Research on exosomes have arisen great interest in the last decades, there are multiple areas where exosomes are contributing such as nanomedicine, cancer research and diagnosis, drug delivery systems, and so on. Furthermore, exosomes are also being widely studied as biomarkers for several diseases. In this sense, in order to exploit their potential and research about these extracellular vesicles it is very important to accurately isolate, detect and characterize them. Looking for achieving this purpose, a wide range of techniques for the isolation and characterization of this type of extracellular vesicles have been developed. In this article, we will be describing one of the most used isolation methods at the moment: the exosome precipitation solutions.
As we have previously mention, one of the main research areas of exosomes right now is the use of these extracellular vesicles as biomarkers for several diseases. In order to address this application, we first have to understand what biomarkers are, and how they are used in research and diagnosis.
Biomarkers are biological molecules found in blood, other body fluids, or tissues that is sign of a biological state, which means that they are indicators that objectively measure and give us information about the normal or abnormal process, or of a condition or disease.
A biomarker may be used to see how well the body responds to a treatment for a disease or condition.
As exosomes have demonstrated to contain a complex cargo of contents derived from the original cell, these vesicles can give us very specific information about the state of these original cells in a precise moment, and this is the reason why the analysis of exosome content can provide proof about what is happening in the cell of origin. Studying the patterns of the different type of exosomes in different samples we will be able to find different molecular profiles related to different diseases, which can provide valuable diagnosis in the future.
In this sense, exosomes have bring out great interest in a wide range of research and diagnosis areas because they have proven high accuracy as biomarkers in plasma and other types of samples. (learn more about the use of exosomes as potential biomarkers)
Precipitation solutions are one of the most common methods for the isolation of exosomes.
A precipitate is a solid produced by a solution because of a chemical or biochemical reaction, this process is called precipitation. When an insoluble substance is formed in a solution, or the solution is supersaturated by some compound, precipitation is formed: if the precipitate is denser than the rest of the solution it falls, if it is less dense, it floats, and if it has equal density, it remains in suspension.
This method is a highly efficient procedure, and it provides a very high capacity to precipitate all extracellular vesicles in the sample. In addition, precipitation combined with protein solvents can improve purity and ensure contaminant-free isolates for therapeutic applications.
However, some of the main disadvantages of this method compared to others are the need to remove the polyethylene glycol or precipitating salt to prevent it from interfering with downstream applications, as well as the need for additional purification steps.
Exosomes are isolated from varied and complex biological samples retaining their physicochemical properties and biological function after isolation.
A recent study has revealed that the choice of extracellular vesicles (EVs) isolation method is crucial because it influences in the depletion of lipoproteins and protein contaminants and EVs recovery. In this sense, exosome precipitation solutions are ultracentrifugation free method, providing intact exosomes and high yield and good for small volume. On the other hand, the purity/recovery ratio in exosome precipitation solutions for high-risk biological plasma samples must be carefully considered.
Furthermore, Immunostep Exosome Precipitation Solutions are very clean exosome preparations, which help reding carry-over of albumins and immunoglobulins, and it also obtain intact exosome suitable for a wide variety of RNA-seq study and protein-sensitive applications.
As we have addressed in previous articles, a wide variety of methods have been introduced to help researchers all over the world in the task of isolating exosomes from biological fluids, precipitation solutions are one of them. Each of these methods has its advantages and disadvantages: time needed to perform the method, the need for specialized equipment, sample starting volume, purity and recovery are key parameters to consider in this regard.
The exosome precipitation solution is framed within the polyethylene glycol-based precipitation method, and its main advantages include its simplicity, speed, reproducibility and high recovery. In this sense, we could say that we can increase biomarker accuracy using our exosome precipitation solution.
But what are the ideal conditions for exosome precipitation solutions? For instance:
At Immunostep we have performed several tests in this regard, with different types of samples and different starting volumes, and we are able to affirm that for a plasma or urine sample with a volume between 1-20 mL, if what we want to perform is a RNA analysis of exosomes contained in the sample, this method is quite effective.
Differential centrifugation is a process widely used in microbiology and cytology, which is used to separate some elements of a cell for further analysis of specific parts of the cell. We can use ultracentrifugation of exosomes for the separation of intact cells in plasma, or other types of samples. To do this, the sample is first filtered or clarified to remove debris and cells, and then subjected to repeated centrifugations, progressively increasing the centrifugal force, up to that exosomes are isolated and the extracellular vesicles fraction to be analyzed is recovered.
Ultracentrifugation is still by far the most widely used primary method, the second more used exosome isolation method is the precipitation solutions. However, ultracentrifugation process is a more time-consuming method that yields aggregated proteins and nucleic acid can sediment and is not good for small volumes.