Reagent for exosome separation and exosome separation method

Abstract

The invention belongs to the technical field of exosome separation and purification, and particularly provides a reagent for exosome separation, which comprises alcohol, carbohydrates and protein, wherein in terms of alcohol mass, the mass percentage of the carbohydrates is 1-5%, and the mass percentage of the protein is 0.5-2%. The reagent for exosome separation provided by the invention can wrap the exosome while removing impure proteins, so that the exosome is prevented from being damaged, and the extraction efficiency, purity and recovery rate of the exosome are improved. The invention further provides an exosome separation method, the reagent is combined with a micro-fluidic chip, the integration degree is high, the operation steps are simplified, the operation difficulty and cost are reduced, and the exosome separation method is suitable for non-professionals to use. Besides, by utilizing a buffer system arranged in the chip, under the action of an internal oscillator, an additional manual washing operation step is omitted, and the exosomes in the blood are extracted in a high-throughput manner.

Description

Technical Field

[0001] The present invention belongs to the technical field of separation and purification of exosomes, and specifically relates to a reagent for separating exosomes and a method for separating exosomes. Background Art

[0002] Exosomes are vesicles with a lipid bilayer structure, with a diameter of 30 to 150 nm. They are secreted by cells into body fluids and carry lipids, proteins, DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and other substances of parent cells. Exosomes participate in the exchange of information and substances between cells and play an important role in intercellular communication. The exosomes of tumor cells contain a large amount of tumor-related mRNA, microRNA, and rRNA, which have important research and application value in the mechanism of cancer occurrence and early detection of cancer.

[0003] At present, there are many separation and purification technologies for exosomes, and the quality of separated exosome products is also uneven. A single separation technology is not suitable for exosomes from various sources. Therefore, it is particularly important to develop a variety of different separation technologies to effectively extract exosomes with different physiological properties. The current main methods include ultracentrifugation (UC), immunoaffinity, polymer precipitation, nanomembrane filtration, size exclusion chromatography, etc.

[0004] Among them, ultracentrifugation is the "gold standard" for the separation and purification of exosomes, but this method is time-consuming, low in yield, and relies on expensive precision instruments and equipment. At the same time, multiple ultracentrifugation and high speed will damage the structure of exosomes, which is not conducive to the downstream analysis and application of exosomes. Other separation technologies based on physical properties such as exosome size, density, solubility, and surface electrical properties also have problems such as low yield, low purity, and long time. Although the exosomes extracted by immunoaffinity method have high purity and high separation efficiency, the cost of specific antibodies is high, and the operation is complicated and the process is cumbersome. The principle of polymer co-precipitation technology is that hydrophilic polymers interact with the hydrophilic bonds of exosomes in the sample to form a hydrophobic microenvironment around the exosomes, thereby forming a precipitate to extract exosomes. However, this method of extraction is prone to exosome contamination, and polymers are not very friendly to cell experiments and have certain toxic effects on cells. Microfluidic chip technology has the advantages of fast analysis speed, high degree of integration, and use by non-professionals, and is expected to be widely used in the field of exosome analysis and detection. By using microfluidic chips and rationally designing the chip structure, the main separation and purification steps can be integrated on the chip, reducing the dependence on equipment. However, the blood components are complex and rich in impurities, which seriously affects the purity of exosomes separated and purified by microfluidic chips. Summary of the invention

[0005] The purpose of the present invention is to improve the extraction efficiency, purity and recovery rate of exosomes.

[0006] To this end, the present invention provides a reagent for isolating exosomes, comprising alcohol, carbohydrates and proteins; wherein, based on the mass of alcohol, the mass percentage of the carbohydrates is 1-5%, and the mass percentage of the proteins is 0.5-2%.

[0007] Specifically, the alcohol includes any one of methanol, ethanol and mannitol, with a volume fraction of 60-80%.

[0008] Specifically, the above-mentioned sugar substances include any one of trehalose, sucrose, glucose, lactose and fructose.

[0009] Specifically, the above-mentioned protein includes any one of albumin and skimmed milk powder.

[0010] The present invention also provides a method for isolating exosomes, based on the above-mentioned reagent for isolating exosomes, comprising the following steps:

[0011] (1) adding a reagent for isolating exosomes to a blood sample, incubating, and centrifuging to obtain a supernatant;

[0012] (2) The supernatant is added to a microfluidic chip, filtered under pressure, and the exosome sample is recovered.

