CN109517793B - Establishment method for NK cell and gamma delta T cell co-culture - Google Patents
Establishment method for NK cell and gamma delta T cell co-culture Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0646—Natural killers cells [NK], NKT cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2302—Interleukin-2 (IL-2)
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- C12N2502/00—Coculture with; Conditioned medium produced by
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- C12N2502/00—Coculture with; Conditioned medium produced by
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Abstract
The invention relates to the technical field of cell culture, in particular to a method for establishing co-culture of NK cells and gamma delta T cells, wherein the method is used for in-vitro co-amplification culture of the NK cells and the gamma delta T cells and mainly comprises the following steps: s1, selecting a reagent; s2, culturing the PBMC through the culture medium for 3 days; s3, supplementing a culture medium, maintaining the concentration and culturing for 4 days; s4, centrifuging after 7 days, removing supernatant, transferring to a T25 cell culture bottle, and then replacing an EX culture medium; s5, supplementing the medium to maintain the concentration after day 10; s6, continuously supplementing the culture medium to maintain the concentration after the 12 th day; s7, detecting the proportion of NK and gamma delta T cells and the total number of the cells after 14 days; the co-culture establishing method provided by the invention can be used for culturing two anti-tumor immune cells with similar properties at one time, so that the cell culture efficiency is improved, and compared with the method for independently culturing various immune cells to obtain NK cells and gamma delta T cells, the co-culture establishing method can be used for greatly reducing the culture cost and also reducing the culture technical difficulty.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to an establishment method for co-culture of NK cells and gamma delta T cells, wherein the NK cells and the gamma delta T cells can be co-amplified and cultured in vitro.
Background
Natural killer cells (NK) are important immune cells of the body, closely related to anti-tumor immunity technology, and can be used as adjuvant cell therapy for tumor therapy by first culturing NK cells in vitro and then self-reinfusing; the in vitro culture of NK cells is a key link of the cell therapy, the NK cells have affinity receptors in IL-2 and can generate a proliferation reaction under the stimulation of IL-2, the existing in vitro culture of the NK cells is mainly obtained by adding a culture medium containing the IL-2 for amplification, and at present, a plurality of commercial culture media are provided for selection.
The gamma delta T cell is a T cell for executing the inherent immune function, the TCR of the gamma delta T cell consists of gamma and delta chains, and the gamma delta T cell is an immune cell which can kill cancer cells and tumor stem cells and can recognize cancer antigens; the existing research shows that a subtype of the gamma delta T cell can be quickly activated and proliferated under the stimulation of phospholipid molecules, the self-transfusion of the subtype gamma delta T cell can also be used as the adjuvant therapy application of tumor therapy, and the technology of amplifying the gamma delta T cell in vitro through the phospholipid molecules has important clinical value; currently, few commercial media are selected for expansion of γ δ T cells, and γ δ T cells are usually obtained by adding phospholipid molecules to T cell media.
Both the gamma delta T cells and the NK cells have tumor cell killing activity and can be used for adjuvant therapy of tumor patients, and if the two cells can be synchronously amplified and applied to the patients, the method has important significance, however, the method capable of synchronously amplifying the two cells is not established at present, the in vitro amplification culture of the NK cells and the gamma delta T cells is independently performed at present, the establishment method of the synchronous amplification culture is still to be developed, and the significance value of the method is still to be verified.
Disclosure of Invention
In order to solve the problems, the invention aims to disclose the technical field of cell culture, in particular to an establishment method for co-culture of NK cells and gamma delta T cells, which can realize in-vitro co-amplification culture of the NK cells and the gamma delta T cells, so as to realize the in-vitro culture purpose of synchronously culturing the NK cells and the gamma delta T cells.
In order to achieve the purpose, the invention adopts the technical scheme that: a method for establishing a co-culture of NK cells and gamma delta T cells, which is characterized by comprising the following steps:
s1, reagent preparation comprising: IL2 cytokine, AlyS505NK-AC medium, EX medium, HMBPP antigen, CD3 antibody, CD56 antibody, γ 9 antibody and PBMC peripheral blood mononuclear cells;
s2, selecting a 12-hole plate, mixing each milliliter with 1.5 multiplied by 106Cells of PBMC peripheral blood mononuclear cells are placed in a pore plate; serum-free AlyS505NK-AC culture medium activation, IL2 cytokine and HMBPP antigen are added in sequence; culturing and observing for 3 days;
s3, day 3, 2mL serum-free AlyS505NK-AC medium, IL2 cytokine and HMBPP antigen were supplemented in 12-well plates to a final concentration equal to the original concentration in a total volume of 3mL, followed by culture in an incubator for 4 days;
s4, centrifuging the cells at a speed of 500g after 7 days, centrifuging for 5min, discarding the supernatant, transferring the cells in a 12-well plate into a T25 cell culture bottle respectively, and then replacing 5mL of EX culture medium for amplification culture: sequentially adding serum-free EX culture medium, IL2 cytokine and HMBPP antigen;
s5, day 10 later, 5mL serum-free EX medium, IL2 cytokine and HMBPP antigen were supplemented in T25 cell culture flasks to a final concentration consistent with the original concentration in a total volume of 10 mL;
s6, after day 12, continuously supplementing 10mL serum-free EX culture medium, IL2 cytokine and HMBPP antigen to make the final concentration consistent with the original concentration and the total volume 20 mL;
s7, day 14 later, the NK and γ δ T cell ratios and the total cell numbers were examined.
