A method for large-scale extraction of exosomes in fresh milk

Abstract

The present invention discloses a method for large-scale extraction of exosomes in fresh milk, which uses a hydrophilic and lipophobic nano-interface mesh to filter the fresh milk to remove cell fragments and fat particles in the fresh milk, and then ultrafiltration is performed through a tangential flow ultrafiltration device with an ultrafiltration membrane of pore size 100kDa to 300kDa to concentrate the clarified fresh milk; the concentrated solution is mixed with a two-phase aqueous solution prepared by combining polyethylene glycol and dextran, and then phase separation is performed, and the enriched exosome solution in the lower phase is collected, and the exosome concentration is repeated 1 to 2 times; and the concentrated exosomes are further separated and other impurities are removed through a highly cross-linked agarose filler column, and exosomes and exovesicles of different diameters are separated. The purification process of the present invention is simple, does not require high-speed centrifugation, can process more than dozens of liters of fresh milk at a time, has high extraction purity and can effectively separate exovesicles of different particle sizes, has little damage to exosomes, and can achieve large-scale extraction.

Description

Technical Field

[0001] The invention belongs to the technical field of extraction technology, and particularly relates to a method for large-scale extraction of exosomes in fresh milk. Background Art

[0002] Exosomes are a class of metabolic products of lipid bilayer membranes released by cells. They are round or cup-shaped under transmission electron microscopy, with a diameter of 30 to 150 nm. A large number of studies have shown that exosomes contain a variety of proteins, nucleic acids, lipids and other components. From current research, it is found that exosomes have a very bright application prospect. They contain rich markers and can be used in disease monitoring and treatment. Moreover, exosomes from different tissues and cells have the characteristics of their source tissues and cells, and play a key role in the process of intercellular material and information exchange mediated by exosomes. At the same time, exosomes have broad application prospects in clinical treatment, carrier drugs, clinical testing, etc. The components such as proteins, lipids, RNA, miRNA in tumor cell exosomes can be used as cancer diagnosis and omens.

[0003] A large number of exosomes have also been found in the fresh milk currently being studied. Studies have shown that fresh milk exosomes help regulate immunity, regulate intestinal development, and alleviate some diseases. Domestic research on fresh milk exosomes mainly focuses on the extraction and identification of exosomes and their use as drug carriers and disease screening markers. Foreign research on bovine milk exosomes is more about the risks of the development of chronic diseases such as the human immune system, intestinal development, cancer prevention, obesity, and diabetes. Recently, the research on fresh milk exosomes has become more and more in-depth. As a product, research direction, and functional research demand are also increasing, so the method of extracting a large number of fresh milk exosomes needs to be solved.

[0004] There are many technologies for extracting exosomes, mainly ultracentrifugation and kit method. Sucrose density gradient centrifugation is the most commonly used method for extracting and purifying exosomes. These methods can only extract a limited amount of exosomes, the process is time-consuming, the equipment requirements are high, the recovery rate is limited, and the purity does not meet the requirements. None of the above methods can meet the requirements of large-scale extraction and purification, which limits the scope of application of exosome industrialization.

[0005] There are not many reports on the separation and extraction methods of exosomes in fresh milk. The existing various extraction methods also have various advantages and disadvantages, especially in large-scale extraction, which makes it difficult to achieve mass production. Moreover, the milk fat in fresh milk is dispersed in the milk plasma in the form of fat globules, which are generally 0.1-10 μm in diameter, and the vast majority are 2-5 μm. The biggest obstacle to the extraction of exosomes is fat particles, which are generally 3%-5% in fresh milk and are insoluble in water. They are dispersed in fresh milk in the form of microspheres to form an emulsion that cannot be removed by ordinary filtration. Therefore, it is particularly important to find a low-cost, efficient, and widely applicable method for large-scale extraction of fresh milk exosomes and the removal of fat particles at the same time. Summary of the invention

[0006] The purpose of the present invention is to provide a method for large-scale extraction of exosomes from fresh milk in view of the problems existing in the fresh milk exosome extraction technology.

