CN115011561A - Chimeric antigen receptor macrophage and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a chimeric antigen receptor macrophage and a preparation method and application thereof, wherein the chimeric antigen receptor macrophage is a mouse bone marrow-derived macrophage which is modified by a chimeric antigen receptor and expresses the chimeric antigen receptor. The chimeric antigen receptor macrophage provided by the invention is prepared from mouse bone marrow-derived macrophage (BMDM), and has the following advantages: as primary macrophages, the condition of CAR-M can be truly reflected; the cell source is not limited, and the number of cells which can be researched is large; when some properties of CAR-M in vivo are studied, corresponding experiments in mice with mouse CAR-BMDM as the subject can reflect some conditions of CAR-M in vivo more truly.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a chimeric antigen receptor macrophage and a preparation method and application thereof.
Background
Chimeric antigen receptor macrophages (CAR-M) are cells that have a CAR loaded on them to specifically kill/phagocytose tumor cells. CAR-M has advantages over CAR-T, in addition to killing tumor cells directly: can improve the immune microenvironment of solid tumor; can present antigen as antigen presenting cells after phagocytosis of tumor cells; the tumor can be infiltrated into the tumor more easily, and the tumor can be infiltrated by other immune cells in a synergistic manner. Therefore, the method has very important significance for the research of CAR-M.
Currently, regarding the preparation of CAR-M, the used underpan cells mainly use mouse or human cell lines (such as RAW 264.7; J774A.1; THP-1 cells), while immortalized or tumor cell lines do not represent the properties of primary cells well, and the CAR-M research provides limited effects. In addition, there are also researchers who use human primary macrophages as basal disc cells to prepare CAR-M cells, but the human primary macrophages are limited in source, firstly, the blood cells of healthy volunteers are inconvenient to source, and the proportion of monocytes (for differentiation into macrophages) in healthy human PBMCs is low, about 10%, while almost not being able to expand. Therefore, the human primary macrophage is used as the source of the CAR-M, the number of cells is limited, and the research of the CAR-M is greatly influenced.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a chimeric antigen receptor macrophage and a preparation method and application thereof.
The specific technical scheme is as follows:
the invention provides a chimeric antigen receptor macrophage, wherein the chimeric antigen receptor macrophage is a mouse bone marrow-derived macrophage which is modified by a chimeric antigen receptor and expresses the chimeric antigen receptor.
Further, the modification comprises introducing a nucleic acid or vector encoding a polynucleotide sequence of a chimeric antigen receptor into a mouse bone marrow-derived macrophage;
preferably, the nucleic acids are located on different viral vectors; the virus vector is a lentivirus vector, an adenovirus vector or a retrovirus vector;
preferably, the vector is a transposon or an mRNA vector.
Further, the mouse bone marrow-derived macrophages are mouse primary bone marrow-derived macrophages.
The second aspect of the present invention provides a method for preparing a chimeric antigen receptor macrophage, comprising the steps of:
(1) obtaining primary bone marrow-derived macrophages of a mouse;
(2) and (3) introducing nucleic acid or a vector of a polynucleotide sequence for coding the chimeric antigen receptor into primary mouse bone marrow-derived macrophages to obtain the chimeric antigen receptor.
Further, the method for obtaining the primary bone marrow-derived macrophages of the mice comprises the following steps:
1) extracting bone marrow cells of the mice;
2) adding the mouse bone marrow cells into a DMEM cell culture medium for culturing, changing the culture medium after three days of culture, and completely changing the culture medium after seven days to obtain adherent cells, namely BMDM cells;
the DMEM cell culture medium is a DMEM high-glucose medium containing 10-20% of FBS, 10-20 ng/mL of M-CSF, 1% of penicillin and streptomycin;
preferably, the DMEM cell culture medium is a DMEM high glucose medium containing 20% FBS, 20ng/mL M-CSF, and 1% penicillin and streptomycin;
preferably, the mouse bone marrow cells are cultured in non-TC treated or TC treated T25 cell culture flasks;
preferably, the mouse bone marrow cells are cultured in a non-TC treated T25 cell culture flask;
preferably, step 2) further comprises digesting adherent cells with an ACCUTASE digest.
