CN109517793A - A kind of method for building up of NK cell and gamma delta T cells co-cultivation - Google Patents

A kind of method for building up of NK cell and gamma delta T cells co-cultivation Download PDF

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CN109517793A
CN109517793A CN201811454667.3A CN201811454667A CN109517793A CN 109517793 A CN109517793 A CN 109517793A CN 201811454667 A CN201811454667 A CN 201811454667A CN 109517793 A CN109517793 A CN 109517793A
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黄朝峰
柏川
陈焕鹏
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Zhuhai Basso Cell Science And Technology Co ltd
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Guangzhou Changfeng Biotechnology Co Ltd
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Abstract

The present invention relates to a kind of technical field of cell culture, the method for building up that espespecially a kind of NK cell and gamma delta T cells are capable of a kind of NK cell of external coamplification culture and gamma delta T cells co-culture specifically includes that S1, reagent selection；S2, PBMC is cultivated by culture medium, the time is 3 days；S3, supplement culture medium, maintain concentration and cultivate 4 days；S4, centrifugation is abandoned supernatant and is then gone in T25 Tissue Culture Flask after the 7th day, rear to replace EX culture medium；S5, culture medium maintenance concentration is augmented after the 10th day；S6, continue to augment culture medium maintenance concentration after the 12nd day；S7, NK and gamma delta T cells ratio and total number of cells are detected after the 14th day；Co-cultivation method for building up of the invention, disposably turn out two kinds of antineoplastic immune cells with similarity, improve cell culture efficiency, toxigenic capacity can be greatly lowered compared to independent cultivate for various immunocytes obtain NK cell and gamma delta T cells, also reduce culture technique difficulty.

