CN115109742A - A clinical-grade high-purity blood or urine exosome isolation and purification kit - Google Patents

Abstract

The invention relates to a clinical-grade separation and purification kit for exosomes in high-purity blood or urine, which combines differential centrifugation and ultrafiltration centrifugation, and adds the steps of dilution and ultrafiltration concentration to separate and purify exosomes so as to greatly improve the purity of exosomes and reduce the loss of exosomes. Also comprises the application of the kit in gastric cancer diagnosis. Compared with the prior art, the invention obtains a large amount of high-purity stable exosomes with the average size of 30-150 nm, complete teacup saucer-like structure and uniform particle size distribution, and carries the marker proteins CD9, CD81 and CD63 on the surface, thereby overcoming the defects of low purity, low yield, low expression quantity of characterization proteins and the like in the prior art.

Description

A clinical-grade high-purity blood or urine exosome isolation and purification kit

technical field

The invention belongs to the field of nano biotechnology, in particular to a clinical-grade high-purity exosome separation and purification kit in blood or urine.

Background technique

Exosomes are 30-150 nm "cup holder" phospholipid bilayer vesicles secreted by many types of cells, with a density of 1.13-1.19 g/mL, carrying a variety of proteins, lipids and nucleic acids. It is involved in immune regulation, regulation of extracellular matrix remodeling, activation of cell signaling pathways, etc. The isolated exosomes can be further detected for tumor markers, such as miRNA, which can be used for tumor screening and early diagnosis. So far, there have been some reports on the method of exosome isolation, but there are some problems. The purity of exosomes obtained by traditional ultracentrifugation is acceptable, but the yield is not high; the ultrafiltration designed according to the principle of exosome size, Methods such as dialysis cannot remove impurities that are consistent with the size of exosomes. The polyethylene glycol (PEG) precipitation method and the immunomagnetic bead method both introduce exogenous proteins that are not suitable for clinical treatment to varying degrees. sexual substances. There is a huge clinical demand for the development of new high-purity exosome isolation kits.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kit for separating and purifying exosomes in clinical-grade high-purity blood or urine in order to overcome the above-mentioned defects in the prior art, so as to generate a large number of clinical-grade high-purity exosomes.

Further, another object of the present invention is to provide an application of the above-mentioned kit.

The object of the present invention is achieved by the following technical solutions: a kit for separating and purifying exosomes in clinical grade high-purity blood or urine, using a combination of differential centrifugation and ultrafiltration centrifugation, adding dilution and ultrafiltration concentration steps to separate and purify exosomes exosomes to greatly improve their purity and reduce the loss of exosomes, including the following steps:

(1) Collect at least 2ml of the patient's blood sample, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution; and/or

Collect at least 2ml of the patient's urine sample, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution;

(2) Centrifuge the diluent obtained in step (1) at a speed of 2000-3000 rpm at 4°C, save the supernatant, and discard the precipitate;

(3) Centrifuge the supernatant obtained in step (2) at a speed of 8000-12000 rpm at 4°C, save the supernatant, and discard the precipitate;

(4) Dilute the supernatant obtained in step (3) with phosphate buffer at 4°C, and continue to centrifuge with a 100 kDa-150 kDa ultrafiltration tube at a speed of 3000-4000 rpm for 10 min to remove small molecular impurities. At the same time, the concentrated supernatant is obtained to reduce the workload and cost of the subsequent ultracentrifugation;

(5) Centrifuge the concentrated supernatant obtained in step (4) at a speed of 8000 g for 60 min at 4°C. After the centrifugation, discard the supernatant and keep the precipitate;

(6) The precipitate obtained in step (5) is resuspended with phosphate buffer and transferred to a dialysis bag, and dialyzed for 3 hours to obtain the desired target solution of high-purity serum or exosomes in urine.

Wherein, the phosphate buffer solution formula: KCL 200 mg/L; NaCL 8 g/L; KH ₂ PO ₄ 240 mg/L; Na ₂ HPO ₄ 1.44 g/L.

On the basis of the above scheme, the dilution volume ratio of the phosphate buffer to the supernatant in the step (1) is 1:1.

Further, in the step (2), the centrifugation time is 8-10 min; in the step (3), the centrifugation time is 15-25 min.