[0007] To achieve the above object, the method for large-scale extraction of exosomes from fresh milk adopted by the present invention comprises the following steps:

[0008] 1. Filter the fresh milk through a hydrophilic and lipophobic nano-interface membrane to remove fat particles in the fresh milk and collect the filtrate.

[0009] 2. The filtrate collected in step 1 is ultrafiltered through a tangential flow ultrafiltration device with an ultrafiltration membrane having a pore size of 100 kDa to 300 kDa, and the filtrate after the fat particles are removed is concentrated.

[0010] 3. The concentrated fresh milk collected in step 2 and the two-phase aqueous solution are fully mixed and centrifuged, the upper phase is discarded, and the lower phase is collected; the lower phase is repeated 1 to 2 times to further concentrate the exosome solution; the two-phase aqueous solution is prepared by dissolving polyethylene glycol and dextran in distilled water or PBS buffer, and the concentration of polyethylene glycol in the two-phase aqueous solution is 20 to 40 g / L, and the concentration of dextran is 14 to 20 g / L.

[0011] 4. The concentrated exosome solution in step 3 was diluted with a diluent and further separated by a gel column, wherein the gel column used highly cross-linked agarose as a filler, and the diluent was a 20 mM pH = 7.2 Tris-HCl buffer containing 150 mM NaCl.

[0012] In the above step 1, the fresh milk includes any one or more of fresh cow's milk, fresh goat's milk, fresh human milk and camel's milk.

[0013] In the above step 1, the coating of the hydrophilic and lipophobic nano-interface mesh is a polyvinyl alcohol / silicon dioxide nanocomposite film, and the contact angle between the film and water is 0. 0 ~25 0 The contact angle with fat in fresh milk is 600 ~130 0 , which can be used to prevent the fat particles in fresh milk from passing through the membrane.

[0014] In the above step 3, the concentrated fresh milk collected in step 2 and the aqueous two-phase solution are preferably fully mixed at a volume ratio of 1:3 to 3:1 and then centrifuged.

[0015] In the above step 3, the molecular weight of the polyethylene glycol is 5 kDa to 35 kDa, and the molecular weight of the dextran is 7 kDa to 65 kDa.

[0016] In the above step 3, the centrifugal force of the centrifugal separation is 1000-3000g, and the centrifugal time is 10-40 minutes.

[0017] In the above step 4, the molecular weight of the highly cross-linked agarose is 600 kDa to 800 kDa, and the particle size is 75 to 95 μm.

[0018] The beneficial effects of the present invention are as follows:

[0019] 1. The present invention first filters the fresh milk through a hydrophilic and lipophobic nano-interface mesh, collects the clarified liquid, removes the fat particles in the fresh milk, and solves the effect of fat on purification; then ultrafiltration is performed using a tangential flow ultrafiltration device with an ultrafiltration membrane with a pore size of 100kDa to 300kDa, and the first concentration is performed to remove some small molecular proteins and water, and the clarified fresh milk is concentrated by 10 to 20 times; the clarified filtrate of the fresh milk after the concentration treatment is centrifuged with a two-phase aqueous solution prepared by a combination of polyethylene glycol and dextran, and the second concentration and preliminary purification are performed, the exosomes are compressed into the dextran phase, and the upper phase is removed. After this process is repeated 1 to 2 times, the clarified exosomes have been concentrated by 60 to 150 times, so that the production-level amount is changed to the laboratory-level amount, and the exosomes are highly concentrated, making it possible to use a gel column for separation; finally, a gel column with highly cross-linked agarose as a filler is used to separate and remove other impurities and apoptotic bodies, purify exosomes and extracellular vesicles of different particle sizes, and realize the large-scale extraction of exosomes in fresh milk.