Further, the concrete operation of extracting the mouse bone marrow cells in the step 1) is as follows: separating and taking down the femur and tibia of the mouse, placing into a cell culture dish containing 75% alcohol, further separating and removing the tissue around the bone, then transferring the tissue into another cell culture dish containing 1 XPBS for cleaning, and finally transferring the tissue into another cell culture dish containing DMEM cell culture medium; removing two ends of the bone of femur and tibia, flushing bone marrow cells from one broken end of the bone into a sterile centrifuge tube by using a DMEM cell culture medium, centrifuging the collected cell suspension at 1500r/min at 4 ℃ for 8min, and removing the supernatant; adding erythrocyte lysate, repeatedly blowing with a suction tube, and standing on ice for 5 min; adding 4-5 times of PBS of erythrocyte lysate to terminate, centrifuging at 1500r/min at 4 deg.C for 8min, and removing supernatant; 5ml of MEM cell culture medium was added to resuspend the cells, and then the cells were filtered through a 70 μm cell screen; centrifuging at 4 deg.C for 8min at 1500r/min, removing supernatant, and repeating for 3 times to obtain the final product;
preferably, the DMEM cell culture medium is a DMEM high-glucose medium containing 10-20% of FBS, 10-20 ng/mL of M-CSF, 1% of penicillin and streptomycin.
Further, the nucleic acids are located on different viral vectors; the virus vector is a lentivirus vector, an adenovirus vector or a retrovirus vector;
the vector is a transposon or an mRNA vector;
the means of introduction include electroporation, transduction, or transfection.
Further, preferably, the nucleic acid encoding the polynucleotide sequence of the chimeric antigen receptor is introduced into mouse primary bone marrow-derived macrophages; the nucleic acid is located on a lentiviral vector;
preferably, the preparation method of the lentivirus vector comprises the following steps: transfecting 293T cells with a lentiviral vector encoding a polynucleotide sequence of a chimeric antigen receptor, a psPAX2 plasmid and a pMD2.G plasmid according to a mass ratio of (10-15): (7.5-10): 3-5) to obtain the lentivirus.
Further, the lentivirus vector is transferred into primary mouse bone marrow-derived macrophages in a transfection mode;
preferably, MOI of the lentivirus vector is 1-1000 when the lentivirus vector transfects primary mouse bone marrow-derived macrophages.
The third aspect of the invention provides the chimeric antigen receptor macrophage or the chimeric antigen receptor macrophage prepared by the preparation method, which is used for researching the property of the chimeric antigen receptor macrophage in a mouse body or used for researching the chimeric antigen receptor macrophage in treating tumors and indications thereof;
preferably, the tumor is a hematological or solid tumor;
preferably, the tumor is leukemia, multiple myeloma, malignant lymphoma, brain glioma, liver cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer or breast cancer;
preferably, the indication is liver fibrosis, lung fibrosis or senile dementia.
The invention has the beneficial effects that:
the chimeric antigen receptor macrophage provided by the invention is prepared from mouse bone marrow-derived macrophage (BMDM), and has the following advantages: (1) as primary macrophages, the condition of CAR-M can be truly reflected; (2) the cell source is not limited, and the number of cells which can be researched is large; (3) when some properties of CAR-M in vivo are studied, corresponding experiments in mice with mouse CAR-BMDM as the subject can reflect some conditions of CAR-M in vivo more truly. The chimeric antigen receptor macrophage lays a foundation for the subsequent research of CAR-M, particularly the in-vivo research of CAR-M, and is also convenient for the research in the treatment of tumors and indications thereof.
Drawings
FIG. 1: positive rate detection of CAR-M cell CAR expression.
