Application of coenzyme in suspension culture of adherent cells

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of coenzyme in suspension culture of adherent cells. Based on the fact that the inventor finds that coenzyme can promote the growth mode of adherent cells to be converted from adherent to suspension, the invention discloses application of the coenzyme in suspension culture of the adherent cells and induction of differentiation of mononuclear cells into suspension macrophages for the first time, and provides a culture medium. The culture medium is a basic culture medium (culture medium A) containing a coenzyme or a basic culture medium (culture medium B) containing a macrophage inducer and a coenzyme, the culture medium A can greatly improve the harvesting efficiency and yield of the adherent cells, and can maintain good stability and long-time high activity of the adherent cells; the culture medium B can greatly improve the harvesting efficiency and yield of the macrophages, can maintain good stability and long-time high activity of the macrophages, provides favorable conditions for subsequent preparation preparation of macrophage products, and has a great application prospect in clinical research.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to the application of coenzymes in suspension cultured adherent cells. Background Art

[0002] Macrophages are a type of nonspecific immune cell derived from the myeloid lineage. They play an important role in the immune system and participate in regulating and controlling immune responses. In recent years, more and more evidence has shown that macrophages play an important role in the occurrence and development of tumors. On the one hand, macrophages can inhibit tumor growth by directly engulfing and killing tumor cells; on the other hand, macrophages can also activate and regulate immune responses through antigen presentation, presenting tumor antigens to T lymphocytes, thereby activating the immune response of specific T lymphocytes and B lymphocytes.

[0003] Macrophages are mainly differentiated from monocytes in the blood. When cultured in vitro, monocytes are differentiated into macrophages by adding granulocyte-macrophage colony-stimulating factor (GM-CSF). However, the content of monocytes in white blood cells is low, and neither monocytes nor macrophages can proliferate in vitro. Therefore, if you want to make macrophages into cell therapy drugs, the first factor is to increase the yield of macrophages. Because monocytes or macrophages are adherent when cultured in vitro, they need to be digested by adding cell dissociation reagents when harvesting. This operation is cumbersome and not conducive to increasing the yield of macrophages. Therefore, it is necessary to develop a method for suspending and culturing adherent cells. Summary of the invention

[0004] The first aspect of the present invention aims to provide the application of coenzyme.

[0005] The second aspect of the present invention aims to provide a culture medium.

[0006] The third aspect of the present invention aims to provide a method for culturing adherent cells in suspension.

[0007] The fourth aspect of the present invention aims to provide a method for inducing monocytes to differentiate into suspended macrophages.

[0008] The purpose of the fifth aspect of the present invention is to provide a method for preparing CAR (chimeric antigen receptor)-macrophages.

[0009] The sixth aspect of the present invention aims to provide cells obtained by the method of the third aspect of the present invention.

[0010] The seventh aspect of the present invention aims to provide macrophages obtained by the method of the fourth aspect of the present invention.

[0011] The purpose of the eighth aspect of the present invention is to provide CAR-macrophages obtained by the method of the fifth aspect of the present invention.

[0012] The purpose of the ninth aspect of the present invention is to provide the use of the cell of the sixth aspect of the present invention, the macrophage of the seventh aspect of the present invention, or the CAR-macrophage of the eighth aspect of the present invention in the preparation of anti-tumor drugs.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] The first aspect of the present invention provides the use of a coenzyme in any one of a1) to a4):

[0015] a1) Suspension culture of adherent cells;

[0016] a2) preparing products for suspension culture of adherent cells;

[0017] a3) Inducing monocytes to differentiate into suspended macrophages;

[0018] a4) preparing a product for inducing monocytes to differentiate into suspended macrophages.

[0019] In some embodiments, the coenzyme comprises at least one of adenosine triphosphate (ATP) or a salt thereof, and reduced nicotinamide adenine dinucleotide phosphate (NADPH); further adenosine triphosphate (ATP) or a salt thereof; and further ATP disodium.

[0020] In some embodiments, the adherent cells include at least one of monocytes and macrophages; further monocytes.

