Exosome separation and purification method

A technology for separation and purification of exosomes, applied in the biological field, can solve the problems of small processing volume, low cost, poor reproducibility of sample quality attributes, etc., and achieve the effect of high recovery rate and low cost

Inactive Publication Date: 2021-09-28
天九再生医学(天津)科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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 me

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] step one

[0065] 1.1 Acid precipitation

[0066] Take 1L of milk and use hydrochloric acid to adjust the pH to 4.6. During the pH adjustment process, use a magnetic stirrer to stir at room temperature, and then let the acid settle for 60 minutes.

[0067] 1.2 Sample clarification

[0068] Place the sample after acid precipitation in a centrifuge cup, use a centrifuge to centrifuge at a centrifugal force of 3500g for 30min, and take the supernatant after centrifugation. Or use a bag filter with a cut-off pore size of 10 μm to filter with a filter speed of 50 ml / min, and take the filtered supernatant solution.

[0069] 1.3 Depth filtering

[0070] The centrifuged or filtered supernatant solution is further clarified and filtered using a depth filter with a cut-off pore size of 0.5-10 μm at a filtration rate of 10 ml / min to obtain a supernatant solution.

[0071] 1.4 Ultrafiltration concentrated liquid exchange

[0072] Ultrafiltration membrane bag: 100KD ultrafiltra...

Embodiment 2

[0112] step one

[0113] 1.1 Impurity removal

[0114] Take 1L of milk and put it in a beaker, add 0.5mol / L calcium chloride solution to the above solution to a final concentration of 0.001mol / L, then add rennet according to the final concentration of 5SU / ml, and use a magnetic stirrer at 30°C Stir well.

[0115] 1.2 Sample clarification

[0116] Place the sample after rennet-precipitated casein into a centrifuge cup, use a centrifuge to centrifuge at a centrifugal force of 4000g for 30min, and take the centrifuged supernatant. Or use a bag filter with a cut-off pore size of 10 μm to filter with a filter speed of 50 ml / min, and take the filtered supernatant solution.

[0117] 1.3 Depth filtering

[0118] The centrifuged or filtered supernatant solution is further clarified and filtered using a depth filter with a cut-off pore size of 0.5-10 μm at a filtration rate of 10 ml / min to obtain a supernatant solution.

[0119] 1.4 Ultrafiltration concentrated liquid exchange

[...

Embodiment 3

[0160] step one

[0161] 1.1 Acid precipitation

[0162] Take 10L of milk and use lactic acid to adjust the pH to 4.6. During the pH adjustment process, turn on the stirrer to stir at room temperature, and then let the acid settle for 60 minutes.

[0163] 1.2 Sample clarification

[0164] Place the sample after acid precipitation in a centrifuge cup, use a centrifuge to centrifuge at a centrifugal force of 4000g for 30min, and take the supernatant after centrifugation. Or use a Cobetter bag filter with a cut-off pore size of 10 μm for filtration at a filtration rate of 200ml / min, and take the supernatant solution after filtration.

[0165] 1.3 Depth filtering

[0166] The supernatant solution after centrifugation or filtration is further clarified and filtered with a depth filter with a cut-off pore size of 0.5-10 μm, and the filtration speed is 100 L / m 2 / h to obtain a supernatant solution.

[0167] 1.4 Ultrafiltration concentrated liquid exchange

[0168] Ultrafiltrati...

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Abstract

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

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates 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. They have a typical lipid bilayer structure and a saucer-shaped structure. They are mainly secreted in cell culture supernatant, milk, serum, plasma, In saliva, urine, amniotic fluid, and plant tissues; exosomes carry important information such as a variety of proteins, lipids, and RNA. Carriers of drugs and early diagnostic markers for some diseases. [0003] Milk exosomes are an evolutionarily conserved unique class of microvesicles that maintain the integrity of their contained nucleic acids and proteins as they pass through the stomach and gastrointestinal tract, where they can act locally or be transported into the circulatory system. In addition, milk exosomes are more stable than other naturally occ...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2509/00
Inventor 葛啸虎王淼陆路杜焕青韩春乐
Owner 天九再生医学(天津)科技有限公司
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