Cell culture medium and cell culture method
By using cell culture medium containing specific components and isolation steps, safety and uncertainty issues in existing NK cell culture methods are solved, and efficient NK cell proliferation and activity improvements are achieved.
Patent Information
- Application Number
- CN202411514338.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-29
- Publication Date
- 2024-12-20
AI Technical Summary
The existing NK cell culture methods have complex serum components and may be contaminated, resulting in uncertainty and insecurity, and lack of safe and efficient cell culture media and methods.
NK cells were isolated and inoculated through specific steps using cell culture medium containing DMEM/F12 medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, ginkgolactone, nitrogen acetylcysteine. Promote its proliferation in vitro.
It significantly improves the proliferation ability, viability and purity of NK cells, promotes cytokine secretion, and enhances the activity of NK cells.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of immune cell culture, and in particular to a cell culture medium and a cell culture method. Background Art
[0002] Lymphocytes mainly include T lymphocytes, B lymphocytes, and NK cells. Natural killer cells (NK) are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases. They can recognize target cells and kill mediators. NK cells are different from T and B cells. They are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization.
[0003] At present, there are many methods to promote NK cell proliferation in vitro, including using animal serum for culture, and adding anti-2B4 antibody, anti-NKG2D antibody, dextran, α-mannan peptide, hyaluronic acid, etc. to the culture medium to expand and culture NK cells. Although the above methods can stimulate the efficient expansion of NK cells, the composition of animal serum is complex, and mycoplasma, virus and other contaminants may be introduced during the sampling process. In addition, there are many components in the culture medium, which causes uncertainty and insecurity to NK cell culture.
[0004] Therefore, it is necessary to develop a safe and efficient NK cell culture medium and NK cell culture method to provide a new direction for the in vitro proliferation of NK cells. Summary of the invention
[0005] In view of the above-mentioned defects in the prior art, the present invention provides a cell culture medium and a cell culture method to solve the problems raised by the above-mentioned background technology.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0008] The contents of puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide and acetylcysteine in the DMEM / F12 culture medium are: 30-50 μg / L puerarin, 25-40 μg / L butylphthalide, 12-20 μg / L glutamine dipeptide, 10-15 μg / L bilobalide, 6-12 μg / L IL-34 and 3-8 μg / L acetylcysteine.
[0009] Preferably, the contents of puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and acetylcysteine in DMEM / F12 culture medium are: 40 μg / L puerarin, 30 μg / L butylphthalide, 15 μg / L glutamine dipeptide, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L acetylcysteine.
[0010] Preferably, the components of DMEM / F12 medium are: anhydrous calcium chloride 116.6 mg / L, L-leucine 59.05 mg / L, linoleic acid 0.042 mg / L, copper sulfate pentahydrate 0.0013 mg / L, L-lysine hydrochloride 91.25 mg / L, lipoic acid 0.105 mg / L, ferric nitrate nonahydrate 0.05 mg / L, L-methionine 17.24 mg / L, phenol red 8.1 mg / L, ferrous sulfate heptahydrate 0.417 mg / L, L-phenylalanine 35.48 mg / L, 1,4-butanediamine dihydrochloride 0.081 mg / L, potassium chloride 311.8 mg / L, L-serine 26.25 mg / L, sodium pyruvate 55 mg / L, magnesium chloride 28.64 mg / L, L-threonine 53.45 mg / L, vitamin H 0.0035mg / L, anhydrous magnesium sulfate 48.84mg / L, L-alanine 4.45mg / L, D-calcium pantothenate 2.24mg / L, sodium chloride 6999.5mg / L, L-asparagine 7.5mg / L, choline chloride 8.98mg / L, anhydrous sodium dihydrogen phosphate 54.35mg / L, L-aspartic acid 6.65mg / L, folic acid 2.65mg / L, disodium hydrogen phosphate 71.02mg / L, L-cysteine hydrochloride 17.56mg / L, i-inositol 12.6mg / L, zinc sulfate heptahydrate 0.432mg / L, L-glutamic acid 7.35mg / L, niacinamide 2.02mg / L, L-arginine hydrochloride 14 7.5mg / L, L-proline 17.25mg / L, pyridoxal hydrochloride 2mg / L, L-cystine hydrochloride 31.29mg / L, L-tryptophan 9.02mg / L, pyridoxine hydrochloride 0.031mg / L, L-glutamine 365mg / L, L-tyrosine 38.4mg / L, riboflavin 0.219mg / L, glycine 18.75mg / L, L-valine 52.85mg / L, thiamine hydrochloride 2.17mg / L, L-histidine hydrochloride 31.48mg / L, D-glucose 3151mg / L, thymidine 0.365mg / L, L-isoleucine 54.47mg / L, hypoxanthine 2mg / L, vitamin B12 0.68mg / L.