FIG. 2: the killing rate of each group was counted after 48 hours of killing (effector cells: target cells ═ 3).
Detailed Description
In order that the invention may be more clearly understood, it will now be further described with reference to the following examples and the accompanying drawings. The examples are for illustration only and do not limit the invention in any way. In the examples, each raw reagent material is commercially available, and the experimental method not specifying the specific conditions is a conventional method and a conventional condition well known in the art, or a condition recommended by an instrument manufacturer.
The culture medium and the reagent adopted by the invention are as follows:
the DMEM cell culture medium adopted in the invention is a DMEM high-glucose medium (Gibco), 10-20% FBS (fetal bovine serum), 10-20 ng/mL M-CSF + 1% double antibody (penicillin-streptomycin). In a specific example, in the step of obtaining BMDM cells, the DMEM cell culture medium is DMEM high glucose medium (Gibco) + 20% FBS (fetal bovine serum) +20ng/mL M-CSF + 1% diabase (penicillin-streptomycin). In the BMDM cell culture step, the DMEM cell culture medium is DMEM high glucose medium (Gibco) + 20% FBS (fetal bovine serum) +20ng/mL M-CSF + 1% double antibody (penicillin-streptomycin).
Erythrocyte lysate, purchased from Biyuntian corporation, product number: C3702.
Opti-MEM medium, purchased from Satemer Feishel science, cat #: 31985088.
ACCUTASE digest, available from Biolegend.
Example 1
This example provides a method for preparing CAR-M using mouse Bone Marrow Derived Macrophages (BMDM), as follows:
(1) acquisition of BMDM cells
C57BL/6N mouse is killed by cervical dislocation method, sterilized fully by 75% alcohol solution, and the mouse is fixed; the femur and tibia of the mouse were separated and removed under sterile conditions, taking care not to break the bone, and then placed 75cm containing 75% alcohol beforehand 2 The cell culture dish of (1); transferring into biological safety cabinet, separating to remove periosseous tissue, transferring into another cell culture dish containing 1 × PBS, washing, and transferring to another 75cm 2 Waiting for the next step of treatment in a cell culture dish (containing DMEM cell culture medium); removing two ends of the femur and tibia with ophthalmic tissue, washing bone marrow cells from one broken end of the bone with a syringe containing 2mL of DMEM cell culture medium into a 50mL sterile centrifuge tube, and repeating for 3 times; centrifuging the collected cell suspension at 4 ℃ for 8min at 1500r/min, and removing the supernatant; adding erythrocyte lysate (1 mL/piece), repeatedly blowing with a suction tube, and standing on ice for 5 min; stop with 4-5 fold lysis of erythrocytes in PBS, 15Centrifuging at 4 ℃ for 8min at 00r/min, and removing supernatant; adding 5mL of DMEM cell culture medium to resuspend the cells, and then filtering the cells by using a 70-micron cell screen; centrifuging at 4 deg.C for 8min at 1500r/min, discarding supernatant, and repeating for 3 times. Mouse bone marrow cells were obtained.
(2) BMDM cell culture
And adding the prepared mouse bone marrow cells into a prepared DMEM cell culture medium to induce the bone marrow cells to differentiate into macrophages. Counting cells, adjusting the cell concentration to 1-3 × 10 6 Culturing the cells/mL in a T25 cell culture bottle (6 mL/bottle) which is not subjected to TC treatment and TC treatment by respectively using two prepared DMEM cell culture media (M-CSF factors with the concentration of 20ng/mL and 10ng/mL are distinguished), culturing the cells in a 5% carbon dioxide incubator at 37 ℃, replacing the culture medium after three days of culture, completely replacing the culture medium after seven days, and obtaining adherent cells which are BMDM cells. The purity was then determined by flow cytometry using ACCUTASE digest to compare the number of BMDM cells obtained from different factor concentrations in the medium and different flasks and F4/80 staining. The results show that the yield of BMDM cells cultured by 20ng/mL M-CSF is higher, and the purity is not obviously different from that of a 10ng/mL group. BMDM cells cultured in non-TC treated T25 cell culture flasks were obtained in higher yields and in a purity comparable to that of the TC treated T25 cell culture flask group.