[0013] Specifically, in the above step (1), the volume ratio of the reagent to the blood sample is 1:(0.5-2).

[0014] Specifically, the incubation time in the above step (1) is 10-60s.

[0015] Specifically, in the above step (2), the microfluidic chip includes an upper cover plate, a lower cover plate and a waste liquid pool; a first filter channel is provided at the bottom of the upper cover plate; a mixing chamber and a recovery chamber are provided on the upper cover plate; two ends of the first filter channel are respectively connected to the mixing chamber and the recovery chamber; a second filter channel corresponding to the first filter channel is provided at the top of the lower cover plate; the upper cover plate and the lower cover plate are bonded to form a filter chamber; the first filter channel and the second filter channel are isolated by a filter membrane; and the second filter channel is connected to the waste liquid pool.

[0016] Specifically, the above-mentioned microfluidic chip also includes an oscillator for oscillating the filter cavity.

[0017] Specifically, the above step (2) is specifically as follows: adding the supernatant into the mixing chamber, pressurizing the filtration chamber, so that the exosomes in the supernatant flow into the recovery chamber through the first filtration channel for recovery, and the impurities pass through the filter membrane and flow into the waste liquid pool through the second filtration channel.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] The reagent for exosome separation provided by the present invention can encapsulate exosomes while removing foreign proteins, thereby preventing exosomes from being destroyed and improving the extraction efficiency, purity and recovery rate of exosomes. The exosome separation method provided by the present invention combines the reagent with a microfluidic chip, has a high degree of integration, simplifies the operation steps, reduces the difficulty and cost of operation, and is suitable for use by non-professionals. In addition, by using the built-in buffer system of the chip, under the action of the internal oscillator, the additional manual washing operation steps are omitted, and exosomes in blood are extracted with high throughput.

[0020] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram of the NTA detection results in Example 1 of the present invention.

[0022] Figure 2 This is a diagram of the BCA test results in Example 1 of the present invention.

[0023] Figure 3 This is a graph showing the purity test results of exosomes in Example 1 of the present invention.

[0024] Figure 4 This is a diagram of the NTA detection results in Example 2 of the present invention.

[0025] Figure 5 This is a diagram of the BCA test results in Example 2 of the present invention.

[0026] Figure 6 This is a graph showing the purity test results of exosomes in Example 2 of the present invention.

[0027] Figure 7 This is an exploded view of the microfluidic chip provided in Example 3 of the present invention.

[0028] Figure 8 It is a graph of the NTA test results in Example 3 of the present invention and Comparative Example 1.

[0029] Fig. 9 3 is a graph showing the BCA test results in Example 3 of the present invention and Comparative Example 1.

[0030] Fig.10 3 is a graph showing the purity test results of exosomes in Example 3 of the present invention and Comparative Example 1.

[0031] Fig.11 It is the TEM result figure of Example 3 of the present invention and Comparative Example 1.

[0032] Figure numerals: 1. upper cover plate; 2. first filter channel; 3. mixing chamber; 4. recovery chamber; 5. filter membrane; 6. lower cover plate; 7. second filter channel; 8. waste liquid tank; 9. oscillator. DETAILED DESCRIPTION

[0033] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0034] The present invention provides a reagent for separating exosomes, comprising alcohol, sugar substances and protein; wherein, based on the mass of alcohol, the mass percentage of the sugar substances is 1-5%, and the mass percentage of the protein is 0.5-2%.

[0035] Among them, the alcohol includes any one of methanol, ethanol, and mannitol, with a volume fraction of 60-80%, preferably 80% ethanol; the sugar substance includes any one of trehalose, sucrose, glucose, lactose, and fructose, preferably 2% trehalose is added; the protein includes any one of albumin and skimmed milk powder, preferably 0.5% albumin is added.

[0036] The present invention also provides a method for isolating exosomes, based on the above-mentioned reagent for isolating exosomes, comprising the following steps:

[0037] (1) Add the blood sample to the container, and use an automatic injection needle to add the reagent for exosome separation at a flow rate of 100μl / 5s. If the flow rate is too fast, the sample will be mixed unevenly, and if it is too slow, the upper sample will have the middle protein denatured by the reagent, while the lower sample has not yet contacted the reagent. The volume ratio of the reagent to the blood sample is 1: (0.5-2), preferably 1: 1. The blood is incubated with the reagent, and after the impurities are denatured, the impurities are removed under the action of low-speed centrifugal force to obtain a supernatant.