[0021] In some embodiments, the adherent cells are CAR-adherent cells.

[0022] In some embodiments, the macrophages in a3) and a4) are CAR-macrophages.

[0023] In some embodiments, the product is culture medium.

[0024] The second aspect of the present invention provides a culture medium.

[0025] (1) A culture medium (referred to as culture medium A), which is a basal culture medium containing coenzymes.

[0026] In some embodiments, the culture medium consists of coenzymes and basal medium.

[0027] In some embodiments, the coenzyme comprises at least one of adenosine triphosphate (ATP) or a salt thereof, and reduced nicotinamide adenine dinucleotide phosphate (NADPH); further adenosine triphosphate (ATP) or a salt thereof; and further ATP disodium.

[0028] In some embodiments, the basal culture medium comprises at least one of Advanced Dulbecco's Modified Eagle Medium (AdvancedDMEM), DMEM, RPMI (Roswell Park Memorial Institute) 1640 medium, X-VIVO medium (hematopoietic medium) and William's E medium; further X-VIVO medium; and further X-VIVO15.

[0029] In some embodiments, the concentration of the coenzyme in the culture medium is 10-800 μg / mL; further 30-750 μg / mL; further 140-160 μg / mL.

[0030] In some embodiments, the basal medium is a basal medium containing serum, preferably fetal bovine serum (FBS).

[0031] In some embodiments, the concentration of the serum in the basal medium is 5-25% by volume; further 5-15%; further 9-11% by volume.

[0032] In some embodiments, the culture medium (referred to as culture medium A) is used for suspension culture of adherent cells.

[0033] In some embodiments, the adherent cells include at least one of monocytes and macrophages; further monocytes.

[0034] In some embodiments, the adherent cells are CAR-adherent cells.

[0035] In some embodiments, the culture medium further comprises instructions, which describe a method for culturing adherent cells in suspension.

[0036] In some embodiments, the method for culturing adherent cells in suspension is the method for culturing adherent cells (preferably CAR-adherent cells) in suspension according to the third aspect of the present invention.

[0037] (ii) A culture medium (referred to as culture medium B), which is a basic culture medium containing a macrophage inducer and coenzymes.

[0038] In some embodiments, the culture medium consists of a macrophage inducer, a coenzyme, and a basal medium.

[0039] In some embodiments, the macrophage inducing agent comprises at least one of granulocyte-macrophage colony stimulating factor (GM-CSF) and phorbol ester (PMA); further GM-CSF; and further recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF).

[0040] In some embodiments, the coenzyme comprises at least one of adenosine triphosphate (ATP) or a salt thereof, and reduced nicotinamide adenine dinucleotide phosphate (NADPH); further adenosine triphosphate (ATP) or a salt thereof; and further ATP disodium.

[0041] In some embodiments, the basal culture medium comprises at least one of Advanced Dulbecco's Modified Eagle Medium (AdvancedDMEM), DMEM, RPMI (Roswell Park Memorial Institute) 1640 medium, X-VIVO medium (hematopoietic medium) and William's E medium; further X-VIVO medium; and further X-VIVO15.

[0042] In some embodiments, the concentration of the macrophage inducer in the culture medium is 25-150 ng / mL; further 25-75 ng / mL; further 46-54 ng / mL.

[0043] In some embodiments, the concentration of the coenzyme in the culture medium is 10-800 μg / mL; further 30-750 μg / mL; further 140-160 μg / mL.

[0044] In some embodiments, the basal medium is a basal medium containing serum, preferably fetal bovine serum (FBS).

[0045] In some embodiments, the concentration of the serum in the basal medium is 5-25% by volume; further 5-15%; further 9-11% by volume.

[0046] In some embodiments, the culture medium (referred to as culture medium B) is used to induce monocytes to differentiate into suspended macrophages.

[0047] In some embodiments, the culture medium further comprises instructions describing a method for inducing monocytes to differentiate into suspension macrophages.

[0048] In some embodiments, the macrophage is a CAR-macrophage.

