CN117866893A - NK cell in vitro amplification method - Google Patents
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Abstract
The invention discloses an in vitro amplification method of NK cells, which comprises the following steps: resuspension of mononuclear cells using an activation medium, adding the suspension to a cell culture plate coated with CD16 and NKp46 antibodies, culturing for more than 21 days, culturing by using the activation medium on days 1-4, and culturing by using an amplification medium for fluid replacement from day 5; the activation culture medium is obtained by adding autologous plasma, IL2, IL15, IL18, IL21 and IL27 on the basis of an NK cell amplification serum-free culture medium; the amplification culture medium is obtained by adding autologous plasma and IL2 on the basis of an NK cell amplification serum-free culture medium. According to the invention, the CD16 and NKp46 antibodies are combined and coated on the cell culture plate, and simultaneously, five cytokines such as IL2 and IL15 are added to synergistically stimulate NK cells to induce NK cells to differentiate and mature, so that the obtained NK cells have high purity and high killing performance, and have high clinical application value.
Description
Technical Field
The invention relates to an in-vitro amplification method of NK cells, and belongs to the technical field of cell culture.
Background
Tumors have been the first problem to afflict human health, and effective treatments have not been found due to their complexity, variability and heterogeneity. In the past decade, immunotherapy has made great progress in cancer treatment, and the principle is to collect lymphocytes from human body, and to obtain a great number of lymphocytes with high killing performance after in vitro activation, amplification, screening or modification, and then to infuse the lymphocytes back into the patient, so as to kill tumor cells. Although most immunotherapeutic strategies are based on the use of T cells, studies have shown that Natural Killer (NK) cells have tremendous immunotherapeutic potential.
NK cells belong to lymphocytes, account for 10% -20% of human peripheral blood lymphocytes and account for 5% of umbilical cord blood lymphocytes, and can kill tumor cells and virus infected cells in a nonspecific manner without antigen pre-sensitization. Notably, the human body has a limited content of NK cells itself, and NK cells cannot naturally produce cytokines necessary for in vivo expansion, survival, and cytotoxic effects. Therefore, obtaining a sufficient number of NK cells while maintaining a high purity and killing ability of NK cells is a key technical hurdle for clinical application of NK cells. At present, the in-vitro amplification of NK cells is mainly divided into two main technical methods, and the trophoblast cells are taken as the main technical method, so that the method has the characteristics of rapid amplification and high efficiency, but the clinical application of the method is limited because the trophoblast cells have the risks of tumorigenicity, pollution and the like; the second technical method is a cytokine method, and the common method is to stimulate mononuclear cells through cytokines such as IL2, IL7 and the like to induce the mononuclear cells to differentiate and amplify towards the NK direction, so that the method has high safety, but the amplified NK cells have low purity, generally 55-80 percent and low killing performance.
Disclosure of Invention
Aiming at the prior art, the invention provides an in-vitro amplification method of NK cells, and the NK cells obtained by the method are high-efficiency NK cells, high in purity and high in killing capacity.
The invention is realized by the following technical scheme:
an in vitro amplification method of NK cells comprises the following steps: single nuclear cells were resuspended using activation medium to adjust cell density to 1.0X10 6 ～2.0×10 6 Adding the cells per mL into a cell culture plate coated with CD16 and NKp46 antibodies at a volume of 2 mL/hole, placing the cell culture plate in a carbon dioxide incubator for culturing for more than 21 days, wherein the cells are cultured by using an activation medium on days 1-4, are subjected to fluid replacement culture by using an amplification medium on day 5, and then are subjected to fluid replacement once every 2 days, and the NK cell density is regulated to be 1.0x10 while the fluid replacement is carried out 6 ～1.2×10 6 individual/mL; by day 21, a large number of NK cells of high purity and high killing ability were harvested.
