CN106754705B - NK cell culture medium and method for in-vitro amplification of NK cells - Google Patents
NK cell culture medium and method for in-vitro amplification of NK cells Download PDFInfo
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Abstract
The invention discloses NK cell culture media, which comprise a basic culture medium and additives, wherein the additives comprise 8-12g/L of human serum albumin, 2.5-7.5mmol/L of nicotinamide, 0.5-2.0 mu mol/L of lenalidomide and 8-16mmol/L of HEPES.
Description
Technical Field
The invention relates to the field of cell culture, in particular to NK cell culture media and a method for amplifying NK cells in vitro.
Background
NK cells, as the th line of defense of the body, can not only exert their main tumoricidal effect in the natural immune system, but also secrete different cytokines and various chemokines to regulate the adaptive immune response in the early stage of the immune response, and are indispensable effector cells for the body to exert the immune effect.
The content of NK cells in peripheral blood is far from meeting clinical requirements, and at present, NK cells are expanded mainly by stimulation of cytokines, but the difference between the expanded NK cells and the clinical requirements still exists in .
Disclosure of Invention
The invention mainly solves the technical problem of providing NK cell culture media and a method for amplifying NK cells in vitro, and the NK cells with large quantity, high activity and strong killing property can be amplified.
In order to solve the technical problems, the invention provides NK cell culture media, which comprise a basic culture medium and additives, wherein the additives comprise 8-12g/L of human serum albumin, 2.5-7.5mmol/L of nicotinamide, 0.5-2.0 mu mol/L of lenalidomide and 8-16mmol/L of HEPES.
Wherein the basic culture medium is MEM-alpha culture medium.
Wherein, the additive comprises the following components in percentage by concentration: 9-11g/L human serum albumin, 3.5-6.5mmol/L nicotinamide, 0.8-1.5 mu mol/L lenalidomide and 9-15mmol/L HEPES.
Wherein, the additive comprises the following components in percentage by concentration: 10g/L human serum albumin, 5mmol/L nicotinamide, 1.0. mu. mol/L lenalidomide, 10mmol/L HEPES.
To solve the above technical problems, the present invention provides methods for in vitro expansion of NK cells, comprising the steps of suspending prepared NK cells in an NK cell culture medium and transferring the NK cell suspended medium to a cell culture flask, the NK cell density being 4.5X 106-5.5×106Each/20 ml; adding 15-25% of autologous plasma and 5-15 mug/ml of anti-Her-2 monoclonal antibody, 950-1050U/ml of recombinant human IL-2, 25-35ng/ml of recombinant human IL-15 and 5-15ng/ml of recombinant human IL-21 into a cell culture bottle in a volume ratio, placing the cell culture bottle at 37 ℃ and 5% CO2Culturing in an incubator; supplementing an NK cell culture medium and 950-1050U/ml recombinant human IL-2, 25-35ng/ml recombinant human IL-15 and 5-15ng/ml recombinant human IL-21 at preset intervals, and transferring the cell suspension into a cell culture bag for culture when the culture system is 90-110 ml; obtaining expanded NK cells after a predetermined number of days of culture; wherein, NK cell culture medium includes basal medium and additive, and each component of additive and concentration are: 8-12g/L human serum albumin, 2.5-7.5mmol/L nicotinamide, 0.5-2.0. mu. mol/L lenalidomide, 8-16 mmol/LHEPES.
Wherein the density of NK cells initially suspended in the NK cell culture medium is 5X 106Each 20 ml.
Wherein, the concentration of autologous plasma is 20 percent by volume, the concentration of the anti-Her-2 monoclonal antibody is 10 mu g/ml, the concentration of the recombinant human IL-2 is 1000U/ml, the concentration of the recombinant human IL-15 is 30ng/ml, and the concentration of the recombinant human IL-21 is 10 ng/ml.
Wherein the basic culture medium is MEM-alpha culture medium.
Wherein, the additive comprises the following components in percentage by concentration: 10g/L human serum albumin, 5mmol/L nicotinamide, 1.0. mu. mol/L lenalidomide, 10mmol/L HEPES.