[0049] In some embodiments, the method of inducing monocytes to differentiate into suspended macrophages is the method of inducing monocytes to differentiate into suspended macrophages of the fourth aspect of the present invention or the method of preparing CAR-macrophages (preferably suspended CAR-macrophages) of the fifth aspect of the present invention.

[0050] The third aspect of the present invention provides a method for culturing adherent cells in suspension, wherein the adherent cells are cultured using the culture medium A in the second aspect of the present invention.

[0051] In some embodiments, the adherent cells include at least one of monocytes and macrophages; further monocytes.

[0052] In some embodiments, the culture conditions are 33-40° C., 3-7% CO 2 ; further 36-38° C., 4-6% CO 2 .

[0053] In some embodiments, the culture time is 1-3 days; further 1.5-2.5 days.

[0054] In some embodiments, the ratio of the adherent cells to the medium A is 3×10 6 Pieces: 2-4mL.

[0055] A method for culturing CAR-adherent cells in suspension, wherein the adherent cells are cultured in a system comprising an adenovirus expressing CAR and the culture medium A in the second aspect of the present invention.

[0056] In some embodiments, the adherent cells include at least one of monocytes and macrophages; further monocytes.

[0057] In some embodiments, the ratio of the adenovirus to the adherent cells is 2000 IFU: 1-4 (MOI is 500-2000).

[0058] In some embodiments, the culture conditions are 33-40° C., 3-7% CO 2 ; further 36-38° C., 4-6% CO 2 .

[0059] In some embodiments, the culture time is 1-3 days; further 1.5-2.5 days.

[0060] In some embodiments, the ratio of the adherent cells to the medium A is 3×10 6 Pieces: 2-4mL.

[0061] A fourth aspect of the present invention provides a method for inducing monocytes to differentiate into suspended macrophages, wherein the monocytes are cultured using the culture medium B in the second aspect of the present invention.

[0062] In some embodiments, the culture conditions are 33-40° C., 3-7% CO 2 ; further 36-38° C., 4-6% CO 2 .

[0063] In some embodiments, the culture time is 5-10 days; further 6-8 days.

[0064] In some embodiments, the medium is changed once on days 1-3 of culture.

[0065] In some embodiments, the ratio of the mononuclear cells to the medium B is 3×10 6 Pieces: 2-4mL.

[0066] The fifth aspect of the present invention provides a method for preparing CAR-macrophages (preferably suspended CAR-macrophages), wherein the monocytes are cultured using the culture medium B in the second aspect of the present invention, and during the culture process, the nucleic acid molecule encoding the CAR, or an expression cassette or vector containing the nucleic acid molecule is delivered to the macrophages.

[0067] In some embodiments, the culture conditions are 33-40° C., 3-7% CO 2 ; further 36-38° C., 4-6% CO 2 .

[0068] In some embodiments, the culture time is 5-10 days; further 6-8 days.

[0069] In some embodiments, the medium is changed once on days 1-3 of culture.

[0070] In some embodiments, the ratio of the mononuclear cells to the medium B is 3×10 6 Pieces: 2-4mL.

[0071] In some embodiments, the method of delivering the nucleic acid molecule encoding the CAR, or the expression cassette or vector comprising the nucleic acid molecule to the macrophage is to add an adenovirus expressing the CAR.

[0072] In some embodiments, the CAR-expressing adenovirus is added on day 4-5.5 of culture.

[0073] In some embodiments, the dosage ratio of the adenovirus to the monocytes is 2000 IFU: 1-4 (MOI is 500-2000).

[0074] The sixth aspect of the present invention provides cells obtained by the method of the third aspect of the present invention.

[0075] The seventh aspect of the present invention provides macrophages obtained by the method of the fourth aspect of the present invention.

[0076] The eighth aspect of the present invention provides CAR-macrophages obtained by the method of the fifth aspect of the present invention.

[0077] The ninth aspect of the present invention provides the use of the cell of the sixth aspect of the present invention, the macrophage of the seventh aspect of the present invention, or the CAR-macrophage of the eighth aspect of the present invention in the preparation of an anti-tumor drug.