Description

A kind of method for building up of NK cell and gamma delta T cells co-cultivation
Technical field
The present invention relates to a kind of technical field of cell culture, espespecially a kind of NK cell and gamma delta T cells can expand jointly in vitro Increase the method for building up of a kind of the NK cell and gamma delta T cells co-cultivation of culture.
Background technique
Natural killer cells (natural killer cell, NK) is the important immunocyte of body, with antineoplastic immune Technology is closely related, and the in vitro culture NK cell auxiliary cell treatment that then self feedback can be used as oncotherapy first is answered With；NK cell injuring model is the key link of this cell therapy, and NK cell has affinity receptor in IL-2, in IL-2 Stimulation is lower can to occur breeder reaction, existing NK cell injuring model mainly by add the culture medium containing IL-2 expand come, There is more commercial culture medium selective at present.
Gamma delta T cells are the T cells for executing inherent immunity function, and TCR is made of γ and δ chain, gamma delta T cells be it is a kind of both Cancer cell, tumor stem cell can be killed, and can identify the immunocyte of cancer antigen；It is existing research shows that gamma delta T cells an Asia Type can under the stimulation of phospholipid molecule quick activationa and proliferation, and the hypotype gamma delta T cells feed back self and can also be used as tumour and control The adjuvant treatment application for the treatment of has important clinical value by phospholipid molecule amplification in vitro gamma delta T cells technology；Currently used for The commercial culture medium selection of gamma delta T cells amplification is few, usually thin to obtain gamma delta T with T cell culture medium addition phospholipid molecule Born of the same parents.
Gamma delta T cells and NK cell all have tumor cytotoxicity active, can be used for the adjuvant treatment of tumour patient, If can will be of great significance with both cells of synchronous amplification and applied to patient, however there is presently no can synchronize The method for expanding two kinds of cells is established, and the amplification in vitro culture of NK cell and gamma delta T cells individually carries out at present, synchronous amplification The method for building up of culture is still yet-to-be developed, and meaning value is also still to be verified.
Summary of the invention
To solve the above problems, the present invention is directed to disclose a kind of technical field of cell culture, espespecially a kind of NK cell and γ δ T cell is capable of the method for building up of a kind of the NK cell and gamma delta T cells co-cultivation of external coamplification culture, to realize synchronization Cultivate the in vitro culture purpose of NK cell and gamma delta T cells.
To achieve the above object, the technical solution adopted by the present invention is that: what a kind of NK cell and gamma delta T cells co-cultured builds Cube method, which is characterized in that the method for building up includes:
S1, reagent preparation include: IL2 cell factor, AlyS505NK-AC culture medium, EX culture medium, HMBPP antigen, CD3 anti- Body, CD56 antibody, 9 antibody of γ and PBMC peripheral blood mononuclear cells；
S2,12 orifice plates are selected, by every milliliter 1.5 × 106The cell of a PBMC peripheral blood mononuclear cells is placed in orifice plate；According to Secondary addition serum-free AC culture medium activation, IL2 cell factor and HMBPP antigen；Culture observation 3 days；
S3, the 3rd day augment 2mL serum-free AC culture medium, IL2 cell factor and HMBPP antigen, so that finally in 12 orifice plates Concentration is identical as original concentration, total volume 3mL, then cultivates 4 days in incubator；
S4, after the 7th day, the centrifugation of 500g speed is carried out to cell, abandons supernatant after being centrifuged 5min, then by the cell in 12 orifice plates point It does not go in T25 Tissue Culture Flask, subsequently, replacement 5mL EX culture medium carries out amplification cultivation: sequentially adding serum-free EX training Support base, IL2 cell factor and HMBPP antigen；
S5, after the 10th day, it is anti-that 5mL serum-free EX culture medium, IL2 cell factor and HMBPP are augmented in T25 Tissue Culture Flask Original, so that ultimate density is consistent with original concentration, total volume 10mL；
S6, after the 12nd day, continue to augment 10 mL serum-free EX culture mediums, IL2 cell factor and HMBPP antigen, so that final dense Spend, total volume 20mL consistent with original concentration；
S7, after the 14th day, NK and gamma delta T cells ratio and total number of cells are detected.
Preferably, the IL2 cell factor the concentration of culture medium may be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL, in the culture medium of the culture medium and subsequent supplement that are added in same orifice plate for the first time, IL2 cell factor Concentration remains consistent.
Preferably, in the step S2, the culture medium of different IL2 cytokine concentrations is respectively arranged at least two holes.
Preferably, the CD3 antibody, CD56 antibody, 9 antibody of γ are in the detection of step S7.
Preferably, the method for building up further includes that detection method and culture are applied.
The beneficial effects of the present invention are embodied in: the present invention establishes an equal proportion amplification cultivation NK cell and gamma delta T cells Co-culture method；It overcomes and is only capable of both cells of independent amplification in the prior art, but because respective condition of culture is variant And can not two kinds of immunocytes of synchronous amplification technological deficiency；And the present invention is established by the unlimited screening and optimizing of condition, Cong Zhongjian The method for co-culturing amplification has been found, can be good at expanding both cells.
It is thin disposably to turn out two kinds of antineoplastic immunes with similarity to co-cultivation method for building up of the invention Born of the same parents improve cell culture efficiency, for the various immunocytes acquisition NK cells of independent culture and gamma delta T cells Toxigenic capacity can be greatly reduced, simplify operating procedure, also reduce culture technique difficulty.
Detailed description of the invention
Fig. 1 is the total number of cells bar chart of different condition of culture.
Fig. 2 is the expression figure that cell surface molecule marks under different condition of culture.
Fig. 3 is that the total cell of different condition of culture expands bar chart.