The activation medium is obtained by adding autologous plasma, IL2, IL15, IL18, IL21 and IL27 on the basis of a serum-free medium for NK cell amplification, wherein the addition amount of the autologous plasma is 8% -12% (volume percentage, the same applies below), preferably, the final concentration of the IL2, IL15, IL18, IL21 and IL27 is 45-55U/mL, 45-55 ng/mL, 90-110 ng/mL, 4-6 ng/mL and 4-6 ng/mL respectively, preferably, the final concentration of the IL2, IL15, IL18, IL21 and IL27 is 50U/mL, 50ng/mL, 100ng/mL, 5ng/mL and 5ng/mL respectively. The NK cell amplification serum-free medium is a commercial medium existing in the prior art and can be purchased in the market.
The amplification culture medium is obtained by adding autologous plasma and IL2 on the basis of a serum-free culture medium for NK cell amplification, wherein the addition amount of the autologous plasma is 8% -12%, preferably 10%, and the final concentration of the IL2 is 45-55U/mL, preferably 50U/mL.
Further, the CD16 and NKp46 antibody coated cell culture plates were obtained by: coating TC-treated cell culture plates with antibody coating liquid containing CD16 and NKp46, placing the cell culture plates at 4 ℃ for incubation for 10-14 hours, and washing with PBS buffer solution for later use; in the antibody coating solution, the concentration of CD16 is 0.2-0.6. Mu.g/mL, preferably 0.5. Mu.g/mL, and the concentration of NKp46 is 0.2-0.6. Mu.g/mL, preferably 0.5. Mu.g/mL.
Preferably, after the mononuclear cells are resuspended using the activation medium, the cell density is adjusted to 1.5X10 6 And each mL.
Further, the culture conditions in the carbon dioxide incubator are: 37 ℃,5% CO 2 Saturated humidity.
Further, when the culture is performed using the activation medium, the fluid is supplemented once on day 2 or 3.
Further, the autologous plasma is obtained by the following method: centrifuging umbilical cord blood to obtain blood plasma and whole blood cells, inactivating the blood plasma in a water bath at 56 deg.C for 30min, cooling at 4 deg.C for 30min, and centrifuging.
Further, the mononuclear cells are isolated by the following method: the umbilical cord blood is centrifugally separated to obtain plasma and whole blood cells, the whole blood cells are uniformly mixed with normal saline, the mixture is slowly added into the upper layer of lymphocyte separation liquid, the lymphocyte is obtained through separation by a density gradient centrifugation method, the normal saline is added, and the mononuclear cells are obtained after centrifugal washing for 3 times.
According to the NK cell in-vitro amplification method, the CD16 and NKp46 antibodies are combined and coated on the cell culture plate, and simultaneously five cytokines, namely IL2, IL15, IL18, IL21 and IL27 are added to synergistically stimulate the NK cells to induce the differentiation and maturation of the NK cells, so that the obtained NK cells are high in purity and high in killing performance. The NK cell in-vitro amplification method is simple to operate, high in safety, capable of directly culturing mononuclear cells without magnetic bead separation, inducing and amplifying a large number of NK cells with high purity and high killing performance, and has high clinical application value.
Drawings
Fig. 1: the purity flow assay results of NK cells are schematically shown.
Fig. 2: results of double positive flow assays for CD56 and CD16 of NK cells are shown.
Fig. 3: the in vitro killing detection result of NK cells on tumor cells is schematically shown (target cells are Raj i), wherein the method refers to NK cells obtained by culturing by the method; "PC-1" refers to NK cells cultured using a commercial NK induction culture kit PC-1; "PC-2" refers to NK cells cultured using a commercial NK induction culture kit PC-2.
Fig. 4: the in vitro killing detection result of NK cells on tumor cells is schematically shown (the target cells are K562), wherein the method refers to NK cells obtained by culturing by the method; "PC-1" refers to NK cells cultured using a commercial NK induction culture kit PC-1; "PC-2" refers to NK cells cultured using a commercial NK induction culture kit PC-2.
