Exosome separation and purification method

Abstract

The invention relates to an exosome separation and purification method which comprises the following steps: taking a body fluid or a culture solution containing exosome, and carrying out anion exchange chromatography, cation exchange chromatography and molecular sieve chromatography to obtain a purified exosome solution. By means of the purification method, high-purity exosomes can be obtained with low cost and high efficiency even in large-scale exosome requirements; compared with the existing exosome purification methods such as a centrifugation method, a PEG polymer precipitation method, an affinity chromatography method, an ultrafiltration method and a molecular sieve size exclusion chromatography method, the method disclosed by the invention can be used for purifying exosomes with different volumes (100ml to 500L), especially milk-derived products with more impurity proteins, and also can be used for obtaining the milk-derived exosomes with the purity of 99 percent or more; meanwhile, the method has the advantages of low cost and high recovery rate, and can meet the requirements of industrial production.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for separating and purifying exosomes. Background technique [0002] Exosomes are small vesicles with a diameter of about 30-200 nm secreted by living cells, with a typical lipid bilayer structure and saucer-type structure, mainly secreted in cell culture supernatant, milk, serum, plasma, In saliva, urine, amniotic fluid and plant tissue; exosomes carry a variety of proteins, lipids, RNA and other important information, which not only play an important role in the transfer of substances and information between cells, but are also expected to become a variety of therapeutic agents. Drug carriers and early diagnostic markers for some diseases. [0003] Milk exosomes are a unique class of evolutionarily conserved microvesicles that maintain the integrity of the nucleic acids and proteins they contain during their passage through the stomach and gastrointestinal tract, where th...

AI Technical Summary

Problems solved by technology

Centrifugation is currently the most commonly used method for extracting and separating exosomes, and is considered to be the "gold standard" for exosomes. Its advantages are simple operation and high purity of isolated exosomes, but the whole process takes longer than At the same time, the sample volume is limited by the centrifuge and the processing volume is small. The type, quantity and quality of the isolated exosomes are greatly affected by the parameters of the centrifugation process (centrifugal force and centrifugation time), and the reproducibility of the sample quality attributes is poor.

[0006] The second is the PEG polymer precipitation method. This method mainly achieves the purpose of isolating exosomes by co-precipitating polymers, hydrophobic proteins and lipid molecules. Exosome production, but the purity of exosomes obtained is low, usually mixed with a large amount of co-precipitated protein and nucleic acid impurities

[0007] The third is affinity chromatography, including immunoaffinity chromatography and phosphatidylserine affinity chromatography. This method is a method that passes through membrane proteins (CD9, CD81, CD63, TIM4) on the surface of exosomes and their corresponding antibodies. Or phosphatidylserine affinity adsorption to achieve the separation of exosomes. The exosomes separated by this method have high specificity and purity, and the shape of exosomes is complete. However, the affinity chromatography medium is expensive and the cost of purity is high, so it cannot be used in the industry. Large scale separation and purification

In particular, the process of affinity chromatography needs to absorb exosomes first, and then separate them by elution, which is more complicated in terms of operation.

[0008] The fourth is the ultrafiltration method. This method separates and purifies exosomes and impurity molecules in the sample according to their size through ultrafiltration membranes with different molecular weight cut-offs. It has the advantages of simple operation process, large sample processing capacity and high concentration multiple. However, the purity of isolated exosomes is low

[0010] To sum up, the current separation and purification methods of exosomes cannot meet the problems of high purity, low cost, high recovery rate and industrial production at the same time, especially when dealing with a large number of raw materials, the purity and yield of exosomes obtained are even more need to pay a great price

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