CN114196628B - Culture medium and culture method of NK cells - Google Patents

Culture medium and culture method of NK cells Download PDF

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CN114196628B
CN114196628B CN202111572220.8A CN202111572220A CN114196628B CN 114196628 B CN114196628 B CN 114196628B CN 202111572220 A CN202111572220 A CN 202111572220A CN 114196628 B CN114196628 B CN 114196628B
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CN114196628A (en
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俞英豪
彭昉
王冶陶
蔡燕萍
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Hangzhou Life Ark Biomedical Technology Co ltd
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Abstract

The invention relates to the technical field of cell culture, in particular to a culture medium and a culture method of NK cells. The average purity of the obtained NK cells (CD 3-CD56+) reaches more than 80 percent, the average amplification factor reaches 4076 times (calculated by the initial culture NK cells accounting for 7.5 percent of PBMC), and the cell killing activity (the effective target ratio is 20:1) is killed for more than 80 percent for 5 hours.

Description

Culture medium and culture method of NK cells
Technical Field
The invention relates to the technical field of cell culture, in particular to a culture medium and a culture method of NK cells.
Background
NK cells are the most important immune cells known at present for killing tumor cells subjected to tumor immune editing, and are one of important means for clinically carrying out tumor cell immunotherapy, and clinical research shows that allogeneic and autologous NK cell immunotherapy is safe. In 2009, the ministry of health issued "regulations on the management of autoimmune cell (T cell, NK cell) therapy techniques (solicited opinion), and the NK cell immunotherapy technique was listed as a third type of new medical technique in the scope of clinical therapy. However, NK cell therapy has not been widely used in hospitals for decades, and in some previous clinical trials, the clinical application of autologous NK cell therapy has not been obvious, wherein the root cause is that a sufficient number and activity of NK immune cells cannot be obtained to affect the therapeutic effect of NK cells, and it is conceivable that in patients with a large tumor burden, if 90% or more of the reinfused NK cells are in a silent state, but not in an activated state, it cannot definitely have a very good killing effect on tumor cells, and in addition, according to in vitro experiments, the killing efficiency can reach 80% or more when the effective target ratio of NK cells to tumor cells is 10:1, but if the amount of reinfused NK cells is far smaller than the amount of tumor cells in vivo, the therapeutic effect is weakened. Therefore, how to realize large-scale expansion culture of NK cells in vitro is a key problem of the current NK cell treatment.
Currently, two common methods for large-scale expansion of NK cells in vitro:
1. trophoblast (K562 cell) stimulation method
Constructing trophoblasts such as CD8 alpha-CD 137-K562, 4-1BBL-K562 and the like by using a genetic engineering means, and obtaining NK cells by using the method has higher purity and larger multiple, but the cost and the steps are more complex due to the fact that (1) gene transfection is involved; (2) The K562 cells (a tumor cell) are needed, and users have certain concern on the safety; (3) When NK cells or CAR-NK cells register medicines, safety evaluation needs to be carried out on the NK cells or the CAR-NK cells, and the trophoblasts are complete cells, so that the related evaluation contents are very complex, and the difficulty and expenditure of security evaluation are greatly increased.
2. Factor stimulation method
The traditional NK cell in-vitro amplification method utilizes some cytokines or factor combinations to stimulate PBMC or purified NK cells in vitro, the NK cells obtained by the method have low general purity (the purity of the NK cells is low, for example, the content of other miscellaneous cells is high, such as T cells, and the T cells are specific cells, if the NK cells are infused back into a human body, GVHD reaction is easy to generate), the multiple is low (the multiple is low, for example, the number of the NK cells in the final product is small, the aim of treating or killing target cells cannot be achieved, and the number of the NK cells is small), and extremely high-purity effector cells can be obtained, but the amplification multiple is extremely low, and even if the culture time is prolonged, the clinically required number cannot be achieved.
Disclosure of Invention
In view of the above, the present invention provides a medium and a method for culturing NK cells, by which over 80% of NK cells can be obtained within 13 days, and the average expansion is over 4000 times without decreasing the killing activity.
In order to achieve the above object, the present invention provides the following technical solutions:
a culture medium comprising water, component 1 and component 2;
the component 1 consists of amino acids and cytokines;
the amino acid consists of arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, valine, hydroxyproline and taurine;
the cytokines consist of IL2, IL12, IL15, IL17, IL21, IL7, IL18, CD80, CD86 and CD 137;
the component 2 is composed of at least two of inorganic salts, saccharides and vitamins.