[0020] 2. The whole operation process of the present invention is simple, avoiding the limitation of high-speed centrifugation on the scale of extraction, and is suitable for large-scale extraction and concentration. Through nearly 100 times high concentration, the problem of limited sample loading on the gel column is overcome, and the difficulty of large-scale extraction is solved. The whole experimental process does not introduce chemical reagents with strong denaturing ability and violent reactions, and the damage to exosomes is small. Ultracentrifuges and immunomagnetic beads are not required, and industrial production can be achieved without complicated processes. A large number of exosomes from different sources can be processed. The extracted exosomes are subjected to a series of identification and verification such as activity identification, electron microscopy detection, particle size analysis, nanoparticle tracing analysis, and protein content detection. The damage rate is small, the mutation rate is small, and the properties are stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 These are microscope observation pictures of fresh milk before fat particles are removed (a), after fat particles are removed by hydrophilic and lipophobic nano-interface mesh membrane (b), and after fat particles are removed by chemical reagent precipitation method (c).

[0022] Figure 2 This is a particle size distribution diagram obtained by particle size analysis (NTA) verification after ultrafiltration and concentration using a tangential flow ultrafiltration device in Example 1.

[0023] Figure 3 This is a particle size distribution image obtained by performing particle size analysis (NTA) on the exosomes extracted from the aqueous two-phase solution in Example 1.

[0024] Figure 4 This is a transmission electron microscopy (TEM) verification image of exosomes extracted from the gel column in Example 1.

[0025] Figure 5 This is a particle size distribution image obtained by performing particle size analysis (NTA) verification on the exosomes extracted from the gel column in Example 1.

[0026] Figure 6 This is a distribution diagram of vesicles of different sizes after separation by the highly cross-linked agarose gel column in Example 1.

[0027] Figure 7 yes Figure 6 Peak 1 and peak 2 substances were validated using the exosomal protein marker CD63 (western blot). DETAILED DESCRIPTION

[0028] First, the present invention takes fresh milk as an example to compare the effects of separating and removing fat particles by a hydrophilic and lipophobic nano-interface mesh (coated with a polyvinyl alcohol / silicon dioxide composite film) and removing fat particles by precipitation with chemical reagents. The specific experiment is as follows: 1000mL of fresh milk is filtered through a hydrophilic and lipophobic nano-interface mesh coated with a polyvinyl alcohol / silicon dioxide composite film to remove fat particles in the fresh milk, and the permeate is collected; another 1000mL of fresh milk is placed in a beaker, heated to 40°C, and a 0.2M sodium acetate buffer solution with a pH of 4.6 is added while shaking, and the pH value is adjusted to 4.8 with a 1% NaOH aqueous solution by mass or a 10% acetic acid aqueous solution by volume, cooled to room temperature, left for 5 minutes, filtered with filter paper to remove precipitates, and then filtered with a microfiltration (MF) membrane with a pore size of 1.4μm to remove most of the fat particles, and the permeate is collected. Comparison Figure 1 As can be seen from Figures a, b, and c, the polyvinyl alcohol / silicon dioxide composite film removes fat particles more cleanly and effectively.

[0029] Next, the present invention takes fresh milk as an example to investigate the effects of ordinary ultrafiltration and tangential flow ultrafiltration on the exosome concentration effect. The specific experiment is: 1000mL of fresh milk is filtered through a hydrophilic and lipophobic nanointerface mesh coated with a polyvinyl alcohol / silicon dioxide composite film to remove fat particles in the fresh milk and collect the filtrate. A portion of the collected filtrate is ultrafiltered through an ordinary 0.45μm pore size ultrafiltration membrane (the pressure is smaller than 0.22μm), the ultrafiltrate is collected, and then filtered with an ultrafiltration membrane with a pore size of 100kDa to remove some small molecules of protein and water, and the concentrated liquid on the ultrafiltration membrane is collected. The liquid is concentrated 15 times, but the pressure of the filtration process increases, and subsequent filtration is difficult to carry out. The solution blocks the filter membrane and the filter needs to be changed halfway. The other part of the collected filtrate was ultrafiltered through a tangential flow ultrafiltration device with a pore size of 100kDa polyethersulfone ultrafiltration membrane to remove some small molecular proteins and water. The concentrated liquid on the ultrafiltration membrane was concentrated by nearly 10 times, and the concentration time was shortened by more than half compared with ordinary ultrafiltration; the concentrated liquid was further mixed with a biphasic solution (prepared by fully dissolving polyethylene glycol with a molecular weight of 35kDa and dextran with a molecular weight of 50kDa in distilled water, wherein the concentration of polyethylene glycol was 33.5g / L and the concentration of dextran was 16.5g / L) at a volume ratio of 1:1, centrifuged at 3000g for 20 minutes, discarded the upper phase, collected the lower phase solution, and then mixed the collected lower phase with the biphasic solution at a volume ratio of 1:1, repeated centrifugation once, removed about 90% of the liquid in the upper phase, and finally the remaining liquid was concentrated about 125 times relative to fresh milk. The exosome solution after biphasic concentration was identified by electron microscopy and particle size analysis, and it basically met the requirements of the experiment.

