Exosome purification method, prepared exosome and application

Abstract

The invention discloses an exosome purification method, a prepared exosome and application. The method comprises the following steps: firstly, forming a crude exosome by utilizing the charged difference of substances such as exosome, impure protein and endotoxin and adopting an acid-base precipitation and ultrafiltration combined technology; then fine purification is performed by adopting a composite chromatography method, impurities are efficiently removed, and the high-purity exosome is rapidly obtained. According to the method, the defects of long time consumption, more impurities, small treatment capacity and incomplete protein removal of the traditional method are overcome, and low-cost and high-yield high-purity exosome production is realized. The obtained exosome significantly promotes cell migration, especially human fibroblasts, has positive effects on cell repair and skin wound healing, has the potential of being applied to the field of cosmetics and accelerating epidermis inflammation and scar repair, and promotes commercialized application of high-purity exosome.

Description

Technical Field

[0001] The present invention relates to a method for purifying exosomes, prepared exosomes and applications, and belongs to the field of biotechnology. Background Art

[0002] Exosomes are nanomaterials secreted by cells with a size of about 40-200nm. They are a type of extracellular vesicles with a structure similar to that of cells. There are multiple specific membrane proteins on the surface of the phospholipid layer. Exosomes are rich in cholesterol and sphingomyelin and have a lipid bilayer structure. Almost all exosomes have tubulin, actin-binding protein, and tetraspanin. The protein content of exosomes can be divided into specific and non-specific types. Specific proteins play an important role as scaffold proteins in exosomes used as drug carriers and are key components in drug delivery systems. Exosomes are widely distributed in various body fluids such as blood, urine, cerebrospinal fluid, saliva, milk, and bile.

[0003] Exosomes have the advantages of lower immunogenicity, higher biocompatibility, stronger biological barrier penetration and preferred targeting carriers. Exosomes also have the advantages of wide sources, low cost and low side effects, and can be the preferred carriers of herbal medicines, chemical drugs and nanomedicines. The substances carried by exosomes are related to the cells from which they are derived. It is this heterogeneity of exosomes that makes it possible to diagnose and monitor diseases. Exosomes can mediate the body's immune response, antigen presentation, cell migration, cell differentiation, tumor invasion and other aspects. Therefore, exosomes can be used to repair scratch injuries on the skin, promote wound healing and early diagnosis of diseases.

[0004] The extraction and purification technologies of exosomes mainly include density gradient centrifugation, ultrafiltration, ultracentrifugation, size exclusion, polymer precipitation, etc. Density gradient centrifugation uses density differences to achieve separation. Exosomes and media have different densities and different distributions; ultracentrifugation separates different substances in the sample based on different centrifugal forces. Exosomes, proteins, cell fragments, organelles and other substances do not have to have sedimentation rates, and exosomes can be extracted from the supernatant; size exclusion is a method of extracting exosomes using chromatography based on the different sizes of exosomes and the different paths they flow through; polymer precipitation uses polymers to create a hydrophobic microenvironment by hijacking the water molecules around the exosomes, thereby forcing the exosomes to leave the solution and precipitate under low-speed centrifugation. Commonly used polymers include polyethylene glycol (PEG), lectin, protamine and sodium acetate.

[0005] In summary, although there are many methods for extracting and purifying exosomes, these methods have great limitations in actual extraction and purification. Free proteins and fats will affect the purification of exosomes. The main limitations are that they take a long time, produce more impurities in exosomes, have a relatively small processing volume, and have insufficient protein removal. They cannot simultaneously meet the requirements of high purity, low cost, and high yield, and cannot be used for commercial production of high-purity exosomes. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for purifying exosomes and application in cosmetics, the steps are as follows: S1. Centrifuge the biological sample to be purified, discard the precipitate, and retain the supernatant.

[0007] This centrifugation step is used to remove larger insoluble particles and aggregates.

[0008] S2. The pH value of the supernatant obtained in step S1 is adjusted to acidic, centrifuged, the precipitate is discarded, and the supernatant is retained.

[0009] This centrifugation step is used to precipitate contaminating proteins with lower isoelectric points.

[0010] S3. The pH value of the supernatant obtained in step S2 is adjusted to alkaline, centrifuged, the precipitate is discarded, and the supernatant is retained.

