CN111808811A - Composite magnetic particle, method for removing red blood cells in peripheral blood or bone marrow cells and application of composite magnetic particle - Google Patents
Composite magnetic particle, method for removing red blood cells in peripheral blood or bone marrow cells and application of composite magnetic particle Download PDFInfo
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Abstract
The invention provides a composite magnetic particle, a method for removing red blood cells in peripheral blood or bone marrow cells and application thereof, and relates to the technical field of specific cell separation. The surface of the composite magnetic particle is coupled with a monoclonal antibody or polyclonal antibody which can specifically identify the surface antigen of the erythrocyte, and the composite magnetic particle can further adsorb and remove the erythrocyte in peripheral blood or marrow cells through the antigen-antibody reaction specificity due to the surface of the composite magnetic particle is coupled with the monoclonal antibody or polyclonal antibody which can specifically identify the surface antigen of the erythrocyte, thereby effectively relieving the problems of complex process and high preparation cost of the existing method for removing the erythrocyte in the marrow cells, and being widely applied to the preparation process of the monocyte.
Description
Technical Field
The invention relates to the technical field of specific cell separation, in particular to a composite magnetic particle, a method for removing red blood cells in peripheral blood or bone marrow cells and application thereof.
Background
Monocytes (monocytes) are the largest blood cells in the blood and the largest volume of leukocytes, and are an important component of the body's defense system. In the production process of the conventional African swine fever live vaccine, mononuclear cells in bone marrow cells are required to be used for production, but the bone marrow cells contain a large amount of red blood cells besides the mononuclear cells. During the production process, the erythrocytes are removed first to prepare monocytes.
The existing method for removing the red blood cells from the bone marrow cells has the defects of time and labor waste and high cost, and is not suitable for industrial production. Taking bone marrow cells in a 50mL centrifuge tube as an example, the bone marrow rinse was collected in a 50mL centrifuge tube through a Falcon single cell strainer, centrifuged at 1500rpm for 5min at 4 ℃ and the supernatant removed. Resuspending the cell pellet with an equal volume of 1 Xerythrocyte lysate, adding 25ml erythrocyte lysate, mixing, rapidly placing in a 25 deg.C water bath for 2min to lyse erythrocytes (mixing by gently turning upside down every minute during placement), adding 25ml marrow rinse solution after 2min to stop lysis, centrifuging at 4 deg.C at 1500rpm5min, and collecting the pellet by discarding the supernatant. Adding bone marrow washing solution to wash cells, centrifuging at 1500rpm for 5min at 4 deg.C, discarding supernatant, and collecting precipitate. The washing was repeated 2 times. Adding appropriate amount of bone marrow rinse solution at 3 times, centrifuging at 1500rpm for 5min at 4 deg.C, discarding supernatant, collecting precipitate, and collecting cell precipitate as monocyte. The method needs 40min for completing the operation, red blood cell lysate needs to be purchased or prepared, and the operation steps are easy to pollute bacteria, thereby causing potential risks. In addition, scale production is not suitable for obtaining monocytes using centrifuges. Meanwhile, the separation of mononuclear cells in peripheral blood requires the purchase of a commercial lymphocyte separation solution and centrifugation, and is not suitable for the industrial production of mononuclear cells in terms of cost and production mode.
Therefore, it is necessary and urgent to develop a method for removing red blood cells from peripheral blood or bone marrow cells, which has a simple process and is economical, and to efficiently prepare monocytes.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first object of the present invention is to provide composite magnetic particles, which are coupled with monoclonal antibodies or polyclonal antibodies that specifically recognize erythrocyte surface antigens on the surfaces thereof, and which can specifically adsorb and remove erythrocytes in peripheral blood or bone marrow cells by antigen-antibody reaction.
A second object of the present invention is to provide a method for producing composite magnetic fine particles.
The third objective of the invention is to provide a method for removing red blood cells from peripheral blood or bone marrow cells.
The fourth purpose of the present invention is to provide an application of the composite magnetic particle and the method for removing red blood cells in peripheral blood or bone marrow cells in a monocyte preparation process.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides a composite magnetic particle, which comprises a magnetic particle and a specific monoclonal antibody or polyclonal antibody;
the specific monoclonal antibody or polyclonal antibody comprises a monoclonal antibody or polyclonal antibody which specifically recognizes erythrocyte surface antigen;
the specific monoclonal antibody or polyclonal antibody is coupled to the surface of the magnetic particle.
Further, the composite magnetic fine particles include magnetic fine particles having carboxyl groups on the surface, and are preferably Magnosphere manufactured by JSR CorpTMMagnetic particles of MS 300/Caboxyl.