In some embodiments, the concentrations of the components in component 1 are as follows:
the amino acid consists of 250-350 mg/L of arginine, 56-60 mg/L of cystine, 8-12 mg/L of histidine, 25-35 mg/L of isoleucine, 25-35 mg/L of leucine, 45-55 mg/L of lysine, 8-12 mg/L of methionine, 18-24 mg/L of phenylalanine, 45-55 mg/L of proline, 8-12 mg/L of threonine, 6-10 mg/L of tryptophan, 22-28 mg/L of tyrosine, 16-20 mg/L of valine, 10-18mg/L of hydroxyproline and 18-22 mg/L of taurine;
the cytokine consists of IL2 40-60 KIU/L, IL12 4-6 KIU/L, IL 15-25 KIU/L, IL17 0.8-1.2 KIU/L, IL 85-95 KIU/L, IL7 2-4 KIU/L, IL 18.5-2.5 KIU/L, CD80 0.8-1.2 KIU/L, CD86 0.8-1.2 KIU/L and CD137 0.8-1.2 KIU/L.
In some embodiments, the concentrations of the components in component 1 are as follows:
the amino acid consists of 300mg/L arginine, 58mg/L cystine, 10mg/L histidine, 30mg/L isoleucine, 30mg/L leucine, 50mg/L lysine, 10mg/L methionine, 21mg/L phenylalanine, 50mg/L proline, 10mg/L threonine, 8mg/L tryptophan, 25mg/L tyrosine, 18mg/L valine, 18mg/L hydroxyproline and 20mg/L taurine;
the cytokine consists of IL 250 KIU/L, IL12 5KIU/L, IL 20KIU/L, IL17 1KIU/L, IL21 90KIU/L, IL7 3KIU/L, IL18 2KIU/L, CD80 1KIU/L, CD86 1KIU/L and CD1371 KIU/L.
In some embodiments, in the component 2, the saccharide consists of 100 to 1000mg/L of hydroxyethyl starch and 200 to 2000mg/L of glucose;
the vitamin consists of 0.05-0.15 mg/L of vitamin H, 0.1-0.3 mg/L of vitamin B, 1.5-2.5 mg/L of folic acid, 1.5-2.5 mg/L of vitamin E, 1.5-2.5 mg/L of vitamin B, 0.4-0.6 mg/L of vitamin B and 0.001-0.03 mg/L of vitamin B;
the inorganic salt is prepared from 250-350 mg/L potassium chloride and 3500-4500 mg/L, na sodium chloride 2 HPO 4 1500-2500 mg/L and 150-250 mg/L of magnesium sulfate.
In some embodiments, in component 2, the saccharide consists of 1000mg/L hydroxyethyl starch and 2000mg/L glucose;
the vitamins comprise 0.1mg/L of vitamin H, 0.2mg/L of vitamin B, 2mg/L of folic acid, 2mg/L of vitamin E, 62 mg/L of vitamin B, 0.5mg/L of vitamin B and 0.002mg/L of vitamin B12;
the inorganic salt consists of 300mg/L potassium chloride and 4000mg/L, na sodium chloride 2 HPO 4 2000mg/L and 200mg/L of magnesium sulfate.
The invention also provides application of the culture medium in the separation and/or culture of PBMC or NK cells.
The invention also provides a culture method of the NK cells, which comprises the step of inoculating PBMC or the NK cells into the culture medium for culture.
In some embodiments, the culture method specifically comprises the steps of:
inoculating PBMC or NK cells into the culture medium, culturing for 3-7 days until the color of the cells is yellowish, and centrifuging;
taking the cell sediment, adding the cell sediment into the culture medium, carrying out warm bath for 30min at 37 ℃, transferring the cell sediment into a cell culture bag, and adding the culture medium for culturing for 5-9 days.
In some embodiments, the centrifugation is 400g centrifugation for 3 to 15 minutes. In some embodiments, the centrifugation time may be 3min/5min, 10min, or 15min.