[0011] Preferably, a cell culture method comprises the following steps:
[0012] (1) NK cell isolation;
[0013] (2) Inoculating NK cells into the cell culture medium according to claim 1 or claim 2 and culturing them for 10 to 15 days to obtain proliferated NK cells.
[0014] Preferably, the specific steps of step (1) are: using a blood collection tube added with 0.2g of sodium heparin to collect 30mL of fresh adult human peripheral blood, obtaining plasma after centrifugation, adding an equal volume of physiological saline to the plasma for dilution, and then adding 1mL of lymphocyte separation solution, obtaining peripheral blood mononuclear cells after centrifugation and washing; adjusting the concentration of peripheral blood mononuclear cells with diluent to form a preincubation solution; placing the preincubation solution in a plastic culture dish and culturing it for 1 to 3 hours and then passing it through a nylon cotton column; adding anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads in sequence for negative sorting, and collecting a cell suspension rich in NK cells.
[0015] Preferably, the diluent is 10% FCS-RPMI-1640, and RPMI-1640 complete medium (containing 10% FCS) is purchased from Shanghai Yuchun Biotechnology Co., Ltd.
[0016] Preferably, the concentration of peripheral blood mononuclear cells after adjustment with the diluent is 2×10 6 ~4×10 6 Pieces / mL.
[0017] Preferably, the culture conditions required for placing in a plastic culture dish are: a temperature of 37° C. and a carbon dioxide concentration of 5%.
[0018] Preferably, the step (1) is more specifically as follows:
[0019] (1.1) Isolation of peripheral blood mononuclear cells (PBMC): see Ficoll-diatrizoate separation method;
[0020] (1.2) The concentration of mononuclear cells was adjusted to 2×10 6 ~4×10 6 pcs / ml, added to a sterile plastic culture dish and incubated at 37°C with 5% CO 2 After culturing in the environment for 2 h, monocytes and B cells adhered to the plastic were removed;
[0021] (1.3) Non-adherent cells were pre-incubated with 10% FCS-RPMI-1640 solution for 1 h and then passed through a nylon cotton column to remove B cells and remaining monocytes adhering to the nylon cotton column. The column was washed with 1% BSA-PBS washing solution and the washed T cell and NK cell suspensions were collected;
[0022] (1.4) Separation of T cells and NK cells using magnetic field: Label and separate cells using sterile operation. Newly isolated T cells and NK cells are labeled in a water bath, and each 1×10 6 The cell suspension of 100 μl / mL was incubated with 10ul anti-CD3 antibody for 10min, incubated with 100ul goat anti-mouse antiserum labeled with biotin for 10min, 25ul of streptavidin labeled with FITC (fluorescein isothiocyanate) was added, reacted for 8min, and 100ul of biotin-labeled magnetic particles were added for 8min. After each step of the above reaction, the cells were washed twice with 10 times the volume of 1% BSA-PBS washing solution and centrifuged at 3000r / min for 8min to terminate the reaction;
[0023] (1.5) Resuspend the cell suspension labeled with magnetic complexes in 1% BSA-PBS washing solution and adjust the cell concentration to 5×10 7 ~1×10 8 / ml. Pre-incubate the separation column with 1% BSA-PBS washing solution for 30 minutes, pre-cool at 4℃ and place it in the magnetic field of a permanent magnet, add 80-100ml of the labeled cell suspension. Take the separation column out of the magnetic field, use 50ml of 1% BSA-PBS washing solution under sterile conditions with a syringe to gently pressurize the eluted labeled cells, and centrifuge the precipitated cells, which are NK cells.