[0011] This centrifugation step is used to precipitate contaminating proteins with higher isoelectric points.

[0012] S4. The supernatant obtained in step S3 is filtered in two steps, the first step is to use a large pore filter for filtration, and the second step is to use a small pore filter for filtration to obtain a filtered supernatant.

[0013] S5. The supernatant obtained in step S4 is ultrafiltered and concentrated, and then diafiltered to obtain a diafiltered supernatant.

[0014] S6. The supernatant obtained in step S5 is subjected to composite chromatography to obtain a pure exosome product.

[0015] Preferably, in step S1, the centrifugal force is 5000 g to 10000 g, the temperature is 4° C. to 10° C., and the time is 40 min to 60 min.

[0016] Preferably, the concentration of the acid used for acid adjustment in step S2 is 3 mol / L to 4 mol / L, and the acid may be one or more of phosphoric acid, hydrochloric acid, acetic acid, and citric acid. The pH value adjustment range is 4 to 6. The centrifugal force is 5000 g to 10000 g, the temperature is 4° C. to 10° C., and the time is 40 min to 60 min.

[0017] Preferably, the concentration of the base used for alkali adjustment in step S3 is 2 mol / L to 3 mol / L, and the base can be one or more of Tris base, sodium bicarbonate, and disodium hydrogen phosphate. The pH value is adjusted in the range of 8 to 9. The centrifugal force is 5000 g to 10000 g, the temperature is 4° C. to 10° C., and the time is 40 min to 60 min.

[0018] Preferably, in step S4, the large-pore filter is made of PP material with a pore size of 0.2 μm to 5.0 μm, and the small-pore filter is made of PES material with a pore size of 0.2 μm to 0.4 μm.

[0019] Preferably, in step S5, the supernatant is ultrafiltered and concentrated by an ultrafiltration membrane package or a hollow fiber column, the ultrafiltration membrane package has a molecular weight cutoff of 100 kDa to 1000 kDa, and the hollow fiber column has a molecular weight cutoff of 100 kDa to 1000 kDa.

[0020] Preferably, the composite chromatography in step S6 is a composite of weak anion exchange chromatography, strong anion exchange chromatography, weak cation exchange chromatography, strong cation exchange chromatography and molecular sieve chromatography.

[0021] Preferably, the number of diafiltrations after ultrafiltration to fully remove foreign proteins is 5 to 10 times.

[0022] Preferably, the matrix of the composite chromatography is agarose microspheres, the ligand is a composite ligand, and the average particle size of the filler ranges from 30 μm to 100 μm.

[0023] Preferably, the milk source is fresh milk, low-fat milk, low-sugar milk or frozen milk.

[0024] Preferably, the milk source is one or more of cow's milk, goat's milk, horse's milk or milk of other animals.

[0025] Preferably, the amount of the initial exosome milk processed is 50 mL to 500 L.

[0026] Preferably, the buffer solution for diafiltration is 10mmol / L~50mmol / L, pH 8.0~9.0 PB buffer, 0.9% sodium chloride or 10mmol / L~50mmol / L HEPES buffer.

[0027] Preferably, the exosome sample purified after chromatography is collected as the flow-through.

[0028] The present invention provides an application of exosomes in repairing skin scratches.

[0029] The present invention provides a method for purifying exosomes, which has a stable process and high quality attributes. The high-purity exosomes obtained from dairy products can be used for skin scratch repair. By adding protein concentrations of 2.5 μg / mL, 5 μg / mL, and 10 μg / mL, the particle numbers are 1.71×10 9 / mL, 3.42×10 9 / mL, 6.85×10 9 The exosomes were used to treat HSF cells with 100 μg / mL of exosomes, and the migration ability of the cells was detected by scratch test, which is helpful for the migration and repair of human skin fibroblasts. Adding it to various cosmetics can help repair the skin.

[0030] Due to the adoption of the above technical scheme, the present invention has the following advantages compared with the prior art: it is easy to operate, has a large processing volume, a short time, good quality attributes and higher purity. The purity of the purified exosomes is as high as 99% or above, which enables commercial production, and has a more obvious effect and versatility in skin scratch repair applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is an electron micrograph of exosomes in Example 1 of the present invention.