The invention provides a preparation method of the composite magnetic particles, which comprises the following steps:
providing a solution containing magnetic particles, then adding a monoclonal antibody or polyclonal antibody and a coupling agent which can specifically recognize erythrocyte surface antigens, and coupling to obtain the composite magnetic particles.
Further, the coupling agent comprises at least one of glutaraldehyde, ammonium sulfate, carbodiimide/N-hydroxysuccinimide (EDC/NHS), and an NHS ester-containing antibody, preferably a water-soluble carbodiimide;
preferably, the mass ratio of the magnetic particles to the monoclonal antibody or polyclonal antibody is 5-15 mg: 2.5-7.5mg, preferably, 5-10 mg: 3.5-4.5mg, more preferably, 8-10 mg: 4-5.5 mg.
Further, the coupling is oscillation coupling; the reaction condition of the concussion coupling at least meets at least one of the following conditions:
the temperature of the oscillation coupling is 35-40 ℃, the time is 1.5-2.5 h, the oscillation power is 25-35W, and the frequency is 30-40 rpm.
More preferably, the reaction conditions of the concussion coupling at least satisfy at least one of the following:
the temperature of the coupling by oscillation is 37 ℃, the time is 2h, the oscillation power is 30 watts, and the frequency is 35 rpm.
Further, the preparation method also comprises the step of collecting the composite magnetic particles;
preferably, the collecting comprises the steps of: adsorbing the composite magnetic particles by using a magnet, then discarding the cleaning precipitate, and suspending the precipitate in a solution containing a sealing agent;
more preferably, the blocking agent is ProClin (TM) 300, manufactured by Sigma.
The invention provides a method for removing red blood cells in peripheral blood or bone marrow cells, which comprises the following steps:
(A) providing peripheral blood or bone marrow cells, adding a buffer solution, and uniformly mixing to obtain peripheral blood or bone marrow cell suspension;
(B) and adding the composite magnetic particles into peripheral blood or bone marrow cell suspension, uniformly mixing, standing, and taking supernatant to obtain a solution with red blood cells removed.
Further, the volume ratio of the peripheral blood or bone marrow cells to the buffer solution in the step (A) is 1: 1-2, preferably 1: 1;
preferably, the buffer of step (A) comprises one of 0.9% physiological saline, MEM or D-Hanks; preferably a PBS solution;
preferably, the volume ratio of the peripheral blood or bone marrow cell suspension to the composite magnetic particles in the step (B) is 2-6: 1-4, preferably 2: 1.
Preferably, the standing time of the step (B) is 3-8 min, preferably 5 min.
Further, the method comprises the following steps:
(A) providing peripheral blood or bone marrow cells, and then uniformly mixing the peripheral blood or bone marrow cells and a buffer solution according to the volume ratio of 1: 1-2 to obtain peripheral blood or bone marrow cell suspension;
(B) adding the composite magnetic particles into peripheral blood or bone marrow cell suspension according to the volume ratio of 2-6: 1-4, uniformly mixing, standing for 3-8 min, and taking supernatant to obtain a solution with red blood cells removed.
Preferably, the method comprises the steps of:
(A) providing peripheral blood or bone marrow cells, and subsequently contacting the peripheral blood or bone marrow cells with a buffer in a ratio of 1:1 to obtain peripheral blood or bone marrow cell suspension;
(B) and adding the composite magnetic particles into peripheral blood or bone marrow cell suspension according to the volume ratio of 2:1, uniformly mixing, standing for 5min, and taking supernatant to obtain a solution with red blood cells removed.
The invention provides the composite magnetic particles and the application of the method for removing red blood cells in peripheral blood or bone marrow cells in preparing mononuclear cells.
Compared with the prior art, the invention has the beneficial effects that:
the surface of the composite magnetic particle is coupled with a monoclonal antibody or polyclonal antibody which can specifically recognize erythrocyte surface antigen. The surface of the composite magnetic particle is coupled with a monoclonal antibody or polyclonal antibody which can specifically recognize the surface antigen of the erythrocyte, so that the erythrocyte in peripheral blood or marrow cells can be adsorbed and removed through the antigen-antibody reaction specificity.
The preparation method of the composite magnetic particle provided by the invention comprises the steps of adding a monoclonal antibody or polyclonal antibody and a coupling agent for specifically recognizing erythrocyte surface antigen into a solution containing the magnetic particle, and then performing oscillation coupling to obtain the composite magnetic particle. The preparation method has the advantages of simple process and easy operation, and is very suitable for large-scale industrial production.