[0078] In some embodiments, the tumor comprises at least one of a solid tumor and a hematological tumor; preferably a solid tumor.

[0079] In some embodiments, the solid tumor comprises liver cancer, colorectal cancer, bladder cancer, breast cancer, cervical cancer, prostate cancer, glioma, melanoma, pancreatic cancer, nasopharyngeal cancer, lung cancer, gastric cancer, adrenocortical carcinoma, pararenal cortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumor, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, heart tumor, bile duct epithelial carcinoma, chordoma, colorectal cancer, craniopharyngioma, ductal carcinoma in situ, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, intracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, fallopian tube cancer, gallbladder cancer, head and neck cancer, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, Langerhans cell At least one of histiocytosis, laryngeal cancer, lip cancer, oral cancer, Merkel cell carcinoma, malignant mesothelioma, multiple endocrine neoplasia syndrome, mycosis fungoides, sinonasal cancer, neuroblastoma, non-small cell lung cancer, ovarian cancer, pancreatic neuroendocrine tumors, islet cell tumors, papillomatosis, paraganglioma, sinonasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, retinoblastoma, salivary gland tumor, sarcoma, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, testicular cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, endometrial and uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer and single myeloma.

[0080] In some embodiments, the blood tumor is selected from at least one of B-cell acute lymphoid leukemia, T-cell acute lymphoid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell-follicular lymphoma, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, non-Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia and preleukemia.

[0081] The beneficial effects of the present invention are:

[0082] The present invention is based on the inventor's discovery that coenzymes can promote the transformation of the growth mode of adherent cells from adherence to suspension, and further discloses for the first time the use of coenzymes in suspension culture of adherent cells and inducing monocytes to differentiate into suspended macrophages, and provides a culture medium, which is a basal culture medium (culture medium A) containing coenzymes or a basal culture medium (culture medium B) containing macrophage inducers and coenzymes. Culture medium A can greatly improve the harvesting efficiency and yield of adherent cells, and can also maintain good stability and long-term high activity of adherent cells; culture medium B can greatly improve the harvesting efficiency and yield of macrophages, and can also maintain good stability and long-term high activity of macrophages, and also provides favorable conditions for the subsequent preparation of macrophage products, and has great application prospects in clinical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 The viability and yield of cells induced to differentiate into macrophages and CAR-macrophages by using the culture medium of Example 1 and Comparative Example 1 are shown (n=4).

[0084] Figure 2 The cell morphology of macrophages and CAR-macrophages prepared using the culture medium of Example 1 and Comparative Example 1 is shown (scale is 1:100).

[0085] Figure 3 The cell viability and cell yield of CAR-monocytes prepared by culturing with the culture medium of Example 4, Example 5, Example 6 and Comparative Example 2 are shown (n=4).

[0086] Figure 4 The cell morphology of CAR-monocytes prepared by culturing with the culture medium of Example 4, Example 5, Example 6 and Comparative Example 2 is shown (scale is 1:100). DETAILED DESCRIPTION

[0087] definition

[0088] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0089] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0090] Adherent cells refer to cells that must have a support surface to attach to in order to grow. They rely on attachment factors secreted by themselves or provided in the culture medium to grow and reproduce on the surface.

[0091] Monocytes are a subset of leukocytes that play a key role in maintaining body homeostasis, pathogen recognition and clearance, and inflammation. After generation and development from myeloid progenitor cells, monocytes circulate in the vasculature, bone marrow, and spleen; they do not proliferate in steady state. Once monocytes migrate to peripheral tissues, either during normal development or due to pathogen attack, they differentiate into dendritic cells or macrophages.

