Method for extracting plasma exosome based on ultracentrifugation method

Abstract

The invention discloses a method for extracting plasma exosomes based on an ultracentrifugation method, which belongs to the technical field of biomedicine, and comprises the following main steps: firstly, carrying out differential centrifugation to remove visible components, cell debris and large vesicles in blood, ensuring no platelet residue, reducing the possibility that large particulate matters carry exosomes to settle, and extracting exosomes from the exosomes; the subsequent exosome recovery amount is increased; secondly, low-temperature operation is carried out in the whole process, so that the biological activity and the structure of the exosome can be better guaranteed, the degradation of exosome protein and RNA is effectively avoided, and the sample purity is high. According to the method for extracting the plasma exosomes based on the ultracentrifugation method, impurities such as cell debris and protein particles in the plasma are effectively removed by using a 0.22-micron filter for filtration, the purity of the exosomes is improved, and the method for extracting the plasma exosomes based on the ultracentrifugation method is short in separation time, high in repeatability, low in operation cost, large in sample treatment capacity, not prone to introducing personal errors and capable of being used for primary separation of a large number of samples.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for extracting plasma exosomes based on ultracentrifugation. Background Art

[0002] Exosomes are low-immunogenic nanovesicles with membrane structures secreted by living cells. Their diameter is generally between 30-150 nanometers, with strong penetrability and great difficulty in separation. They are widely present in body fluids such as blood, urine, cerebrospinal fluid and breast milk. The "cargo" carried by exosomes includes nucleic acids (mRNA, miRNA and DNA, etc.), proteins and lipid components, etc. They are widely involved in biological activities such as intercellular signal transduction, antigen presentation, genetic material transport, cell microenvironment transformation, and are related to a variety of tumors / diseases. Various cells are autosecreted, with a lipid bilayer membrane structure, and are not easily degraded.

[0003] The existing exosome extraction methods require differential centrifugation, ultracentrifugation, immune capture and other steps, which are costly, complicated and cumbersome, have high technical requirements for operators, are prone to human errors, and are difficult to ensure the biological activity and purity of exosomes. Therefore, a method for extracting plasma exosomes based on ultracentrifugation is proposed.

[0004] The above information disclosed in this background technology is only used to increase the understanding of the background technology of the present invention and therefore, it may include information that does not constitute the prior art known to ordinary technicians in this field. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems of the prior art in that the existing methods for extracting exosomes are complicated to operate, costly, and difficult to ensure the activity and purity of exosomes. To this end, one object of the present invention is to propose a method for extracting plasma exosomes based on ultracentrifugation.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] The method for extracting plasma exosomes based on ultracentrifugation comprises the following steps:

[0008] Step 1: First, collect peripheral blood samples from volunteers using anticoagulant tubes containing EDTA and store at room temperature for 10 minutes;

[0009] Step 2: After the sample is balanced, centrifuge it at 500 x g for 10 min to remove the formed elements in the blood, collect the supernatant, and then use a pipette tip to transfer the supernatant to a 2 mL sterile enzyme-free EP tube;

[0010] Step 3: After balancing the EP tube, centrifuge the supernatant at 3000 x g and 4° for 20 min in a low-temperature centrifuge to ensure that there are no platelets left. Then, use a pipette tip to transfer the supernatant to a 2 mL sterile enzyme-free EP tube;

[0011] Step 4: After balancing the EP tube, centrifuge the supernatant at 12000 x g at 4°C for 20 min to remove cell debris and large vesicles. Then, use a pipette to collect the supernatant and transfer it to a 2 mL sterile enzyme-free EP tube and freeze it at -80°C to isolate exosomes.

[0012] Step 5: Take out the frozen sample in step 4, thaw it on ice, balance the EP tube after the sample is completely thawed, and then centrifuge it in a low-temperature centrifuge at 12000x g at 4° for 20 minutes to remove large vesicles and apoptotic bodies. Then, use a pipette tip to collect the supernatant in a centrifuge tube;

[0013] Step 6: Add 1 volume of 4° PBS to dilute the supernatant, shake and mix, use a syringe to draw up the liquid, connect a 0.22 μm filter, and filter out any large vesicles and apoptotic bodies that may remain in the supernatant;

[0014] Step 7: Use a new syringe to transfer the filtered liquid along the tube wall into a disposable ultracentrifuge tube. After balancing, sealing, and labeling, place it in an ultracentrifuge at 100,000xg and 4° for 2 hours to collect exosomes.

[0015] Step 8: Remove the supernatant in step 7, add 4° PBS, and use the pipette tip to gently blow the exosomes on the tube wall 20-30 times to dissolve all the exosomes in PBS;

[0016] Step 9: Use a new syringe to absorb the PBS dissolved with exosomes in step 8, transfer it along the tube wall to a disposable ultracentrifuge tube, balance, seal, and label, and centrifuge at 100,000xg 4° for 1 hour to collect the exosomes.

[0017] As a further optimization scheme of the present invention, in step 2, step 3, and step 4, the centrifugal force is gradually increased, thereby reducing the possibility of large particles carrying exosomes to settle and increasing the subsequent exosome recovery amount.

[0018] As a further optimization scheme of the present invention, in step three, step four, step five, step seven, and step nine, the centrifugation temperature is 4°, which can better ensure the biological activity and structure of the exosomes and effectively avoid the degradation of exosome proteins and RNA.