The method for removing the red blood cells in the peripheral blood or the bone marrow cells comprises the steps of firstly preparing a suspension of the peripheral blood or the bone marrow cells, then adding the composite magnetic particles into the suspension, uniformly mixing, specifically adsorbing the red blood cells in the suspension of the peripheral blood or the bone marrow cells by the composite magnetic particles, then sinking under the action of gravity, and extracting a supernatant to obtain a solution after removing the red blood cells. The method for removing the red blood cells in the peripheral blood or the bone marrow cells is simple and convenient to operate, and can effectively remove the red blood cells from the peripheral blood or the bone marrow cells.
The composite magnetic particles and the method for removing red blood cells in peripheral blood or bone marrow cells provided by the invention can be widely applied to the preparation process of mononuclear cells.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the effect of the monocyte expansion culture prepared in example 5 of the present application and comparative example 1, which is provided in effect example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
According to one aspect of the present invention, a composite magnetic particle comprising a magnetic particle and a specific mono-or polyclonal antibody;
the specific monoclonal antibody or polyclonal antibody comprises a monoclonal antibody or polyclonal antibody which specifically recognizes erythrocyte surface antigen;
the specific monoclonal antibody or polyclonal antibody is coupled to the surface of the magnetic particle.
The composite magnetic particle provided by the invention is a magnetic particle with a surface coupled with a specific monoclonal antibody or polyclonal antibody, wherein the specific monoclonal antibody or polyclonal antibody comprises a monoclonal antibody or polyclonal antibody which specifically recognizes erythrocyte surface antigen. The surface of the composite magnetic particle is coupled with monoclonal antibody or polyclonal antibody which can specifically recognize erythrocyte surface antigen, and then the composite magnetic particle can specifically adsorb erythrocyte through antigen-antibody reaction, thereby separating erythrocyte in peripheral blood or marrow cells, and providing a foundation for the preparation of late monocyte.
In a preferred embodiment of the present invention, the composite magnetic particles include magnetic particles having carboxyl groups on the surface thereof, preferably Magnosphere manufactured by JSR, JapanTMMagnetic particles of MS 300/Caboxyl.
In a preferred embodiment, the magnetic fine particles of the present invention may further contain a plurality of active groups, and the protein or polypeptide may be chemically crosslinked to the surface of the magnetic fine particles. Preferably, the reactive group comprises a hydroxyl, carboxyl, sulfonyl or amino reactive group. The magnetic particles containing reactive groups may be prepared by techniques conventional in the art or may be directly commercially available.
According to an aspect of the present invention, a method for preparing the above composite magnetic fine particles comprises the steps of:
providing a solution containing magnetic particles, then adding a monoclonal antibody or polyclonal antibody and a coupling agent which can specifically recognize erythrocyte surface antigens, and coupling to obtain the composite magnetic particles.
The preparation method of the composite magnetic particle provided by the invention comprises the steps of adding a monoclonal antibody or polyclonal antibody and a coupling agent for specifically recognizing erythrocyte surface antigen into a solution containing the magnetic particle, and then performing oscillation coupling to obtain the composite magnetic particle. The preparation method has the advantages of simple process and easy operation, and is very suitable for large-scale industrial production.
In a preferred embodiment of the invention, the coupling agent comprises at least one of glutaraldehyde, ammonium sulfate, carbodiimide/N-hydroxysuccinimide (EDC/NHS) and NHS ester-containing antibodies, preferably a water-soluble carbodiimide;
as a preferred embodiment, the water-soluble carbodiimide (EDC), also known as 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, has a good carboxyl-activating effect, and when used as a coupling agent, can significantly improve the coupling effect of the composite magnetic fine particles of the present application.
In a preferred embodiment of the present invention, preferably, the mass ratio of the magnetic particles to the monoclonal antibody or polyclonal antibody is 5-15 mg: 2.5-7.5mg, preferably, 5-10 mg: 3.5-4.5mg, more preferably, 8-10 mg: 4-5.5 mg.
In a preferred embodiment of the invention, the coupling is a concussive coupling; the reaction condition of the concussion coupling at least meets at least one of the following conditions:
the temperature of the oscillation coupling is 35-40 ℃, the time is 1.5-2.5 h, the oscillation power is 25-35W, and the frequency is 30-40 rpm. Under the condition, the combination of the antibody and the carrier is maximally activated.
Preferably, the reaction conditions of the concussive coupling at least satisfy at least one of the following:
the temperature of the coupling by oscillation is 37 ℃, the time is 2h, the oscillation power is 30 watts, and the frequency is 35 rpm.
In a preferred embodiment of the present invention, the preparation method further comprises a step of collecting the composite magnetic fine particles.