[0092] Macrophages are specialized, long-lived, phagocytic cells of the innate immune system. Together with neutrophils, they are the first responders to infection. Macrophages are involved in the recognition, phagocytosis, and degradation of cell debris and pathogens. Macrophages also play a role in presenting antigens to T cells and inducing other antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. In addition, at the beginning of inflammation, macrophages play an important role by releasing cytokines and chemokines, which in turn recruit other immune cells to the site of inflammation. Macrophages are present in most tissues and therefore have different functions. In addition to initiating immune and inflammatory responses to pathogens, macrophages also play a role in maintaining tissue homeostasis and repairing and remodeling tissues. Macrophages also appear to play a key role in the tumor microenvironment, especially in matrix remodeling, angiogenesis, metastasis, and tumor progression. There are many sources of macrophages. Tissue-resident macrophages can differentiate from circulating monocytes or be generated during embryonic development in the fetal liver, yolk sac, or embryonic regions near the dorsal aorta and thus exist independently of monocytes in adulthood.

[0093] The present invention is further described in detail below through specific examples.

[0094] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0095] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the examples are commercially available unless otherwise specified. The manufacturers of the reagents used are indicated, and similar products from other manufacturers are substitutes.

[0096] The preparation method of monocytes used in the following examples / comparative examples is as follows:

[0097] Reagents and consumables: Single blood collection (Leukopak, with relevant ethical approval), Ficoll-Paque TM PREMIUMsterile solution (Cytiva), D-PBS (without calcium or magnesium ions, Yeasen), autoMACS Running Buffer (Miltenyi), CD14 MicroBeads Human (Miltenyi), X-VIVO 15 (Lonza), FBS (gibco), rhGM-CSF (Xiamen Tebao), adenosine triphosphate disodium injection (Guangdong Nanguo), various types of pipettes (Corning), various types of centrifuge tubes (Corning), various types of sterile containers (Corning), 6-well cell culture plates (TC treated, Corning).

[0098] Equipment: biological safety cabinet (BSC), micropipette, electric pipette, centrifuge, cell counter, CO2 incubator, inverted biological microscope.

[0099] 1. Single blood collection to isolate peripheral blood mononuclear cells (PBMC)

[0100] 1.1 (All operations are performed in BSC) Dilute Leukopak (the amount of Leukopak depends on the desired number of monocytes. If the number of monocytes is 1, the amount of Leukopak required is 10) with D-PBS in a certain ratio (range Leukopak: D-PBS = 1:1);

[0101] 1.2 Prepare Ficoll tubes, add 20 mL Ficoll to a 50 mL centrifuge tube, add 30 mL / tube of diluted Leukopak to the Ficoll tube, and perform density gradient centrifugation at 400 g, 30 min, 18-22 °C, ramp rate 1, ramp rate 0;

[0102] 1.3 After centrifugation, the solution in the centrifuge tube can be seen to be divided into three layers: the upper layer is plasma and D-PBS, the lower layer is mainly red blood cells and granulocytes, and the middle layer is lymphocyte separation solution; at the interface between the upper and middle layers, there is a white film layer mainly composed of mononuclear cells (PBMC) (PBMC includes lymphocytes and monocytes, and also contains platelets), absorb the white film layer;

[0103] 1.4 Wash PBMC to remove platelets, wash three times, wash solution: D-PBS, centrifugation parameters are: 550g × 10min, 400g × 10min, 300g × 10min;

[0104] 1.5 After washing, discard the supernatant, add Running buffer to suspend PBMC, take a small amount of PBMC for counting, and then make the density of suspended live cells: 1×10 8 cells / mL;

[0105] 2. Isolation of Monocytes

[0106] 2.1 According to the instructions of CD14 MicroBeads Human (Miltenyi), add CD14 MicroBeads to incubate PBMCs to allow them to bind to the target cells - monocytes;

[0107] 2.2 After incubation, add Running Buffer to terminate incubation and centrifuge to remove unbound CD14 MicroBeads;

[0108] 2.3 Sort CD14+ cells, i.e. monocytes, according to the LS column instructions.

[0109] Example 1. A culture medium for inducing monocytes to differentiate into suspended macrophages

[0110] A culture medium for inducing monocytes to differentiate into suspended macrophages, which is a basic culture medium containing 50 ng / mL rhGM-CSF (recombinant human granulocyte-macrophage colony stimulating factor) and 150 μg / mL adenosine triphosphate disodium (ATP disodium, Nan Guo); wherein the basic culture medium is X-VIVO 15 (Lonza) containing 10% v / v FBS (gibco).