Wherein the cell culture bag is a breathable cell culture bag.
The invention has the beneficial effects that: different from the situation of the prior art, the NK cell culture medium comprises a basic culture medium and additives, wherein the basic culture medium is an MEM-alpha culture medium, and the additives comprise the following components in percentage by weight: 8-12g/L human serum albumin, 2.5-7.5mmol/L nicotinamide, 0.5-2.0 μmol/L lenalidomide, and 8-16mmol/L HEPES. By amplifying the NK cells in vitro by the culture medium, the NK cells with large quantity, high activity and strong killing property can be amplified.
Drawings
FIG. 1 is a graph showing the flow results of in vitro amplified NK cells of the present invention;
FIG. 2 is a second image of the flow-type result of NK cells amplified in vitro according to the present invention.
Detailed Description
Example 1
The method for in vitro expansion of NK cells is as follows: suspending the prepared NK cells in NK cell culture medium at a density of 5 × 106Per 20ml, then transferring the NK cell-suspended medium to a cell culture flask; adding 20% volume ratio of autologous plasma, 10 μ g/ml anti-Her-2 monoclonal antibody, 1000U/ml recombinant human IL-2, 30ng/ml recombinant human IL-15, 10ng/ml recombinant human IL-21 into cell culture flask, placing the cell culture flask at 37 deg.C and 5% CO2Culturing in an incubator; supplementing an NK cell culture medium, 1000U/ml recombinant human IL-2, 30ng/ml recombinant human IL-15 and 10ng/ml recombinant human IL-21 every 3 days, and transferring the cell suspension into a 1.9L cell culture bag for continuous culture when the culture system is 100 ml; after 14 days of culture, the cells were removed from the culture bag to obtain expanded NK cells. Wherein the cell culture bag is a breathable cell culture bag.
In this example, the NK cell culture medium includes a basic medium and additives, the basic medium is MEM-alpha medium, and the components and concentrations of the additives are as follows: 10g/L human serum albumin, 5mmol/L nicotinamide, 1.0. mu. mol/L lenalidomide, 10mmol/L HEPES.
In this example, the cell culture flask was a T75 flask.
Referring to FIG. 1, FIG. 1 is a flow chart of NK cells amplified in vitro according to the present example, as shown in FIG. 1, since the purity of prepared NK cells is not 100%, other cells are also amplified. Fig. 1 is divided into 4 quadrants: on the upper left, CD (16 + 56) positive and CD3 negative, the cell is NK cell, accounting for 84.7%; on the right, the cell is positive for CD3 and positive for CD (16 + 56), namely the NKT cell (with T cell phenotype and NK cell phenotype) accounting for 10.8%; in the lower right, CD3 positive and CD (16 + 56) negative, the cells are T cells, accounting for 4.27%; bottom left, heterocyte, 0.161%. As can be seen from FIG. 1, the amplification effect of this example is very considerable.
Example 2
The method for in vitro expansion of NK cells is as follows: suspending the prepared NK cells in NK cell culture medium at a density of 5 × 106Per 20ml, then transferring the NK cell-suspended medium to a cell culture flask; adding 20% volume ratio of autologous plasma, 10 μ g/ml anti-Her-2 monoclonal antibody, 1000U/ml recombinant human IL-2, 30ng/ml recombinant human IL-15, 10ng/ml recombinant human IL-21 into cell culture flask, placing the cell culture flask at 37 deg.C and 5% CO2Culturing in an incubator; supplementing an NK cell culture medium, 1000U/ml recombinant human IL-2, 30ng/ml recombinant human IL-15 and 10ng/ml recombinant human IL-21 every 3 days, and transferring the cell suspension into a 1.9L cell culture bag for continuous culture when the culture system is 100 ml; after 14 days of culture, the cells were removed from the culture bag to obtain expanded NK cells. Wherein the cell culture bag is a breathable cell culture bag.