Preferably, the concentration of the IL2 cytokine in the culture medium can be set to 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL, 80ng/mL, and the concentration of IL2 cytokine in the first addition of culture medium and the subsequent supplementation of culture medium in the same well plate is maintained consistent.
Preferably, in the step S2, the culture media with different concentrations of the IL2 cytokine are respectively provided with at least two wells.
Preferably, the CD3 antibody, CD56 antibody, γ 9 antibody are used in the detection of step S7.
Preferably, the establishing method further comprises a detection method and a culture application.
The invention has the following beneficial effects: the invention establishes a co-culture method for amplifying and culturing NK cells and gamma delta T cells in equal proportion; overcomes the technical defect that the two types of cells can only be independently amplified but the two types of immune cells can not be synchronously amplified due to different culture conditions in the prior art; the invention establishes a co-culture amplification method through the optimization establishment of condition infinite screening, and can well amplify the two cells.
The co-culture establishing method of the invention cultures two anti-tumor immune cells with similar properties at one time, improves the cell culture efficiency, greatly reduces the culture cost, simplifies the operation steps and reduces the culture technical difficulty compared with independently culturing various immune cells to obtain NK cells and gamma delta T cells.
Drawings
FIG. 1 is a bar graph of the total number of cells for different culture conditions.
FIG. 2 is a graph showing the expression of cell surface molecular markers under different culture conditions.
FIG. 3 is a bar graph of total cell expansion for different culture conditions.
FIG. 4 is a bar graph of total number of NK cells for different culture conditions.
Fig. 5 is a bar graph of the total number of γ δ T cells for different culture conditions.
FIG. 6 is a bar graph comparing the percentage of two cells for different culture conditions.
FIG. 7 is a bar graph comparing the total number of two cells for different culture conditions.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings:
a method for establishing a co-culture of NK cells and gamma delta T cells, wherein the establishing method comprises the following steps:
s1, reagent preparation comprising: IL2 cytokine, AlyS505NK-AC medium, EX medium, HMBPP antigen, CD3 antibody, CD56 antibody, γ 9 antibody and PBMC peripheral blood mononuclear cells;
s2, selecting a 12-hole plate, mixing each milliliter with 1.5 multiplied by 106Cells of PBMC peripheral blood mononuclear cells are placed in a pore plate; serum-free AlyS505NK-AC culture medium activation, IL2 cytokine and HMBPP antigen are added in sequence; culturing and observing for 3 days; the concentration of the IL2 cytokine in the culture medium can be set to be 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL and 80ng/mL, and the concentration of the IL2 cytokine in the culture medium added for the first time in the same pore plate and the culture medium supplemented for the subsequent time is kept consistent; in the step S2, at least two holes are respectively arranged on the culture media with different concentrations of IL2 cytokines;
s3, day 3, 2mL serum-free AlyS505NK-AC medium, IL2 cytokine and HMBPP antigen were supplemented in 12-well plates to a final concentration equal to the original concentration in a total volume of 3mL, followed by culture in an incubator for 4 days;
s4, centrifuging the cells at a speed of 500g after 7 days, centrifuging for 5min, discarding the supernatant, transferring the cells in a 12-well plate into a T25 cell culture bottle respectively, and then replacing 5mL of EX culture medium for amplification culture: sequentially adding serum-free EX culture medium, IL2 cytokine and HMBPP antigen;
s5, day 10 later, 5mL serum-free EX medium, IL2 cytokine and HMBPP antigen were supplemented in T25 cell culture flasks to a final concentration consistent with the original concentration in a total volume of 10 mL;
s6, after day 12, continuously supplementing 10mL serum-free EX culture medium, IL2 cytokine and HMBPP antigen to make the final concentration consistent with the original concentration and the total volume 20 mL;
s7, detecting the proportion of NK and gamma delta T cells and the total number of the cells after 14 days; the CD3 antibody, CD56 antibody, γ 9 antibody were used in the step S7 assay.
The establishing method also comprises a detection method and culture application.