[0111] Example 2. A culture medium for inducing monocytes to differentiate into suspended macrophages

[0112] A culture medium for inducing monocytes to differentiate into suspended macrophages, which is a basic culture medium containing 25 ng / mL rhGM-CSF (recombinant human granulocyte-macrophage colony stimulating factor) and 30 μg / mL ATP disodium (adenosine triphosphate disodium, Nan Guo); wherein the basic culture medium is X-VIVO15 (Lonza) containing 10% v / v FBS (gibco).

[0113] Example 3. A culture medium for inducing monocytes to differentiate into suspended macrophages

[0114] A culture medium for inducing monocytes to differentiate into suspended macrophages, which is a basic culture medium containing 150 ng / mL rhGM-CSF (recombinant human granulocyte-macrophage colony stimulating factor) and 750 μg / mL ATP disodium (adenosine triphosphate disodium, Nan Guo); wherein the basic culture medium is X-VIVO15 (Lonza) containing 10% v / v FBS (gibco).

[0115] Example 4. A culture medium for suspension culture of adherent cells

[0116] A culture medium for suspending adherent cells is a basic culture medium containing 150 μg / mL adenosine triphosphate disodium (ATP disodium, Nan Guo); wherein the basic culture medium is X-VIVO15 (Lonza) containing 10% v / v FBS (gibco).

[0117] Example 5. A culture medium for suspension culture of adherent cells

[0118] A culture medium for suspending adherent cells is a basic culture medium containing 30 μg / mL adenosine triphosphate disodium (ATP disodium, Nan Guo); wherein the basic culture medium is X-VIVO15 (Lonza) containing 10% v / v FBS (gibco).

[0119] Example 6. A culture medium for suspension culture of adherent cells

[0120] A culture medium for suspending adherent cells is a basic culture medium containing 750 μg / mL adenosine triphosphate (ATP, Nan Guo); wherein the basic culture medium is X-VIVO15 (Lonza) containing 10% v / v FBS (gibco).

[0121] Comparative Example 1. A culture medium for inducing monocytes to differentiate into macrophages

[0122] A culture medium for inducing monocytes to differentiate into macrophages is a basic culture medium containing 50 ng / mL rhGM-CSF (recombinant human granulocyte-macrophage colony stimulating factor); wherein the basic culture medium is X-VIVO15 (Lonza) containing 10% v / v FBS (gibco).

[0123] Comparative Example 2. A culture medium for culturing adherent cells

[0124] A culture medium for culturing adherent cells is X-VIVO 15 (Lonza) containing 10% v / v FBS (gibco).

[0125] Effect Example

[0126] 1. A method for preparing suspended macrophages and CAR-macrophage culture

[0127] 1) A method for preparing suspended macrophages (UTD) from monocytes, comprising the following steps: dividing the monocytes into 8 portions, each portion containing 3×10 6 cells, 3 mL of the culture medium of Example 1 (n=4) or Comparative Example 1 (n=4) was added to each portion to resuspend the monocytes, and 4 portions of cells of Example 1 were inoculated into a 6-well cell culture plate, with 1 well for each portion; 4 portions of cells of Comparative Example 1 were inoculated into a 6-well cell culture plate (recorded as day0), and cultured at 37°C and 5% CO2; on the second day of culture (day2), the medium was changed (the same culture medium as before).

[0128] 2) A method for preparing suspended CAR-macrophages (CAR) from monocytes, comprising the following steps: dividing the monocytes into 8 portions, each portion containing 3×10 6 cells, 3 mL of the culture medium of Example 1 (n=4) or Comparative Example 1 (n=4) was added to each portion to resuspend the monocytes, and the 4 portions of cells of Example 1 were inoculated into a 6-well cell culture plate, with 1 well for each portion; the 4 portions of cells of Comparative Example 1 were inoculated into a 6-well cell culture plate (recorded as day0), and cultured at 37°C and 5% CO2; on the second day of culture (day2), the medium (the same culture medium as before) was replaced, and on the fifth day of culture (day5), adenovirus expressing CAR (the preparation method refers to Example 1 of patent document CN118652355A) was added for transfection (MOI: 1000).

