In recent years, the research on foreign bodies is in full swing. Exosomes play an important role in immune antigen presentation, tumor growth and migration, tissue injury repair and other physiological and pathological aspects. At the same time, exosomes secreted by different cells have different components and functions, which can be used as biomarkers for disease diagnosis. Cui, a teacher from Union Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology, has accumulated her own experience in exocrine extraction and published her own research results in Bioactive Materials. The following is teacher Cui's sharing.

In order to study exosomes, extraction and purification of exosomes have always been a very concerned issue. Whether enough foreign bodies with high purity can be obtained is directly related to the follow-up research. Although there is no extraction method that can ensure the content, purity and biological activity of foreign bodies at the same time, we can do some discussion and experience summary on the extraction of foreign bodies in combination with the current actual situation.

Ultracentrifugation (differential centrifugation) is the most commonly used method to extract foreign bodies at present, that is, low-speed centrifugation and high-speed centrifugation alternately can separate vesicle particles with similar sizes. Ultraseparation method is popular because of its simple operation and large number of vesicles. My recently published paper also adopted this method.

The specific steps are familiar to everyone. Whether extracting foreign bodies from serum or from cell conditioned medium, everyone is used to collecting serum or conditioned medium first, then storing it at -80℃, and then ultracentrifugation in batches after reaching a certain amount.

In this case, you will find a very serious problem, especially when shooting an electron microscope-there are many impurities in the background, and many vesicles are not typical in shape, not the typical double-concave disk or saucer described in the literature.

Through the comparison of several experiments, I probably found the reason, mainly because low-speed centrifugation and ordinary high-speed centrifugation were not carried out when the culture medium was collected by freezing.

Low-speed centrifugation can remove non-adherent cells, dead cells and large cell fragments in the medium, while ordinary high-speed centrifugation can remove large extracellular vesicles. No matter non-adherent cells, dead cells, large cell fragments or large extracellular vesicles, they will rupture during freezing and rewarming at -80℃, resulting in small cell membrane fragments.

In this case, the background of electron microscope is very chaotic, and it is difficult to meet the requirements of high-level papers and magazines. In addition, it will also lead to such a contradictory phenomenon, that is, the output of exosomes calculated in protein quantification is artificially raised, while the content of exosomes marker protein in western blot is not high.

So, I'm here to recommend a little trick.

That is, when collecting conditioned medium or serum to extract exosomes, the steps of low-speed centrifugation and ordinary high-speed centrifugation must be done in advance, especially when the extracted exosomes are used for transmission electron microscopy or western blot experiments. We must pay attention to this. Although this process is time consuming, it is more important to produce useful results.

As for other foreign body extraction methods, such as density gradient centrifugation, chromatographic column method, ultrafiltration centrifugation, microfluidic chip method, magnetic bead immunoassay, polymer precipitation method, I think few people in school have tried it, after all, it is more troublesome. But different methods have their own advantages and disadvantages, so you can choose carefully according to your own experiments.