Extracellular vesicles (EV) are all kinds of vesicle structures with membrane structure released by cells, and the diameters of these vesicles can range from 30 and 40nm to 8 and 9um. Extracellular vesicles have different subgroups, and exosomes are the most popular subgroup at present.
However, because it is difficult to purify a very pure subset of exosomes at present, people usually purify vesicles with a diameter less than 200nm, so more and more people begin to call them sEV (small extracellular vesicle). For the sake of rigor, today we also call these membrane vesicles extracellular vesicles. Unless otherwise specified herein, extracellular vesicles mainly refer to exosomes and microcapsules.
Today, the main contents include the research significance of extracellular vesicles, the classification of extracellular vesicles, the components of extracellular vesicles, the preparation of cell culture supernatant, the preservation of extracellular vesicles, and the experimental requirements for the identification of extracellular vesicles. All contents refer to the comments published so far, not Zhang Faming. So please feel free to study for new friends.
Research significance of extracellular vesicles
At present, the function of extracellular vesicles has not been fully clarified. It is reported that they can regulate host-pathogen interaction, participate in the pathological process of infectious and inflammatory diseases, nervous system diseases and cancer, and also play an important role in mediating intercellular communication in normal physiological processes. It is reported that extracellular vesicles also play an important role in development. Extracellular vesicles also have a bright application prospect in clinical medicine, mainly because they contain rich biomarkers, which can be used to monitor clinical status, treatment response, disease progress and so on. At the same time, because they have the function of delivering biomolecules, they still have the potential to develop into clinical drug delivery carriers.
Classification of extracellular vesicles
Extracellular vesicle-a term coined by the International Extracellular Vesicle Society (ISEV). Vesicles can be classified according to their biosynthetic or release pathways: the diameter of exosomes is 30- 150nm, which originates from endocytosis, and its density is about1.1. Particles/microbubbles are released directly from plasma membrane, and the diameter is about100-1000 nm. The diameter of apoptotic bodies/filtering vesicles is about 50nm-2μm, which is caused by apoptosis. The diameter of tumor vesicle is about 1- 10μm, which is produced by the release of tumor cells. And various other EV subgroups. Due to the differences in the size of different EV subgroups and the biomolecules they contain, several groups have begun to characterize the composition of EV subgroups. Recent papers claim that extracellular vesicles are successfully classified into subgroups based on general surface proteomic analysis or transcription profile of individual EV population. At present, different extracellular vesicle subsets can be roughly separated by various means, such as differential centrifugation, filtration, immunoaffinity, chromatography, flow cytometry and density gradient centrifugation. However, these methods can not completely purify specific subsets, and the isolated subsets are usually rich in other extracellular vesicle subsets.
Components contained in extracellular vesicles
The composition of extracellular vesicles is not random, and each extracellular vesicle carries specific molecular information. In fact, these nano-sized extracellular vesicles can carry bioactive molecules such as protein, lipid, nucleic acid and sugar to transmit signals between cells, and the unique molecular composition of their packaging determines the type of extracellular signals to be transmitted to recipient cells. There is a complex classification system to determine which molecules can enter extracellular vesicles.
Preparation of cell culture supernatant
At present, there are two strategies to prepare cell culture supernatant for extracting extracellular vesicles. Cells were cultured in fetal bovine serum (FBS) medium without extracellular vesicles, and cells (serum hungry cells) were cultured in FBS-free medium. But at present, some high-level articles usually adopt the former method. In addition, some articles began to pay attention to the influence of free RNA in fetal bovine serum from which extracellular vesicles were removed on the experimental results, but it has not been understood yet. If it is necessary to preserve the culture supernatant before it is used for extracellular vesicle separation, it is necessary to remove dead cells and cell debris from the system in advance.
Preservation of extracellular vesicles
l？ Rincz et al. stored extracellular vesicles from neutrophils under different conditions and then analyzed the characteristics of these vesicles. They found that although the number and shape of vesicles in extracellular vesicle samples did not change significantly, even extracellular vesicles stored at -20 or -80 degrees Celsius significantly increased the eversion of phosphatidylserine. But they also found that extracellular vesicles derived from urine did not have these changes. Kalra et al. isolated extracellular vesicles from colorectal cancer cells, kept them under different conditions for a certain period of time, and then stained them with PKH67 to track the uptake of extracellular vesicles. It was found that these vesicles could still be absorbed by cells. As far as the current situation is concerned, scholars have not reached a consensus on whether storage conditions affect extracellular vesicles. Therefore, it is suggested that we should separate extracellular vesicles as soon as possible and use them in experiments to minimize the storage process.
Identification and experimental requirements of extracellular vesicles
According to the guide manual (MISEV) published by International Extracellular Vesicle Association 20 14, it is necessary to identify whether there is a marker protein of extracellular vesicles in the sample by WB, observe the morphological characteristics of extracellular vesicles in the sample by electron microscope, and analyze the population characteristics (particle concentration, diameter distribution, etc. ) of extracellular vesicles.
Let's call it a day. As a new field of biological research, the field of extracellular vesicles has attracted more and more attention from scholars and biological companies, and more and more research reports have appeared. However, we should also be soberly aware that the research technology in this field is not perfect, and the understanding of extracellular vesicles is not enough. The study of extracellular vesicles is still in its infancy and there is still a long way to go.