CN106754705A - A kind of method of NK cell culture mediums and amplification in vitro NK cells - Google Patents
A kind of method of NK cell culture mediums and amplification in vitro NK cells Download PDFInfo
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Abstract
The invention discloses a kind of NK cell culture mediums, including basal medium and additive, additive each component and concentration are：8 12g/L human serum albumins, 2.5 7.5mmol/L niacinamides, 0.5 2.0 μm of ol/L lenalidomides, 8 16mmol/L HEPES.Invention additionally discloses a kind of method of amplification in vitro NK cells.Using NK cell culture mediums of the invention and the method for amplification in vitro NK cells, it is amplifiable go out quantity is big, high, the lethal strong NK cells of activity.
Description
Technical field
The present invention relates to field of cell culture, the side of more particularly to a kind of NK cell culture mediums and amplification in vitro NK cells
Method.
Background technology
NK（Natural killer cell, NK）It is the important immunocyte of body, is mainly distributed on outer
In all blood.NK cells not only can play it and mainly kill as the first line of defence of body defenses system in natural immune system
Knurl effect, and different cell factor and various chemotactic factor (CF)s can be secreted early stage immune response obtain adjusting body
Property immune response, is the indispensable effector cell of physical exertion immunological effect.
Content of the NK cells in peripheral blood far from meeting clinical needs, the main stimulation using cell factor at present come
Amplification NK cells, but the NK cells of above method amplification still suffer from a certain distance with clinical demand.How providing one kind can expand
Increase the culture medium for a large amount of and the NK cells of high activity, be one of NK cell expansion ex vivos field problem demanding prompt solution.
The content of the invention
A kind of method the present invention solves the technical problem of NK cell culture mediums and amplification in vitro NK cells is provided,
It is amplifiable go out quantity is big, high, the lethal strong NK cells of activity.
In order to solve the above technical problems, the present invention provides a kind of NK cell culture mediums, including basal medium and additive,
Additive each component and concentration are：8-12g/L human serum albumins, 2.5-7.5mmol/L niacinamides, 0.5-2.0 μm of ol/L
Lenalidomide, 8-16mmol/L HEPES.
Wherein, basal medium is MEM-alpha culture mediums.
Wherein, additive each component and concentration are：9-11g/L human serum albumins, 3.5-6.5mmol/L niacinamides,
0.8-1.5 μm of ol/L lenalidomides, 9-15mmol/L HEPES.
Wherein, additive each component and concentration are：10g/L human serum albumins, 5mmol/L niacinamides, 1.0 μm of ol/L
Lenalidomide, 10mmol/L HEPES.
In order to solve the above technical problems, the present invention provides a kind of method of amplification in vitro NK cells, comprise the following steps：Will
The NK cells of preparation are suspended from NK cell culture mediums, and outstanding will have media transfer a to Tissue Culture Flask of NK cells, and NK is thin
The density of born of the same parents is 4.5 × 106-5.5×106Individual/20ml；It is the autologous plasma of 15%-25% that volume ratio is added in Tissue Culture Flask
And the anti-Her-2 monoclonal antibodies of 5-15 μ g/ml, 950-1050U/ml recombinant human il-2s, 25-35ng/ml recombined humans IL-15,5-
15ng/ml recombinant human il-2s 1, and Tissue Culture Flask is placed in 37 DEG C, 5%CO2Incubator in cultivate；The interval scheduled time mends
Plus NK cell culture mediums and 950-1050U/ml recombinant human il-2s, 25-35ng/ml recombined human IL-15,5-15ng/ml recombined humans
IL-21, when cultivating system is 90-110ml, cell suspension is transferred in cell culture bags and is cultivated；After culture predetermined number of days,
Obtain the NK cells of amplification；Wherein, NK cell culture mediums include basal medium and additive, additive each component and concentration
For：8-12g/L human serum albumins, 2.5-7.5mmol/L niacinamides, 0.5-2.0 μm of ol/L lenalidomides, 8-16mmol/L
HEPES。
Wherein, the density of the NK cells being initially suspended from NK cell culture mediums is 5 × 106Individual/20ml.
Wherein, the concentration of autologous plasma is volume ratio 20%, and the concentration of anti-Her-2 monoclonal antibodies is 10 μ g/ml, restructuring
The concentration of human IL-2 is 1000U/ml, and the concentration of recombined human IL-15 is 30ng/ml, and the concentration of recombinant human il-2 1 is 10ng/ml.
Wherein, basal medium is MEM-alpha culture mediums.
