CN117327653A - NK cell in-vitro culture method for enhancing cell killing activity - Google Patents
NK cell in-vitro culture method for enhancing cell killing activity Download PDFInfo
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Abstract
The invention discloses an NK cell in-vitro culture method for enhancing cell killing activity. Auranofin is a medicine for treating rheumatoid arthritis, can activate an NK cell Nrf2 signal path, improves the tolerance level of NK cells to active oxygen, and further improves the capacity of NK cells in resisting oxidative stress in tumor microenvironment, thereby enhancing the killing activity of NK cells. The invention creatively introduces the auranofin in the NK cell in-vitro culture process, and discloses a method for adding the auranofin, which can obviously enhance the NK cell killing activity and has higher application value.
Description
Technical Field
The invention belongs to the technical field of NK cell culture, and relates to an NK cell in-vitro culture method for enhancing cell killing activity.
Background
Natural killer cells (Natural killer cell, NK cells) are a subset of lymphocytes, an important component of innate immunity in humans, and also a first line of defense against viral infection and tumor cells. NK cells can directly kill virus-infected self cells or tumor cells without the need for pre-stimulation and activation of antigens, and are therefore called natural killer cells.
NK cells can function spontaneously without prior sensitization. Their ability to rapidly produce IFN-gamma and TNF-alpha upon cell triggering results in early responses to oncogenic transformation. Thus, their immediate intervention in tumor cells, including their effect in promoting inflammation, is related to their active participation in immune monitoring of tumors.
However, in tumor microenvironments, NK cell killing activity is inhibited, especially the level of oxidative stress in the tumor microenvironment results in a significant decrease in NK cell killing, which results in uncontrolled proliferation and migration of tumor cells to other sites.
Many cellular immunotherapy is now to extract NK cells from Peripheral Blood Mononuclear Cells (PBMC) and to expand them in large amounts in vitro for reinfusion in tumor patients, but it is difficult to expand NK cells having high killing activity in vitro without feeder cells because of the ability to enhance NK cell killing by using feeder cells as artificial antigen presenting cells such as irradiated Peripheral Blood Mononuclear Cells (PBMC), genetically modified K562 cells and lymphoblastic cell line (EBV-LCL) transformed with Epstein-Barr virus. In particular, expression of K562-based artificial antigen presenting cells IL-21 induces NK cell culture techniques with PBMC as the starter cells. However, some researchers have recognized that NK cell expansion with feeder cells is problematic. For example, there is a concern that the culture time of NK cells is as long as 14 to 21 days, and after infusion, the in vivo survival time of NK cells is shortened and the cytotoxic effect is reduced because the feeder cells have been removed from the optimal environment for NK cell culture. For this reason, researchers developed NK culture methods with pure factors, eliminating feeder cells, but with the consequent disadvantage of low NK cell killing activity.
In order to solve the problems, the invention provides an NK cell in-vitro culture method for enhancing the NK cell killing activity, so as to solve the problem of low killing activity in the NK cell in-vitro culture process.
Auranofin is a medicine for treating rheumatoid arthritis, can activate an NK cell Nrf2 signal path, improves the tolerance level of NK cells to active oxygen, and further enhances the capacity of NK cells in resisting oxidative stress in tumor microenvironment, thereby improving the killing activity of NK cells. The invention creatively introduces the auranofin in the NK cell in-vitro culture process, and discloses a method for adding the auranofin, which can improve the NK cell killing activity by more than 80 percent and has higher application value.
Disclosure of Invention
The invention aims to overcome the defects of the existing NK cell culture and provide an NK cell in-vitro culture method for enhancing the NK cell killing activity, which can enhance the NK cell killing activity.
In order to achieve the above object, the present invention provides the following technical solutions:
an NK cell in vitro culture method for enhancing cell killing activity, comprising the steps of:
(1) Isolating PBMCs with lymphocyte separation fluid;
(2) Adding the separated mononuclear cells into a culture medium A for activation culture for 3 days, wherein the culture medium A is a serum-free immune cell culture medium added with CD16 antibodies, IL-2, IL-15, IL-21 and autologous plasma;
(3) From day 3 to day 8, culture medium B, which is serum-free immunocyte medium supplemented with IL-2, IL-15, IL-21 and autologous plasma, is supplemented every 2 days;
(4) From day 9 to day 13, medium C, which is serum-free immune cell medium supplemented with auranofin, IL-2, IL-15, IL-21 and autologous plasma, was supplemented every 2 days;
(5) Cells were harvested on day 14 and tested.