In some embodiments, the inoculation has a density of (1-10) x 10 5 cell/mL; in some embodiments, the seed density may be 1×10 5 cell/mL、5×10 5 cell/mL、6×10 5 cell/mL or 1×10 6 cell/mL
In some embodiments, the culturingIs 37 ℃ and CO 2 The concentration is 5-25%; in some embodiments, the CO 2 The concentration is 5%, 10%, 15%, 20% or 25%.
The average purity of the obtained NK cells (CD 3-CD56+) reaches more than 80 percent, the average amplification factor reaches more than 4000 times (calculated by the NK cells accounting for 7.5 percent of PBMC), and the cell killing activity (the effective target ratio is 20:1) is killed for more than 80 percent in 5 hours after the NK cells are cultured for 13 days by adopting the culture medium provided by the invention.
Drawings
FIG. 1 shows the purity of NK cells obtained after culturing PBMC with the medium of example 1;
FIG. 2 shows the purity of NK cells obtained after culturing PBMC in control group 7 medium;
FIG. 3 shows the purity of NK cells obtained after culturing PBMC in the medium of example 4.
Detailed Description
The invention provides a culture medium and a culture method of NK cells. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
Unless otherwise specified, the test materials adopted in the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
The medium consisted of water and the components of table 1:
TABLE 1
The reagents 1 to 4 were dissolved in deionized water, and mixed well, and the reagent 5 was added before use, and the final concentrations of the components in the medium are shown in Table 1.
PBMC cells were isolated and cultured according to conventional methods, with the experimental set-up control and experimental groups:
experimental group: PBMC cells were grown at 6X 10 5 The cell/mL density was inoculated to the medium of example 1, cultured at 37℃for 3-7 days (specific time was determined according to the color of the cell suspension), when the color of the cells was yellowish, the medium was added to the cell pellet in an appropriate amount (400 g), the cell pellet was incubated at 37℃for 30min, transferred to a cell culture bag, sufficient medium was added, and the culture was continued for 13 days, and NK cells were collected.
Control group 1: the culture medium is as follows: RPMI-1640 medium+cytokine of reagent 5 of example 1 of the present invention.
Culture method of control group 1: PBMC cells were grown at 6X 10 5 Inoculating cell/mL density to control group culture medium, culturing at 37deg.C for 3-7 days, centrifuging to obtain a liquid (400 g), adding appropriate amount of the culture medium into cell sediment, incubating at 37deg.C for 30min, transferring to cell culture bag, adding enough culture medium, continuously culturing for 13 days, and collecting NK cells.
Amplified NK cells were counted with an automatic cytometer and plotted. The purity of NK cells was measured using a multi-color flow cytometer, and the results are shown in tables 2 and 3, wherein the number of cells and the purity are both the average value of 30 batches of experiments. The purity test results of a certain experimental batch are shown in fig. 1.
TABLE 2
TABLE 3 culture results for control group 1
As can be seen from tables 2 and 3, when PBMC cells are cultured by using the culture medium of the present invention, more than 90% of NK cells can be obtained in 13 days, and the average expansion is 5457 times; and the killing activity is not reduced, and the effective target ratio is 20:1, and the killing rate is more than 80% after 5 hours. About 40% of NK cells were obtained in the medium of the control group 1 for 13 days, and the average expansion was 140-fold.
Examples 2 to 4
The present example examined the effect of different components in the medium on NK cell culture, with experimental and control groups, in which:
experimental group: the media of examples 2-4;
control group: 2-12 culture mediums of a control group;
the composition of each medium is shown in Table 4, wherein the specific compositions of reagents 1 to 5 are the same as in example 1.
The culture method comprises the following steps: PBMC cells were grown at 6X 10 5 inoculating/mL of the culture medium at 37 ℃ for 3-7 days, centrifuging, changing the liquid (400 g), adding a proper amount of the culture medium into cell sediment, carrying out warm bath for 30min at 37 ℃, transferring to a cell culture bag, adding enough culture medium, continuously culturing for 13 days, and collecting NK cells.
PBMC cells were cultured according to the above medium composition and culture method, the initially cultured PBMC cells were cultured for 13 days as in example 1, NK cells were collected, the amplified NK cells were counted by an automatic cell counter, and the purity of the NK cells was measured by a multi-color flow cytometer, and the results are shown in Table 4. The purity of control group 7 (NK cell purity: 41.02%) was measured as shown in FIG. 2, and the purity of example 4 was measured as shown in FIG. 3 (NK cell purity: 83.01%).
