most widely used method for isolating exosomes and has been successfully used in a variety of biological fluids and cell culture media 62, 63. During differential ultracentrifugation, exosomes are separated based on their density and size. Thus, contamination from other vesicles, molecules or particles that overlap in these parameters is expected. To reduce the presence of cell debris and large vesicles, cleaning steps are needed before pelleting the exosomes 64, 65, 66.”
“According to these results, a specific g‐force does not pellet exosomes with the same efficiency in different types of rotors, and the centrifugation time needs to be properly considered. Otherwise, sample purity, protein content and exosomal yield will be compromised 68.”
“It should be noted that the g‐force used during ultracentrifugation protocols has a significant effect on the purity and yield of exosomes 75. Moreover, exosome sedimentation efficiency has been found to vary among cell lines.”
“However, as it has been mentioned, this method is not specific and contamination with other extracellular vesicles is unavoidable. If the protocol is not well standardized and adapted (in terms of time and gravitational force) to the characteristics of the equipment being used, exosome isolation will not be consistent, and losses will occur inevitably 59.”
2.2. Filtration‐based strategies
“Ultrafiltration is another available technique to obtain exosome enriched samples from a variety of sources including cell culture medium and biological fluids 91. In this process, extracellular vesicles suspended in a solution can be separated by size or molecular weight. Usually, different forces are applied to make them pass through (or be retained on) a selective membrane. Centrifugal force, pressure or vacuum are usually applied for ultrafiltration through a membrane that is commonly built from low protein affinity materials.”
“Nevertheless, ultrafiltration, a simple protocol, is incapable of isolating only exosomes, as microvesicles and apoptotic bodies will also be present in the resulting product. Moreover, large amounts of highly abundant proteins, that mimic exosome size or molecular weight, will also be found in the resulting solution. Such contamination arises from the physical limits of the procedure and the overlapping properties of the particles in the matrix being processed. Furthermore, the effects of the applied force and the contact with the membrane on the exosomes need to be further studied. Potential deformation and exosome losses due to extrusion and membrane binding are expected 104.”
“Exosome precipitation is a recently developed technique that is now the second most used isolation method after ultracentrifugation, most likely because it is fairly easy to perform and does not require specialized or expensive equipment 68. This technique was originally developed to isolate viruses and other macromolecules, but exosomes can also be settled from biological fluids using the same principles 115. Most precipitation methods consist on mixing the sample, which can be either a biological fluid or cell culture medium, with a hydrophilic polymer. After mixing, the sample is incubated overnight at 4ºC and afterwards low speed centrifugation is used to precipitate the exosomes which are later resuspended in the preferred buffer for further analysis 116. Protamine, sodium acetate, and organic solvents can also be used for precipitation procedures 117, 118.”
“There appears to be a consensus that precipitation‐based methods yield the highest number of extracellular vesicles but with low purity, as it was found when compared to column‐based methods 116. Thus, another approach consists on the incorporation of these methods after precipitation to further purify the sample.”
“Nonetheless, low purity is a key disadvantage. Coisolation of non‐vesicular contaminants such as lipoproteins and ribonucleoprotein complexes, albumin, immunoglobulins and other soluble proteins is unavoidable 126. Contamination with other vesicles is also expected. Unfortunately, this contamination may interfere with further biochemical and immunological analysis. In this context, the need for further purification has produced modified protocols that include pre‐ and post‐isolation cleaning steps, lengthening originally fast procedures 127, and increasing workload and costs as well.”
“In addition to coisolation of nonvesicular contaminants and other vesicles, recent studies have found exosome aggregation after precipitation 128, 129. Exosome aggregates may also interfere with downstream analysis.”
3. The future of exosome isolation procedures
“Exosome isolation remains a challenge for biomedical research. There is still no consensus over which purification technique produces the best results and there is intense competition within the field. Moreover, an accurate comparison between methods cannot be easily made because of the inherent exosome complexity.”
“Moreover, coisolation of contaminants should be minimum since contamination is the most common complication of current isolation techniques 58. Almost invariably, coisolation of other vesicles and non‐vesicular molecules occurs, interfering with data comparison between research laboratories 123.