[0024] Preferably, the specific steps of step (2) are:
[0025] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of (3-5):1, centrifuging for 5-10 minutes, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0026] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 2×10 6 ~4×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0027] S3. Adding the cell culture medium to the expanded cells to initiate proliferation culture, and replacing the cell culture medium with new one every 48 to 72 hours to promote the proliferation process.
[0028] Preferably, the speed of the centrifugal separation in step S1 is 800-1000 r / min.
[0029] Preferably, it is characterized in that the proliferation culture time in step S3 is 14 days.
[0030] Compared with the prior art, the present invention has the following beneficial effects:
[0031] The present invention provides a cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and nitrogen acetylcysteine. Puerarin and bilobalide added in the cell culture medium can synergistically improve the proliferation ability of NK cells; butylphthalide added in the cell culture medium is beneficial to improving the viability of NK cells, and the addition of glutamine dipeptide can significantly improve the purity of NK cells; the cytokine content test results also prove that the cell culture medium provided by the present invention can also promote NK cells to secrete cytokines, thereby enhancing the activity of NK cells. DETAILED DESCRIPTION
[0032] The technical solution of the present invention is further described in detail below in conjunction with specific implementation methods.
[0033] Example 1
[0034] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0035] The contents of the components in the DMEM / F12 culture medium are: 30 μg / L puerarin, 25 μg / L butylphthalide, 12 μg / L glutamine dipeptide, 10 μg / L bilobalide, 6 μg / L IL-34, and 3 μg / L nitrogen acetylcysteine.
[0036] A cell culture method, comprising the following steps:
[0037] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 2×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 1 hour, and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting, and a cell suspension rich in NK cells was collected;
[0038] (2) inoculating the NK cells into a cell culture medium and culturing them for 10 days to obtain proliferated NK cells; the specific steps are as follows:
[0039] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 3:1, centrifuging at 800 r / min for 5 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0040] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 2×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0041] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 48 hours to promote the proliferation process.
[0042] Example 2
[0043] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0044] The contents of the components in the DMEM / F12 culture medium are: 50 μg / L puerarin, 40 μg / L butylphthalide, 20 μg / L glutamine dipeptide, 15 μg / L bilobalide, 12 μg / L IL-34, and 8 μg / L nitrogen acetylcysteine.
[0045] A cell culture method, comprising the following steps:
[0046] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 4×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 3 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0047] (2) inoculating the NK cells into a cell culture medium and culturing them for 15 days to obtain proliferated NK cells; the specific steps are as follows:
[0048] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 5:1, centrifuging at 1000 r / min for 10 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0049] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 4×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0050] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 72 hours to promote the proliferation process.
[0051] Example 3
[0052] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0053] The contents of the components in the DMEM / F12 culture medium are: 40 μg / L puerarin, 30 μg / L butylphthalide, 15 μg / L glutamine dipeptide, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L nitrogen acetylcysteine.
[0054] A cell culture method, comprising the following steps:
[0055] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0056] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0057] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0058] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0059] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0060] Comparative Example 1
[0061] The difference between this comparative example and Example 3 is that puerarin is replaced by syringesterol, and the other ingredients and steps remain unchanged.
[0062] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, syringesterol, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0063] The contents of the components in the DMEM / F12 culture medium are: 40 μg / L syringesterol, 30 μg / L butylphthalide, 15 μg / L glutamine dipeptide, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L nitrogen acetylcysteine.
