CN115074326A - Culture method for in-vitro amplification of NK cells by combining CD16 antibody with cytokine - Google Patents
Culture method for in-vitro amplification of NK cells by combining CD16 antibody with cytokine Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2315—Interleukin-15 (IL-15)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2321—Interleukin-21 (IL-21)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Abstract
The invention relates to the field of methods for amplifying NK cells in vitro, in particular to a culture method for amplifying NK cells in vitro by combining CD16 antibodies with cytokines. The invention inoculates peripheral blood mononuclear cells into pre-coated RetroNectin and mouse anti-human CD16 cell culture flasks, and adds a culture medium containing IL-2, IL-15, IL-21 and autologous plasma. The anti-CD 16 antibody is combined with the CD16 site of NK cell surface cells to enhance the stimulation of IL-15 and IL-2 to NK cells and the killing effect of ADCC to tumor cells, so that the NK cells are induced to be activated, and the NK cells with higher amplification efficiency and purity are obtained. The technology does not use magnetic beads to sort cells, does not use a trophoblast cell activation method, and has the advantages of simple operation, quick response, low preparation cost and high safety. Can efficiently amplify NK cells, and the cells are amplified by 148 times within 14 days. The NK cells have high purity and high clinical value.
Description
Technical Field
The invention relates to the field of methods for amplifying NK cells in vitro, in particular to a culture method for amplifying NK cells in vitro by combining CD16 antibodies with cytokines.
Background
In the existing technology for amplifying NK cells in vitro, a cell culture method adopts Herceptin, Herceptin and PHA, RetroNectin + Merocin + mouse anti-human CD161 antibody clad plate, hyaluronic acid + CD16+
RetroNectin et al, as well as classical methods using trophoblasts. The method is easy to introduce safety problems in the operation process (for example, the introduction of various exogenous factors or reagents can increase the difficulty of cell quality control, thereby increasing the risk of clinical application), and has the problems of unstable process and higher cost due to complex operation.
Disclosure of Invention
In order to solve the problems in the prior art, a culture method for in vitro expansion of NK cells by combining a CD16 antibody and a cytokine is provided. The invention inoculates peripheral blood mononuclear cells into pre-coated RetroNectin and mouse anti-human CD16 cell culture flasks, and adds a culture medium containing IL-2, IL-15, IL-21 and autologous plasma. The method does not use magnetic beads to sort cells, does not use a trophoblast cell activation method, and has the advantages of simple operation, quick response, low preparation cost and high safety. Can efficiently amplify NK cells, and the cells are amplified by 148 times within 14 days.
The invention provides a culture method for in vitro amplification of NK cells by combining CD16 antibody with cytokines, which comprises the following steps:
s1, separating mononuclear cells from peripheral blood;
s2, inoculating peripheral blood mononuclear cells into a precoated RetroNectin and mouse anti-human CD16 cell culture bottle, and adding a culture medium containing IL-2, IL-15, IL-21 and autologous plasma;
s3, continuously culturing for 14-17 days to obtain high-purity high-activity NK cells.
Preferably, the coating solution is prepared before the culture.
Preferably, the coating solution comprises 10ml PBS, 35ug Retrocectin and 35ug CD16, and is prepared by mixing.
Preferably, the specific steps comprise:
A. coating: adding the coating solution into a culture bottle, uniformly spreading and flatly placing, and keeping out of the sun overnight at the temperature of 4 ℃;
B. blood collection: collecting 15ml of peripheral blood, and adding the peripheral blood into a first centrifugal tube for later use;
C. separating lymphocyte separating medium: adding PBS with the same volume as the PBS to 30ml into a centrifuge tube, uniformly mixing, transferring to a centrifuge tube II added with 15ml of lymph separation liquid in advance, centrifuging at 1800rpm/800g at room temperature for 20min, and setting the speed of acceleration and deceleration as 0;
D. washing PBMC: sucking out the white PBMC layer after centrifugation by using a suction tube, adding 18-22ml of PBS into the centrifuge tube III for cleaning, centrifuging for 6min at 2000rpm, and discarding the supernatant; washing with 20ml PBS again, centrifuging at 1800rpm for 6min, and discarding the supernatant; resuspending, mixing and sampling by using a culture medium, counting by adopting typan blue staining, centrifuging for 6min at 1800rpm, and discarding the supernatant;
E. preparing a cell suspension: suspending the cells with 40ml of culture medium, precipitating and mixing;
F. inoculating cells: adding 40ml of the cell suspension into a culture bottle, adding IL-15 with the final concentration of 30ng/ml, IL-21 with the final concentration of 100ng/ml, autologous plasma with the final concentration of 5% and IL-2 with the final concentration of 6000U/ml, and culturing the inoculated culture bottle in a carbon dioxide incubator;
G. flasks were grown at 4, 6, 8, 11 and 14 days during which time they were examined and cells recovered on day 17.
Preferably, the culture conditions of the carbon dioxide incubator are as follows: at a temperature of 37 ℃ and CO 2 The concentration was 5%.