[0030] Then, the present invention takes fresh milk as an example to investigate the effect of ultrafiltration membrane pore size changes on the exosome concentration effect during ultrafiltration in a tangential flow ultrafiltration device. The specific experiment is as follows: 2000mL of fresh milk is taken from each group and filtered through a hydrophilic and lipophobic nano-interface mesh membrane coated with a polyvinyl alcohol / silicon dioxide composite film to remove fat particles in the fresh milk, collect the filtrate, and then ultrafilter the collected filtrate through a tangential flow ultrafiltration device with polyethersulfone ultrafiltration membranes of different pore sizes shown in Table 1 to remove some small molecules of protein and water. The ultrafiltration effect is shown in Table 1.

[0031] Table 1 Comparison of ultrafiltration results of ultrafiltration membranes with different pore sizes

[0032]

[0033] From the experimental results in Table 1 above, it can be seen that the concentration effect is best when the pore size of the ultrafiltration membrane is 300kDa. As the pore size of the ultrafiltration membrane decreases, the ultrafiltration time is prolonged and the efficiency becomes worse, but it has little effect on the extraction results and can meet the experimental requirements. When the ultrafiltration pore size is 10kDa, in addition to the above characteristics, the particle size of the extracted exosomes increases, and the number of miscellaneous particles increases after detection. Therefore, the present invention selects an ultrafiltration membrane with a pore size of 100kDa to 300kDa.

[0034] Finally, the present invention takes fresh milk as an example to investigate the effect of a two-phase aqueous solution composed of polyethylene glycol (PEG) and dextran (DEX) of different molecular weights on the exosome concentration effect. The specific experiment is: 200mL of fresh milk is taken from each group and filtered through a hydrophilic and lipophobic nanointerface mesh coated with a polyvinyl alcohol / silicon dioxide composite film to remove fat particles in the fresh milk, collect the filtrate, and then pass the collected filtrate through a tangential flow ultrafiltration device with a pore size of 300kDa polyethersulfone ultrafiltration membrane to ultrafilter, remove some small molecules of protein and water, collect the concentrated liquid on the ultrafiltration membrane, and mix the concentrated liquid with a two-phase aqueous solution composed of PEG and DEX of different molecular weights shown in Table 2 (prepared by fully dissolving PEG and DEX in distilled water, wherein the concentration of PEG is 33.5g / L and the concentration of DEX is 16.5g / L) at a volume ratio of 1:1, centrifuged at 3000g for 20 minutes, discarded the upper phase, and collected the exosome solution recovered in the lower phase dextran. The recovery rate of exosomes is shown in Table 2.