[0038] The incubation time of blood and reagent is 10-60s, preferably 30s. The content of impurity proteins in blood is greater than that of target exosomes. If the incubation time is less than 30s, impurity proteins are not completely removed, and if it exceeds 30s, there is a certain risk of damage to exosomes. Through multiple experimental comparisons, it was found that at 30s, impurity proteins can be effectively removed and the degree of damage to exosomes is small. ...

[0039] The preferred centrifugation condition is 3000g for 10 minutes. Under this centrifugation condition, cell debris and large particle impurities can be removed. If the condition is lower than this, the impurities may not be removed completely, and if the condition is higher than this, it is a waste of time and is unnecessary.

[0040] (2) According to actual needs, select from existing microfluidic chips, preferably using a microfluidic chip with the following structure: including an upper cover plate, a lower cover plate and a waste liquid pool; a first filter channel is opened at the bottom of the upper cover plate; a mixing chamber and a recovery chamber are arranged on the upper cover plate; the two ends of the first filter channel are respectively connected to the mixing chamber and the recovery chamber; a second filter channel corresponding to the first filter channel is opened at the top of the lower cover plate; the upper cover plate and the lower cover plate are bonded to form a filter chamber; the first filter channel and the second filter channel are separated by a filter membrane; the second filter channel is connected to the waste liquid pool. The microfluidic chip preferably uses a PDMS chip, and the overall chip width is designed according to needs, generally 3 cm. The width of the filter channel inside the chip is 10-200μm, preferably 50μm; the depth is 5-100μm, preferably 25μm; under this size condition, the filter membrane can be better attached to the PDMS material, which is more conducive to chimerism and ensures better sealing. If the size is too small, the sample will be blocked on the membrane, the channel pressure will suddenly increase, and the integrity of the exosomes will be destroyed; if the size is too large, the membrane contact of the channel will not fit well, and it will be difficult for impurities to pass through the membrane. The pore size of the filter membrane is in the range of 10-500KD, preferably 100KD.

[0041] When in use, a buffer solution is placed in the mixing chamber, and an exosome preservation reagent is placed in the recovery chamber. The supernatant obtained in step (1) is sucked into the mixing chamber of the chip by the injection needle connected to the chip, and the chip is pressurized. Impurities are filtered through the filter membrane to the second filter channel and then flow into the waste liquid pool. The exosomes enter the recovery chamber through the first filter channel, and the exosome sample is recovered.

[0042] The buffer is preferably PBS buffer, which can effectively dilute the reagent and reduce its damage to exosomes.

[0043] The pressure inside the chip is generally 1Pa to 0.5MPa absolute pressure (gauge pressure -101kPa to 0.6MPa), preferably 0.05MPa.

[0044] Furthermore, the microfluidic chip also includes an oscillator for vibrating the filter chamber. The oscillator can be set at the bottom of the microfluidic chip body, and the vibration frequency is 1-10 Hz, preferably 5 Hz. The exosomes intercepted in the filter chamber are shaken down by shaking, eliminating the additional operation steps of manually blowing the chip. The use of physical shaking can also reduce the loss of exosomes during liquid transfer, which is beneficial to improve the recovery rate of exosomes.

[0045] The effects of the reagent for isolating exosomes and the method for isolating exosomes of the present invention are studied through specific examples below.

[0046] Embodiment 1:

[0047] Prepare multiple sets of reagents according to the following recipes.

[0048] Experimental group 1: Based on the mass of the ethanol solution, 2% trehalose and 0.5% albumin were added thereto; the volume fraction of the ethanol solution was 80%;

[0049] Experimental group 2: Based on the mass of the ethanol solution, 1% trehalose and 2% albumin were added thereto; the volume fraction of the ethanol solution was 70%;

[0050] Experimental group 3: Based on the mass of the methanol solution, 5% trehalose and 0.5% albumin were added thereto; the volume fraction of the methanol solution was 60%;

[0051] Experimental group 4: Based on the mass of the methanol solution, 2% trehalose and 1% albumin were added thereto; the volume fraction of the methanol solution was 70%;

[0052] Control group 1: Prepare 80% ethanol solution;

[0053] Control group 2: 2.5% trehalose was added to the ethanol solution by mass; the volume fraction of the ethanol solution was 80%;

[0054] Control group 3: based on the mass of the ethanol solution, 2.5% of albumin was added thereto; the volume fraction of the ethanol solution was 80%.