[0064] A cell culture method, comprising the following steps:
[0065] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0066] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0067] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0068] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0069] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0070] Comparative Example 2
[0071] The difference between this comparative example and Example 3 is that puerarin is not added to the cell culture medium, and the other components and steps remain unchanged.
[0072] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0073] The contents of the components in the DMEM / F12 culture medium are: 30 μg / L butylphthalide, 15 μg / L glutamine dipeptide, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L nitrogen acetylcysteine.
[0074] A cell culture method, comprising the following steps:
[0075] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0076] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0077] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0078] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0079] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0080] Comparative Example 3
[0081] The difference between this comparative example and Example 3 is that bilobalide is not added to the cell culture medium, and the other components and steps remain unchanged.
[0082] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, and N-acetylcysteine;
[0083] The contents of the components in the DMEM / F12 culture medium are: 40 μg / L puerarin, 30 μg / L butylphthalide, 15 μg / L glutamine dipeptide, 10 μg / L IL-34, and 6 μg / L nitrogen acetylcysteine.
[0084] A cell culture method, comprising the following steps:
[0085] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0086] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0087] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0088] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0089] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0090] Comparative Example 4
[0091] The difference between this comparative example and Example 3 is that butylphthalide is not added to the cell culture medium, and the other components and steps remain unchanged.
[0092] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine;
[0093] The contents of the components in the DMEM / F12 culture medium are: 40 μg / L puerarin, 15 μg / L glutamine dipeptide, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L N-acetylcysteine.
[0094] A cell culture method, comprising the following steps:
[0095] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0096] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0097] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0098] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0099] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0100] Comparative Example 5
[0101] The difference between this comparative example and Example 3 is that the glutamine dipeptide in the cell culture medium is replaced with polylysine, and the other components and steps remain unchanged.
[0102] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, polylysine, IL-34, bilobalide, and N-acetylcysteine;
[0103] The contents of the components in the DMEM / F12 culture medium are: 40 μg / L puerarin, 30 μg / L butylphthalide, 15 μg / L polylysine, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L N-acetylcysteine.
[0104] A cell culture method, comprising the following steps:
[0105] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0106] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0107] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0108] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0109] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0110] Comparative Example 6
[0111] The difference between this comparative example and Example 3 is that glutamine dipeptide is not added to the cell culture medium, and the other components and steps remain unchanged.
[0112] A cell culture medium for promoting the proliferation of NK cells in vitro, the cell culture medium comprising the following components: DMEM / F12 culture medium, puerarin, butylphthalide, IL-34, bilobalide, and N-acetylcysteine;
[0113] The contents of the components in the DMEM / F12 culture medium are: 40 μg / L puerarin, 30 μg / L butylphthalide, 12 μg / L bilobalide, 10 μg / L IL-34, and 6 μg / L N-acetylcysteine.
[0114] A cell culture method, comprising the following steps:
[0115] (1) NK cell separation: Fresh adult human peripheral blood was collected using a blood collection tube supplemented with sodium heparin, and plasma was obtained after centrifugation. An equal volume of physiological saline was added to the plasma for dilution, and then lymphocyte separation fluid was added to collect the lymphocyte layer. After centrifugation and PBS washing, peripheral blood mononuclear cells were obtained. The peripheral blood mononuclear cells were diluted with 10% FCS-RPMI-1640 diluent to adjust the cell concentration to 3×10 6 / mL to form a pre-incubation solution; the pre-incubation solution was placed in a plastic culture dish at a temperature of 37°C and a carbon dioxide concentration of 5% for 2 hours and then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting to collect a cell suspension rich in NK cells;
[0116] (2) inoculating the NK cells into a cell culture medium and culturing them for 14 days to obtain proliferated NK cells; the specific steps are as follows:
[0117] S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of 4:1, centrifuging at 900 r / min for 8 min, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured;
[0118] S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 3×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells;
[0119] S3. Add the cell culture medium to the expanded cells to start proliferation culture, and replace the cell culture medium with new one every 60 hours to promote the proliferation process.