As a preferred embodiment, the collecting comprises the steps of: the composite magnetic particles are adsorbed by a magnet, and then the washing and washing precipitate is discarded, and then the precipitate is suspended in a solution containing a blocking agent.
Preferably, the blocking agent is ProClin manufactured by SigmaTM300。
According to one aspect of the present invention, a method of removing red blood cells from peripheral blood or bone marrow cells, the method comprising the steps of:
(A) providing peripheral blood or bone marrow cells, adding a buffer solution, and uniformly mixing to obtain peripheral blood or bone marrow cell suspension;
(B) and adding the composite magnetic particles into peripheral blood or bone marrow cell suspension, uniformly mixing, standing, and taking supernatant to obtain a solution with red blood cells removed.
The method for removing the red blood cells in the peripheral blood or the bone marrow cells comprises the steps of firstly preparing a suspension of the peripheral blood or the bone marrow cells, then adding the composite magnetic particles into the suspension, uniformly mixing, specifically adsorbing the red blood cells in the suspension of the peripheral blood or the bone marrow cells by the composite magnetic particles, then sinking under the action of gravity, and extracting a supernatant to obtain a solution after removing the red blood cells. The method for removing the red blood cells in the peripheral blood or the bone marrow cells is simple and convenient to operate, and can effectively remove the red blood cells from the peripheral blood or the bone marrow cells.
In a preferred embodiment of the present invention, the volume ratio of the peripheral blood or bone marrow cells to the buffer in step (a) is 1: 1-2, preferably 1: 1;
in a preferred embodiment of the present invention, the buffer of step (A) comprises one of 0.9% physiological saline, MEM or D-Hanks; preferably a PBS solution;
in a preferred embodiment of the present invention, the volume ratio of the peripheral blood or bone marrow cell suspension to the composite magnetic particles in step (B) is 2-6: 1-4, preferably 2: 1. When the volume ratio of the peripheral blood or the bone marrow cell suspension to the composite magnetic particles is 2:1, the supernatant only contains about 3-5% of red blood cells, and the process requirement can be met. When the volume ratio of the peripheral blood or bone marrow cell suspension to the composite magnetic particles exceeds 2:1, the red blood cells cannot be effectively adsorbed, and more than 5 percent of the red blood cells exist, so that the conditions of downstream immune cells or producing microorganisms are influenced.
Preferably, the standing time of the step (B) is 3-8 min, preferably 5 min.
In a preferred embodiment of the invention, the method comprises the steps of:
(A) providing peripheral blood or bone marrow cells, and then uniformly mixing the peripheral blood or bone marrow cells and a buffer solution according to the volume ratio of 1: 1-2 to obtain peripheral blood or bone marrow cell suspension;
(B) adding peripheral blood or bone marrow cell suspension into the composite magnetic particles in a volume ratio of 2-6: 1-4, uniformly mixing, standing for 3-8 min, and taking supernatant to obtain a solution with red blood cells removed.
Preferably, the method comprises the steps of:
(A) providing peripheral blood or bone marrow cells, and subsequently contacting the peripheral blood or bone marrow cells with a buffer in a ratio of 1:1 to obtain peripheral blood or bone marrow cell suspension;
(B) and adding the composite magnetic particles into peripheral blood or bone marrow cell suspension according to the volume ratio of 2:1, uniformly mixing, standing for 5min, and taking supernatant to obtain a solution with red blood cells removed.
In a preferred embodiment of the present invention, the method further comprises the step of recovering the composite magnetic particles.
As a preferred embodiment, the step of recovering the composite magnetic particles can be recovery by a magnetic method, the recovered composite magnetic particles can be adsorbed by a new batch of red blood cells, and the red blood cells are eluted by repeated adsorption, and the composite magnetic particles can be reused for more than 100 times, so that the production cycle and yield of the existing mononuclear cells are greatly improved, and the production cost is reduced.
According to one aspect of the invention, the composite magnetic particles and the method for removing red blood cells from peripheral blood or bone marrow cells are used for preparing mononuclear cells.
The composite magnetic particles and the method for removing red blood cells in peripheral blood or bone marrow cells provided by the invention can be widely applied to the preparation process of mononuclear cells.
The mononuclear cells further prepared by the invention can be induced into the required cells by stimulating factors. For example, colony-macrophage colony stimulating factor is mainly induced into macrophage, and granulocyte-macrophage colony stimulating factor is mainly induced into dendritic cell. The cells induced by different inducers can proliferate the immune cells for the treatment of diseases; the induced cells can also be used as carriers for the cultivation of microorganisms.