[0055] The blood samples were divided into 7 parts and added to each EP tube. The reagents prepared by the experimental groups 1-4 and the control groups 1-3 were added to each sample at a flow rate of 100 μl / 5s. The volume ratio of the sample to the reagent was 1:1. After the sample and the reagent were incubated for 30s, they were centrifuged at 3000g for 10min. An equal amount of supernatant was taken from each group for NTA and BCA detection, and the purity of the obtained samples was analyzed. The results are shown in Figure 1-3 shown.

[0056] From the above data analysis, it can be seen that when alcohol substances are not added with sugar or protein, the number and purity of exosome particles recovered are low. However, in the experimental group, the synergistic effect of protein and sugar can greatly increase the number and purity of exosome particles.

[0057] Embodiment 2:

[0058] The blood sample was divided into three portions and added to each EP tube. The reagent prepared in Experimental Group 1 of Example 1 was added to each sample at a flow rate of 100 μl / 5 s, and the volume ratio of the sample to the reagent was 1: 1. The three samples were incubated with the reagent for 5 s, 45 s, and 85 s, respectively, and centrifuged at 3000 g for 10 min after the incubation.

[0059] Equal amounts of supernatant were taken from each group for NTA and BCA assays. Figure 4-6 shown.

[0060] From the above data analysis, it can be seen that if the co-incubation time is too short, the impurity proteins in the sample will have less contact with alcohol substances, and the impurity proteins will not be completely denatured during this process, so the amount of impurity proteins in the exosome samples obtained later will increase, resulting in reduced purity. If the incubation time is too long, there is a risk of denaturation of the lipid bilayer liquid on the exosomes. If the exosomes appear in a denatured state at this time, the recovery rate of exosomes in the later stage will be reduced.

[0061] Embodiment 3:

[0062] This embodiment provides a microfluidic chip. Figure 7 , comprising an upper cover plate, a lower cover plate, a waste liquid pool and an oscillator; a first filter channel is provided at the bottom of the upper cover plate; a mixing chamber and a recovery chamber are provided on the upper cover plate; two ends of the first filter channel are respectively connected with the mixing chamber and the recovery chamber; a second filter channel corresponding to the first filter channel is provided at the top of the lower cover plate; the upper cover plate and the lower cover plate are bonded to form a filter chamber; the first filter channel and the second filter channel are isolated by a filter membrane; the waste liquid pool is installed at the bottom of the lower cover plate, and the second filter channel is connected with the waste liquid pool; the oscillator is installed at the bottom of the waste liquid pool.

[0063] The upper cover and the lower cover are made of PDMS with a width of 3 cm. The first filter channel and the second filter channel inside the chip have a filter channel width of 50 μm and a depth of 25 μm, and the filter membrane pore size is 100 KD.

[0064] Based on the above microfluidic chip, this embodiment provides a method for isolating exosomes, comprising the following steps:

[0065] (1) The blood sample was added to an EP tube, which was inserted into the chamber of a centrifuge. The reagent of Experimental Group 1 in Example 1 was added thereto using an automatic injection needle at a flow rate of 100 μl / 5 s, and the volume ratio of the reagent to the blood sample was 1:1. The blood and the reagent were incubated for 30 s, and the centrifuge was started. The supernatant was removed by centrifugation at 3000 g for 10 min to remove impurities.

[0066] (2) PBS buffer is placed in the mixing chamber of the microfluidic chip, and the exosome preservation reagent is placed in the recovery chamber. The supernatant is sucked into the mixing chamber of the chip by the injection needle connected to the microfluidic chip, the chip is pressurized to 0.05MPa, the oscillator is started, the vibration frequency is 5 Hz, the impurities are filtered through the filter membrane to the second filter channel, and then flow into the waste liquid pool. The exosomes enter the recovery chamber through the first filter channel, and the exosome sample is recovered.