[0129] On the 7th day of culture (day 7), the morphology of the macrophages (UTD) or CAR-macrophages (CAR) of Example 1 and Comparative Example 1 was photographed under a microscope (eg Figure 2As shown: In Example 1, UTD and CAR cells are basically in suspension at day 7, while in Comparative Example 1, UTD and CAR are more attached to the wall at day 7). Then the cells are harvested by blowing the cells (without trypsin digestion), and the cells are counted by a cell counter after harvesting. The viability of macrophages (displayed by the counter), live macrophages (obtained according to the live cell concentration (displayed by the counter) × volume), and then the yield cell yield (cell yield = live macrophages / number of inoculated monocytes) are calculated. The results are shown in FIG. Figure 1 As shown: the cell viability of the method using the culture medium of Example 1 is equivalent to that of the method using Comparative Example 1, and its cell yield is much higher than that of the method using Comparative Example 1, that is, adding coenzymes (such as; disodium ATP) in the system of inducing monocyte differentiation and culture into macrophages or CAR-macrophages can improve the harvest efficiency of suspended macrophages or CAR-macrophages (i.e., yield cell yield) while maintaining good stability and long-term high activity of macrophages or CAR-macrophages (i.e., viability cell viability). The effects (cell viability, cell viability) of the methods using the culture medium of Examples 2 and 3 are similar to those of Example 1.

[0130] 2. A method for preparing suspended CAR-monocyte culture, comprising the following steps:

[0131] The mononuclear cells were divided into 16 portions, each containing 3 × 10 6 cells, 3 mL of the culture medium of Example 4, Example 5, Example 6 or Comparative Example 2 (n = 4) was added to each portion to resuspend the monocytes, and 12 portions of cells of Example 4 (n = 4), Example 5 (n = 4) and Example 6 (n = 4) were inoculated into a 6-well cell culture plate, with 1 well per portion; 4 portions of cells of Comparative Example 2 were inoculated into a 6-well cell culture plate (recorded as day 0), and 16 portions of cells were transfected with adenovirus expressing CAR (the preparation method refers to Example 1 of patent document CN118652355A) (MOI: 1000), and cultured at 37° C. and 5% CO2.

[0132] On the second day of culture (day 2), the morphology of the CAR-monocytes of Example 4, Example 5, Example 6 and Comparative Example 2 was photographed under a microscope (eg, Figure 4As shown: In Example 4, the CAR-monocytes were all in suspension at day 2, followed by Example 5 and Example 6. In Comparative Example 2, most of the monocytes were already attached to the wall at day 2. ), then 16 cells were harvested, the cells were harvested by blowing (without trypsin digestion), and the cells were counted by a cell counter after harvesting. The viability of the CAR-monocytes (displayed by the counter), the number of live CAR-monocytes (obtained according to the live cell concentration (displayed by the counter) × volume), and then the yield cell yield was calculated (cell yield = number of live CAR-monocytes / number of inoculated monocytes), the results are as follows Figure 3 As shown: the cell viability of CAR-monocytes using the culture medium of Example 4, Example 5 and Example 6 is equivalent to that of the method using Comparative Example 2, while the cell yield is higher than that of the method using Comparative Example 2, that is, adding coenzymes (such as; disodium ATP) to the system of culturing and transfecting monocytes can improve the harvesting efficiency of monocytes (i.e., yield cell yield) while maintaining good stability and long-term high activity of macrophages (i.e., viability cell viability).

[0133] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Application of coenzyme in any one of a1) to a4): a1) Suspension culture of adherent cells; a2) preparing products for suspension culture of adherent cells; a3) Inducing monocytes to differentiate into suspended macrophages; a4) preparing a product for inducing monocytes to differentiate into suspended macrophages; Preferably, the coenzyme comprises at least one of adenosine triphosphate or a salt thereof, and reduced nicotinamide adenine dinucleotide phosphate; further adenosine triphosphate or a salt thereof; Preferably, the adherent cells include at least one of monocytes and macrophages; Preferably, the product is a culture medium.