In this example, the NK cell culture medium includes a basic medium and additives, the basic medium is MEM-alpha medium, and the components and concentrations of the additives are as follows: 11g/L human serum albumin, 6mmol/L nicotinamide, 1.2. mu. mol/L lenalidomide, 12mmol/L HEPES.
In this example, the cell culture flask was a T75 flask.
Referring to FIG. 2, FIG. 2 is a flow chart of NK cells amplified in vitro according to the present example, as shown in FIG. 2, since the purity of prepared NK cells is not 100%, other cells are also amplified. Fig. 2 is divided into 4 quadrants: on the upper left, CD (16 + 56) positive and CD3 negative, the cell is NK cell, accounting for 84.9%; on the right, the cell is positive for CD3 and positive for CD (16 + 56), namely the NKT cell (with T cell phenotype and NK cell phenotype) accounting for 10.9%; on the lower right, CD3 positive and CD (16 + 56) negative, the cell is a T cell, accounting for 4.05%; bottom left, heterocyte, accounting for 0.159%. As can be seen from FIG. 2, the amplification effect of this example is very considerable.
Comparative example 1
The method for in vitro expansion of NK cells is as follows: suspending the prepared NK cells in NK cell culture medium at a density of 5 × 106Per 20ml, then transferring the NK cell-suspended medium to a cell culture flask; placing the cell culture flask at 37 deg.C and 5% CO2Culturing in an incubator; supplementing the NK cell culture medium every 3 days, and when the culture system is 100ml, transferring the cell suspension into a 1.9L cell culture bag for continuous culture; after 14 days of culture, the cells were removed from the culture bag to obtain expanded NK cells.
Wherein, the NK cell culture medium comprises 1000U/ml recombinant human IL-2, 30ng/ml recombinant human IL-15 and 10ng/ml recombinant human IL-21.
Comparative example 2
The amplification method is the same as that of the example 1, and the NK cell culture medium used for amplification comprises a basic culture medium and additives, wherein the basic culture medium is an MEM-alpha culture medium, and the components and the concentrations of the additives are as follows: 10g/L human serum albumin, 5mmol/L nicotinamide, 10mmol/L HEPES.
Comparative example 3
The amplification method is the same as that of the example 2, and the NK cell culture medium used for amplification comprises a basic culture medium and additives, wherein the basic culture medium is an MEM-alpha culture medium, and the components and the concentrations of the additives are as follows: 11g/L human serum albumin, 6mmol/L nicotinamide and 12mmol/L HEPES.
Experimental example 1
The cells cultured in example 1, example 2, comparative example 1, comparative example 2 and comparative example 3 were collected on days 0, 7 and 14, respectively, and were stained with trypan blue, and then counted, and the total number of cells on the day of counting was divided by the number of cells on day 0 to obtain the fold expansion.
TABLE 1 NK cell expansion fold
As shown in Table 1, the numbers of NK cells amplified in examples 1 and 2 were significantly larger than those amplified in comparative examples 1, 2 and 3.
Experimental example 2
NK cells amplified in example 1, example 2, comparative example 1, comparative example 2 and comparative example 3 were used as effector cells with human leukemia cell K562 in the logarithmic growth phase as a target cell, and the killing activity of NK cells was examined by the LDH method. The method specifically comprises the following steps: mixing the effector cells and the target cells according to the effective target ratio of 1:1, 10:1 and 20:1, simultaneously setting effector cell holes and target cell holes, and detecting the OD value at 450nm by using an enzyme labeling instrument. Wherein, the kill rate calculation formula is as follows: killing rate = [1- (experimental well OD value-effector cell well OD value/target cell well OD value) ] × 100%, experimental wells are wells containing effector cells and target cells.
TABLE 2 NK cell killing Rate
As shown in Table 2, the killing activity of the NK cells amplified in the examples 1 and 2 on K562 is higher than that of the NK cells amplified in the comparative examples 1, 2 and 3 under the condition of different effective target ratios.
