CN115521914A - Human primary natural killer cell in-vitro amplification system and method - Google Patents
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Abstract
The invention provides an in-vitro amplification system and method for human primary natural killer cells, which belong to the technical field of biochemistry, and the system comprises 1640 cell culture medium, 90-110U/mL IL-2, 180-220ng/mL NK cell-derived exosome, recombinant expression protein mixed freeze-dried powder and matrix glue. The in vitro amplification method of the primary natural killer cells can solve the safety problems that allogeneic cells with biosafety risks are easily introduced into the existing in vitro amplification method of the NK cells, excessive inflammatory reaction is easily caused, and the like, and the technical problems of high cost, complex operation, poor amplification efficiency and the like.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly discloses an in-vitro amplification system and method for human primary natural killer cells.
Background
Natural Killer (NK) cells are a class of innate immune cells distinct from T cells and B cells, present in lymph nodes, spleen, liver, lung, bone marrow and peripheral blood, and as core cells of the innate immune system, NK cells play a vital role in maintaining human health, resisting cancer and infection. Unlike T cell and B cell, NK cell has three functions of immune monitoring, immune response and immune memory, and its natural killing activity needs neither antigen pre-sensitization nor antibody and no MHC limitation, and can participate in immune response in the first time.
NK cells kill target cells and exert their natural killing function mainly through the following four pathways: (1) A series of cytokines (IFN- γ, TNF- α, IL-10), growth factors (GM-CSF), chemokines (CCL 3, CCL4, CCL5, XCL 1) are produced, and an immune response is developed by modulating the interaction between NK cells and dendritic cells, macrophages and T cells. (2) Antibody-dependent cell-mediated cytotoxicity (ADCC): the Fab segment of IgG antibody of NK cell surface CD16 is combined with the antigen epitope of target cell specifically, and mediates the specific recognition of NK cell and kills target cell. (3) Perforin (PRF)/Granzyme (Granzyme, GZM) pathway: NK cells produce lytic granules containing perforin, granzyme and the like, induce apoptosis of target cells or directly destroy target cell membranes, resulting in target cell rupture. (4) Fas/FasL pathway and TRAILR/TRAIL pathway: activated NK cells express various Tumor Necrosis Factor (TNF) superfamily members, such as FASL and TRAIL, on their surfaces, which induce apoptosis in target cells by binding to their corresponding receptors (FAS or TRAILR).
NK cells play an extremely important role in the fields of bacteria elimination, virus resistance, aging delay, cancer resistance and the like as a first defense line for maintaining human health. Based on the broad-spectrum cytotoxic effect of NK cells, the research of applying NK cells to treat various diseases shows the blowout-type development in recent years.
Researches prove that the low cytotoxic activity of NK cells is related to the increase of cancer risk, and the good activity of the NK cells is closely related to the better cancer treatment effect and the lower cancer recurrence risk. To date, NK cell-based immunotherapy has gradually become a fourth tumor treatment method following radiotherapy, chemotherapy, and surgical treatment, wherein NK cell adoptive immunotherapy achieves tumor treatment effect by directly or indirectly killing tumor cells by infusing NK cells cultured by in vitro induction back to a patient. As for hematological tumors, NK cells have a surprising effect on the treatment of acute lymphocytic leukemia, acute myelogenous leukemia, lymphoma and the like. In the aspect of solid tumors, the therapeutic effect is not as ideal as that of blood tumors, but researchers find that NK cells have positive effects on neuroblastoma, lung cancer, breast cancer, prostate cancer, gastric cancer and the like, and the application prospect is bright.
NK cells also take an important position in defending against viral invasion. Clinical studies have shown that NK cells have a good anti-viral effect on viruses that can produce immune escape, such as hepatitis B virus, cytomegalovirus, HIV and the like, and also have the same effect on world-wide virus (SARS-CoV-2), and Sorrent pharmaceutical company and Celularity cell therapy company have announced that clinical cooperation is started in combination in 2020, and NK cells are planned to be applied to treatment and prevention of SARS-CoV-2 infection.