Finally, when the BMDM cells infected by the lentivirus are cultured, a non-TC-treated T25 cell culture flask is adopted, and the DMEM cell culture medium is a DMEM high-glucose medium (Gibco) + 20% FBS (fetal bovine serum) +20ng/mL M-CSF + 1% double antibody (penicillin-streptomycin).
(3) Preparation of lentiviruses
Preparing a target plasmid and a packaging plasmid: extracting the required target plasmid and the second generation packaging plasmids psPAX2 and pMD2.G by using a plasmid macroextraction kit, and detecting the concentration of the corresponding plasmid by using a Nano Drop instrument.
Preparing 293T cells: recovering 293T cells for 2-3 generations; the day before the virus was coated, 293T cells were trypsinized and counted, and 3.5X 10 cells were inoculated 6 The left and right cells were plated on 10cm dishes with DMEM complete medium to 10 mL; when the cell fusion degree reaches about 70-80%, replacing 5mL of new antibiotic-free DThe virus coating is ready after MEM culture.
③ Liposome transfection: add 20. mu.L Lipofectamine 3000 to 500. mu.L Opti-MEM medium in a 1.5mL EP tube, mix gently with a gun without air bubbles, and let stand at room temperature for 5 min; 10-15. mu.g of the plasmid of interest (either pHR CD19-FcR γ CAR or pHR CD19-Megf-10 CAR plasmid, both containing GFP tags), 7.5-10. mu.g of psPAX2 plasmid, 3-5. mu.g of pMD2.G plasmid were then added to a 500. mu.L Opti-MEM culture medium in another 1.5mL EP tube, followed by addition of 20. mu. L P3000 reagent and gently mixed with a gun without air bubbles; mixing the two mixed solutions, blowing the mixed solutions uniformly by using a gun with light force for multiple times, standing the mixed solutions for 15min at room temperature, transferring the mixed solutions into 293T cell culture solution for later use after liquid change is finished by using the gun, and slightly shaking the culture dish without shaking the 293T cells to separate the 293T cells from the bottom of the culture dish; finally, placing the culture dish into a 37 ℃ and 5% carbon dioxide incubator, sucking out the culture medium containing the liposome after 6-8h, and changing the culture medium into a normal DMEM complete culture medium;
fourthly, toxin collection (operation in the biological safety cabinet): sucking out the culture solution supernatant of the 293T cells cultured for 48h into a 15mL centrifugal tube, and then supplementing 10mL293T cell culture solution into the culture dish (continuously culturing the cells, and recovering the virus once after 24 h); then placing the supernatant of the culture fluid in a centrifuge for 10min at 3000rpm, sucking the supernatant by using a syringe, and filtering the supernatant by using a 0.45 mu m filter to obtain CD19-FcR gamma CAR and CD19-Megf-10 CAR lentivirus for later use (the lentivirus can be directly used or can be stored in a refrigerator at-80 ℃).
(4) Lentiviral infection of BMDM cells
Firstly, separating and culturing mouse BMDM according to the method for obtaining the BMDM cells, sucking a culture solution of the BMDM cells with good growth state till the seventh day of culture, and washing the BMDM cells once by using DMEM without antibiotics; ② adding the collected CD19-FcR gamma CAR or CD19-Megf-10 CAR lentivirus into BMDM cells respectively, wherein Blank group is added with equal amount of Blank culture medium. Sealing the mouth of the culture bottle by using a sealing film, and putting the culture bottle into a 5% carbon dioxide incubator at 37 ℃ for culture; ③ 48h later, the culture medium containing the virus liquid is removed, and a new BMDM culture medium is added (the discarded supernatant, the used centrifuge tube and the like must be sterilized under high pressure and cannot be thrown away, and 75 percent ethanol is sprayed for disinfection after the centrifuge is used); and fourthly, after 72 hours of infection, obtaining FcR gamma CAR-M and Megf10 CAR-M cells, and detecting the positive rate of the cells by a fluorescence microscope or a flow cytometer.