The technical solution of the present invention will be further described with reference to the following examples.
Note: the magnetic fine particles used in the examples of the present application are Magnosphere, a product number of JSR, JapanTMMagnetic particles of MS 300/Caboxyl.
Example 1
Preparing monoclonal antibody or polyclonal antibody for specifically recognizing erythrocyte surface antigen:
(1) taking out bone marrow of pig bone or peripheral anticoagulation, taking 2ml of the anticoagulation, uniformly mixing with 1:1 physiological saline, and carefully adding the anticoagulation onto the liquid surface of 6ml of cell separation liquid; centrifuge at 400g (about 1500rpm, 15cm radius horizontal rotor) for 20 minutes. The tube bottom red blood cells were collected and the above steps were repeated again with the cell separation medium. Washing the red blood cells with normal saline for 2 times to obtain the needed red blood cells.
(2) Diluting the red blood cells to a specific volume by PBS or normal saline, mixing ISA206 adjuvant according to the mass ratio of 1:1, stirring for 10min, and performing sterile inspection to obtain a qualified product for later use.
(3) And taking the emulsified red blood cell preparation, and immunizing BALB/c mice subcutaneously by 300 microliters per mouse. The immunization was carried out twice 21 days after the first immunization and three times 21 days after the second immunization. 7 days after immunization, the mice were aseptically killed and splenocytes taken from the mice were fused with myeloma cells. After fusion, single cells are selected for culture, after culture and expansion, the supernatant is taken to respectively carry out agglutination reaction with erythrocytes of pigs, chickens, sheep, cattle, rabbits and mice, monoclonal antibodies which are not agglutinated with the erythrocytes of the pigs are removed, monoclonal antibodies which are cross agglutinated with other species of animals are removed, 6 specific monoclonal antibodies are selected, and after expansion culture, the monoclonal antibodies are purified by ProtienA and then can be used for preparing composite magnetic beads.
Example 2
A method of making composite magnetic particles, the method comprising the steps of:
(1) taking 1mL of solution containing magnetic particles with the concentration of 10mg/mL, washing the solution for 2 times by using 2-morpholine ethanesulfonic acid (MES) buffer solution, and finally suspending the solution in 1mL of 2-morpholine ethanesulfonic acid buffer solution to obtain solution A;
the concentration of the 2-morpholine ethanesulfonic acid (MES) buffer solution is 0.1mol/L, and the pH value is 5.0;
(2) adding 500 mu g of monoclonal antibody or polyclonal antibody prepared in the embodiment 1 into the solution A and mixing uniformly to obtain a solution B;
(3) dissolving 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) in 0.1mol/L MES buffer solution having a pH of 5.0 to give an EDC concentration of 10 mg/mL;
(4) adding 100 mu L of EDC solution prepared in the step (3) into the solution B prepared in the step (2), and carrying out oscillation reaction for 2 hours at 37 ℃;
(5) adsorbing with magnet, discarding supernatant, washing the precipitate with PBS solution containing 0.1% Tween-20 for 3 times, suspending in PBS solution containing 1% BSA, adding 0.1% ProClin TM300, and mixing to obtain composite magnetic particles.
Wherein the PBS solution is a PBS solution with the concentration of 0.01mol/L, pH of 7.4; ProClin (TM) 300 was purchased from Sigma under the designation 48914-U.
Example 3
A method of making composite magnetic particles, the method comprising the steps of:
(1) taking 1mL of solution containing magnetic particles with the concentration of 10mg/mL, washing the solution for 2 times by using 2-morpholine ethanesulfonic acid (MES) buffer solution, and finally suspending the solution in 1mL of 2-morpholine ethanesulfonic acid buffer solution to obtain solution A;
the concentration of the 2-morpholine ethanesulfonic acid (MES) buffer solution is 0.1mol/L, and the pH value is 5.0;
(2) adding 50 mu g of monoclonal antibody or polyclonal antibody prepared in the embodiment 1 into the solution A and mixing uniformly to obtain a solution B;
(3) dissolving 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) in 0.1mol/L MES buffer solution having a pH of 5.0 to give an EDC concentration of 10 mg/mL;
(4) adding 100 mu L of EDC solution prepared in the step (3) into the solution B prepared in the step (2), and carrying out oscillation reaction for 1.5 hours at 35 ℃;
(5) adsorbing with magnet, discarding supernatant, washing the precipitate with PBS solution containing 0.1% Tween-20 for 3 times, suspending in PBS solution containing 1% BSA, adding 0.1% ProClin TM300, and mixing to obtain composite magnetic particles.