In addition to anti-viral and anti-tumor, NK cells also play an important role in the body's fight against bacterial, fungal and parasitic infections. And the NK cells have great application potential in the aspects of autoimmune disease treatment (diabetes, rheumatoid arthritis and multiple sclerosis), anti-aging health care and the like. In addition, NK cells are proved to play an important role in the pathological process of nervous system diseases (stroke, alzheimer disease, parkinson disease, brain tumor and the like), play the roles of enhancing immunity and participating in neuroprotection, and have obvious improvement effects on fatigue and sleep quality of sub-health people.
In conclusion, the NK cells have great significance to human health, and cell treatment means based on the NK cells have extremely wide bright prospect, thereby bringing new hopes for improving the quality of life of human beings.
Currently, the mainstream methods for in vitro amplification of NK cells include: 1. and (3) a trophoblast cell culture mode. Namely: obtaining trophoblast cells that do not divide and proliferate but remain metabolically active stimulates NK cell proliferation. The trophoblasts are generally transformed by a genetic engineering technology, and then irradiated by cobalt 60, the cell membrane surface stably expresses a plurality of cytokines, and NK cells in peripheral blood mononuclear cells are directionally activated and amplified under the synergistic action of the cytokines. 2. Pure factor cell culture mode. Namely, the differentiation of peripheral blood mononuclear cells into NK cells is induced by using corresponding cytokines, and the NK cells are proliferated in a large amount by matching with a corresponding cell culture medium.
However, the above culture method still has certain defects, for example, the trophoblasts are generally made of tumor cell lines, if the trophoblasts are not completely inactivated, the amplified NK cells may contain tumor cells with proliferation activity, and if the trophoblasts are used in a human body, the risk of biosafety is brought; the cytokine culture method cannot achieve the optimal stimulation effect due to single cytokine, and is easy to cause over-activation of NK cells to cause inflammatory reaction; meanwhile, for some individuals with low immunity, because the functions of NK cells are seriously damaged, the effective amplification effect is difficult to achieve under the normal stimulation environment.
Disclosure of Invention
The first purpose of the invention is to provide an in vitro amplification system of human primary natural killer cells, which solves the safety problems of inflammation and the like easily caused in the current NK cell amplification and the technical problems of poor amplification effect.
The second objective of the present invention is to provide an in vitro amplification method of primary natural killer cells, which is simple in operation, does not require a complex instrument, and has a low operation cost.
The invention is realized by the following technical scheme:
the invention provides a human primary natural killer cell in-vitro amplification system, which comprises 1640 cell culture medium, IL-2 with the final concentration of 90-110U/mL, NK cell-derived exosome with the final concentration of 180-220ng/mL, recombinant expression protein freeze-dried powder and matrix glue, wherein the weight ratio of the recombinant expression protein freeze-dried powder to the matrix glue is 1 (9-11). Wherein the 1640 cell culture medium is used for providing basic nutrients for the in vitro amplification culture of the NK cells; IL-2 is capable of maintaining the growth of NK cells and is a cytokine necessary for maintaining the growth of the NK cells; the NK cell-derived exosome carries various regulatory factors such as nucleic acid and protein and can provide necessary regulatory signals for the in vitro growth of NK cells; the matrigel can provide necessary skeleton support for the NK cells and simulate the biomechanical stimulation of the NK cells in an in vivo tissue environment; meanwhile, the recombinant expression protein freeze-dried powder can effectively stimulate the activation of NK cells.
Secondly, the invention also provides an in vitro amplification method of the primary natural killer cells, which comprises the following steps: separating peripheral blood mononuclear cells from peripheral blood; mixing the matrix glue, the recombinant expression protein freeze-dried powder and a 1640 cell culture medium, paving the mixture on a six-hole plate, and then placing the six-hole plate for 28-32min at 36-38 ℃ to obtain a culture plate; peripheral blood mononuclear cells, an NK cell culture medium and NK cell-derived exosomes were sequentially added to a culture plate, cultured at 36-38 ℃ and observed. The method is simple and convenient to operate, can greatly reduce the operation difficulty of operators, thereby improving the operation efficiency, does not need to use particularly complex instruments, reagents and the like, and reduces the operation cost.