Among the matrix reagents selected: the IL2 cytokine can be selected from human-IL2 and IL2 (interleukin-2) from RD company, is applied to promoting the activation and proliferation of T cells and NK cells, and can stimulate the proliferation of the NK cells, increase the cytotoxic effect and stimulate the NK cells to secrete various cytokines;
AlyS505NK-AC culture medium and EX culture medium can be selected from Kyoto Betula cells; HMBPP may be selected from sigma corporation; the CD3 antibody, CD56 antibody and γ 9 antibody can be selected from BD; PBMCs may be selected from blood stations;
the NK and gamma delta T cell proportion and the total cell number are detected by a CD3 antibody, a CD56 antibody and a gamma 9 antibody, and the verification process comprises the following steps:
1) as shown in fig. 1, the total number of cells (unit: millions of cells), wherein the total number of cells is greatest at a concentration of IL2 of 20ng/mL, followed by a concentration of 10ng/mL, and less at the remaining concentrations;
2) as shown in FIG. 2, the flow cytometry is used for detecting the expression of cell surface molecular markers under different culture conditions, wherein CD56 is an NK cell surface molecular marker, CD3 is a T cell surface molecular marker, and gamma 9 is a gamma delta T cell surface molecular marker. NK cells were represented by CD56 positive CD3 negative cells (upper panel inner box cell population), γ δ T cells by γ 9 positive cells (lower panel inner box cell population), and the numbers in the figure represent the percentage of the total number of cells in the box;
3) as in fig. 3, the total cell expansion fold for different culture conditions (unit: fold), wherein the fold expansion of total cells is highest at a concentration of IL2 of 20ng/mL, followed by a concentration of 10ng/mL, and lower at the remaining concentrations;
4) as shown in fig. 4, the total number of NK cells (unit: million cells), wherein the total number of NK cells is greatest at IL-2 concentration of 20ng/mL, followed by concentrations of 10ng/mL and 80ng/mL, and the remaining concentrations are lesser;
5) as shown in fig. 5, the total number of γ δ T cells (unit: million cells), wherein the total number of γ δ T cells is greatest at a concentration of IL2 of 20ng/mL, followed by a concentration of 10ng/mL, with the remaining concentrations all being less;
6) FIG. 6 shows the results of comparison of the percentages of two cells in different culture conditions, wherein the ratio of NK cells to γ δ T cells is closer and more closely different at IL-2 concentration of 20ng/mL, and the ratio of NK cells to γ δ T cells is the greatest at 10 ng/mL;
7) as shown in fig. 7, the results of comparison of the total number of two cells for different culture conditions (unit: millions of cells), wherein the total number of NK cells and γ δ T cells is greatest and closer in number at a concentration of IL2 of 20ng/mL, and second to a lesser number and a greater difference in number at a concentration of 10 ng/mL.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the technical scope of the present invention, and those skilled in the art may make modifications and variations within the spirit of the present invention, and all modifications, equivalents and modifications of the above embodiments according to the technical spirit of the present invention are within the scope of the present invention.
Claims (3)
1. A method for establishing a co-culture of NK cells and gamma delta T cells, which is characterized by comprising the following steps:
s1, reagent preparation comprising: IL2 cytokine, AlyS505NK-AC medium, EX medium, HMBPP antigen, CD3 antibody, CD56 antibody, γ 9 antibody and PBMC peripheral blood mononuclear cells;
s2, selecting a 12-hole plate, mixing each milliliter with 1.5 multiplied by 106Cells of PBMC peripheral blood mononuclear cells are placed in a pore plate; serum-free AlyS505NK-AC culture medium activation, IL2 cytokine and HMBPP antigen are added in sequence; culturing and observing for 3 days; wherein the concentration of the IL2 cytokine in the culture medium is 10ng/mL, 20ng/mL, 40 ng/mL, 60 ng/mL or 80ng/mL, and the concentration of the IL2 cytokine in the first addition of culture medium and the subsequent supplementation of culture medium in the same well plate is maintained consistent;
s3, day 3, 2mL serum-free AlyS505NK-AC medium, IL2 cytokine and HMBPP antigen were supplemented in 12-well plates to a final concentration equal to the original concentration in a total volume of 3mL, followed by culture in an incubator for 4 days;
s4, centrifuging the cells at a speed of 500g after 7 days, centrifuging for 5min, discarding the supernatant, transferring the cells in a 12-well plate into a T25 cell culture bottle respectively, and then replacing 5mL of EX culture medium for amplification culture: sequentially adding serum-free EX culture medium, IL2 cytokine and HMBPP antigen;
s5, day 10 later, 5mL serum-free EX medium, IL2 cytokine and HMBPP antigen were supplemented in T25 cell culture flasks to a final concentration consistent with the original concentration in a total volume of 10 mL;
s6, after day 12, continuously supplementing 10mL serum-free EX culture medium, IL2 cytokine and HMBPP antigen to make the final concentration consistent with the original concentration and the total volume 20 mL;
s7, detecting the proportion of NK and gamma delta T cells and the total number of the cells after 14 days; the CD3 antibody, the CD56 antibody and the gamma 9 antibody are used for detection.
2. The method of claim 1, wherein the culture medium with different concentrations of IL2 cytokine in step S2 has at least two wells.
3. The method of claim 1, wherein the method further comprises a detection method and a culture application.
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| 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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