(5) Detection of the efficiency of lentivirus infection of BMDM
After 72h, virus-infected BMDM cells were collected using ACCUTASE cell digest and CAR expression and virus infection efficiency of BMDM cells were examined using flow cytometry. Results as shown in figure 1, CARs were successfully expressed on the cell surface of BMDM.
(6) Detection of killing of target cells by CAR-M
The cell killing experiment is carried out by collecting and analyzing the fluorescence intensity by using a SPECTROStar Omega enzyme-labeling instrument. BMDM, FcR gamma CAR-M and Megf10 CAR-M cells were first treated at 2-5X 10 4 And/well inoculating to a lightproof 96-well cell culture plate, each group comprises 5-10 multiple wells, and standing for 24-48 h. After 24h according to effector cell: target cells 3: 1 Raji cells stably expressing luciferase (serum-free, M-CSF-free DMEM medium) were added and co-cultured with each group of macrophages. The experiments were divided into 4 groups: raji blank, BMDM, FcR gamma CAR-M and Megf10 CAR-M cells.
After culturing for 24-48h in a 5% carbon dioxide incubator at 37 ℃, adding D-luciferin and potassiumsalt substrates with the concentration of 100-:
% cell Lysis (lysine%) [ 1- (fluorescence signal of co-cultured cells-background fluorescence signal)
(culture of PC-3 cells alone-background fluorescence Signal) ]100
Killing results are shown in figure 2, and compared with a BMDM control group, the prepared FcR gamma CAR-M and Megf10 CAR-M cells can obviously kill Raji target cells, which shows that the FcR gamma CAR-M and Megf10 CAR-M cells with the target killing function are successfully prepared.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A chimeric antigen receptor macrophage, wherein said chimeric antigen receptor macrophage is a mouse bone marrow-derived macrophage modified with and expressing a chimeric antigen receptor.
2. The chimeric antigen receptor macrophage according to claim 1, wherein the modification comprises introducing a nucleic acid or vector encoding a polynucleotide sequence of the chimeric antigen receptor into mouse bone marrow-derived macrophages;
preferably, the nucleic acids are located on different viral vectors; the viral vector is a lentivirus vector, an adenovirus vector or a retrovirus vector;
preferably, the vector is a transposon or an mRNA vector.
3. The chimeric antigen receptor macrophage according to claim 1, wherein the mouse bone marrow-derived macrophage is a mouse primary bone marrow-derived macrophage.
4. A method for preparing a chimeric antigen receptor macrophage, comprising the steps of:
(1) obtaining primary bone marrow-derived macrophages of a mouse;
(2) and (3) introducing nucleic acid or a vector of a polynucleotide sequence for coding the chimeric antigen receptor into primary mouse bone marrow-derived macrophages to obtain the chimeric antigen receptor.
5. The method of claim 4, wherein the primary bone marrow-derived macrophages are obtained by:
1) extracting mouse bone marrow cells;
2) adding mouse bone marrow cells into a DMEM cell culture medium for culturing, changing the culture medium after culturing for three days, and completely changing the culture medium after culturing for seven days to obtain adherent cells, namely BMDM cells;
the DMEM cell culture medium is a DMEM high-glucose medium containing 10-20% of FBS, 10-20 ng/mL of M-CSF, 1% of penicillin and streptomycin;
preferably, the DMEM cell culture medium is a DMEM high glucose medium containing 20% FBS, 20ng/mL M-CSF, and 1% penicillin and streptomycin;
preferably, the mouse bone marrow cells are cultured in non-TC treated or TC treated T25 cell culture flasks;
preferably, the mouse bone marrow cells are cultured in a non-TC treated T25 cell culture flask;
preferably, step 2) further comprises digesting adherent cells with an ACCUTASE digest.