Compared with the prior art, the invention at least has the following advantages and positive effects:
the invention provides an in vitro amplification system and method of human primary natural killer cells, which have the following advantages: 1. the use is convenient, and the amplification efficiency is good; 2. the preparation is convenient and the cost is low; 3. no biosafety risks such as allergy, serious inflammation and tumor are caused; 4. can achieve better amplification and activation effects on NK cells with poor activity; 5. has good biological safety, and has great industrialization potential and application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a diagram showing in vitro expansion culture of NK cells in the present invention (A is a diagram showing the culture immediately after isolation, B is a diagram showing the culture after one week of isolation, and C is a diagram showing the culture after two weeks of isolation);
FIG. 2 is an analysis chart of NK cell purity after amplification in the present invention (A is NK cell purity before amplification, B is NK cell purity after amplification);
FIG. 3 is a graph showing cell counts before and after amplification and amplification efficiencies in the present invention (wherein A is the NK cell counts before and after amplification, and B is an amplification efficiency graph);
fig. 4 shows the killing activity of cells before and after expansion (target ratio 5:1) (n = 5) in the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are conventional products which are not indicated by manufacturers and are commercially available.
It should be noted that, in the present application, the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
The invention provides an in-vitro amplification system of human primary natural killer cells, which comprises 1640 cell culture medium, IL-2 with the final concentration of 90-110U/mL, NK cell-derived exosome with the final concentration of 180-220ng/mL, recombinant expression protein freeze-dried powder and matrix gel, wherein the weight ratio of the recombinant expression protein freeze-dried powder to the matrix gel is 1 (9-11). Wherein the 1640 cell culture medium is used for providing basic nutrients for the in vitro amplification culture of the NK cells; IL-2 is capable of maintaining the growth of NK cells and is a cytokine necessary for maintaining the growth of the NK cells; the NK cell derived exosome carries various regulatory factors such as nucleic acid and protein and can provide necessary regulatory signals for the in vitro growth of NK cells; the stroma glue can provide necessary skeleton support for the NK cells and simulate the biomechanical stimulation of the NK cells in an in vivo tissue environment; meanwhile, the recombinant expression protein freeze-dried powder can effectively stimulate the activation of NK cells. The proportion interval can enable the recombinant expression protein to be fully connected with the matrigel, so that the recombinant expression protein can effectively stimulate the activation of NK cells, when the content of the recombinant expression protein is too high, over stimulation can be caused, the safety is influenced, the safety coefficient is reduced, when the content of the recombinant expression protein is too low, the stimulation effect is possibly poor, and the amplification efficiency of the NK cells is reduced.
The in-vitro amplification system comprises 1640 cell culture medium, IL-2 with the final concentration of 100U/mL, NK cell-derived exosome with the final concentration of 200ng/mL, recombinant expression protein freeze-dried powder and matrix gel. The concentration is the optimal concentration of the amplification system, and the concentration can further improve the in-vitro amplification effect of the NK cells and prevent the excessive activation of the NK cells, thereby improving the safety.
The above 1640 cell culture medium comprises penicillin at a final concentration of 90-110. Mu.g/mL, streptomycin at a final concentration of 90-110. Mu.g/mL, 1640 basic medium, and fetal bovine serum at a final concentration of 9-11% by weight. Penicillin and streptomycin are added to resist infectious microbes, and the concentration range of penicillin and streptomycin can prevent NK cells in the culture medium from being polluted by the infectious microbes and can avoid the influence on the NK cells.
The recombinant expression protein freeze-dried powder comprises IL-5, IL-18 and 4-1BBL, wherein the weight ratio of the IL-5 to the IL-18 to the 4-1BBL is 1 (1.5-2.5) to 1.
The weight ratio of the mixture of the recombinant expression protein freeze-dried powder and the matrix glue to the 1640 cell culture medium is 1 (4.5-5.5).
Secondly, the invention also provides an in vitro amplification method of the primary natural killer cells, which comprises the following steps: separating peripheral blood mononuclear cells from peripheral blood; mixing the matrix glue, the recombinant expression protein freeze-dried powder and a 1640 cell culture medium, paving the mixture on a porous plate, and standing the porous plate at 36-38 ℃ for 28-32min to obtain a culture plate; and adding the peripheral blood mononuclear cells, the NK cell culture medium and the NK cell-derived exosome into a culture plate in sequence, culturing at 36-38 ℃ and observing. The method is simple and convenient to operate, can greatly reduce the operation difficulty of operators, thereby improving the operation efficiency, does not need to use particularly complex instruments, reagents and the like, and reduces the operation cost.