Wherein, additive each component and concentration are：10g/L human serum albumins, 5mmol/L niacinamides, 1.0 μm of ol/L
Lenalidomide, 10mmol/L HEPES.
Wherein, cell culture bags are ventilative cell culture bags.
The beneficial effects of the invention are as follows：The situation of prior art is different from, NK cell culture mediums of the invention include basis
Culture medium and additive, basal medium are MEM-alpha culture mediums, and additive each component and concentration are：8-12g/L human serums
Albumin, 2.5-7.5mmol/L niacinamides, 0.5-2.0 μm of ol/L lenalidomides, 8-16mmol/L HEPES.By above-mentioned
Culture medium amplification in vitro NK cells, it is amplifiable go out quantity is big, high, the lethal strong NK cells of activity.
Brief description of the drawings
Fig. 1 is the NK cell streamings result figure one of amplification in vitro of the present invention；
Fig. 2 is the NK cell streamings result figure two of amplification in vitro of the present invention.
Specific embodiment
Embodiment 1
The method of amplification in vitro NK cells is as follows：The NK cells of preparation are suspended from NK cell culture mediums, the density of NK cells is 5
×106Individual/20ml, in then outstanding having media transfer a to Tissue Culture Flask of NK cells；Body is added in Tissue Culture Flask
Product is than the autologous plasma for 20% and the anti-Her-2 monoclonal antibodies of 10 μ g/ml, 1000U/ml recombinant human il-2s, 30ng/ml restructuring
Human IL-15,10ng/ml recombinant human il-2s 1, are then placed in 37 DEG C, 5%CO by Tissue Culture Flask2Incubator in cultivate；Every 3
It adds NK cell culture mediums and 1000U/ml recombinant human il-2s, 30ng/ml recombined humans IL-15,10ng/ml recombinant human il-2 1,
When cultivating system is 100ml, cell suspension is transferred in 1.9L cell culture bags and continues to cultivate；After culture 14 days, from training
Cell is taken out in foster bag, the NK cells of amplification are obtained.Wherein, cell culture bags are ventilative cell culture bags.
In the present embodiment, NK cell culture mediums include basal medium and additive, and basal medium is MEM-alpha
Culture medium, additive each component and concentration are：10g/L human serum albumins, 5mmol/L niacinamides, 1.0 μm of ol/L carry out that degree
Amine, 10mmol/L HEPES.
In the present embodiment, Tissue Culture Flask is T75 blake bottles.
Fig. 1 is referred to, Fig. 1 is the NK cell streaming result figures of the present embodiment amplification in vitro, as shown in figure 1, due to preparing
NK cell purities without 100%, therefore also in the presence of the amplification of other cells.Fig. 1 is divided into 4 quadrants：Upper left, CD（16+56）Sun
Property, CD3 it is negative, this cell is NK cells, accounts for 84.7%；Upper right, CD3 is positive, CD（16+56）The positive, this cell is NKT
Cell（Existing T cell phenotype, there is NK cell phenotypes again）, account for 10.8%；Bottom right, CD3 is positive, CD（16+56）Feminine gender, this cell
As T cell, accounts for 4.27%；Lower-left, heteroproteose cell accounts for 0.161%.As seen from Figure 1, the expanding effect of the present embodiment very may be used
See.
Embodiment 2
The method of amplification in vitro NK cells is as follows：The NK cells of preparation are suspended from NK cell culture mediums, the density of NK cells is 5
×106Individual/20ml, in then outstanding having media transfer a to Tissue Culture Flask of NK cells；Body is added in Tissue Culture Flask
Product is than the autologous plasma for 20% and the anti-Her-2 monoclonal antibodies of 10 μ g/ml, 1000U/ml recombinant human il-2s, 30ng/ml restructuring
Human IL-15,10ng/ml recombinant human il-2s 1, are then placed in 37 DEG C, 5%CO by Tissue Culture Flask2Incubator in cultivate；Every 3
It adds NK cell culture mediums and 1000U/ml recombinant human il-2s, 30ng/ml recombined humans IL-15,10ng/ml recombinant human il-2 1,
When cultivating system is 100ml, cell suspension is transferred in 1.9L cell culture bags and continues to cultivate；After culture 14 days, from training
Cell is taken out in foster bag, the NK cells of amplification are obtained.Wherein, cell culture bags are ventilative cell culture bags.
In the present embodiment, NK cell culture mediums include basal medium and additive, and basal medium is MEM-alpha
Culture medium, additive each component and concentration are：11g/L human serum albumins, 6mmol/L niacinamides, 1.2 μm of ol/L carry out that degree
Amine, 12mmol/L HEPES.