Wherein the PBS solution is a PBS solution with the concentration of 0.01mol/L, pH of 7.4; ProClin (TM) 300 was purchased from Sigma under the designation 48914-U.
Example 4
A method of making composite magnetic particles, the method comprising the steps of:
(1) taking 1mL of solution containing magnetic particles with the concentration of 10mg/mL, washing the solution for 2 times by using 2-morpholine ethanesulfonic acid (MES) buffer solution, and finally suspending the solution in 1mL of 2-morpholine ethanesulfonic acid buffer solution to obtain solution A;
the concentration of the 2-morpholine ethanesulfonic acid (MES) buffer solution is 0.1mol/L, and the pH value is 5.0;
(2) adding 300 mu g of monoclonal antibody or polyclonal antibody prepared in the embodiment 1 into the solution A and mixing uniformly to obtain a solution B;
(3) dissolving 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) in 0.1mol/L MES buffer solution having a pH of 5.0 to give an EDC concentration of 10 mg/mL;
(4) adding 100 mu L of EDC solution prepared in the step (3) into the solution B prepared in the step (2), and carrying out oscillation reaction for 2.5 hours at 40 ℃;
(5) adsorbing with magnet, discarding supernatant, washing the precipitate with PBS solution containing 0.1% Tween-20 for 3 times, suspending in PBS solution containing 1% BSA, adding 0.1% ProClin TM300, and mixing to obtain composite magnetic particles.
Wherein the PBS solution is a PBS solution with the concentration of 0.01mol/L, pH of 7.4; ProClin (TM) 300 was purchased from Sigma under the designation 48914-U.
Example 5
A method for preparing monocytes, comprising the steps of:
(A) taking 75ml of pig bone marrow cells aseptically, and diluting the bone marrow cells to 150ml by using a PBS buffer solution to obtain a pig bone marrow cell suspension;
(B) adding the composite magnetic particles prepared in the embodiment 2 into the pig bone marrow cell suspension according to the volume ratio of the bone marrow cell suspension to the composite magnetic particles of 2-6: 1-4, preferably 2:1, slightly shaking for 40S, standing for 3min to enable the composite magnetic particles after adsorbing red blood cells to sink, and separating to obtain composite magnetic particle sediment and supernatant; the supernatant is clear, and a small amount of sample is taken to observe the ratio of the double-concave-shaped red blood cells under a microscope to be less than 5%.
(C) Pouring the supernatant into a sterile container, centrifuging at 1500RPM 4 deg.C for 5min, discarding the supernatant, collecting the precipitate, resuspending the precipitate with RPM1640 culture medium containing 10% fetal calf serum, detecting cell viability rate, which is not less than 95%, adjusting cell density to 1 × 106Putting the mononuclear cell into a cell culture bottle, and putting the cell culture bottle into an incubator containing 5% carbon dioxide for culture to obtain mononuclear cells;
meanwhile, adding the composite magnetic particle precipitate into 100ml of PBS (phosphate buffer solution) with the concentration of 15mm/L and the pH value of 5.5, eluting the red blood cells attached to the magnetic beads, adding the phosphate buffer solution with the concentration of 0.01M and the pH value of 7.6 into the composite magnetic beads after 2 times of elution, standing for 2 minutes, discarding supernatant, repeating the steps again, removing the balance solution after ensuring that the pH value of the magnetic beads after balance is 7.0 +/-0.5, adding the phosphate buffer solution with the concentration of 0.01M and the pH value of 7.2, and recovering to obtain the composite magnetic particles for adsorbing new red blood cells.
Example 6
A method for preparing monocytes, comprising the steps of:
(A) aseptically taking 75ml of anticoagulated peripheral blood, adding 150ml of PBS buffer solution into the anticoagulated peripheral blood, and uniformly mixing to obtain peripheral blood cell suspension;
(B) adding the composite magnetic particles prepared in the embodiment 3 into peripheral blood suspension in a volume ratio of 2-6: 1-4, preferably 2:1, slightly shaking for 20S, standing for 8min to enable the composite magnetic particles after adsorbing red blood cells to sink, and separating to obtain composite magnetic particle sediment and supernatant;
(C) the procedure in this example (C) is the same as in example 5.
Example 7
A method for preparing monocytes, comprising the steps of:
(A) taking 75ml of pig bone marrow cells aseptically, and diluting the bone marrow cells to 75ml by using a PBS buffer solution to obtain a pig bone marrow cell suspension;
(B) adding the composite magnetic particles prepared in the embodiment 4 into the pig bone marrow cell suspension in a volume ratio of 2-6: 1-4, preferably 2:1, slightly shaking for 30 seconds, standing for 5min to enable the composite magnetic particles after adsorbing red blood cells to sink, and separating to obtain composite magnetic particle sediment and supernatant;
(C) the procedure in this example (C) is the same as in example 5.