Each well of the above plate contains 1.5-2.5mL of amplification system.
And (3) sequentially adding the peripheral blood mononuclear cells, the NK cell culture medium and the NK cell exosomes into the culture plate, and replacing the NK cell culture medium and the NK cell exosomes every 2-3 days.
The NK cell culture medium includes 1640 basic medium, IL-2 at a final concentration of 90-110U/mL, 9-11% by weight of fetal bovine serum, 80-120. Mu.g/mL of penicillin and 80-120. Mu.g/mL of streptomycin.
Example 1
1. Obtaining 10mL of peripheral blood of a healthy volunteer, separating peripheral blood mononuclear cells by utilizing lymphocyte separation liquid: a. the same amount of the separation solution as the blood sample is added to the sterile centrifuge tube, and the fresh anticoagulated blood sample is carefully aspirated by the pipette above the surface of the separation solution. 860g, and centrifuging for 30min. c. After the centrifugation is finished, the centrifugal tube sequentially comprises a plasma layer, an annular milky white lymphocyte layer, a transparent separation liquid layer and a red blood cell layer from top to bottom. Carefully sucking the annular milky white lymphocyte layer into another sterile centrifugal tube, adding 3 times of normal saline, blowing, beating and mixing uniformly. d.250g, centrifuged for 15min and the supernatant was discarded. e. 5mL of physiological saline was added to resuspend the cells. 250g, centrifuged for 10min and the supernatant was discarded. f. Adding 1mL of NK cell culture solution to resuspend peripheral blood mononuclear cells, sucking 10. Mu.L of cell suspension, counting cells, placing at 37 ℃ and 5% CO 2 The cells were cultured overnight in a constant temperature incubator.
2. Corning Matrigel was mixed with a recombinant expression protein lyophilized powder (IL-15. The culture medium can be placed in an incubator at 37 ℃ for 30 minutes for subsequent use.
3. The counted PBMCs were added to a six-well plate plated with matrigel, 2mL of NK cell medium (1640 medium, 100U/mL IL-2, 10% fetal bovine serum; 100. Mu.g/mL of penicillin and streptomycin) per well, NK cell exosomes were added at a concentration of 200ng/mL, and the mixture was cultured in an incubator at 37 ℃.
4. The cells in the six-well plates were half-changed every 2 days (about 1mL NK cell medium per change) while being supplemented with NK cell exosomes (200 ng).
5. The cultured cells were observed every 7 days, and the observation results were recorded.
Example 2
1. Obtaining 10mL of peripheral blood of a healthy volunteer, separating peripheral blood mononuclear cells by using a lymphocyte separation solution: a. an equal amount of separation medium to the blood sample is added to the sterile centrifuge tube and fresh anticoagulated blood sample is carefully aspirated up the surface of the separation medium with a pipette. 860g, and centrifuging for 30min. c. After the centrifugation is finished, the centrifugal tube sequentially comprises a plasma layer, an annular milky white lymphocyte layer, a transparent separation liquid layer and a red blood cell layer from top to bottom. Carefully sucking the annular milky white lymphocyte layer into another sterile centrifugal tube, adding 3 times of normal saline, blowing, beating and mixing uniformly. d.250g, centrifuge for 15min, and discard the supernatant. e. 5mL of physiological saline was added to resuspend the cells. 250g, centrifuge for 10min and discard the supernatant. f. Adding 1mL of NK cell culture solution to resuspend peripheral blood mononuclear cells, sucking 10. Mu.L of cell suspension, counting cells, placing at 37 ℃ and 5% CO 2 The cells were cultured overnight in a constant temperature incubator.
2. Corning Matrigel was mixed with a recombinant expression protein lyophilized powder (IL-15. The culture medium can be placed in an incubator at 37 ℃ for 30 minutes for subsequent use.
3. The counted PBMCs were added to a six-well plate plated with matrigel, 1.5mL of NK cell medium (1640 medium, 90U/mL IL-2,9% fetal bovine serum; 80. Mu.g/mL penicillin and streptomycin) was added to each well at a concentration of 180ng/mL, and the mixture was cultured in an incubator at 37 ℃.