In the present embodiment, Tissue Culture Flask is T75 blake bottles.
Fig. 2 is referred to, Fig. 2 is the NK cell streaming result figures of the present embodiment amplification in vitro, as shown in Fig. 2 due to preparing
NK cell purities without 100%, therefore also in the presence of the amplification of other cells.Fig. 2 is divided into 4 quadrants：Upper left, CD（16+56）Sun
Property, CD3 it is negative, this cell is NK cells, accounts for 84.9%；Upper right, CD3 is positive, CD（16+56）The positive, this cell is NKT
Cell（Existing T cell phenotype, there is NK cell phenotypes again）, account for 10.9%；Bottom right, CD3 is positive, CD（16+56）Feminine gender, this cell
As T cell, accounts for 4.05%；Lower-left, heteroproteose cell accounts for 0.159%.As seen from Figure 2, the expanding effect of the present embodiment very may be used
See.
Comparative example 1
The method of amplification in vitro NK cells is as follows：The NK cells of preparation are suspended from NK cell culture mediums, the density of NK cells is 5
×106Individual/20ml, in then outstanding having media transfer a to Tissue Culture Flask of NK cells；By Tissue Culture Flask be placed in 37 DEG C,
5%CO2Incubator in cultivate；NK cell culture mediums were added every 3 days, when cultivating system is 100ml, cell suspension is turned
Continuation is cultivated in moving on to 1.9L cell culture bags；After culture 14 days, cell is taken out from culture bag, obtain the NK cells of amplification.
Wherein, NK cell culture mediums include 1000U/ml recombinant human il-2s, 30ng/ml recombined humans IL-15,10ng/ml weight
Group human IL-2 1.
Comparative example 2
Amplification method is same as Example 1, expands used NK cell culture mediums, including basal medium and additive, base
Basal culture medium is MEM-alpha culture mediums, and additive each component and concentration are：10g/L human serum albumins, 5mmol/L Buddhist nun gram
Acid amides, 10mmol/L HEPES.
Comparative example 3
Amplification method is same as Example 2, expands used NK cell culture mediums, including basal medium and additive, base
Basal culture medium is MEM-alpha culture mediums, and additive each component and concentration are：11g/L human serum albumins, 6mmol/L Buddhist nun gram
Acid amides, 12mmol/L HEPES.
Experimental example 1
Respectively 0,7,14 days Examples 1, embodiment 2, comparative example 1, comparative example 2, the culture of comparative example 3 cell, expected with platform
Counted after indigo plant dyeing, by the TCS on the day of counting divided by the cell number of the 0th day, obtain amplification times.
The NK cells expandeds of table 1
According to table 1, embodiment 1, the NK cell quantities of the amplification of embodiment 2 are significantly greater than comparative example 1, comparative example 2, comparative example
The NK cell quantities of 3 amplifications.
Experimental example 2
It is target cell with the Leukemia K562 cell in exponential phase, by embodiment 1, embodiment 2, comparative example 1, contrast
Example 2, the NK cells of the amplification of comparative example 3 detect the killing activity of NK cells using LDH methods as effector cell.Specially：By 1:
1、10:1、20:1 effect target ratio mixes effector cell and target cell, while setting effector cell hole, Target cell wells, uses enzyme mark
OD values during instrument detection 450nm.Wherein, killing rate computing formula is：Killing rate=[1-（Experimental port OD values-effector cell hole OD
Value/Target cell wells OD values）] × 100%, experimental port is the hole containing effector cell and target cell.
The NK cell killing rates of table 2
According to table 2, in the case of different effect target ratios, the killing of embodiment 1, the NK cells of the amplification of embodiment 2 to K562
Activity is above comparative example 1, comparative example 2, the killing activity of the NK cells of the amplification of comparative example 3.
In sum, using NK cell culture mediums of the invention and the method for amplification in vitro NK cells, it is amplifiable go out quantity
Greatly, high, the lethal strong NK cells of activity.It is pointed out that NK cell culture mediums of the invention are for amplification in vitro NK
The serum-free cell culture medium of cell.
Embodiments of the invention are the foregoing is only, the scope of the claims of the invention is not thereby limited, it is every to utilize this hair
Equivalent structure or equivalent flow conversion that bright specification and accompanying drawing content are made, or directly or indirectly it is used in other related skills
Art field, is included within the scope of the present invention.