6. The method according to claim 5, wherein the step 1) of extracting bone marrow cells of mice comprises the following steps: separating and taking down the femur and tibia of the mouse, placing into a cell culture dish containing 75% alcohol, further separating and removing the tissue around the bone, then transferring the tissue into another cell culture dish containing 1 XPBS for cleaning, and finally transferring the tissue into another cell culture dish containing DMEM cell culture medium; removing two ends of the bone of femur and tibia, flushing bone marrow cells from one broken end of the bone into a sterile centrifuge tube by using a DMEM cell culture medium, centrifuging the collected cell suspension at 1500r/min at 4 ℃ for 8min, and removing the supernatant; adding erythrocyte lysate, repeatedly blowing with a suction tube, and standing on ice for 5 min; adding 4-5 times of PBS of erythrocyte lysate to terminate, centrifuging at 1500r/min and 4 deg.C for 8min, and discarding supernatant; 5ml of MEM cell culture medium was added to resuspend the cells, and then the cells were filtered through a 70 μm cell screen; centrifuging at 4 deg.C for 8min at 1500r/min, discarding supernatant, and repeating for 3 times to obtain the final product;
preferably, the DMEM cell culture medium is a DMEM high-glucose medium containing 10-20% of FBS, 10-20 ng/mL of M-CSF, 1% of penicillin and streptomycin.
7. The method of claim 4, wherein the nucleic acids are located on different viral vectors; the virus vector is a lentivirus vector, an adenovirus vector or a retrovirus vector;
the vector is a transposon or an mRNA vector;
the means of introduction include electroporation, transduction, or transfection.
8. The method of claim 4, wherein the nucleic acid encoding the polynucleotide sequence of the chimeric antigen receptor is preferably introduced into mouse primary bone marrow-derived macrophages; the nucleic acid is located on a lentiviral vector;
preferably, the preparation method of the lentiviral vector comprises the following steps: transfecting 293T cells with a lentiviral vector encoding a polynucleotide sequence of a chimeric antigen receptor, a psPAX2 plasmid and a pMD2.G plasmid according to a mass ratio of (10-15): (7.5-10): 3-5) to obtain the lentivirus.
9. The method of claim 8, wherein the lentiviral vector is transfected into primary bone marrow-derived macrophages in mice;
preferably, MOI of the lentivirus vector is 1-1000 when the lentivirus vector transfects primary mouse bone marrow-derived macrophages.
10. The use of the chimeric antigen receptor macrophage of any one of claims 1-3 or the chimeric antigen receptor macrophage prepared by the preparation method of any one of claims 4-9 for studying the properties of the chimeric antigen receptor macrophage in mice or for studying the chimeric antigen receptor macrophage in the treatment of tumors and indications thereof;
preferably, the tumor is a hematological or solid tumor;
preferably, the tumor is leukemia, multiple myeloma, malignant lymphoma, brain glioma, liver cancer, lung cancer, stomach cancer, colon cancer, pancreatic cancer or breast cancer;
preferably, the indication is liver fibrosis, lung fibrosis or senile dementia.
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| CN202210710127.7A CN115011561A (en) | 2022-06-22 | 2022-06-22 | Chimeric antigen receptor macrophage and preparation method and application thereof |
| PCT/CN2022/137819 WO2023246003A1 (en) | 2022-06-22 | 2022-12-09 | Chimeric antigen receptor macrophage, method for preparing same, and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116240240A (en) * | 2023-03-02 | 2023-06-09 | 赛元生物科技(杭州)有限公司 | Methods for improving efficiency of adenovirus transfection of macrophages using combination of MPLA and IFN-gamma |
| WO2023246003A1 (en) * | 2022-06-22 | 2023-12-28 | 深圳先进技术研究院 | Chimeric antigen receptor macrophage, method for preparing same, and use thereof |
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| WO2023246003A1 (en) | 2023-12-28 |
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