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Exosome is a kind of tiny vesicle secreted by most cells. It has a lipid bilayer structure and distributes in peripheral blood, urine, saliva, ascites, amniotic fluid, and other body fluids. Exosome carries important information such as proteins, lipids, and nucleic acids, especially non-coding RNA (miRNA, lncRNA, circRNA, etc.), which makes it more meaningful. Exosomes not only play an important role in cell-to-cell material and information transmission but also are expected to become early diagnostic markers for a variety of diseases. Exosome RNA sequencing can quickly and efficiently obtain comprehensive information of exosome RNA, which is an ideal means of disease diagnosis and follow-up. At present, researches on RNA based on exosomes mainly focus on miRNA, lncRNA, circRNA, mRNA and other types.
Creative Biostructure can provide various methods for exosome isolation, including traditional differential centrifugation, density gradient centrifugation, and later developed ultrafiltration, polymer precipitation, immune separation, isolation screening, volume exclusion chromatography, etc.
Figure 1. Extracellular vesicle (EV) biogenesis, subpopulations, exosome isolation methods (Karim Sidhom, et al. 2020)
Differential centrifugation is still one of the most common exosome separation techniques. The method consists of several steps, including
Exosomes can be extracted from cell culture supernatant, serum, plasma, urine, cerebrospinal fluid, and other body fluids (ascites, amniotic fluid, emulsion, saliva, etc.), and the obtained high-purity exosome granules can be used for subsequent NTA, TEM, WB analysis, etc.
According to size-exclusion chromatography, SEC separated macromolecules based on size rather than molecular weight. The technique uses columns filled with porous polymer microspheres. Molecules move through the microspheres according to their diameters. Molecules with smaller radii take longer to migrate through the pores of the column, while larger molecules elute from the column earlier. SEC has been shown to have more advantages than centrifugation because exosomes separated by chromatography are not affected by shear forces. Currently, SEC is a widely accepted technique for isolating exosomes from blood and urine. We have a set of chromatography-based standard procedures for the extraction and purification of exosomes to ensure high recovery rates.
Exosome precipitation from biological fluids could be made by altering their solubility or dispersibility. In general, samples are incubated with a precipitation solution containing PEG which forces less soluble components out of the solution. The precipitate containing exosomes is isolated by low-speed centrifugation or filtration after overnight incubation. This method is easy to use and requires no specialized equipment, also it is scalable for a large amount of samples. Several kinds of precipitation kits are available upon your request.
Density gradient centrifugation refers to the combination of hyper centrifugation with sucrose density gradient to achieve separation of exosomes from non-vesicular particles, such as proteins and protein/RNA aggregates. Therefore, this method separates the vesicles from particles of different densities and enables the extraction of exosomes with low content. However, proper centrifugation time is important, otherwise, contaminant particles can still be found in the exosomes if they have similar densities.
Immunoaffinity capture using the beads coated with antibodies, which targeted exosomal surface proteins to isolate specific vesicle populations. The experimental procedures include sample treatment, beads incubation and binding, low-speed centrifugation, and biological analysis of isolated exosomes.
Microfluidics (MF) technology uses chips with specific antibody-mediated binding to capture exosomes. The Microfluidics-based technique can be used in exosome isolation with specific surface markers (microfluidics combined with immunoaffinity capture) and in exosome isolation with specific size populations (microfluidics combined with membrane filtration). With a microfluidics-based platform, we can provide high throughput isolation and purification services for our customers.
Ultrafiltration (UF) is dependent on a filter with different pore sizes that are used to separate exosomes from proteins and other macromolecules.
Our service content:
We have rich experience in exosome identification and can provide exosome transmission electron microscopy identification results, accurate exosome particle size analysis, and exosome surface biomarker expression identification. If you are interested in our exosome isolation services, please feel free to contact us. We are looking forward to cooperating with you.