Comparative example 1
(A) 10ml of diluted bone marrow cells are added with 100ml of erythrocyte lysate for even mixing, quickly placed in a 27 ℃ water bath for 3 minutes to lyse the erythrocytes, and the lysis is repeated with the period of upside down. After the lysis, 50ml PBS was added, centrifuged at 1500rpm for 5min at 4 ℃ and the precipitate was collected.
(B) 30ml PBS was added to resuspend the cells, centrifuged at 1500rpm for 5min at 4 ℃ and the supernatant was discarded to collect the pellet. The washing was repeated 2 times.
(C) The appropriate amount of PBS was added at 3 rd time and sampled and counted. Centrifuging at 1500rpm for 5min at 4 deg.C, removing supernatant, collecting precipitate, and collecting cell precipitate as bone marrow mononuclear cell.
The whole operation process is about 40min, centrifugal equipment is needed, and industrialization is difficult to realize.
Effect example 1
The monocytes prepared in example 5 and comparative example 1 of the present application were adjusted to a cell density of 1X 106The results of cell proliferation comparison of each cell in MEM medium (containing 10% FBS) for 7 days are shown in FIG. 1.
As can be seen from FIG. 1, the activity and amplification performance of the monocytes prepared in the present application are significantly higher than those of the monocytes prepared in comparative example 1 by the conventional method.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A composite magnetic particle, wherein the composite magnetic particle comprises a magnetic particle and a specific monoclonal antibody or polyclonal antibody;
the specific monoclonal antibody or polyclonal antibody comprises a monoclonal antibody or polyclonal antibody which specifically recognizes erythrocyte surface antigen;
the specific monoclonal antibody or polyclonal antibody is coupled to the surface of the magnetic particle.
2. The composite magnetic particle according to claim 1, wherein the composite magnetic particle comprises a magnetic particle having a carboxyl group on the surface, preferably Magnosphere manufactured by JSR, JapanTMMagnetic particles of MS 300/Caboxyl.
3. A method for producing the composite magnetic fine particle according to claim 1 or 2, characterized by comprising the steps of:
providing a solution containing magnetic particles, then adding a monoclonal antibody or polyclonal antibody and a coupling agent which can specifically recognize erythrocyte surface antigens, and coupling to obtain the composite magnetic particles.
4. The method of producing composite magnetic fine particles according to claim 3, wherein the coupling agent comprises at least one of glutaraldehyde, ammonium sulfate, carbodiimide/N-hydroxysuccinimide (EDC/NHS), and an antibody containing NHS ester, preferably water-soluble carbodiimide;
preferably, the mass ratio of the magnetic particles to the monoclonal antibody or polyclonal antibody is 5-15: 2.5 to 7.5.
5. The method of producing composite magnetic fine particles according to claim 3, wherein the coupling is oscillation coupling;
preferably, the reaction conditions of the concussive coupling at least satisfy at least one of the following:
the temperature of the oscillation coupling is 35-40 ℃, the time is 1.5-2.5 h, the oscillation power is 25-35W, and the frequency is 35-50 rpm;
more preferably, the reaction conditions of the concussion coupling at least satisfy at least one of the following:
the temperature of the oscillation coupling is 37 ℃, the time is 2 hours, the oscillation power is 25-35 watts, and the frequency is 30-40 rpm.
6. The method of producing composite magnetic fine particles according to claim 3, further comprising a step of collecting the composite magnetic fine particles;
preferably, the collecting comprises the steps of: adsorbing the composite magnetic particles by using a magnet, then discarding the cleaning precipitate, and suspending the precipitate in a solution containing a sealing agent;
more preferably, the blocking agent is ProClin manufactured by SigmaTM300。
7. A method for removing red blood cells from peripheral blood or bone marrow cells, said method comprising the steps of:
(A) providing peripheral blood or bone marrow cells, adding a buffer solution, and uniformly mixing to obtain peripheral blood or bone marrow cell suspension;
(B) adding the composite magnetic particles according to claim 1 or 2 to peripheral blood or a bone marrow cell suspension, uniformly mixing, standing, and taking a supernatant to obtain a solution from which erythrocytes are removed.