Compared with the prior art, the invention has the following beneficial technical effects:
the invention inoculates peripheral blood mononuclear cells into cell culture flasks precoated with RetroNectin and mouse anti-human CD16, and adds a culture medium containing IL-2, IL-15, IL-21 and autologous plasma. The anti-CD 16 antibody is combined with a CD16 site of an NK cell epitope to enhance the stimulation of IL-15 and IL-2 to NK cells and the killing effect of ADCC to tumor cells, so that the NK cells are induced to be activated, and the NK cells with higher amplification efficiency and purity are obtained. The technology does not use magnetic beads to sort cells, does not use a trophoblast cell activation method, and has the advantages of simple operation, quick response, low preparation cost and high safety. Can efficiently amplify NK cells, and the cells are amplified by 148 times within 14 days. The NK cells have high purity and high clinical value.
Drawings
FIG. 1 is a schematic flow chart of a method according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of NK cell expansion within 14 days;
FIG. 3 is a diagram showing the results of flow assay of NK cells.
Detailed Description
Example one
The invention provides a culture method for in vitro amplification of NK cells by combining CD16 antibody and cytokine, which comprises the following steps:
s1, separating mononuclear cells from peripheral blood;
s2, inoculating the peripheral blood mononuclear cells into a precoated RetroNectin and mouse anti-human CD16 cell culture bottle, and adding a culture medium containing IL-2, IL-15, IL-21 and autologous plasma;
s3, continuously culturing for 14-17 days to obtain high-purity high-activity NK cells.
Example two
The invention provides a culture method for in vitro amplification of NK cells by combining CD16 antibodies and cytokines, which comprises the following steps:
s1, separating mononuclear cells from peripheral blood;
s2, inoculating peripheral blood mononuclear cells into a precoated RetroNectin and mouse anti-human CD16 cell culture bottle, and adding a culture medium containing IL-2, IL-15, IL-21 and autologous plasma;
s3, continuously culturing for 14-17 days to obtain high-purity high-activity NK cells.
Furthermore, a coating solution is prepared before culturing.
Further, the coating solution was prepared by mixing 10ml of PBS, 35ug of Retrocin and 35ug of CD 16.
EXAMPLE III
As shown in figure 1, the culture method for the in vitro amplification of NK cells by the CD16 antibody and the cytokine comprises the following steps:
A. coating: adding the coating solution into a T75 culture flask, uniformly spreading, and keeping flat at 4 ℃ in a dark overnight;
B. blood collection: collecting 15ml of peripheral blood, and adding the peripheral blood into a 50ml centrifuge tube I for later use;
C. separating lymphocyte separating medium: adding PBS with the same volume as the PBS to 30ml into a centrifuge tube, uniformly mixing, transferring to a centrifuge tube II added with 15ml of lymph separation liquid in advance, centrifuging at 1800rpm/800g at room temperature for 20min, and setting the speed of acceleration and deceleration as 0;
D. washing PBMC: sucking out the white PBMC layer after centrifugation by using a Pasteur pipette, adding 18-22ml of PBS into a centrifuge tube III for washing, centrifuging for 6min at 2000rpm, and discarding the supernatant; washing with 20ml PBS again, centrifuging at 1800rpm for 6min, and discarding the supernatant; resuspending in 20ml culture medium, mixing, sampling, counting with typan blue stain, centrifuging at 1800rpm for 6min, and discarding the supernatant;
E. preparing a cell suspension: resuspending cells with 40ml GT-T551H3 medium, precipitating, and mixing;
F. inoculating cells: adding 40ml of the cell suspension into a culture bottle, adding IL-15 with the final concentration of 30ng/ml, IL-21 with the final concentration of 100ng/ml, autologous plasma with the final concentration of 5% and IL-2 with the final concentration of 6000U/ml, and culturing the inoculated culture bottle in a carbon dioxide incubator; the culture conditions of the carbon dioxide incubator are as follows: at a temperature of 37 ℃ and CO 2 The concentration is 5%;
G. culturing for 4, 6, 8, 11 and 14 days in separate flasks, detecting during the culture period, and recovering cells on day 17;
H. and (3) detecting the surface markers CD3, CD56, CD16 and CD45 of the NK cells by adopting a flow detection method. As a result, as shown in FIG. 3, it was found that CD45 was 63.47% and CD3-CD56+ was 62.63% after 14 days of cell culture.