Claims (10)
1. a kind of NK cell culture mediums, it is characterised in that including basal medium and additive, the additive each component and dense
Spend and be：8-12g/L human serum albumins, 2.5-7.5mmol/L niacinamides, 0.5-2.0 μm of ol/L lenalidomides, 8-
16mmol/L HEPES。
2. NK cell culture mediums according to claim 1, it is characterised in that the basal medium is MEM-alpha trainings
Support base.
3. NK cell culture mediums according to claim 2, it is characterised in that the additive each component and concentration are：9-
11g/L human serum albumins, 3.5-6.5mmol/L niacinamides, 0.8-1.5 μm of ol/L lenalidomides, 9-15mmol/L
HEPES。
4. NK cell culture mediums according to claim 3, it is characterised in that the additive each component and concentration are：
10g/L human serum albumins, 5mmol/L niacinamides, 1.0 μm of ol/L lenalidomides, 10mmol/L HEPES.
5. a kind of method of amplification in vitro NK cells, it is characterised in that comprise the following steps：
The NK cells of preparation are suspended from NK cell culture mediums, and outstanding there will be the media transfer of NK cells to Tissue Culture Flask
In, the density of NK cells is 4.5 × 106-5.5×106Individual/20ml；
Add volume ratio anti-for the autologous plasma and the anti-Her-2 monoclonals of 5-15 μ g/ml of 15%-25% in the Tissue Culture Flask
Body, 950-1050U/ml recombinant human il-2s, 25-35ng/ml recombined humans IL-15,5-15ng/ml recombinant human il-2 1, and will be described
Tissue Culture Flask is placed in 37 DEG C, 5%CO2Incubator in cultivate；
The interval scheduled time adds the NK cell culture mediums and 950-1050U/ml recombinant human il-2s, 25-35ng/ml recombined humans
IL-15,5-15ng/ml recombinant human il-2 1, when cultivating system is 90-110ml, cell culture bags is transferred to by cell suspension
Middle culture；
After culture predetermined number of days, the NK cells of amplification are obtained；
Wherein, the NK cell culture mediums include basal medium and additive, and the additive each component and concentration are：8-
12g/L human serum albumins, 2.5-7.5mmol/L niacinamides, 0.5-2.0 μm of ol/L lenalidomides, 8-16mmol/L
HEPES。
6. the method for amplification in vitro NK cells according to claim 5, it is characterised in that be initially suspended from NK cell culture mediums
In NK cells density be 5 × 106Individual/20ml.
7. the method for amplification in vitro NK cells according to claim 6, it is characterised in that the concentration of the autologous plasma is
Volume ratio 20%, the concentration of the anti-Her-2 monoclonal antibodies is 10 μ g/ml, and the concentration of the recombinant human il-2 is 1000U/
The concentration of ml, the recombined human IL-15 is 30ng/ml, and the concentration of the recombinant human il-2 1 is 10ng/ml.
8. the method for amplification in vitro NK cells according to claim 7, it is characterised in that the basal medium is MEM-
Alpha culture mediums.
9. the method for amplification in vitro NK cells according to claim 8, it is characterised in that the additive each component and dense
Spend and be：10g/L human serum albumins, 5mmol/L niacinamides, 1.0 μm of ol/L lenalidomides, 10mmol/L HEPES.
10. the method for amplification in vitro NK cells according to claim 8, it is characterised in that the cell culture bags are
Gas cell culture bags.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192865A (en) * | 2017-12-29 | 2018-06-22 | 吉林省拓华生物科技有限公司 | NK cell expansion ex vivos method and the kit for this method |
| CN112300992A (en) * | 2020-10-27 | 2021-02-02 | 杜德(江门)生物科技有限公司 | NK cell culture solution and multistage activation NK cell culture method |
| 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 |
| 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 |
| CN114891742B (en) * | 2022-06-23 | 2024-06-04 | 杭州中赢生物医疗科技有限公司 | Culture medium of NK cells with strong killing property and in-vitro amplification method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108192865A (en) * | 2017-12-29 | 2018-06-22 | 吉林省拓华生物科技有限公司 | NK cell expansion ex vivos method and the kit for this method |
| 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 |
| CN112300992A (en) * | 2020-10-27 | 2021-02-02 | 杜德(江门)生物科技有限公司 | NK cell culture solution and multistage activation 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 |
| CN114891742B (en) * | 2022-06-23 | 2024-06-04 | 杭州中赢生物医疗科技有限公司 | Culture medium of NK cells with strong killing property and in-vitro amplification method |
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| CN106754705B (en) | 2020-01-31 |
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