[0120] Experimental example: NK cell index detection
[0121] The control group was the same as Example 3 except that puerarin, butylphthalide, glutamine dipeptide and bilobalide were not added. That is, the culture medium components of the control group were DMEM / F12 culture medium, IL-34 and N-acetylcysteine.
[0122] 1. Cell proliferation multiples and cell viability
[0123] The cultured NK cells were stained with trypan blue, and the number of NK cells obtained in Examples 1 to 3 and Comparative Examples 1 to 6 was counted using a cell counter, and the cell proliferation times and cell viability were calculated. The proliferation times = (the number of NK cells obtained / the number of cells before the experiment). The results are shown in Table 1:
[0124] Table 1 NK cell proliferation and cell viability test results
[0125] Group Multiplication times Cell viability Example 1 348 99.1％ Example 2 346 99.3％ Example 3 351 99.5％ Comparative Example 1 305 99.0％ Comparative Example 2 271 97.7％ Comparative Example 3 264 98.8％ Comparative Example 4 320 85.1％ Comparative Example 5 331 97.3％ Comparative Example 6 325 98.3％ Control group 211 70.2％
[0126] As can be seen from Table 1, the cell culture media of Examples 1 to 3 can make NK cells proliferate more than 340 times, indicating that the cell culture media has good proliferation and growth-promoting ability, especially Example 3. The proliferation multiples of NK cells in Examples 1 to 3 are significantly higher than those in Comparative Examples 1 to 6. It can be seen from the comparison between Example 3 and Comparative Examples 1 to 3 that the absence of puerarin and bilobalide in the cell culture media will significantly affect the proliferation ability of NK cells, and even the use of syringaresinol to replace puerarin will reduce the proliferation ability of NK cells; it can be seen from the comparison between Example 3 and Comparative Examples 4 to 6 that the absence of butylphthalide and glutamine dipeptide in the cell culture media will reduce the proliferation ability of NK cells, but the reduction effect is not obvious. From the cell viability of Comparative Example 4, it can be seen that the addition of butylphthalide to the cell culture media is beneficial to improving the cell viability, and the absence of butylphthalide will significantly reduce the NK cell viability.
[0127] 2. Cell purity analysis
[0128] The NK cells collected after culture were resuspended in PBS buffer and the density was adjusted to 10 6 FITC-labeled CD16 and PE-labeled CD56 antibodies were added, incubated at room temperature in the dark for 30 minutes, and then detected by flow cytometry to detect the proportion of cells with corresponding immune phenotypes (%). The test results are shown in Table 2:
[0129] Table 2 Cell purity test results
[0130] Group <![CDATA[CD16 + CD56 + ]]> Example 1 96.9±0.50 Example 2 94.7±0.48 Example 3 95.2±0.51 Comparative Example 1 93.1±0.45 Comparative Example 2 92.4±0.40 Comparative Example 3 90.7±0.48 Comparative Example 4 90.3±0.49 Comparative Example 5 81.1±0.37 Comparative Example 6 75.6±0.34 Control group 70.1±0.33
[0131] As shown in Table 2, the purity of NK cells in each experimental group was higher than that in the control group, and glutamine dipeptide had a greater effect on cell purity.
[0132] 3. Cytokine content analysis
[0133] The supernatants after the NK cell culture of Examples 1 to 3 and Comparative Examples 1 to 6 were collected, and the IFN-γ and TNF-ɑ contents in the supernatants were detected by ELISA method. The results are shown in Table 3:
[0134] Table 3 IFN-γ, TNF-ɑ content
[0135]
[0136]
[0137] As can be seen from Table 3, not adding puerarin, butylphthalide, glutamine dipeptide and bilobalide to the culture medium will lead to a decrease in the content of NK cell factors IFN-γ and TNF-ɑ, indicating that the culture medium provided by the present invention can promote NK cells to secrete cytokines, thereby providing NK cell activity.