The serum-free immune cell culture medium comprises a culture medium of Daidae NK cells, a culture medium of Corning 581 and a culture medium of LonzaXVI VO 15.
Preferably, the ratio of the components in the NK cell culture medium is as follows:
more preferably, the ratio of the components in the NK cell culture medium is as follows:
preferably, step (1) comprises:
1) Transferring peripheral blood into a centrifuge tube, centrifuging for 10min at 800g, and sucking the upper layer part to obtain autologous plasma for later use;
2) Diluting normal saline and blood cell sediment, blowing and mixing uniformly, slowly adding the suspension onto the Ficoll layer, and centrifuging at room temperature of 700g for 30min;
3) Sucking out the centrifuged white membrane layer, re-suspending by PBS buffer solution, centrifuging for 10min at room temperature by 500g, repeating for two times, and centrifuging and precipitating to obtain peripheral blood mononuclear cells;
4) Autologous plasma is placed at 56 ℃ for 40min, and complement in the plasma is inactivated; standing at 4 ℃ for 20min, centrifuging at 1200g for 15min, discarding the precipitate, and transferring the supernatant to a new centrifuge tube for later use.
Preferably, NK cells in step (2) -step (4) are all cultured by resting in a 5% CO2 incubator at 37 ℃.
Preferably, in step (2), the cell density is adjusted to 2X 10 with medium A 6 /ml。
Preferably, in step (3), if the cell density is>1×10 6 Per ml, cell density was adjusted to 1X 10 with medium B 6 /ml。
Preferably, in step (4), if the cell density is>1×10 6 Per ml, cell density was adjusted to 1X 10 with medium C 6 /ml。
The innovation of the invention is that: the inventive introduction of auranofin stimulates NK cells, so that the killing power of the obtained NK cells is obviously enhanced.
Drawings
FIG. 1 is a graph showing the results of flow cytometry detection of NK cell purity of experimental group 1 of the present invention.
FIG. 2 is a graph showing the results of flow cytometry detection of the purity of NK cell of control group 1 of example 1 of the present invention.
FIG. 3 is a graph showing comparison of NK cell killing activity of experimental group 1 of the present invention with that of control group 1.
FIG. 4 is a graph comparing NK cell killing activity of experimental group 2 and control group 2 according to example 2 of the present invention.
FIG. 5 is a graph comparing NK cell killing activity of experimental group 3 of the present invention with that of control group 3.
Detailed Description
The present invention is described in conjunction with the following specific embodiments, which are given as examples of the present invention and are not to be construed as limiting the invention in any way, and any person skilled in the art can, with reference to the present invention, suitably modify the process parameters. Any simple modification of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention, without departing from the scope of the present invention.
Both the consumables and the reagents used in the examples provided herein are commercially available; and the different sources have no significant effect on the product performance.
The following examples of the present application were carried out using the following media:
corning 581 Medium, cat# 88-581-CM, basic culture method is common for NK cells using CD16/IL-2/IL-15/IL-21 cytokine activation and amplification method;
the culture medium of Lonza (Longsha) XVO 15, the product number BE02-060F, the basic culture method is a common method for activating and amplifying NK cells by using CD16/IL-2/IL-15/IL-21 cytokines;
the daceae is NK cell serum-free medium, product number 6113031, and the basic culture method is a common method for activating and amplifying NK cells by using CD16/IL-2/IL-15/IL-21 cytokines.