[0035] Table 2 Recovery rate of exosomes extracted by aqueous two-phase solutions composed of different molecular weight PEG and DEX

[0036] Recovery rate % PEG5kDa PEG8kDa PEG10kDa PEG20kDa PEG25kDa PEG30kDa PEG35kDa DEX7kDa 50％ 52％ 54％ 55％ 64％ 60％ 55％ DEX10kDa 53％ 53％ 56％ 57％ 66％ 67％ 58％ DEX45kDa 55％ 60％ 60％ 65％ 64％ 70％ 70％ DEX50kDa 53％ 55％ 58％ 62％ 64％ 68％ 75％ DEX65kDa 52％ 55％ 58％ 60％ 57％ 59％ 59％

[0037] Note: The recovery rate in the table refers to the volume concentration of exosomes in the lower phase.

[0038] It can be seen from Table 2 above that the recovery rate of exosomes extracted by the aqueous biphasic solution prepared by the combination of PEG35kDa and DEX50kDa is the highest, and the recovery rates of the other combinations gradually decrease with the change of molecular weight, but all meet the use requirements.

[0039] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments, but the protection scope of the present invention is not limited thereto.

[0040] Example 1

[0041] Taking fresh milk as an example, extracting exosomes from fresh milk

[0042] 1. Filter 2000 mL of fresh milk through a hydrophilic and lipophobic nano-interface membrane coated with a polyvinyl alcohol / silicon dioxide composite film to remove fat particles in the fresh milk and collect the filtrate.

[0043] 2. The filtrate collected in step 1 was ultrafiltered through a tangential flow ultrafiltration device with a pore size of 100 kDa polyethersulfone ultrafiltration membrane to remove some small molecular proteins and water, and about 200 mL of ultrafiltration concentrate was collected. The liquid was concentrated nearly 10 times. Figure 2 It can be seen that the exosomes extracted after ultrafiltration and concentration have three peaks, which have been verified to include exosomes, vesicles and apoptotic bodies.

[0044] 3. Select 13.4g of polyethylene glycol with a molecular weight of 35kDa and 6.6g of dextran with a molecular weight of 50kDa, fully dissolve them in distilled water, and then dilute to 400mL to prepare a two-phase aqueous solution. The concentration of polyethylene glycol in the two-phase aqueous solution is 33.5g / L, and the concentration of dextran is 16.5g / L. Then mix the ultrafiltration concentrate collected in step 2 with the two-phase solution in a volume ratio of 1:1, centrifuge at 3000g for 20 minutes, discard the upper phase, and collect about 40mL of the lower phase solution. Then mix the collected lower phase with the two-phase solution in a volume ratio of 1:1, repeat the centrifugation once, remove 80% of the liquid in the upper phase, and finally leave 13mL of liquid, which is about 150 times concentrated compared to fresh milk. By Figure 3 Particle size analysis shows that after extraction with a two-phase aqueous solution, the first peak is the impurity protein and the second peak is the exosome.

[0045] 4. Connect the chromatograph to a gel column filled with highly cross-linked agarose with a particle size of 600-800 kDa and a particle size of 75-95 μm. Before loading, balance the column with 20 mM pH 7.2 PB buffer containing 0.1 M NaCl at a flow rate of 0.5 mL / min. After the baseline of conductivity and UV absorption value is stable, dilute the remaining 16 mL of exosome solution after concentration in step 3 to 30 mL with 20 mM pH=7.2 Tris-HCl buffer containing 150 mM NaCl before loading. The loading flow rate is 0.5 mL / min until a flow-through peak appears. Collect each separated peak separately, of which the first and second peaks are the target peak exosomes, and the third peak is the extracellular vesicle. After the collection is completed, use a column wash solution (aqueous isopropanol solution containing 1 M NaOH with a volume concentration of 30%) to clean the column. Identify the exosome solutions collected from each peak, and store the rest in aliquots at -80°C. By Figure 4 Electron microscopy and Figure 5 Particle size analysis showed that the average particle size of the extracted exosomes was 125±4.0nm, with high purity. The extracted exosome solution was further passed through a gel column filled with cross-linked agarose to obtain Figure 6 After analysis, peaks 1 and 2 were found to be exosomes, and peak 3 was a vesicle secreted by cells. The exosomes extracted from peaks 1 and 2 were verified by protein marker CD63. Figure 7Western blot showed that peak 1 and peak 2 were exosomes.