On the basis of the above scheme, the amount of phosphate buffer used in step (6) is 1 mL-4 mL, the diameter of the dialysis bag is 30 nm-35 nm, and the dialysate is phosphate buffer.

Preferably, in step (6), 1 mL-4 mL of phosphate buffer is used to resuspend and transfer to a dialysis bag, the dialysis bag has a pore size of 30 nm-35 nm, and phosphate buffer is used as the dialysate, and the dialysis is performed for 3-8 hours. The desired target solution of high-purity serum or exosomes in urine can be obtained.

The target solution obtained in step (6) can be filtered with a 0.22 μm sterile filter.

The present invention also provides the application of the above-mentioned kit in the detection of gastric cancer-related markers, using flow cytometry to detect CD9, CD81, and CD63 markers, which are used for the detection of gastric cancer-related markers such as miRNA markers.

Specifically, remove 5 μl of liquid from the prepared exosomes, add PI dye and mix well, add deionized water to a total volume of 100 μl, add to flow cytometry, and analyze to confirm the presence of CD9, CD81, and CD63 on the surface of exosomes antigenic markers.

Compared with the prior art, the present invention obtains a large number of high-purity and stable exosomes with an average size of 30-150 nm, a complete teacup holder-like structure, uniform particle size distribution, and the surface carries the landmark proteins CD9, CD81, CD63. , overcoming the shortcomings of the existing preparation technology such as low purity, low yield, and low expression of the characteristic protein, the exosomes obtained by the present invention can be directly used for subsequent detection, with a high safety factor, and will not cause the loss of exosome function. , which can be used not only for scientific research, but also for the detection of markers such as miRNA in exosomes, and for the screening and diagnosis of early gastric cancer.

Description of drawings

Figure 1. Transmission electron microscopy (TEM) pictures of exosomes obtained.

Figure 2, Western Blot detection, high expression of exosome marker proteins CD9, CD81, CD63.

Figure 3, Nanoparticle Tracking Analysis (NTA) detection, NTA detection of exosome particle size analysis, the obtained exosomes, the average particle size is 113nm ± 20nm, and the number of exosomes is as high as 6*10 ⁷ .

Detailed ways

Example 1: Isolation and purification of exosomes in serum

A clinical grade kit for separating and purifying exosomes in high-purity blood or urine, including a box body, using a combination of differential centrifugation and ultrafiltration centrifugation, adding dilution and ultrafiltration concentration steps to separate and purify exosomes, so as to greatly improve the Its purity and reduce the loss of exosomes, according to the following steps:

(1) Collect 2ml of blood samples from gastric cancer patients, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution;

(2) Centrifuge the diluent obtained in step (1) at 3000 rpm for 8 min at 4°C, and precipitate into cells, save the supernatant, and discard the precipitate;

(3) Centrifuge the supernatant obtained in step (2) at 9000 rpm for 15 min at 4°C, and collect the supernatant and discard the precipitate;

(4) Dilute the supernatant obtained in step (3) with phosphate buffer at 4 °C, and continue to centrifuge at 3000-4000 rpm for 5 min with a 110 kDa ultrafiltration tube to remove small molecular impurities and concentrate at the same time. supernatant to reduce the workload and cost of subsequent ultracentrifugation;

(5) Centrifuge the concentrated supernatant obtained in step (4) at a speed of 100,000g for 60 min at 4°C. After the centrifugation, discard the supernatant and keep the precipitate;

(6) The precipitate obtained in step (5) is resuspended with phosphate buffer and transferred to a dialysis bag, and dialyzed for 8 hours to obtain the desired target solution of high purity and a large amount of exosomes.

The present invention uses transmission electron microscopy (TEM), Western Blot, and Nanoparticle Tracking Analysis to characterize the obtained exosomes. The specific characterization results are as follows:

Figure 1: Transmission electron microscope (TEM) photo: The characterization method of exosome purity is mainly TEM electron microscope. If the exosome purity is not enough, there will be many impurities, and the exosomes cannot be seen clearly. The TEM pictures of the exosomes obtained in this example were taken, as shown in Figure 1, within the field of view, the exosomes were clear and complete, which indicated that the exosomes obtained by this method were of high purity and the structure of the exosomes was complete.

Figure 2: Western Blot detection: The main characterization method of exosome biological activity obtained in this example is Western Blot detection. It can be seen from Figure 2 that exosomes highly express exosome marker proteins CD9, CD81, and CD63.