4. Cells in six-well plates were half-changed every 2 days (approximately 1mL NK cell medium per change) while being supplemented with NK cell exosomes (180 ng).
5. The cultured cells were observed every 7 days, and the observation results were recorded.
Example 3
1. Obtaining 10mL of peripheral blood of a healthy volunteer, separating peripheral blood mononuclear cells by utilizing lymphocyte separation liquid: a. an equal amount of separation medium to the blood sample is added to the sterile centrifuge tube and fresh anticoagulated blood sample is carefully aspirated up the surface of the separation medium with a pipette. 860g, and centrifuging for 30min. c. After centrifugation is finished, the centrifugal tube sequentially comprises a plasma layer, an annular milky white lymphocyte layer, a transparent separation liquid layer and a red blood cell layer from top to bottom. Carefully sucking the annular milky white lymphocyte layer into another sterile centrifugal tube, adding 3 times of normal saline, blowing, beating and mixing uniformly. d.250g, centrifuge for 15min, and discard the supernatant. e. 5mL of physiological saline was added to resuspend the cells. 250g, centrifuged for 10min and the supernatant was discarded. f. Adding 1mL of NK cell culture fluid to resuspend peripheral blood mononuclear cells, sucking 10 μ L of cell suspension, counting cells, placing at 37 deg.C and 5% CO 2 The cells were cultured overnight in a constant temperature incubator.
2. Corning Matrigel was mixed with a recombinant expression protein lyophilized powder (IL-15. The culture medium can be placed in an incubator at 37 ℃ for 30 minutes for subsequent use.
3. The counted PBMCs were added to a six-well plate plated with matrigel, 2mL of NK cell medium (1640 medium, 110U/mL IL-2, 11% fetal bovine serum; 120. Mu.g/mL of penicillin and streptomycin) per well, NK cell exosomes were added at a concentration of 200ng/mL, and the mixture was cultured in an incubator at 37 ℃.
4. The cells in the six-well plates were half-changed every 2 days (approximately 1mL NK cell medium per change) while being supplemented with NK cell exosomes (220 ng).
5. The cultured cells were observed every 7 days, and the observation results were recorded.
Comparative example
Separating tumor tissue, obtaining trophoblast cells which are not divided and not proliferated but still maintain metabolic activity by a repeated wall-pasting method, irradiating the trophoblast cells with cobalt 60 after directional modification, stably expressing various cell factors on the surface of a cell membrane, directionally activating and amplifying natural killer cells in peripheral blood mononuclear cells under the synergistic action of the various cell factors to obtain amplified NK cells, and detecting that the positive rate of the amplified NK cells is 85.4 percent and is obviously lower than that of the amplified NK cells.
Test example 1
Observation of the effect of in vitro amplification of NK cells in the examples, the observation is shown in FIG. 1, wherein A is PBMC isolated from peripheral blood; b is the condition of 7 days of culture; c is the condition of 14 days of culture; therefore, it was found that the amplification system can efficiently amplify isolated PBMC with optimal amplification effect at 14 days.
Test example 2
NK cells (CD 56) of NK cells of examples were subjected to flow cytometry using CD56-FITC, CD3-PE monoclonal fluorescent antibody labeled cells and flow cytometry + CD3 - ) The results of purity analysis are shown in FIG. 2, and it is clear from FIG. 2 that NK cells (CD 56) in human peripheral blood mononuclear cells were not expanded before expansion + CD3 - ) The proportion is about 4.74%, after the NK cell is amplified for 14 days by the method, the proportion of the NK cell can reach 90.68%, and the amplification efficiency is excellent.
Test example 3
Cells were counted before and after culture (14 days) for the cells cultured (n = 10). The results are shown in FIG. 3, and from the results in FIG. 3, the number of PBMCs obtained from the sample before amplification was about 1.8X 10 6 -4.5×10 6 After expansion, the number of NK cells was about 178.1X 10 6 -263×10 6 The amplification fold is from 43.17 times to 93.9 times, and the average amplification efficiency is 72.22 +/-16.15 times.