[0032] Figure 2 This is an electron micrograph of exosomes in Example 2 of the present invention.

[0033] Figure 3 This is an electron micrograph of exosomes in Example 3 of the present invention.

[0034] Figure 4 This is an electron micrograph of exosomes in Example 4 of the present invention.

[0035] Figure 5 This is an electron microscopic image of exosomes in comparative example 1 of the present invention.

[0036] Figure 6 This is an electron microscopic image of exosomes in comparative example 2 of the present invention.

[0037] Figure 7 This is an electron microscopic image of exosomes in comparative example 3 of the present invention.

[0038] Figure 8 This is the HPLC spectrum of exosomes in Example 1 of the present invention.

[0039] Fig. 9 This is the HPLC spectrum of exosomes in Example 2 of the present invention.

[0040] Fig.10 This is the HPLC spectrum of exosomes in Example 3 of the present invention.

[0041] Fig.11 This is the HPLC spectrum of exosomes in Example 4 of the present invention.

[0042] Fig.12 This is the HPLC spectrum of exosomes in Comparative Example 1 of the present invention.

[0043] Fig.13 This is the HPLC spectrum of exosomes in Comparative Example 2 of the present invention.

[0044] Fig.14 This is the HPLC spectrum of exosomes in comparative example 3 of the present invention.

[0045] Fig.15 Example 5 Cell migration detection results.

[0046] Fig.16 Example 5 Analysis of statistical results of cell migration. DETAILED DESCRIPTION

[0047] Embodiment 1:

[0048] Take 0.5 L of raw milk and purified water in a 2L beaker at a volume ratio of 1:1, and use a magnetic stirrer to stir evenly at room temperature. Use 3 mol / L phosphoric acid to adjust the pH to 4.5. Use a magnetic stirrer to stir at room temperature during the pH adjustment process, and then let the acid precipitate at room temperature for 120 min.

[0049] Place the completely precipitated sample in a centrifuge cup and centrifuge it at 6000 g for 60 min. After centrifugation, keep the supernatant and discard the precipitate.

[0050] The supernatant was treated with alkali, and the pH was further adjusted to 8.5 with 0.5 mol / L disodium hydrogen phosphate. A magnetic stirrer was used to stir at room temperature during the pH adjustment process, and then the solution was allowed to stand at room temperature for 60 min.

[0051] Place the completely precipitated sample in a centrifuge cup and centrifuge it at 6000 g for 60 min. After centrifugation, keep the supernatant and discard the precipitate.

[0052] The supernatant solution after centrifugation was filtered using a 0.2 μm PP capsule filter at a filtration rate of 40 mL / min to obtain a filtered supernatant solution; the supernatant was further filtered using a 0.2 μm PES capsule filter at a filtration rate of 30 mL / min to obtain a supernatant solution, and the final volume was recorded.

[0053] Use 20mmol / L, pH8.5, PB buffer to equilibrate the ultrafiltration membrane package, the molecular weight cutoff of the membrane package is 300KD, and the size is 0.1 square meter, until the pH value at the permeate end is 8.5±0.1, add the sample to the liquid inlet bottle for ultrafiltration concentration and liquid exchange, and start the instrument method; when the concentrated volume reaches 100mL, perform fluid replenishment dialysis, add 100mL of 20mmol / L pH8.5 PB buffer, mix thoroughly, continue to concentrate and exchange the liquid, and perform 5 consecutive liquid exchange dialysis treatments.

[0054] Finally, concentrate to the minimum volume, collect, and add 20-30 mL of 20 mmol / L pH 8.5 PB buffer to the liquid inlet bottle again to clean the pipeline and membrane package. Repeat twice, and finally adjust the pH to 7.2 ± 0.2 with 3 mol / L phosphoric acid solution.

[0055] Filter with a 0.22 μm PES capsule filter to remove bacteria, etc. Finally, record the volume after ultrafiltration, take samples for quality inspection, and store the remaining product in a -80°C refrigerator.

[0056] When using the composite chromatography column for the first time, rinse and clean the composite chromatography column with ultrapure water for 2-5 times the column volume, and then wash it with 1mol / L sodium chloride solution for 2-3 times the column volume.