[0067] The centrifuge and injection needle are centrally controlled by the control system to achieve automatic quantitative sampling and centrifugation.

[0068] The collected exosome samples were tested and analyzed for purity by NTA, TEM and BCA. The results are as follows Figure 8-11 shown.

[0069] Comparative Example 1:

[0070] Take the same blood sample as in Example 3 and perform ultracentrifugation. The specific steps are as follows:

[0071] (1) Serum was centrifuged at 3000 g for 10 min to remove particulate matter;

[0072] (2) Centrifuge the supernatant at 10,000 g for 30 min at 4°C to remove small precipitates, large vesicles and other impurities, and transfer the supernatant to a new centrifuge tube;

[0073] (3) Filter through a 0.22 μm filter to remove residue;

[0074] (4) Centrifugation at 100,000 g for 1 h at 4°C;

[0075] (5) Discard the supernatant, resuspend the white-red precipitate in PBS, and centrifuge at 100,000 g for 1 h at 4°C;

[0076] (6) Discard the supernatant, add 100-200 μl PBS to the exosome precipitate at the bottom, resuspend it, collect it, and store it at -80°C.

[0077] The collected exosome samples were tested and analyzed for purity by NTA, TEM and BCA. The results are as follows Figure 8-11 shown.

[0078] The above data analysis shows that the exosome protein content obtained by ultracentrifugation is higher because lipoproteins are close in size to exosomes and precipitate together under the action of centrifugal force, but the purity of the final product is lower than that of the system. Fig.11 It can be seen intuitively that the background of the exosome sample separated in Example 3 is cleaner, while the background of the sample separated by ultracentrifugation in Comparative Example 1 is contaminated with a lot of miscellaneous proteins. In Example 3, obvious saucer-shaped vesicle structures can be seen, while in addition to vesicles, there are a lot of white liposome contaminations in ultracentrifugation.

[0079] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A reagent for isolating exosomes, characterized in that: It comprises alcohol, sugar substances and protein; wherein, based on the mass of alcohol, the mass percentage of the sugar substances is 1-5%, and the mass percentage of the protein is 0.5-2%.

2. The reagent for isolating exosomes according to claim 1, characterized in that: The alcohol includes any one of methanol, ethanol and mannitol, with a volume fraction of 60-80%.

3. The reagent for isolating exosomes according to claim 1, characterized in that: The sugar substance includes any one of trehalose, sucrose, glucose, lactose and fructose.

4. The reagent for isolating exosomes according to claim 1, characterized in that: The protein includes any one of albumin and skimmed milk powder.

5. A method for isolating exosomes, characterized in that: The reagent for isolating exosomes according to any one of claims 1 to 4, comprising the following steps: (1) adding a reagent for isolating exosomes to a blood sample, incubating, and centrifuging to obtain a supernatant; (2) The supernatant is added to a microfluidic chip, filtered under pressure, and the exosome sample is recovered.

6. The method for isolating exosomes according to claim 5, characterized in that: In step (1), the volume ratio of the reagent to the blood sample is 1:(0.5-2).

7. The method for isolating exosomes according to claim 5, characterized in that: The incubation time in step (1) is 10-60s.

8. The method for isolating exosomes according to claim 5, characterized in that: The microfluidic chip in step (2) includes an upper cover plate, a lower cover plate and a waste liquid pool; a first filter channel is provided at the bottom of the upper cover plate; a mixing chamber and a recovery chamber are provided on the upper cover plate; two ends of the first filter channel are respectively connected to the mixing chamber and the recovery chamber; a second filter channel corresponding to the first filter channel is provided on the top of the lower cover plate; the upper cover plate and the lower cover plate are bonded to form a filter chamber; the first filter channel and the second filter channel are isolated by a filter membrane; and the second filter channel is connected to the waste liquid pool.

9. The method for isolating exosomes according to claim 8, characterized in that: The microfluidic chip also includes an oscillator for oscillating the filter cavity.

10. The method for isolating exosomes according to claim 8, characterized in that: The step (2) is specifically as follows: adding the supernatant into the mixing chamber, pressurizing the filtration chamber, allowing the exosomes in the supernatant to flow into the recovery chamber through the first filtration channel for recovery, and allowing the impurities to pass through the filtration membrane and flow into the waste liquid pool through the second filtration channel.

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