[0046] Example 2

[0047] In step 3 of Example 1, the ultrafiltration concentrate collected in step 2 is mixed with the aqueous biphasic solution at a volume ratio of 3:1, centrifuged at 3000g for 20 minutes, the upper phase is discarded, the lower phase is relatively large, and the collected lower phase solution is about 200mL. The collected lower phase is then mixed with the aqueous biphasic solution at a volume ratio of 3:1, centrifuged twice, the liquid in the upper phase is removed, and finally 33mL of liquid remains, which is about 60 times concentrated relative to fresh milk. The other steps are the same as in Example 1.

[0048] Example 3

[0049] In step 3 of Example 1, the ultrafiltration concentrate collected in step 2 was mixed with the aqueous biphasic solution at a volume ratio of 1:3, centrifuged at 3000g for 20 minutes, the upper phase was discarded, and the collected lower phase solution was about 100 mL. The collected lower phase was then mixed with the aqueous biphasic solution at a volume ratio of 1:3, centrifuged twice, and the liquid in the upper phase was removed. Finally, 20 mL of liquid remained, which was concentrated about 100 times relative to fresh milk. The other steps were the same as in Example 1.

Claims

1. A method for large-scale extraction of exosomes in fresh milk, characterized in that: The method comprises the following steps: (1) filtering fresh milk with a hydrophilic and lipophobic nano-interface mesh to remove fat particles in the fresh milk and collecting the filtrate; the coating of the hydrophilic and lipophobic nano-interface mesh is a polyvinyl alcohol / silicon dioxide nano-composite film, and the contact angle of the film with water is 0. 0 ~25 0 The contact angle with fat in fresh milk is 60 0 ~130 0 ; (2) ultrafiltration of the filtrate collected in step (1) through a tangential flow ultrafiltration device using an ultrafiltration membrane with a pore size of 100 kDa to 300 kDa, and concentrating the filtrate after the fat particles are removed; (3) fully mixing the concentrated fresh milk collected in step (2) and the two-phase aqueous solution, centrifuging and separating the mixture, discarding the upper phase, and collecting the lower phase; Repeat this step 1 to 2 times in the lower phase to further concentrate the exosome solution; The two-phase aqueous solution is prepared by dissolving polyethylene glycol and dextran in distilled water or PBS buffer, wherein the concentration of polyethylene glycol in the two-phase aqueous solution is 20-40 g / L, and the concentration of dextran is 14-20 g / L; (4) The concentrated exosome solution in step (3) is diluted with a diluent and further separated by passing through a gel column, wherein the gel column uses highly cross-linked agarose as a filler and the diluent is a 20 mM pH = 7.2 Tris-HCl buffer containing 150 mM NaCl.

2. The method for large-scale extraction of exosomes from fresh milk according to claim 1, characterized in that: In step (1), the fresh milk includes any one or more of fresh cow's milk, fresh goat's milk, fresh human milk, and camel's milk.

3. The method for large-scale extraction of exosomes in fresh milk according to claim 1, characterized in that: In step (3), the concentrated fresh milk collected in step (2) and the aqueous two-phase solution are fully mixed in a volume ratio of 1:3 to 3:1 and then centrifuged.

4. The method for large-scale extraction of exosomes in fresh milk according to claim 1, characterized in that: In step (3), the molecular weight of the polyethylene glycol is 5 kDa to 35 kDa, and the molecular weight of the dextran is 7 kDa to 65 kDa.

5. The method for large-scale extraction of exosomes from fresh milk according to claim 1 or 3, characterized in that: In step (3), the centrifugal force of the centrifugal separation is 1000-3000 g, and the centrifugal time is 10-40 minutes.

6. The method for large-scale extraction of exosomes from fresh milk according to claim 1, characterized in that: In step (4), the molecular weight of the highly cross-linked agarose is 600 kDa to 800 kDa, and the particle size is 75 to 95 µm.

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