[0138] In summary, the cell culture medium provided by the present invention includes the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and nitrogen acetylcysteine, wherein the addition of puerarin and bilobalide to the cell culture medium is beneficial to improving the proliferation ability of NK cells, the addition of butylphthalide is beneficial to improving the viability of NK cells, and the addition of glutamine dipeptide is beneficial to improving the purity of NK cells. The cytokine content test results also prove that the cell culture medium provided by the present invention can also promote the secretion of cytokines by NK cells, thereby enhancing the activity of NK cells.
[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims
1. A cell culture medium for promoting the proliferation of NK cells in vitro, characterized in that: The cell culture medium comprises the following components: DMEM / F12 culture medium, puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide, and N-acetylcysteine; The contents of puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide and acetylcysteine in the DMEM / F12 culture medium are: 30-50 μg / L puerarin, 25-40 μg / L butylphthalide, 12-20 μg / L glutamine dipeptide, 10-15 μg / L bilobalide, 6-12 μg / L IL-34 and 3-8 μg / L acetylcysteine.
2. A cell culture medium according to claim 1, characterized in that The contents of puerarin, butylphthalide, glutamine dipeptide, IL-34, bilobalide and acetylcysteine in the DMEM / F12 culture medium are: 40 μg / L puerarin, 30 μg / L butylphthalide, 15 μg / L glutamine dipeptide, 12 μg / L bilobalide, 10 μg / L IL-34 and 6 μg / L acetylcysteine.
3. A cell culture method, characterized in that: The method comprises the following steps: (1) NK cell isolation; (2) Inoculating NK cells into the cell culture medium according to claim 1 or claim 2 and culturing them for 10 to 15 days to obtain proliferated NK cells.
4. The cell culture method according to claim 3, characterized in that The specific steps of step (1) are: using a blood collection tube added with sodium heparin to collect fresh adult human peripheral blood, obtaining plasma after centrifugation, adding an equal volume of physiological saline to the plasma for dilution, then adding lymphocyte separation solution, obtaining peripheral blood mononuclear cells after centrifugation and washing; adjusting the concentration of the peripheral blood mononuclear cells with a diluent to form a pre-incubation solution; The pre-incubation solution was placed in a plastic culture dish and cultured for 1 to 3 hours, then passed through a nylon cotton column; anti-CD3 monoclonal antibody, goat anti-mouse antiserum, streptavidin, and magnetic beads were added in sequence for negative sorting, and the cell suspension rich in NK cells was collected.
5. The cell culture method according to claim 4, characterized in that: The diluent was 10% FCS-RPMI-1640.
6. The cell culture method according to claim 4, characterized in that: The concentration of peripheral blood mononuclear cells was adjusted to 2×10 6 ~4×10 6 Pieces / mL.
7. The cell culture method according to claim 4, characterized in that: The culture conditions required for placing in a plastic culture dish are: temperature of 37°C and carbon dioxide concentration of 5%.
8. The cell culture method according to claim 3, characterized in that: The specific steps of step (2) are: S1. Mixing a cell culture medium and a cell suspension rich in NK cells at a volume ratio of (3-5):1, centrifuging for 5-10 minutes, removing the supernatant to obtain a cell precipitate to be cultured, and resuspending the cell precipitate to be cultured with the cell culture medium to obtain a cell resuspension to be cultured; S2, adding the cell resuspension to be cultured into the culture position, so that the culture area per square centimeter of the culture position has 2×10 6 ~4×10 6 cells to be cultured, and performing an amplification process on the cells to be cultured to obtain amplified cells; S3. Adding the cell culture medium to the expanded cells to initiate proliferation culture, and replacing the cell culture medium with new one every 48 to 72 hours to promote the proliferation process.
9. The cell culture method according to claim 8, characterized in that: The speed of the centrifugal separation in step S1 is 800-1000 r / min.
10. The cell culture method according to claim 8, characterized in that: The proliferation culture time in step S3 is 14 days.