TABLE 4 Table 4
Note that: "/" indicates no significant change in NK cell number and no amplification effect.
The results show that the amplification times and the purity of NK cells of the control group are obviously lower than those of the experimental group (examples 2-4), wherein the average purity of NK cells (CD 3-CD56+) of the experimental group (examples 2-4) reaches more than 80 percent, and the average amplification times reach 2695 times; the average purity of the control groups (control groups 2 to 12) was 38 to 40%, and the average amplification factor was 115 times.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. A culture medium comprising water, component 1 and component 2;
the component 1 consists of amino acids and cytokines;
the component 2 consists of at least two of inorganic salts, saccharides and vitamins;
the concentration of each component in the component 1 is as follows:
the amino acid consists of 250-350 mg/L of arginine, 56-60 mg/L of cystine, 8-12 mg/L of histidine, 25-35 mg/L of isoleucine, 25-35 mg/L of leucine, 45-55 mg/L of lysine, 8-12 mg/L of methionine, 18-24 mg/L of phenylalanine, 45-55 mg/L of proline, 8-12 mg/L of threonine, 6-10 mg/L of tryptophan, 22-28 mg/L of tyrosine, 16-20 mg/L of valine, 10-18mg/L of hydroxyproline and 18-22 mg/L of taurine;
the cytokine consists of IL2 40-60 KIU/L, IL12 4-6 KIU/L, IL 15-25 KIU/L, IL17 0.8-1.2 KIU/L, IL 85-95 KIU/L, IL 7-4 KIU/L, IL 18.5-2.5 KIU/L, CD80 0.8-1.2 KIU/L, CD86 0.8-1.2 KIU/L and CD137 0.8-1.2 KIU/L;
in the component (2) of the above-mentioned composition,
the saccharide consists of 100-1000 mg/L of hydroxyethyl starch and 200-2000 mg/L of glucose;
the vitamin consists of 0.05-0.15 mg/L of vitamin H, 0.1-0.3 mg/L of vitamin B, 1.5-2.5 mg/L of folic acid, 1.5-2.5 mg/L of vitamin E, 1.5-2.5 mg/L of vitamin B, 0.4-0.6 mg/L of vitamin B and 0.001-0.03mg/L of vitamin B;
the inorganic salt is prepared from 250-350 mg/L of potassium chloride and 3500-4500 mg of sodium chloride/L、Na 2 HPO 4 1500-2500 mg/L and 150-250 mg/L of magnesium sulfate.
2. The medium of claim 1, wherein the concentration of each component in component 1 is as follows:
the amino acid consists of 300mg/L arginine, 58mg/L cystine, 10mg/L histidine, 30mg/L isoleucine, 30mg/L leucine, 50mg/L lysine, 10mg/L methionine, 21mg/L phenylalanine, 50mg/L proline, 10mg/L threonine, 8mg/L tryptophan, 25mg/L tyrosine, 18mg/L valine, 18mg/L hydroxyproline and 20mg/L taurine;
the cytokine consists of IL 250 KIU/L, IL12 5KIU/L, IL 20KIU/L, IL17 1KIU/L, IL21 90KIU/L, IL7 3KIU/L, IL18 2KIU/L, CD80 1KIU/L, CD86 1KIU/L and CD1371 KIU/L.
3. Use of the medium according to claim 1 or 2 for the isolation and/or culture of PBMC or NK cells.
4. A method for culturing NK cells, characterized in that mononuclear cells or NK cells are inoculated into the medium according to claim 1 or 2 for culturing.
5. The method according to claim 4, comprising the steps of:
inoculating PBMC or NK cells into the culture medium of claim 1 or 2, culturing for 3-7 days until the cells are yellowish, and centrifuging;
and adding the cell sediment into the culture medium, carrying out warm bath for 30min at 37 ℃, transferring the cell sediment into a cell culture bag, and adding the culture medium for culturing for 5-9 days.
6. The culture method according to claim 4 or 5, wherein the centrifugation is 400g for 3 to 15min.
7. The culture method according to claim 4 or 5, wherein the grafting is performedThe density of the seeds is (1-10) multiplied by 10 5 cell/ml。
8. The method according to claim 4 or 5, wherein the conditions for the cultivation are 37℃and CO 2 The concentration is 5-25%.
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