8. The method according to claim 7, wherein the volume ratio of peripheral blood or bone marrow cells to buffer in step (A) is 1: 1-2, preferably 1: 1;
preferably, the buffer of step (A) comprises one of 0.9% physiological saline, MEM or D-Hanks; preferably a PBS solution;
preferably, the volume ratio of the peripheral blood or bone marrow cell suspension to the composite magnetic particles in the step (B) is 2-6: 1-4, preferably 2: 1;
preferably, the standing time of the step (B) is 3-8 min, preferably 5 min.
9. The method according to claim 7, characterized in that it comprises the steps of:
(A) providing peripheral blood or bone marrow cells, and then uniformly mixing the peripheral blood or bone marrow cells and a buffer solution according to the volume ratio of 1: 1-2 to obtain peripheral blood or bone marrow cell suspension;
(B) adding the composite magnetic particles according to claim 1 or 2 into peripheral blood or bone marrow cell suspension according to the volume ratio of 2-6: 1-4, uniformly mixing, standing for 3-8 min, and taking supernatant to obtain a solution with red blood cells removed; if the erythrocytes remain unremoved, the above process can be repeated;
preferably, the method comprises the steps of:
(A) providing peripheral blood or bone marrow cells, and subsequently contacting the peripheral blood or bone marrow cells with a buffer in a ratio of 1:1 to obtain peripheral blood or bone marrow cell suspension;
(B) adding the composite magnetic particles of claim 1 or 2 into peripheral blood or bone marrow cell suspension at a volume ratio of 2:1, mixing uniformly, standing for 5min, and taking supernatant to obtain solution with red blood cells removed.
10. Use of the composite magnetic particle according to claim 1 or 2, the method for removing red blood cells from peripheral blood or bone marrow cells according to any one of claims 7 to 9 for preparing mononuclear cells.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6194204B1 (en) * | 1996-08-02 | 2001-02-27 | Center For Blood Research, Inc. | Enrichment of dendritic cells from blood |
| US20050003559A1 (en) * | 2003-07-03 | 2005-01-06 | Labsoft Diagnostic Ag | Immunomagnetic separation of specific target cells |
| CN101165173A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for carrying cell sorting by using gold magnetism particles |
| CN101469323A (en) * | 2007-12-28 | 2009-07-01 | 英科新创(厦门)科技有限公司 | Method for separating red corpuscle from whole blood |
| CN102105175A (en) * | 2008-05-27 | 2011-06-22 | 香港中文大学 | Nanoparticles, methods of making same and cell labelling using same |
| CN104111333A (en) * | 2013-11-13 | 2014-10-22 | 叶森 | Fast erythrocyte removal method applicable to whole blood detection |
| CN106198963A (en) * | 2016-08-31 | 2016-12-07 | 上海美吉生物医药科技有限公司 | A kind of for immunomagnetic beads capturing leukocyte and preparation method thereof |
| CN111308070A (en) * | 2020-02-28 | 2020-06-19 | 厦门宝太生物科技有限公司 | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood |
-
2020
- 2020-07-20 CN CN202010701057.XA patent/CN111808811A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6194204B1 (en) * | 1996-08-02 | 2001-02-27 | Center For Blood Research, Inc. | Enrichment of dendritic cells from blood |
| US20050003559A1 (en) * | 2003-07-03 | 2005-01-06 | Labsoft Diagnostic Ag | Immunomagnetic separation of specific target cells |
| CN101165173A (en) * | 2006-10-19 | 2008-04-23 | 陕西西大北美基因股份有限公司 | Method for carrying cell sorting by using gold magnetism particles |
| CN101469323A (en) * | 2007-12-28 | 2009-07-01 | 英科新创(厦门)科技有限公司 | Method for separating red corpuscle from whole blood |
| CN102105175A (en) * | 2008-05-27 | 2011-06-22 | 香港中文大学 | Nanoparticles, methods of making same and cell labelling using same |
| CN104111333A (en) * | 2013-11-13 | 2014-10-22 | 叶森 | Fast erythrocyte removal method applicable to whole blood detection |
| CN106198963A (en) * | 2016-08-31 | 2016-12-07 | 上海美吉生物医药科技有限公司 | A kind of for immunomagnetic beads capturing leukocyte and preparation method thereof |
| CN111308070A (en) * | 2020-02-28 | 2020-06-19 | 厦门宝太生物科技有限公司 | Kit for separating red blood cells from whole blood and method for separating red blood cells from whole blood |
Non-Patent Citations (2)
| Title |
|---|
| NILS BOHMER等: "《Removal of Cells from Body Fluids by Magnetic Separation in Batch and Continuous Mode: Influence of Bead Size, Concentration,and Contact Time》", 《ACS APPL. MATER.INTERFACES》 * |
| 李建国主编: "《高等仪器分析》", 30 June 2020, 苏州大学出版社 * |
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