Detailed Description
The invention is further illustrated below with reference to examples. However, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
The instruments, reagents and materials used in the examples below are conventional instruments, reagents and materials known in the art and are commercially available. The experimental methods and detection methods in the following examples are conventional experimental methods and detection methods in the prior art unless otherwise specified.
Example NK cell in vitro expansion
1. Culture plate coating treatment
Coating TC-treated cell culture plates with 1mL of antibody coating solution containing CD16 and NKp46, and incubating for 12 hours in a refrigerator at 4 ℃; after coating, removing coating liquid in the plate, and washing with 2mL of PBS buffer solution for 2 times for standby; in the antibody coating solution, the concentration of CD16 was 0.5. Mu.g/mL, and the concentration of NKp46 was 0.5. Mu.g/mL.
2. Preparation of autologous plasma
2.1 transfer the blood sample with anticoagulant into a centrifuge tube, centrifuge at 1800rpm/min for 15min, centrifuge acceleration was set to 9 liters and 7 liters.
2.2, after centrifugation, the upper layer is blood plasma, the lower layer is whole blood cells, and the upper layer blood plasma is transferred into a new centrifuge tube and is sealed by a sealing film; the plasma was placed in a water bath and inactivated at 56℃for 30min.
2.3 the inactivated plasma was refrigerated in a refrigerator at 4℃for half an hour.
2.4 after the cold storage is finished, taking out the plasma from the refrigerator, and centrifuging at 3000rpm/min for 10min; after centrifugation, the supernatant was transferred to a new centrifuge tube and stored at-20℃until use.
3. Isolation of mononuclear cells
3.1 lower layer whole blood cells after the upper layer plasma is removed are diluted by adding normal saline according to the volume ratio of 1:1, and are gently mixed.
3.2 taking a clean 50ml centrifuge tube, and adding 20ml room temperature lymphocyte separation liquid to the bottom of the centrifuge tube; taking 25ml of diluted whole blood cells by a pipette, slowly adding the diluted whole blood cells by adherence, and placing the whole blood cells on the upper layer of lymphocyte separation liquid; note that lymphocyte isolates are not whippable.
3.3 the centrifuge tube was carefully placed in a centrifuge and centrifuged at 700g for 30min, with the lift acceleration set at 3.
3.4, after the centrifugation is finished, carefully taking out the centrifuge tube, and recording that the centrifuge tube cannot shake; the liquid in the centrifuge tube is divided into 4 layers from top to bottom: the uppermost layer is a plasma layer (light red transparent plasma), the second layer is a lymphocyte layer (thin dense annular layer), the third layer is a lymphocyte separation liquid layer, and the lowest layer is a red cell layer (red sediment).
3.5 transfer the second layer of lymphocytes into a clean 50ml centrifuge tube, aspirate as little of the plasma layer and lymphocyte separation layer as possible.
3.6 adding normal saline into the lymphocyte suspension until the volume is 50ml, gently reversing and uniformly mixing; the centrifuge tube was placed in a centrifuge and centrifuged at 300g for 10min with the lift acceleration set at 6.
3.7, after centrifugation, lymphocytes are distributed on the bottom layer of the centrifuge tube; carefully pouring out the supernatant after centrifugation, adding 10ml of PBS buffer solution into a centrifuge tube, and re-suspending lymphocytes uniformly; continuously adding 40ml PBS buffer solution into the centrifuge tube, and uniformly mixing the solution upside down; the centrifuge tube was placed in a centrifuge and centrifuged at 300g for 10min with the lift acceleration set at 6.
3.8 repeat step 3.7.
3.9, after centrifugation, lymphocytes are distributed on the bottom layer of the centrifuge tube; the centrifuged supernatant was carefully decanted for use.
4. Preparation of activation Medium
To NK cell expansion serum-free medium (manufacturer: ekesai, cat# NE 000-N012) was added 10% autologous plasma, followed by IL2 (50U/mL), IL15 (50 ng/mL), IL18 (100 ng/mL), IL21 (5 ng/mL) and IL27 (5 ng/mL).