Peripheral blood mononuclear cells are inoculated into a precoated RetroNectin and mouse anti-human CD16 cell culture flask, and a culture medium containing IL-2, IL-15, IL-21 and autologous plasma is added. The anti-CD 16 antibody is combined with the CD16 site of NK cell surface cells to enhance the stimulation of IL-15 and IL-2 to NK cells and the killing effect of ADCC to tumor cells, so that the NK cells are induced to be activated, and the NK cells with higher amplification efficiency and purity are obtained. The technology does not use magnetic beads to sort cells, does not use a trophoblast cell activation method, and has the advantages of simple operation, quick response, low preparation cost and high safety. Can efficiently expand NK cells, and the cells expand 148 times within 14 days (shown in figure 2). No CD16 antibody stimulated proliferation by less than 100-fold. Therefore, the NK cells obtained by the method have high purity and high clinical value.
The embodiments of the present invention have been described in detail with reference to the drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present invention.
Claims (5)
- A culture method for in vitro expansion of NK cells by combining CD16 antibody and cytokine, which is characterized by comprising the following steps:s1, separating mononuclear cells from peripheral blood;s2, inoculating peripheral blood mononuclear cells into a precoated RetroNectin and mouse anti-human CD16 cell culture bottle, and adding a culture medium containing IL-2, IL-15, IL-21 and autologous plasma;s3, continuously culturing for 14-17 days to obtain high-purity high-activity NK cells.
- 2. The method for culturing the NK cells expanded in vitro by the CD16 antibody and the cytokine according to claim 1, wherein the coating solution is prepared before culturing.
- 3. The method for culturing the NK cells expanded in vitro by the CD16 antibody and the cytokine according to claim 1, wherein the coating solution comprises 10ml PBS, 35ug Retrocin and 35ug CD16 antibody, and the mixture is prepared.
- 4. The culture method for the in vitro expansion of NK cells by the combination of CD16 antibody and cytokine according to claim 1, comprising the following steps:A. coating: adding the coating solution into a culture bottle, uniformly spreading and flatly placing, and keeping out of the sun overnight at the temperature of 4 ℃;B. blood collection: collecting 15ml of peripheral blood, and adding the peripheral blood into a first centrifugal tube for later use;C. separating lymphocyte separation liquid: adding PBS with the same volume as the PBS to 30ml into a centrifuge tube, uniformly mixing, transferring to a centrifuge tube II added with 15ml of lymph separation liquid in advance, centrifuging at 1800rpm/800g at room temperature for 20min, and setting the speed of acceleration and deceleration as 0;D. washing PBMC: sucking out the white PBMC layer after centrifugation by using a suction tube, adding 18-22ml of PBS into the centrifuge tube III for cleaning, centrifuging for 6min at 2000rpm, and discarding the supernatant; washing with 20ml PBS again, centrifuging at 1800rpm for 6min, and discarding the supernatant; resuspending with culture medium, mixing, sampling, counting with typandlue staining, centrifuging at 1800rpm for 6min, and discarding the supernatant;E. preparing a cell suspension: suspending the cells with 40ml of culture medium, precipitating and mixing;F. inoculating cells: adding 40m of the cell suspension into a culture bottle, adding IL-15 with the final concentration of 30ng/ml, IL-21 with the final concentration of 100ng/ml, autologous plasma with the final concentration of 5% and IL-2 with the final concentration of 6000U/ml, and placing the inoculated culture bottle into a carbon dioxide incubator for culture;G. flasks were grown for 4, 6, 8, 11 and 14 days during which time they were examined and cells were recovered on day 17.
- 5. The culture method for the in vitro expansion of NK cells by the combination of CD16 antibody and cytokine according to claim 1, characterized in that the culture conditions of the carbon dioxide incubator are as follows: at a temperature of 37 ℃ and CO 2 The concentration was 5%.
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Application publication date: 20220920 |