Test example 4
Counting the PBMC before culturing and NK cells after culturing, and adding effector cells and NK cells into a 96-well plate according to the ratio of the effector cells (PBMC/NK cells) to the tumor cells (K562 cells) (effective target ratio) of 5:2Target cells, simultaneously setting single killer cells and single target cell hole controls, and detecting the absorbance (OD) at 450nm of each hole by using a CCK-8 reagent after killing for 4 hours 450 ) According to the formula:
killing rate = [1- (OD) 1 -OD 2 )/OD 3 ]X 100% where OD 1 : effector cell counts + tumor cell readings; OD 2 : (ii) an effector cell readout; OD 3 : tumor cell readings.
The killing rate of NK cells is calculated, and the result shows that the killing rate of PBMC to K562 target cells (the effective target ratio is 5:2) is about 18.69-37.50% and the average killing rate is 26.44% + -7.64% before amplification; after 14 days of amplification, the killing efficiency reaches 70.66% -93.77%, and the average killing rate is 79.37% ± 9.28% (fig. 4, n = 5).
Therefore, the method has better in-vitro amplification effect of the NK cells, and the obtained NK cells have the advantages of large quantity, high purity and strong cytotoxic activity, and can meet the requirements of downstream scientific research and clinical application.
In summary, the following steps:
the invention provides an in vitro amplification system and method of human primary natural killer cells, which have the following advantages: 1. the use is convenient, and the amplification efficiency is good; 2. the preparation is convenient and the cost is low; 3. no biosafety risks such as allergy, severe inflammation and tumor are caused; 4. can achieve better amplification and activation effects on NK cells with poor activity; 5. has good biological safety, and has great industrialization potential and application value.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
Claims (9)
1. An in-vitro amplification system of human primary natural killer cells is characterized by comprising 1640 cell culture medium, IL-2 with the final concentration of 90-110U/mL, NK cell-derived exosome with the final concentration of 180-220ng/mL, recombinant expression protein freeze-dried powder and matrix gel, wherein the weight ratio of the recombinant expression protein freeze-dried powder to the matrix gel is 1 (9-11).
2. The in vitro human primary natural killer cell amplification system of claim 1, comprising 1640 cell culture medium, final concentration of 100U/mLIL-2, final concentration of 200ng/mL NK cell-derived exosome, recombinant expression protein lyophilized powder and matrigel.
3. The in vitro human primary natural killer cell expansion system of claim 1, wherein said 1640 cell culture medium comprises penicillin at a final concentration of 90-110 μ g/mL, streptomycin at a final concentration of 90-110 μ g/mL, 1640 basic medium and 9-11% by weight fetal bovine serum.
4. The in vitro human primary natural killer cell amplification system of claim 1, wherein the lyophilized powder of recombinant expression protein comprises IL-5, IL-18 and 4-1BBL, and the weight ratio of IL-5, IL-18 and 4-1BBL is 1 (1.5-2.5): 1.
5. The in vitro amplification system of human primary natural killer cells according to claim 1 or 2, wherein the weight ratio of the mixture of the lyophilized recombinant expression protein powder and the matrigel to the 1640 cell culture medium is 1 (4.5-5.5).
6. An in vitro amplification method using the in vitro amplification system for human primary natural killer cells according to any one of claims 1 to 5, comprising the steps of: separating peripheral blood mononuclear cells from peripheral blood;
mixing the matrix gel, the recombinant expression protein freeze-dried powder and the 1640 cell culture medium, paving the mixture on a porous plate, and standing the porous plate at 36-38 ℃ for 28-32min to obtain a culture plate;
and sequentially adding the peripheral blood mononuclear cells, the NK cell culture medium and the NK cell-derived exosome into the culture plate, culturing at 36-38 ℃ and observing.
7. The in vitro amplification method of claim 6, wherein each well of said culture plate comprises 1.5-2.5mL of amplification system.
8. The in vitro amplification method according to claim 6, wherein said NK cell culture medium and said NK cell-derived exosomes are replaced every 2-3 days after said peripheral blood mononuclear cells, said NK cell culture medium and said NK cell-derived exosomes are sequentially added to said culture plate.
9. The in vitro amplification method of claim 6, wherein said NK cell culture medium comprises 1640 basal medium, final concentration of 90-110U/mL IL-2, 9-11% wt fetal bovine serum, 80-120 μ g/mL penicillin and 80-120 μ g/mL streptomycin.
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