Example 1
An NK cell in vitro culture method for enhancing cell killing activity, which uses Daidae as an NK cell culture medium as a basic culture medium, comprises the following steps:
(1) Isolating PBMCs with lymphocyte separation fluid;
(2) The isolated mononuclear cells were added to medium A for 3 days for activation culture, and the initial cell density was 2X 10 6 /ml. The culture medium A is a serum-free immune cell culture medium added with CD16 antibody, IL-2, IL-15, IL-21 and heat-inactivated autologous plasma; the final concentration of the CD16 antibody is 50ng/mL, the final concentration of IL-2 is 100ng/mL, the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, and the heat inactivation is carried outThe adding proportion of autologous plasma is 10%, the NK culture medium is the Daidae NK cell culture medium, and cell activation culture is carried out under the culture condition of 5% carbon dioxide at 37 ℃;
(3) Culture medium B was supplemented every 2 days from day 3 to day 8 to adjust cell density to 1×10 6 /ml. The culture medium B is a serum-free immune cell culture medium added with IL-2, IL-15, IL-21 and autologous plasma; the final concentration of IL-2 is 100ng/mL, the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, and the adding proportion of the heat-inactivated autologous plasma is reduced to 1% on the 7 th day;
(4) Medium C was supplemented every 2 days from day 9 to day 13 to adjust the cell density to 1×10 6 /ml. The culture medium C is a serum-free immune cell culture medium added with auranofin, IL-2, IL-15, IL-21 and autologous plasma; the final concentration of the auranofin is 1 mug/mL, the final concentration of the IL-2 is 100ng/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-21 is 50ng/mL, and the adding proportion of the heat-inactivated autologous plasma is 1%, if the heat-inactivated autologous plasma is used up, the heat-inactivated autologous plasma can be not added;
(5) Cells were harvested on day 14 and tested.
The procedure disclosed in example 1 was used as experiment 1 (daceae as NK cell medium), and control 1 was set up as a control experiment, control 1 was not supplemented with auranofin during the whole NK cell culture, and the other steps were the same as in example 1. Cells were harvested on day 14 and tested.
Detecting items: the day 14 test items included: cell viability, fold cell expansion, cell purity, cell killing capacity.
Cell viability: the cell viability of the experimental group 1 on the 14 th day is 92.3%, the cell viability of the control group 1 is 92.9%, and the two are not obviously different, which indicates that the cell viability is not affected by the auranofin.
Cell expansion fold: the cell expansion multiple of the experimental group 1 on the 14 th day is 207 times, the cell expansion multiple of the control group 1 is 206 times, and the experimental group 1 and the control group have no obvious difference, which indicates that the auranofin does not influence the cell expansion multiple.
Cell purity: day 14, experiment group 1 reached 92.88% cell purity (CD 3-CD56+ index) (see FIG. 1), control group 1 was 90.13% (see FIG. 2), and the difference was not large, indicating that auranofin did not affect NK cell purity.
Cell killing activity: the killing effect of NK cells on target cells K562 tumor cells is detected by adopting a Promega Cytotox96 killing detection kit, and the ratio of the NK cells to the target cells is 10: 1. 20: 1. 40:1, the detection method is referred to the instruction book of the kit. The results showed that the NK cells cultured in the experimental group 1 of this example had stronger killing activity than the NK cells cultured in the control group 1 at various ratios (see FIG. 3).
Example 2
An NK cell in vitro culture method for enhancing cell killing activity using Corning 581 medium as a basal medium, other protocols and steps are the same as in example 1, comprising the steps of:
(1) Isolating PBMCs with lymphocyte separation fluid;
(2) The isolated mononuclear cells were added to medium A for 3 days for activation culture, and the initial cell density was 2X 10 6 /ml. The culture medium A is a serum-free immune cell culture medium added with CD16 antibody, IL-2, IL-15, IL-21 and heat-inactivated autologous plasma; the final concentration of the CD16 antibody is 50ng/mL, the final concentration of IL-2 is 100ng/mL, the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, the adding proportion of heat-inactivated autologous plasma is 10%, the NK culture medium is Corning 581 culture medium, and cell activation culture is carried out under the condition of 5% carbon dioxide culture at 37 ℃;
(3) Culture medium B was supplemented every 2 days from day 3 to day 8 to adjust cell density to 1×10 6 /ml. The culture medium B is a serum-free immune cell culture medium added with IL-2, IL-15, IL-21 and autologous plasma; the final concentration of IL-2 is 100ng/mL, the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, and the adding proportion of the heat-inactivated autologous plasma is reduced to 1% on the 7 th day;
(4) Medium C was supplemented every 2 days from day 9 to day 13 to adjust the cell density to 1×10 6 /ml. The culture medium C is a serum-free immune cell culture medium added with auranofin, IL-2, IL-15, IL-21 and autologous plasma; the final concentration of the auranofin is 1 mug/ml, the final concentration of the IL-2 is 100ng/ml,the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, the adding proportion of the heat-inactivated autologous plasma is 1%, and if the heat-inactivated autologous plasma is used up, the heat-inactivated autologous plasma can be not added;
(5) Cells were harvested on day 14 and tested.
