Exosomes are nano-scale vesicles secreted by cells. These microvesicles are usually about 30-150 nanometers in diameter and contain important cellular molecules such as proteins and RNA. Previous studies have shown that exosomes can be used as diagnostic markers for cancer, neurodegenerative disease, and kidney disease. In recent years, exosomes isolation technology has made significant progress and development.
Ultracentrifugation is the most commonly used exosomal purification method. After removing dead cells and cell debris by low speed centrifugation, high-speed centrifugation is used to precipitate vesicle particles of the same size from soluble molecules such as free proteins and protein complexes purified. It is important that the exosomes be subsequently washed at least once with PBS or fresh growth medium to reduce free residual proteins therein. In addition, all centrifugation steps must be performed at 4 ° C to keep the proteases, DNase and RNases inactive.
Usually ultracentrifugation is also used in combination with a sucrose density gradient (its continuous distribution from low to high density) or a sucrose cushion (30% sucrose cushion), that is, centrifuged at 100,000-200,000 xg in a centrifuge (containing exosomes) In 120 minutes, the exosomes in the sample should be enriched in a sucrose density range of 1.13-1.19 g / mL.
Although this powerful method can obtain highly purified exosomes, there are some disadvantages. Indeed, the process of ultracentrifugation is time-consuming and labor-intensive, and requires a lot of raw materials. The biggest drawback is that repeated centrifugation operations are likely to cause damage to exosomal vesicles and reduce their quality, or soluble proteins in the sample may form aggregates and clumps with exosomes to cause contamination.
- Ultrafiltration centrifugation
Considering that exosomes are cystic bodies with a size of several tens of nanometers, which are larger than ordinary proteins, exosomes can also be separated according to their size, such as ultrafiltration and size exclusion chromatography (SEC).
Ultrafiltration is the selective separation of samples using ultrafiltration membranes with different retention molecular weight (MWCO). That is, the solvent, ie, some small molecular substances, is filtered to the other side of the membrane, while high relative molecular mass substances larger than the membrane pore size are retained On the ultrafiltration membrane, the purpose of separating exosomes is achieved.
This method is simple and efficient, and does not affect the biological activity of exosomes. It is the best method for studying exosomal RNA because it produces greater RNA production than ultrafiltration and precipitation methods. It is also possible to pass a nanofiltration concentrator. However, the main disadvantage of ultrafiltration is that exosomes may block the filter pores, resulting in shorter membrane life and lower separation efficiency.
Exosome membranes also adhere to each other, resulting in low separation yields and even erroneous test results. In addition, there is another interference that needs to be resolved in the method of separating exosomes based on the size of the exosomes, which is the existence of a large number of non-exosomal nanovesicles that are similar in size to the exosomes.
In SEC, the porous phase fixed in the column can also be selectively separated based on the molecular size using the principle of gravity flow. Small molecules can pass through the pores and cause later elution, while larger components (including exosomes) can be eluted early, bypassing the pores. This method can greatly maintain the integrity and biological activity of exosomes, and combine with differential centrifugation to obtain highly purified exosomes.
- PEG-base precipitation method
Polyethylene glycol (PEG, 8000 kDa) can competitively bind free water molecules, so that less soluble molecules or exosomes are precipitated from the solution. Earlier this method was used to collect virus from samples such as serum, and now it is also used to precipitate exosomes. Samples are usually incubated overnight at 4 ° C with PEG, and exosomes are then recovered by low-speed centrifugation or filtration.
However, this method also has some problems: for example, the purity and recovery of exosomes are low, false positives (more proteins or some polymers that are difficult to remove), and mechanical or chemical additives that damage the exosomes.
Alternatively, if you know the sugar chain composition of the exosomes, you can use lectins to enrich the exosomes. Lectin is a protein that binds to carbohydrates and can be centrifuged at low speed after agglutinating exosomes. In recent years, exosomes have been separated based on the principle of precipitation. Various commercial exosomal extraction kits have also been developed on the market. The operation is simple, and high-purity and high-recovery exosomes can be obtained without ultracentrifugation.
- Magnetic bead immunoassay
Exosomes are available because they are rich in protein and have many specific marker receptors on their surface, such as CD9, CD81, CD63, CD82, Hsp70, Ras-related protein Rab-5b, cytoskeleton protein actin, and TSG101. Anti-marker antibody-coated magnetic beads can be captured after incubation with exosomes.
Because the heterogeneity of exosomes is consistent with their origin, the abundance of these markers on different exosomes is also different. Therefore, you can capture different types of exosomes from a sample by using specific antibody combinations, and select these exosomes by immobilizing these antibodies on ELISA plates, magnetic or chromatography beads, or microfluidic devices.
Although immunoaffinity technology has the advantages of high specificity, high purity exosomes can be obtained without affecting the morphological integrity of exosomes, it is the preferred method for enriching and characterizing unique exosomes. However, this method is low in efficiency, and the biological activity of exosomal contents is easily affected by pH and salt concentration, which is not conducive to the downstream experiments.
- Phosphatidylserine affinity
This method combines PS (phosphatidylserine) with magnetic beads and uses the principle of affinity to capture PS outside exosomal vesicles. This method is similar to the immunomagnetic bead method, and the exosomes obtained are complete in morphology and highest in purity. Since no denaturant is used and the biological activity of exosomes is not affected, exosomes can be used for cell co-culture and in vivo injection. 2016.9 "Scientific Reports" magazine published the latest data of this method, showing that PS method can extract relatively high purity exosomes.
The exosomes isolated by this method are uniform in size under electron microscopy, but require special equipment and are not widely used.
Exosome isolation is the first step for exosome characterization. The quality of exosome separation directly affects the subsequent researches of exosome qualitative and quantitative as well as applications in disease diagnosis and therapy. With the extensive experience in exosome isolation, Creative Biolabs provides a portfolio of exosome isolation products which can help you with the high-quality exosome isolation from many types of biofluids in an efficient, faster and cheaper way.