In conclusion, the NK cell culture medium and the method for amplifying NK cells in vitro can amplify the NK cells with large quantity, high activity and strong killing property. It is to be noted that the NK cell culture medium of the present invention is a serum-free cell culture medium for in vitro expansion of NK cells.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (7)
1, NK cell culture media, characterized by comprising MEM-alpha culture media and additives, wherein the additives comprise 10g/L human serum albumin, 5mmol/L nicotinamide, 1.0 μmol/L lenalidomide and 10 mmol/LHEPES.
The method for in vitro expansion of NK cells of species, comprising the steps of suspending the prepared NK cells in an NK cell culture medium and transferring the NK cell suspended medium into a cell culture flask, the NK cell density being 4.5X 106-5.5× 106Each/20 ml; adding 15-25% of autologous plasma and 5-15 mu g/ml of anti-Her-2 monoclonal antibody, 950-1050U/ml of recombinant human IL-2, 25-35ng/ml of recombinant human IL-15 and 5-15ng/ml of recombinant human IL-21 into the cell culture bottle, and culturing the cell culture bottle in an incubator at 37 ℃ and 5% CO 2; supplementing the NK cell culture medium and 950-1050U/ml recombinant human IL-2, 25-35ng/ml recombinant human IL-15 and 5-15ng/ml recombinant human IL-21 at preset time intervals, and transferring the cell suspension into a cell culture bag for culture when the culture system is 90-110 ml; obtaining expanded NK cells after a predetermined number of days of culture; the NK cell culture medium comprises a basic culture medium and additives, wherein the additives comprise the following components in percentage by concentration: 8-12g/L human serum albumin, 2.5-7.5mmol/L nicotinamide, 0.5-2.0 μmol/L lenalidomide, and 8-16mmol/L HEPES.
3. The method for the in vitro expansion of NK cells according to claim 2, wherein the density of NK cells initially suspended in NK cell culture medium is 5 x 106Each 20 ml.
4. The method for in vitro expansion of NK cells according to claim 2, wherein the concentration of autologous plasma is 20% by volume, the concentration of the anti-Her-2 monoclonal antibody is 10 μ g/ml, the concentration of the recombinant human IL-2 is 1000U/ml, the concentration of the recombinant human IL-15 is 30ng/ml, and the concentration of the recombinant human IL-21 is 10 ng/ml.
5. The method for in vitro expanding NK cells according to claim 2, wherein the basal medium is MEMalpha medium.
6. The method for in vitro expansion of NK cells according to claim 2, wherein the additives are comprised of: 10g/L human serum albumin, 5mmol/L nicotinamide, 1.0. mu. mol/L lenalidomide, 10mmol/L HEPES.
7. The method for in vitro expanding NK cells according to claim 2, wherein the cell culture bag is a gas permeable cell culture bag.
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| CN108192865B (en) * | 2017-12-29 | 2021-09-21 | 吉林省拓华生物科技有限公司 | NK cell in-vitro amplification method and kit used for same |
| CN113106063A (en) * | 2020-02-25 | 2021-07-13 | 河南省银丰生物工程技术有限公司 | Method for in-vitro amplification of NK immune cells |
| CN112300992B (en) * | 2020-10-27 | 2023-05-23 | 杜德(江门)生物科技有限公司 | NK cell culture solution and multistage activated NK cell culture method |
| CN114891742A (en) * | 2022-06-23 | 2022-08-12 | 杭州中赢生物医疗科技有限公司 | Culture medium and in-vitro amplification method for NK (natural killer) cells with strong killing property |
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| WO2007065016A2 (en) * | 2005-12-02 | 2007-06-07 | Au Jessie L S | Methods and compositions to improve activity and reduce toxicity of stents |
| CN105567634A (en) * | 2016-01-27 | 2016-05-11 | 上海润泉生物技术有限公司 | Culture medium and method for NK cell expansion in vitro |
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| WO2007065016A2 (en) * | 2005-12-02 | 2007-06-07 | Au Jessie L S | Methods and compositions to improve activity and reduce toxicity of stents |
| CN105567634A (en) * | 2016-01-27 | 2016-05-11 | 上海润泉生物技术有限公司 | Culture medium and method for NK cell expansion in vitro |
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