The procedure disclosed in example 2 was used as experiment group 2 (Corning 581 medium group), and control group 2 was set up as a control experiment, and control group 2 was not supplemented with auranofin during the whole NK cell culture, and the other steps were the same as in example 2. Cells were harvested on day 14 and tested.
Detecting items: the day 14 test items included: cell viability, fold cell expansion, cell purity, cell killing capacity.
Cell viability: the cell viability of the experimental group 2 on the 14 th day is 91.1%, the cell viability of the control group 2 is 90.2%, and the two are not obviously different, which indicates that the cell viability is not affected by the auranofin.
Cell expansion fold: on day 14, the cell expansion multiple of the experimental group 2 is 197 times, the cell expansion multiple of the control group 2 is 192 times, and the experimental group 2 and the control group have no obvious difference, which indicates that the auranofin does not influence the cell expansion multiple.
Cell purity: the cell purity (CD 3-CD56+ index) of experiment group 2 reaches 83.30% on day 14, and the cell purity of control group 2 is 82.78%, which is not greatly different, indicating that auranofin does not affect NK cell purity.
Cell killing activity: the killing effect of NK cells on target cells K562 tumor cells is detected by adopting a Promega Cytotox96 killing detection kit, and the ratio of the NK cells to the target cells is 10: 1. 20: 1. 40:1, the detection method is referred to the instruction book of the kit. The results showed that the NK cells cultured in the experimental group 2 of this example had stronger killing activity than the NK cells cultured in the control group 2 at various ratios (see FIG. 4).
Example 3
An NK cell in vitro culture method for enhancing cell killing activity using LonzaXMVO 15 culture medium as basic culture medium, other schemes and steps are the same as in example 1, comprising the following steps:
(1) Isolating PBMCs with lymphocyte separation fluid;
(2) Adding the isolated mononuclear cells to Medium AActivated culture for 3 days, initial cell density of 2×10 6 /ml. The culture medium A is a serum-free immune cell culture medium added with CD16 antibody, IL-2, IL-15, IL-21 and heat-inactivated autologous plasma; the final concentration of the CD16 antibody is 50ng/mL, the final concentration of IL-2 is 100ng/mL, the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, the adding proportion of heat-inactivated autologous plasma is 10%, the NK culture medium is Lonza XVO 15 culture medium, and cell activation culture is carried out under the condition of 37 ℃ and 5% carbon dioxide culture;
(3) Culture medium B was supplemented every 2 days from day 3 to day 8 to adjust cell density to 1×10 6 /ml. The culture medium B is a serum-free immune cell culture medium added with IL-2, IL-15, IL-21 and autologous plasma; the final concentration of IL-2 is 100ng/mL, the final concentration of IL-15 is 50ng/mL, the final concentration of IL-21 is 50ng/mL, and the adding proportion of the heat-inactivated autologous plasma is reduced to 1% on the 7 th day;
(4) Medium C was supplemented every 2 days from day 9 to day 13 to adjust the cell density to 1×10 6 /ml. The culture medium C is a serum-free immune cell culture medium added with auranofin, IL-2, IL-15, IL-21 and autologous plasma; the final concentration of the auranofin is 1 mug/mL, the final concentration of the IL-2 is 100ng/mL, the final concentration of the IL-15 is 50ng/mL, the final concentration of the IL-21 is 50ng/mL, and the adding proportion of the heat-inactivated autologous plasma is 1%, if the heat-inactivated autologous plasma is used up, the heat-inactivated autologous plasma can be not added;
(5) Cells were harvested on day 14 and tested.