Fig. 4 is the NK total number of cells bar chart of different condition of culture.
Fig. 5 is the gamma delta T cells sum bar chart of different condition of culture.
Fig. 6 is that two kinds of cell percentages of different condition of culture compare bar chart.
Fig. 7 is that two kinds of total number of cells of different condition of culture compare bar chart.
Specific embodiment
The embodiment of the invention will now be described in detail with reference to the accompanying drawings:
A kind of method for building up of NK cell and gamma delta T cells co-cultivation, the method for building up include:
S1, reagent preparation include: IL2 cell factor, AlyS505NK-AC culture medium, EX culture medium, HMBPP antigen, CD3 anti- Body, CD56 antibody, 9 antibody of γ and PBMC peripheral blood mononuclear cells；
S2,12 orifice plates are selected, by every milliliter 1.5 × 106The cell of a PBMC peripheral blood mononuclear cells is placed in orifice plate；According to Secondary addition serum-free AC culture medium activation, IL2 cell factor and HMBPP antigen；Culture observation 3 days；The IL2 cell factor exists The concentration of culture medium may be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL, in same orifice plate for the first time In the culture medium of addition and the culture medium of subsequent supplement, IL2 cytokine concentrations remain consistent；In the step S2, different IL2 The culture medium of cytokine concentrations is respectively arranged at least two holes；
S3, the 3rd day augment 2mL serum-free AC culture medium, IL2 cell factor and HMBPP antigen, so that finally in 12 orifice plates Concentration is identical as original concentration, total volume 3mL, then cultivates 4 days in incubator；
S4, after the 7th day, the centrifugation of 500g speed is carried out to cell, abandons supernatant after being centrifuged 5min, then by the cell in 12 orifice plates point It does not go in T25 Tissue Culture Flask, subsequently, replacement 5mL EX culture medium carries out amplification cultivation: sequentially adding serum-free EX training Support base, IL2 cell factor and HMBPP antigen；
S5, after the 10th day, it is anti-that 5mL serum-free EX culture medium, IL2 cell factor and HMBPP are augmented in T25 Tissue Culture Flask Original, so that ultimate density is consistent with original concentration, total volume 10mL；
S6, after the 12nd day, continue to augment 10 mL serum-free EX culture mediums, IL2 cell factor and HMBPP antigen, so that final dense Spend, total volume 20mL consistent with original concentration；
S7, after the 14th day, NK and gamma delta T cells ratio and total number of cells are detected；The CD3 antibody, CD56 antibody, 9 antibody of γ are used In the detection of step S7.
The method for building up further includes that detection method and culture are applied.
In selected matrix agent: IL2 the cell factor optional human-IL2 from RD company, IL2, that is, white thin Born of the same parents interleukin -2 applied to the activation and proliferation of promotion T cell and NK cell, and can stimulate NK cell Proliferation, increase cell toxicant Effect and stimulation NK cell secrete cytokine profiles；
AlyS505NK-AC culture medium, EX culture medium can be selected from Zhuhai shellfish funicular cell company；HMBPP can be selected from sigma company； CD3 antibody, CD56 antibody and 9 antibody of γ can be selected from BD company；PBMC can be selected from blood station；
NK and gamma delta T cells ratio and total number of cells are detected by CD3 antibody, CD56 antibody, 9 antibody of γ, implement verifying Process includes:
1) such as Fig. 1, the total number of cells (unit: million cell numbers) of different condition of culture are shown as, wherein IL2 concentration is 20ng/ Total number of cells are most when mL, and when followed by concentration is 10ng/mL, when remaining concentration, total number of cells are less；
2) such as Fig. 2, flow cytomery difference condition of culture cell surface molecule label expression, wherein CD56 is NK thin Cellular surface molecular labeling, CD3 are T cell surface molecular labels, and γ 9 is gamma delta T cells surface molecular label.With CD56 positive CD3 Negative cells indicate NK cell (upper figure box inner cell mass), represent gamma delta T cells (in following figure box carefully with 9 positive cell of γ Born of the same parents group), scheme interior number and represents the percentage that box inner cell accounts for total cell number；
3) such as Fig. 3, the total cell amplification times (unit: multiple) for different condition of culture are shown as, wherein IL2 concentration is When total cell amplification times highest when 20ng/mL, followed by concentration are 10ng/mL, when remaining concentration, total cell amplification times compared with It is low；
4) such as Fig. 4, the NK total number of cells (unit: million cells) for different condition of culture are shown as, wherein IL2 concentration is NK total number of cells are most when 20ng/mL, when followed by concentration is 10ng/mL and 80 ng/mL, when remaining concentration, and NK total number of cells It is less；
5) such as Fig. 5, it is shown as total (unit: million cells) for the gamma delta T cells of different condition of culture, wherein IL2 concentration is At most, when followed by concentration is 10ng/mL, gamma delta T cells sum is less when remaining concentration for gamma delta T cells sum when 20ng/mL；
6) such as Fig. 6, two kinds of cell percentages comparison results for different condition of culture are shown as, wherein IL2 concentration is 20ng/ When mL, the ratio of NK cell and gamma delta T cells is closer to, and the two gap is finer, when followed by concentration is 10ng/mL, works as concentration When for 80ng/mL, the ratio of NK cell and gamma delta T cells differs maximum；
7) such as Fig. 7, two kinds of total number of cells comparison results (unit: million cell numbers) for different condition of culture are shown as, wherein When IL2 concentration is 20ng/mL, NK cell and gamma delta T cells sum are maximum, and quantity is closer to, when being secondly concentration 10ng/mL, Secondly negligible amounts, and quantity gap is big.
The above is only presently preferred embodiments of the present invention, is not intended to limit the scope of the present invention, current row The technical staff of industry can make some deformations and modification, all technologies according to the present invention under the inspiration of the technical program Essence still falls within the range of technical solution of the present invention to any modification, equivalent variations and modification made by above embodiment It is interior.