5. Preparation of amplification Medium
10% autologous plasma was added to NK cell expansion serum-free medium (manufacturer: ekesai, cat# NE 000-N012) followed by IL2 (50U/mL).
6. Mononuclear cell seed plate
Single nuclear cells were resuspended using activation medium to adjust cell density to 1.5X10 6 The cells/mL were added to a cell culture plate pre-coated with CD16 and NKp46 antibodies at a volume of 2 mL/well, and incubated in a carbon dioxide incubator under the following conditions: 37 ℃ and 5% CO 2 Saturated humidity.
Culture of NK cells
Culturing with an activating culture medium at 1-4 days, and supplementing liquid once at 2 days; the amplification medium was used for the liquid-feeding culture from day 5, after which the liquid was fed once every 2 days, and the NK cell density was adjusted to 1.0X10 while feeding 6 ～1.2×10 6 Number of samples per mL (NK cell density was measured every 2 days); by day 21, a large number of NK cells of high purity and high killing ability were harvested.
8. Control setting
The commercial NK induction culture kits PC-1 and PC-2 are used as controls to culture NK cells (namely, the activation culture medium in the 6 and 7 is replaced by the commercial NK induction culture kits PC-1 and PC-2).
Detection of NK cell purity
NK cells in the above 7 were cultured until day 21, part of NK cells was taken, supernatant was discarded after centrifugation, cell pellet was resuspended in PBS buffer, labeled with CD56 and CD3 antibodies, and the purity of the collected cells was examined by flow cytometry, and the results are shown in FIG. 1. As can be seen from FIG. 1, NK cells can be up to 95% pure.
Detection of double positives of NK cells CD56 and CD16
NK cells in the above 7 were cultured until day 21, part of NK cells was taken, supernatant was discarded after centrifugation, cell pellet was resuspended in PBS buffer, labeled with CD56 and CD16 antibodies, and the expression efficiency of the collected cells CD56 and CD16 was examined by flow cytometry, and the results are shown in FIG. 2. As can be seen from FIG. 2, the double positive expression rate of NK cells CD56 and CD16 can reach more than 90%, which shows that the NK cells cultured and amplified by the method of the invention have high potential of antibody-dependent cell-mediated cytotoxicity (ADCC).
Detection of NK cell killing in vitro
NK cells cultured to day 21 in 7 and 8 above were resuspended in medium using Raji and K562 cells stably transformed with luciferases (luciferases) as target cells, and inoculated into 96-well plates at a ratio of 5:1, 10:1, 20:1 with the target cells Raji-luciferases, and inoculated into 96-well plates at a ratio of 0.5:1, 1:1, 2:1 with the target cells K562-luciferases, respectively. After 4 hours of co-cultivation, bright-Glo was used TM The Luciferase assay kit measures the release level of the target cell luciferases, and further detects the killing capacity of NK cells. The results are shown in FIGS. 3 and 4. As can be seen from the figures 3 and 4, the NK cells obtained by the method can kill Raji cells by more than 90 percent and K562 cells by more than 70 percent; compared with commercial NK induction culture kits PC-1 and PC-2, the NK cells obtained by the method have obvious difference and higher tumor killing capacity.
The foregoing examples are provided to fully disclose and describe how to make and use the claimed embodiments by those skilled in the art, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to a person skilled in the art will be within the scope of the appended claims.