The procedure disclosed in example 3 was used as experiment 3 (Lonza xvvo 15 medium), and control 3 was set up as a control experiment, which did not add auranofin during the whole NK cell culture, and the other steps were the same as in example 3. Cells were harvested on day 14 and tested.
Detecting items: the day 14 test items included: cell viability, fold cell expansion, cell purity, cell killing capacity.
Cell viability: the cell viability of the experimental group 3 on the 14 th day is 90.3%, the cell viability of the control group 3 is 89.9%, and the two are not obviously different, which indicates that the cell viability is not affected by the auranofin.
Cell expansion fold: the cell expansion of the experimental group 3 on the 14 th day is 189 times, the cell expansion of the control group 3 is 181 times, and the experimental group 3 and the control group have no obvious difference, which indicates that the auranofin does not influence the cell expansion.
Cell purity: on day 14, the cell purity (CD 3-CD56+ index) of experiment group 3 reached 81.89% (see FIG. 1), and the cell purity of control group 3 was 81.33% (see FIG. 2), which were not significantly different, indicating that auranofin did not affect NK cell purity.
Cell killing activity: the killing effect of NK cells on target cells K562 tumor cells is detected by adopting a Promega Cytotox96 killing detection kit, and the ratio of the NK cells to the target cells is 10: 1. 20: 1. 40:1, the detection method is referred to the instruction book of the kit. The results showed that the NK cells cultured in the experimental group 3 of this example had stronger killing activity than the NK cells cultured in the control group 3 at various ratios (see FIG. 5).
Compared with the existing NK cell culture method, the method creatively introduces the auranofin in the NK cell culture process, has stronger killing activity of the cultured NK cells and has better clinical application value.
The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. An NK cell in vitro culture method for enhancing cell killing activity, comprising the steps of:
(1) Isolating PBMC cells with lymphocyte separation fluid;
(2) Adding the isolated PBMC cells into a culture medium A for 3 days of activation culture, wherein the culture medium A is a serum-free immune cell culture medium added with CD16 antibodies, IL-2, IL-15, IL-21 and autologous plasma;
(3) From day 3 to day 11, culture medium B, which is serum-free immunocyte medium supplemented with IL-2, IL-15, IL-21 and autologous plasma, is supplemented every 2 days;
(4) On day 13, medium C, which is serum-free immunocyte medium supplemented with auranofin, IL-2, IL-15, IL-21 and autologous plasma, was supplemented once;
(5) Cells were harvested on day 14.
2. The method for in vitro culture of NK cells for enhancing cell killing activity according to claim 1, wherein the ratio of said components in said NK cell culture medium is:
3. the NK cell in vitro culture method for enhancing cell killing activity according to claim 1 or 2, wherein the serum-free immune cell culture medium is any one of daceae NK cell culture medium, corning 581 culture medium, lonza xvvo 15 culture medium.
4. The method of in vitro NK cell culture for enhancing cell killing activity according to claim 1, wherein step (1) comprises:
1) Transferring peripheral blood into a centrifuge tube, centrifuging for 10min at 800g, and sucking the upper layer part to obtain autologous plasma for later use;
2) Diluting normal saline and blood cell sediment, blowing and mixing uniformly, slowly adding the suspension onto the Ficoll layer, and centrifuging at room temperature of 700g for 30min;
3) Sucking out the centrifuged white membrane layer, re-suspending by PBS buffer solution, centrifuging for 10min at room temperature by 500g, repeating for two times, and centrifuging and precipitating to obtain peripheral blood mononuclear cells;
4) Autologous plasma is placed at 56 ℃ for 40min, and complement in the plasma is inactivated; standing at 4 ℃ for 20min, centrifuging at 1200g for 15min, discarding the precipitate, and transferring the supernatant to a new centrifuge tube for later use.
5. The method for in vitro culture of NK cells for enhancing cell killing activity according to claim 1, wherein in step (2) is fineInitial cell culture density of 2X 10 6 /ml。
6. The method for in vitro culture of NK cells having enhanced cell killing activity according to claim 1, wherein the cell density in the steps (3) and (4) is 1X 10 6 /ml。
7. The method for in vitro culture of NK cells for enhancing cell killing activity according to claim 1, wherein in step (2) -step (4) NK cells are cultured by standing at 37℃in a 5% CO2 incubator.
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