Although, ultracentrifugation is currently the gold standard for exosome isolation. There is no ideal method that fits all purposes. The selection of the procedure usually depends on the capabilities and resources of each research team and sacrifices must be made in terms of recovery, purity or work load. Moreover, downstream analysis may be compromised by the isolation technique that is chosen 90. In this sense, the final selection of the most suitable technique for exosome isolation and purification needs to consider the effects that the methodology may exert over the sample integrity particularly for the intended final use. For instance, recovery techniques such as ultracentrifugation and filtration tend to render a population of “saucer‐like” or “deflated‐football” shaped vesicles that might no longer be useful 157. Furthermore, the integrity of the exosomal cargo to unravel exosome‐specific functions and biomarkers should also be considered even when no apparent degradation is present 158. This is especially true for microfluidic techniques or after isolation when exosomes are stored under freezing or other harsh conditions 159.”
4. Concluding remarks
“There are currently different strategies being used in the isolation and purification of exosomes whose selection depends on the intended application for the exosomal extract. To date, it is difficult to identify a strategy that yields the highest quality and properties of the isolated exosomes and usually a combination of the different methodologies is required to achieve the best results.”
- Although there are many different methods used for purification, there is no methodology providing enough robustness regarding purification yield, selectivity, and reproducibility
- Usually a combination of techniques needs to be tailored to achieve the desired purification outcomes
- Exosomes belong to a large domain of extracellular vesicles, some of which present overlapping physicochemical properties
- Moreover, exosomes themselves exhibit high heterogeneity in size, cargo, and surface markers
- The current “golden standard” for exosome isolation is ultracentrifugation
- Contamination from other vesicles, molecules or particles that overlap is expected
- Without proper g‐force, sample purity, protein content and exosomal yield will be compromised
- The g‐force used during ultracentrifugation protocols has a significant effect on the purity and yield of exosomes
- Ultracentrifugation is not specific and contamination with other extracellular vesicles is unavoidable
- Ultrafiltration is incapable of isolating only exosomes, as microvesicles and apoptotic bodies will also be present in the resulting product
- Large amounts of highly abundant proteins, that mimic exosome size or molecular weight, will also be found in the resulting solution
- Such contamination arises from the physical limits of the procedure and the overlapping properties of the particles in the matrix being processed
- Precipitation methods consist on mixing the sample, which can be either a biological fluid or cell culture medium, with a hydrophilic polymer
- There appears to be a consensus that precipitation‐based methods yield the highest number of extracellular vesicles but with low purity
- Low purity is a key disadvantage as coisolation of non‐vesicular contaminants such as lipoproteins and ribonucleoprotein complexes, albumin, immunoglobulins and other soluble proteins is unavoidable and contamination with other vesicles is also expected
- Exosomes aggregate after precipitation which may also interfere with downstream analysis
- Exosome isolation remains a challenge for biomedical research and there is still no consensus over which purification technique produces the best results
- An accurate comparison between methods cannot be easily made because of the inherent exosome complexity
- Contamination is the most common complication of current isolation techniques
- Almost invariably, coisolation of other vesicles and non‐vesicular molecules occurs
- There is no ideal method that fits all purposes and sacrifices must be made in terms of recovery, purity or work load
- To date, it is difficult to identify a strategy that yields the highest quality and properties of the isolated exosomes
Keep in mind that exosomes and “viruses” are nearly identical in every way. In fact, exosomes have been called “non-infectious viruses.” The main difference is that exosome research regularly attempts purification using one or multiple methods whereas Virology does not. That being said, the three methods discussed above all inevitably suffer from contamination from other particles as well as potential damages to the particles in the sample. The forces and methods used on these samples are unlike anything they encounter in reality. There is absolutely no way to say that the resulting particles are in the same form as they were originally at the start of the purification/isolation process.
Purification/isolation of these particles is an impossible task. It may even be an unfair demand to ask for this. However, logic does not deal with fairness. In order to prove a “virus” exists and causes a particular disease, it must be completely purified/isolated from an unaltered sample first.
Unfortunately for Virology/Germ Theory, they have the unenviable responsibility to show that complete purification/isolation of particles believed to be “viruses” can be done. No conclusions about any particle as a “virus” can be made until this logical step occurs. To date, they have failed to do so every time.