Figure 3: Analysis of the particle size of exosomes detected by NTA: The number of exosome cells obtained in this example is characterized by NTA. It can be seen from Figure 3 that the exosomes obtained by the present invention have an average particle size of 113nm±20nm. The number of exosomes is as high as 6*10 ⁷ .

Example 2

A clinical grade kit for separating and purifying exosomes in high-purity blood or urine, including a box body, using a combination of differential centrifugation and ultrafiltration centrifugation, adding dilution and ultrafiltration concentration steps to separate and purify exosomes, so as to greatly improve the Its purity and reduce the loss of exosomes, according to the following steps:

(1) Collect at least 2ml of the patient's urine sample, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution;

(2) Centrifuge the phosphate buffer solution obtained in step (1) at 3000 rpm for 9 min at 4°C, take the supernatant, and discard the precipitate;

(3) Centrifuge the supernatant obtained in step (2) at a speed of 10,000 rpm for 20 min at 4°C, and collect the supernatant and discard the precipitate;

(4) Dilute the supernatant obtained in step (3) with phosphate buffer at 4°C, and continue to centrifuge at 3500 rpm for 5 min with a 110 kDa ultrafiltration tube to remove small-molecule impurities and obtain a concentrated supernatant. liquid to reduce the workload and cost of the subsequent ultracentrifugation;

(5) Centrifuge the concentrated supernatant obtained in step (4) at a speed of 100,000g for 60 min at 4°C. After the centrifugation, discard the supernatant and keep the precipitate;

(6) Resuspend the precipitate obtained in step (5) with phosphate buffer and transfer it to a dialysis bag, and dialyze for 7 hours to obtain the desired target solution of high purity and a large amount of exosomes.

Take 5 μl of the target solution from the prepared exosomes, add PI dye-labeled anti-CD9, CD81, CD63 monoclonal antibodies, after mixing, add deionized water to a total volume of 100 μl, add to flow cytometry, and analyze. It was confirmed that CD9, CD81, and CD63 antigen markers exist on the surface of exosomes.

The two specific embodiments of the present invention have been described in detail above, only to illustrate the technical concept and characteristics of the present invention, the purpose of which is to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and not to limit the present invention. scope of protection. It should be understood that those skilled in the art can make many modifications and changes according to the concept of the present invention without creative efforts. Therefore, any technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the prior art according to the concept of the present invention shall fall within the protection scope determined by the claims.

Claims (10)