[0057] Use 20mmol / L pH7.20 PB buffer to balance the column with 2-5 column volumes, so that the pH and conductivity of the solution flowing out of the column are consistent with the equilibrium solution. After the equilibrium is completed, the sample is loaded, and the volume ratio of the feed liquid to the filler is not higher than 1:10.

[0058] During the loading process, the sample is collected when the UV response rises to more than 20mAU. When the UV response drops to 10mAU, the sample collection is stopped. After the loading is completed, the column is equilibrated with 20mmol / L pH7.20 PB buffer. After equilibration for 2-3 times the column volume, elution is performed. The impurities in the composite column are eluted with 1mol / L sodium chloride solution for 2-5 times the column volume. The final product is the collected flow-through.

[0059] After elution, rinse the composite chromatography column with ultrapure water for 1-2 column volumes, then rinse with 20% isopropanol for 2-5 column volumes, rinse with 0.5 mol / L NaOH solution for 2-5 column volumes, and finally rinse the composite chromatography column with ultrapure water to remove residual sodium hydroxide, and preserve the composite chromatography column with 20% ethanol.

[0060] Embodiment 2:

[0061] This embodiment provides a method for purifying exosomes using charge differences, which is basically the same as that in Embodiment 1, except that the 300 KD ultrafiltration membrane is replaced with a 750 KD hollow fiber column.

[0062] Embodiment 3:

[0063] This embodiment provides a method for purifying exosomes using charge differences, which is basically the same as that in Embodiment 1, except that the 300 KD ultrafiltration membrane package is replaced by a 500 KD ultrafiltration membrane package.

[0064] Embodiment 4:

[0065] This embodiment provides a method for purifying exosomes using charge differences, which is basically the same as that in Embodiment 1, except that the PB buffer system for diafiltration is replaced by a HEPES buffer system for diafiltration.

[0066] Comparative Example 1:

[0067] The commercial exosome extraction kit (emulsion) (Cat. No.: UR52146) of Umibio was used as a comparative purification experiment in the present invention. Comparative Example 1 The exosomes obtained by extraction and purification using the commercial exosome kit were analyzed by HPLC. The purity of the exosomes finally obtained was not high, and there were many impurity peaks at the end.

[0068] Comparative Example 2:

[0069] Take 6 50mL centrifuge tubes, add 45mL of raw milk to each tube, balance, centrifuge at a speed of 200g and a temperature of 4°C for 30min, and transfer the supernatant to a new centrifuge tube.

[0070] The supernatant was centrifuged at 2000 g and 4° C. for 30 min, and the supernatant was transferred to a new centrifuge tube.

[0071] The supernatant was centrifuged at 10,000 g and 4° C. for 30 min, and the supernatant was transferred to a new centrifuge tube.

[0072] The supernatant was filtered with a 0.45 μm syringe filter and transferred to 6 ultracentrifuge tubes. After balancing, ultracentrifugation was performed at a centrifugal force of 120,000 g and a temperature of 4°C for 120 min. The precipitate was retained and the supernatant was discarded. 0.5 mL of PBS solution with an ion concentration of 10 mmol / L and a pH of 6.8-7.2 was added to each centrifuge tube to fully resuspend the precipitate, and exosomes were obtained. The purity test results showed that the purity of the exosomes obtained was not high, and there were many impurity peaks at the end.

[0073] Comparative Example 3:

[0074] This comparative example provides a method for preparing milk exosomes, which is basically the same as Example 1, except that the composite column chromatography step is omitted. The purity test results show that the purity of the exosomes finally obtained is not high, and there are many impurity peaks at the end.

[0075] The results of Examples 1-4 and Comparative Examples 1-3 are shown in the following table. The purity of the exosomes purified in the examples is as high as over 99%.

[0076] project purity Example 1 100% Example 2 100% Example 3 100% Example 4 100% Comparative Example 1 10.75% Comparative Example 2 77.51% Comparative Example 3 68.43%

[0077] Example 5: Cell scratch migration assay.