2. Any one of b1)-b2): b1) a culture medium, wherein the culture medium is a basic culture medium containing coenzymes; b2) A culture medium, which is a basic culture medium containing a macrophage inducer and a coenzyme.

3. The culture medium according to claim 2, characterized in that In b1) and b2), the coenzyme comprises at least one of adenosine triphosphate or a salt thereof, and reduced nicotinamide adenine dinucleotide phosphate.

4. The culture medium according to any one of claims 2-3, characterized in that b2) The macrophage inducing agent comprises at least one of granulocyte-macrophage colony stimulating factor and phorbol ester; Preferably, the basic culture medium in b1) and b2) comprises at least one of Advanced DMEM, DMEM, RPMI1640 culture medium, X-VIVO culture medium and William's E culture medium; Preferably, the basal culture medium in b1) and b2) is a basal culture medium containing serum.

5. The culture medium according to any one of claims 2 to 4, characterized in that In b1) and b2), the concentration of the coenzyme in the culture medium is 10-800 μg / mL; further 30-750 μg / mL; further 140-160 μg / mL.

6. The culture medium according to any one of claims 2 to 5, characterized in that b2) The concentration of the macrophage inducer in the culture medium is 25-150 ng / mL; further 25-75 ng / mL; further 46-54 ng / mL.

7. Any of the methods c1)-c4): c1) A method for culturing adherent cells in suspension, comprising culturing the adherent cells using the culture medium described in b1) in any one of claims 2 to 6; c2) A method for inducing monocytes to differentiate into suspended macrophages, comprising culturing the monocytes using the culture medium described in b2) in any one of claims 2 to 6; c3) A method for preparing CAR-macrophages, comprising culturing the monocytes with the culture medium described in b2) of any one of claims 2 to 6, and delivering the nucleic acid molecule encoding the CAR, or the expression cassette or vector comprising the nucleic acid molecule to the macrophages during the culture process; c4) A method for culturing CAR-adherent cells in suspension, wherein the adherent cells are cultured in a system comprising an adenovirus expressing CAR and the culture medium described in b1) in any one of claims 2 to 6.

8. The method according to claim 7, characterized in that c1), or c4), the adherent cells comprise at least one of monocytes and macrophages; Preferably, the culture conditions in c1), or c4) are 33-40°C and 3-7% CO2; Preferably, the culturing time in c1), or c4) is 1-3 days; Preferably, the ratio of the adherent cells to the culture medium in c1), or c4) is 3×10 6 pcs: 2-4mL; Preferably, the ratio of the adenovirus to the adherent cells in c4) is 500-2000 according to MOI.

9. The method according to claim 7, characterized in that: The culture conditions in c2) or c3) are 33-40°C and 3-7% CO2; Preferably, the culturing time in c2), or c3) is 5-10 days; Preferably, the ratio of the mononuclear cells to the culture medium in c2) and c3) is 3×10 6 pcs: 2-4mL; Preferably, in c3), the method of delivering the nucleic acid molecule encoding the CAR, or the expression cassette or vector comprising the nucleic acid molecule to the macrophage is adding an adenovirus expressing the CAR; Preferably, the CAR-expressing adenovirus is added on day 4-5.5 of culture; Preferably, the ratio of the adenovirus to the monocytes is 500-2000 according to MOI.

10. Any cell type d1)-d3): d1) A cell obtained by the method described in any one of c1) and c4) of claims 7 to 9; d2) macrophages obtained by the method described in c2) of any one of claims 7 to 9; d3) CAR-macrophages obtained by the method described in c3) in any one of claims 7-9.

11. Use of the cell described in d1), the macrophage described in d2), or the CAR-macrophage described in d3) in claim 10 in the preparation of an anti-tumor drug.

Images