[0019] As a further optimization scheme of the present invention, in step 4, freezing the supernatant for 24 hours helps to better remove large vesicles.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention effectively avoids exosome sedimentation and protein and RNA degradation through differential centrifugation, cryopreservation and cryocentrifugation, ensures the biological activity and structure of exosomes, and effectively removes impurities in plasma, such as cell debris, protein particles, etc., by filtering with a 0.22μm filter, thereby improving the purity of exosomes. The method for extracting plasma exosomes based on ultracentrifugation in the present invention has short separation time, high repeatability, low operating cost, large sample processing volume, and is not easy to introduce human errors, and can be used for the initial separation of a large number of samples.

[0022] The above summary is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, they will be easily understood and grasped by examining the drawings and reading the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The figure is a flowchart of the method for extracting plasma exosomes based on ultracentrifugation of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] See also Figure 1 , a method for extracting plasma exosomes based on ultracentrifugation, comprising the following steps:

[0026] Step 1: First, collect peripheral blood samples from volunteers using anticoagulant tubes containing EDTA and store at room temperature for 10 minutes;

[0027] Step 2: After the sample is balanced, centrifuge it at 500 x g for 10 min to remove the formed elements in the blood, collect the supernatant, and then use a pipette tip to transfer the supernatant to a 2 mL sterile enzyme-free EP tube;

[0028] Step 3: After balancing the EP tube, centrifuge the supernatant at 3000 x g and 4° for 20 min in a low-temperature centrifuge to ensure that there are no platelets left. Then, use a pipette tip to transfer the supernatant to a 2 mL sterile enzyme-free EP tube;

[0029] Step 4: After balancing the EP tube, centrifuge the supernatant at 12000 x g at 4°C for 20 min to remove cell debris and large vesicles. Then, use a pipette to collect the supernatant and transfer it to a 2 mL sterile enzyme-free EP tube and freeze it at -80°C to isolate exosomes.

[0030] Step 5: Take out the frozen sample in step 4, thaw it on ice, balance the EP tube after the sample is completely thawed, and then centrifuge it in a low-temperature centrifuge at 12000x g at 4° for 20 minutes to remove large vesicles and apoptotic bodies. Then, use a pipette tip to collect the supernatant in a centrifuge tube;

[0031] Step 6: Add 1 volume of 4° PBS to dilute the supernatant, shake and mix, use a syringe to draw up the liquid, connect a 0.22 μm filter, and filter out any large vesicles and apoptotic bodies that may remain in the supernatant;

[0032] Step 7: Use a new syringe to transfer the filtered liquid along the tube wall into a disposable ultracentrifuge tube. After balancing, sealing, and labeling, place it in an ultracentrifuge at 100,000xg and 4° for 2 hours to collect exosomes.

[0033] Step 8: Remove the supernatant in step 7, add 4° PBS, and use the pipette tip to gently blow the exosomes on the tube wall 20-30 times to dissolve all the exosomes in PBS;

[0034] Step 9: Use a new syringe to absorb the PBS dissolved with exosomes in step 8, transfer it along the tube wall to a disposable ultracentrifuge tube, balance, seal, and label, and centrifuge at 100,000xg 4° for 1 hour to collect the exosomes.

[0035] Parts not involved in the present invention are the same as the prior art or can be implemented by the prior art. Although the embodiments of the present invention have been shown and described, it is understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting plasma exosomes based on ultracentrifugation, characterized in that: The following steps are involved: Step 1: First, collect peripheral blood samples from volunteers using anticoagulant tubes containing EDTA and store at room temperature for 10 minutes; Step 2: After the sample is balanced, centrifuge it at 500 x g for 10 min to remove the formed elements in the blood, collect the supernatant, and then use a pipette tip to transfer the supernatant to a 2 mL sterile enzyme-free EP tube; Step 3: After balancing the EP tube, centrifuge the supernatant at 3000 x g and 4° for 20 min in a low-temperature centrifuge to ensure that there are no platelets left. Then, use a pipette tip to transfer the supernatant to a 2 mL sterile enzyme-free EP tube; Step 4: After balancing the EP tube, centrifuge the supernatant at 12000 x g at 4°C for 20 min to remove cell debris and large vesicles. Then, use a pipette to collect the supernatant and transfer it to a 2 mL sterile enzyme-free EP tube and freeze it at -80°C to isolate exosomes. Step 5: Take out the frozen sample in step 4, thaw it on ice, balance the EP tube after the sample is completely thawed, and then centrifuge it in a low-temperature centrifuge at 12000x g at 4° for 20 minutes to remove large vesicles and apoptotic bodies. Then, use a pipette tip to collect the supernatant in a centrifuge tube; Step 6: Add 1 volume of 4° PBS to dilute the supernatant, shake and mix, use a syringe to draw up the liquid, connect a 0.22 μm filter, and filter out any large vesicles and apoptotic bodies that may remain in the supernatant; Step 7: Use a new syringe to transfer the filtered liquid along the tube wall into a disposable ultracentrifuge tube. After balancing, sealing, and labeling, place it in an ultracentrifuge at 100,000xg and 4° for 2 hours to collect exosomes. Step 8: Remove the supernatant in step 7, add 4° PBS, and use the pipette tip to gently blow the exosomes on the tube wall 20-30 times to dissolve all the exosomes in PBS; Step 9: Use a new syringe to absorb the PBS dissolved with exosomes in step 8, transfer it along the tube wall to a disposable ultracentrifuge tube, balance, seal, and label, and centrifuge at 100,000xg 4° for 1 hour to collect the exosomes.

2. The method for extracting plasma exosomes based on ultracentrifugation according to claim 1, characterized in that: In step 4, the supernatant is frozen for 24 hours.

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