[0078] First, use a marker pen to draw vertical lines on the back of the 6-well plate, with a ruler, one for each well, and divide the vertical lines into three equal parts for marking. Add about 7×10 5 HSF (human skin fibroblast) cells, the specific number varies depending on the cell, and after overnight culture, it can cover 6 wells. The next day, use the tip of the gun to compare with the ruler, and try to hang it down to the vertical scratch on the back. The tip of the gun should be vertical and not tilted. Wash the cells 3 times with PBS, remove the scratched cells, add serum-free culture medium and exosomes, and the working concentrations of exosomes are 0μg / mL, 2.5μg / mL, 5μg / mL, and 10 μg / mL, respectively. Put it in a 37 ℃, 5% CO2 incubator, culture for 0h, 24h and take 3 photos each, and perform statistical analysis on the results.

[0079] The results showed that the effect of promoting cell migration after adding exosomes was quite obvious, which could effectively promote the migration of human fibroblasts, promote cell repair, and contribute to the repair and healing of skin epidermal wounds.

[0080] The embodiments of the present invention are described in detail above, but the contents described are only preferred embodiments of the present invention and cannot be considered to limit the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A method for purifying exosomes, characterized in that: The following steps are involved: S1: centrifuge the biological sample to be purified, discard the precipitate, and keep the supernatant; S2: adjusting the pH value of the supernatant obtained in step S1 to acidic, centrifuging, discarding the precipitate, and retaining the supernatant; S3: adjusting the pH value of the supernatant obtained in step S2 to alkaline, centrifuging, discarding the precipitate, and retaining the supernatant; S4 filtering the supernatant obtained in step S3 in two steps, wherein the first step is filtering using a large pore filter and the second step is filtering using a small pore filter to obtain a filtered supernatant; S5: ultrafiltration and concentration of the supernatant obtained in step S4, followed by diafiltration, to obtain a diafiltration supernatant; S6 The supernatant obtained in step S5 is subjected to composite chromatography to obtain a pure exosome product.

2. The method for purifying exosomes according to claim 1, characterized in that: In step S1, the centrifugal force is 5000 g to 10000 g, the temperature is 4° C. to 10° C., and the time is 40 min to 60 min.

3. The method for purifying exosomes according to claim 1, characterized in that: The concentration of the acid used for acid adjustment in step S2 is 3 mol / L-4 mol / L, the acid can be one or more of phosphoric acid, hydrochloric acid, acetic acid, and citric acid, the pH value adjustment range is 4-6, the centrifugal force is 5000g-10000g, the temperature is 4°C-10°C, and the time is 40min-60min.

4. The method for purifying exosomes according to claim 1, wherein: The concentration of the alkali used for alkali adjustment in step S3 is 2mol / L~3mol / L, the alkali can be one or more of Tris alkali, sodium bicarbonate, and disodium hydrogen phosphate, the pH value is adjusted in the range of 8~9, the centrifugal force is 5000g~10000g, the temperature is 4°C~10°C, and the time is 40min~60min.

5. The method for purifying exosomes according to claim 1, characterized in that: In the step S4, the large-pore filter is made of PP material with a pore size of 0.2 μm to 5.0 μm, and the small-pore filter is made of PES material with a pore size of 0.2 μm to 0.4 μm.

6. The method for purifying exosomes according to claim 1, wherein: In the step S5, the supernatant is ultrafiltered and concentrated through an ultrafiltration membrane package or a hollow fiber column, the ultrafiltration membrane package has a molecular weight cutoff of 100 kDa to 1000 kDa, and the hollow fiber column has a molecular weight cutoff of 100 kDa to 1000 kDa.

7. The method for purifying exosomes according to claim 1, characterized in that: The buffer solution for diafiltration in step S5 is 10mmol / L~50mmol / L, pH value 8.0~9.0 PB buffer, 0.9% sodium chloride or 10mmol / L~50mmol / L HEPES buffer.

8. The method for purifying exosomes according to claim 1, characterized in that: The composite chromatography in step S6 is a composite of weak anion exchange chromatography, strong anion exchange chromatography, weak cation exchange chromatography, strong cation exchange chromatography and molecular sieve chromatography, the matrix of the composite chromatography is agarose microspheres, the ligand is a composite ligand, and the average particle size of the filler ranges from 30 μm to 100 μm.

9. Exosomes prepared by the method for purifying exosomes according to any one of claims 1 to 8.

10. Use of the exosomes as claimed in claim 9 in repairing skin scratches.

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