Claims (10)
1. An in vitro amplification method of NK cells, which is characterized in that: single nuclear cells were resuspended using activation medium to adjust cell density to 1.0X10 6 ～2.0×10 6 Adding the cells into a cell culture plate coated with CD16 and NKp46 antibodies at a volume per mL, placing the cells in a carbon dioxide incubator for culturing for more than 21 days, wherein the cells are cultured by using an activation medium at 1-4 days, are subjected to fluid replacement culture by using an amplification medium at 5 days, and are subjected to fluid replacement once every 2 days, and the density of NK cells is regulated to be 1.0 multiplied by 10 while the fluid replacement is carried out 6 ～1.2×10 6 individual/mL;
the activation culture medium is obtained by adding autologous plasma, IL2, IL15, IL18, IL21 and IL27 on the basis of an NK cell amplification serum-free culture medium, wherein the addition amount of the autologous plasma is 8% -12%, and the final concentration of the IL2, IL15, IL18, IL21 and IL27 is 45-55U/mL, 45-55 ng/mL, 90-110 ng/mL, 4-6 ng/mL and 4-6 ng/mL respectively;
the amplification culture medium is obtained by adding autologous plasma and IL2 on the basis of an NK cell amplification serum-free culture medium, wherein the addition amount of the autologous plasma is 8% -12%, and the final concentration of the IL2 is 45-55U/mL.
2. The method for in vitro expansion of NK cells according to claim 1, characterized in that: the addition amount of autologous plasma in the activation culture medium is 10%, and the final concentrations of IL2, IL15, IL18, IL21 and IL27 are 50U/mL, 50ng/mL, 100ng/mL, 5ng/mL and 5ng/mL respectively.
3. The method for in vitro expansion of NK cells according to claim 1, characterized in that: in the amplification medium, the addition amount of autologous plasma is 10%, and the final concentration of IL2 is 50U/mL.
4. The method of claim 1, wherein the CD16 and NKp46 antibody coated cell culture plates are obtained by: coating TC-treated cell culture plates with antibody coating liquid containing CD16 and NKp46, placing the cell culture plates at 4 ℃ for incubation for 10-14 hours, and washing with PBS buffer solution for later use; in the antibody coating solution, the concentration of CD16 is 0.2-0.6 mug/mL, and the concentration of NKp46 is 0.2-0.6 mug/mL.
5. The method for in vitro expansion of NK cells according to claim 4, characterized in that: in the antibody coating solution, the concentration of CD16 was 0.5. Mu.g/mL, and the concentration of NKp46 was 0.5. Mu.g/mL.
6. The method for in vitro expansion of NK cells according to claim 1, characterized in that: the culture conditions in the carbon dioxide incubator are as follows: 37 ℃,5% CO 2 Saturated humidity.
7. The method for in vitro expansion of NK cells according to claim 1, characterized in that: after resuspension of the mononuclear cells using the activation medium, the cell density was adjusted to 1.5X10 6 And each mL.
8. The method for in vitro expansion of NK cells according to claim 1, characterized in that: the culture is carried out by using an activation culture medium, and the liquid is supplemented once on the 2 nd or 3 rd day.
9. The method for in vitro expansion of NK cells according to claim 1, wherein said autologous plasma is obtained by: centrifuging umbilical cord blood to obtain blood plasma and whole blood cells, inactivating the blood plasma in a water bath at 56 deg.C for 30min, cooling at 4 deg.C for 30min, and centrifuging.
10. The method for in vitro expansion of NK cells according to claim 1, wherein said mononuclear cells are isolated by: the umbilical cord blood is centrifugally separated to obtain plasma and whole blood cells, the whole blood cells are uniformly mixed with normal saline, the mixture is slowly added into the upper layer of lymphocyte separation liquid, the lymphocyte is obtained through separation by a density gradient centrifugation method, the normal saline is added, and the mononuclear cells are obtained after centrifugal washing for 3 times.
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| CN118581043A (en) * | 2024-06-20 | 2024-09-03 | 杭州百瑞竞康生物技术有限公司 | Method for preparing NK cells |
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| CN118389423A (en) * | 2024-05-11 | 2024-07-26 | 广州希灵生物科技有限公司 | A method for culturing NK cells in vitro |
| CN118581043A (en) * | 2024-06-20 | 2024-09-03 | 杭州百瑞竞康生物技术有限公司 | Method for preparing NK cells |
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