Claims (5)

1. the method for building up that a kind of NK cell and gamma delta T cells co-culture, which is characterized in that the method for building up includes:
S1, reagent preparation include: IL2 cell factor, AlyS505NK-AC culture medium, EX culture medium, HMBPP antigen, CD3 anti- Body, CD56 antibody, 9 antibody of γ and PBMC peripheral blood mononuclear cells；
S2,12 orifice plates are selected, by every milliliter 1.5 × 106The cell of a PBMC peripheral blood mononuclear cells is placed in orifice plate；According to Secondary addition serum-free AC culture medium activation, IL2 cell factor and HMBPP antigen；Culture observation 3 days；
S3, the 3rd day augment 2mL serum-free AC culture medium, IL2 cell factor and HMBPP antigen, so that finally in 12 orifice plates Concentration is identical as original concentration, total volume 3mL, then cultivates 4 days in incubator；
S4, after the 7th day, the centrifugation of 500g speed is carried out to cell, abandons supernatant after being centrifuged 5min, then by the cell in 12 orifice plates point It does not go in T25 Tissue Culture Flask, subsequently, replacement 5mL EX culture medium carries out amplification cultivation: sequentially adding serum-free EX training Support base, IL2 cell factor and HMBPP antigen；
S5, after the 10th day, it is anti-that 5mL serum-free EX culture medium, IL2 cell factor and HMBPP are augmented in T25 Tissue Culture Flask Original, so that ultimate density is consistent with original concentration, total volume 10mL；
S6, after the 12nd day, continue to augment 10 mL serum-free EX culture mediums, IL2 cell factor and HMBPP antigen, so that final dense Spend, total volume 20mL consistent with original concentration；
S7, after the 14th day, NK and gamma delta T cells ratio and total number of cells are detected.
2. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described IL2 cell factor may be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80 ng/mL in the concentration of culture medium, In the culture medium of the culture medium and subsequent supplement that are added for the first time in same orifice plate, IL2 cytokine concentrations remain consistent.
3. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described In step S2, the culture medium of different IL2 cytokine concentrations is respectively arranged at least two holes.
4. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described CD3 antibody, CD56 antibody, 9 antibody of γ are in the detection of step S7.
5. the method for building up that a kind of NK cell according to claim 1 and gamma delta T cells co-culture, which is characterized in that described Method for building up further include detection method and culture apply.
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Cited By (8)

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CN109112106A (en) * 2018-09-07 2019-01-01 广州长峰生物技术有限公司 The method for building up of the external model of the primary liver cancer tissue of people
CN110200996A (en) * 2019-05-16 2019-09-06 安徽瑞达健康产业有限公司 A kind of application of cell composition on skin ulcer
CN110283785A (en) * 2019-05-16 2019-09-27 安徽瑞达健康产业有限公司 A kind of method that gamma delta T-NK cell co-cultures
CN110511906A (en) * 2019-05-16 2019-11-29 安徽瑞达健康产业有限公司 A kind of application of cell composition on cancer cell SMMC-7721
CN110559316A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 application of cell composition in cancer cell KALS-1
CN110564683A (en) * 2019-05-16 2019-12-13 安徽瑞达健康产业有限公司 Method for co-culture induced amplification of gamma delta T cells and NK cells
CN113430167A (en) * 2021-07-19 2021-09-24 广州百暨基因科技有限公司 Culture method for simultaneously amplifying gamma delta T and NK
EP4183871A1 (en) * 2021-11-23 2023-05-24 Université d'Aix-Marseille Process for preparing a composition comprising a combined cell population

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