1. A kit for separating and purifying exosomes in clinical-grade high-purity blood or urine, is characterized in that, select differential centrifugation and ultrafiltration centrifugation to combine, increase dilution and ultrafiltration concentration steps to separate and purify exosomes, with large Amplify its purity and reduce the loss of exosomes, including the following steps: (1) Collect at least 2ml of the patient's blood sample, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4 °C to obtain a dilution; and/or Collect at least 2ml of the patient's urine sample, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution; (2) Centrifuge the diluent obtained in step (1) at a speed of 2000-3000 rpm at 4°C, save the supernatant, and discard the precipitate; (3) Centrifuge the supernatant obtained in step (2) at a speed of 8000-12000 rpm at 4°C, save the supernatant, and discard the precipitate; (4) Dilute the supernatant obtained in step (3) with phosphate buffer at 4°C, and continue to centrifuge at 3000-4000 rpm for 5-10min with a 100 kDa-150kDa ultrafiltration tube to remove small molecular impurities At the same time, the concentrated supernatant is obtained to reduce the workload and cost of the subsequent ultracentrifugation; (5) Centrifuge the concentrated supernatant obtained in step (4) at a speed of 8000-100,000g for 60 min at 4°C. After the centrifugation, discard the supernatant and keep the precipitate; (6) The precipitate obtained in step (5) is resuspended with phosphate buffer and transferred to a dialysis bag, and dialyzed for 3 hours to obtain the desired target solution of high-purity serum or exosomes in urine. 2. a kind of clinical grade high-purity blood or urine exosome separation and purification kit according to claim 1, is characterized in that, described phosphate buffered saline formula: KCL 200 mg/L; NaCL 8g/L ; KH ₂ PO ₄ 240 mg/L; Na ₂ HPO ₄ 1.44 g/L. 3. The kit for separating and purifying exosomes in clinical-grade high-purity blood or urine according to claim 1 or 2, characterized in that, in the step (1), the dilution volume of phosphate buffer and supernatant The ratio is 1:1. 4. a kind of clinical grade high-purity blood or urine exosome separation and purification kit according to claim 1, is characterized in that, in described step (2), centrifugation time is 8-10 min; Described step (3) ) for 15-25 min. 5. The kit for separating and purifying exosomes in clinical-grade high-purity blood or urine according to claim 1 or 2, characterized in that in step (6), 1 mL-4 mL of phosphate buffer is used for resuspending Transfer it to a dialysis bag with a pore size of 30 nm-35 nm, and use phosphate buffer as a dialysate. After dialysis for 3-8 hours, the desired target solution of high-purity serum or exosomes in urine can be obtained. 6. a kind of clinical grade high-purity blood or urine exosome separation and purification kit according to claim 1 and 2, is characterized in that, according to the following steps: (1) Collect 2ml of blood samples from gastric cancer patients, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution; (2) Centrifuge the diluent obtained in step (1) at 3000 rpm for 8 min at 4°C, and precipitate into cells, save the supernatant, and discard the precipitate; (3) Centrifuge the supernatant obtained in step (2) at 9000 rpm for 15 min at 4°C, and collect the supernatant and discard the precipitate; (4) Dilute the supernatant obtained in step (3) with phosphate buffer at 4 °C, and continue to centrifuge at 3000-4000 rpm for 5 min with a 110 kDa ultrafiltration tube to remove small molecular impurities and concentrate at the same time. supernatant to reduce the workload and cost of subsequent ultracentrifugation; (5) Centrifuge the concentrated supernatant obtained in step (4) at a speed of 100,000g for 60 min at 4°C. After the centrifugation, discard the supernatant and keep the precipitate; (6) The precipitate obtained in step (5) is resuspended with phosphate buffer and transferred to a dialysis bag, and dialyzed for 8 hours to obtain the desired target solution of high purity and a large amount of exosomes. 7. The kit for separating and purifying exosomes in a kind of clinical grade high-purity blood or urine according to claim 1 and 2, is characterized in that, according to the following steps: (1) Collect at least 2ml of the patient's urine sample, centrifuge to separate the supernatant, transfer it to another centrifuge tube, and dilute the collected supernatant with phosphate buffer at 4°C to obtain a dilution; (2) Centrifuge the phosphate buffer solution obtained in step (1) at 3000 rpm for 9 min at 4°C, take the supernatant, and discard the precipitate; (3) Centrifuge the supernatant obtained in step (2) at a speed of 10,000 rpm for 20 min at 4°C, and collect the supernatant and discard the precipitate; (4) Dilute the supernatant obtained in step (3) with phosphate buffer at 4°C, and continue to centrifuge at 3500 rpm for 5 min with a 110 kDa ultrafiltration tube to remove small-molecule impurities and obtain a concentrated supernatant. liquid to reduce the workload and cost of the subsequent ultracentrifugation; (5) Centrifuge the concentrated supernatant obtained in step (4) at a speed of 100,000g for 60 min at 4°C. After the centrifugation, discard the supernatant and keep the precipitate; (6) The precipitate obtained in step (5) is resuspended with phosphate buffer and transferred to a dialysis bag, and dialyzed for 7 hours to obtain the desired target solution of high purity and a large amount of exosomes. 8. A clinical-grade high-purity blood or urine exosome separation and purification kit according to claim 1, wherein the target solution obtained in step (6) is filtered with a 0.22 μm sterile filter. 9. An application of the kit according to any one of the above claims 1 to 8 in the detection of gastric cancer-related markers, using a flow cytometer to detect CD9, CD81, and CD63 markers, for gastric cancer-related markers Detection of substances such as miRNA markers. 10. The application according to claim 9, wherein the detection of CD9, CD81, and CD63 markers by flow cytometer is as follows: take out 5 μl target liquid from the prepared exosomes, add PI Dye-labeled anti-CD9, CD81, CD63 monoclonal antibodies, after mixing, add deionized water to a total volume of 100 μl, add flow cytometer, and analyze to confirm whether there are CD9, CD81, CD63 antigen markers on the surface of exosomes thing.

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