In-vitro efficient multiplication culture method of NKT cells

Abstract

The invention discloses an in-vitro efficient multiplication culture method of NKT cells, and relates to the technical field of in-vitro multiplication culture of NKT cells, and the method comprises the following steps: coating and cell activation bottle treatment, cell separation culture amplification and cell collection. According to the in-vitro efficient multiplication culture method for the NKT cells, the amplification efficiency of the NKT cells can be remarkably improved through accurate cell activation bottle treatment and optimized cell separation culture amplification steps; from initial cell separation to final cell collection, it is ensured that the NKT cells can obtain optimal growth and amplification conditions in an in-vitro environment, an advanced lymphocyte separation technology is adopted, impurities such as red blood cells and blood platelets are effectively removed, and high purity of the NKT cells is ensured; the high-purity NKT cells not only can reduce possible side effects in clinical application, but also can improve the treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro expansion and culture of NKT cells, and in particular to an in vitro efficient expansion and culture method of NKT cells. Background Art

[0002] NKT cells are a special type of immune cells that can recognize and bind to specific glycolipid antigens, thereby activating downstream immune responses. NKT cells play an important role in immune regulation, anti-infection, and anti-tumor. In the field of biotechnology, the therapeutic application of immune cells is becoming more and more extensive, especially in the fields of cancer treatment and infectious disease prevention and treatment. NKT cells, as a unique subset of immune cells, have unique antigen recognition mechanisms and powerful immunoregulatory capabilities, and have become one of the hot spots in immune cell therapy research in recent years. Due to the limited number of NKT cells in the body, and their differentiation and expansion are restricted by many factors, such as cytokines, antigen stimulation, etc., the in vitro expansion and culture of NKT cells has become a technical problem.

[0003] At present, the in vitro expansion and culture of NKT cells mainly rely on methods such as cytokine and antigen stimulation. However, these methods have many limitations: first, the concentration and ratio of cytokine and antigen stimulation are difficult to accurately control, which can easily lead to the differentiation direction of NKT cells deviating from expectations; second, the existing in vitro expansion and culture methods often need to be carried out under specific culture conditions, such as specific temperature, humidity, pH value, etc., which are difficult to fully achieve in ordinary laboratories; finally, the existing in vitro expansion and culture methods often have the risk of cell contamination, which affects the purity and activity of NKT cells. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides an in vitro efficient expansion and culture method for NKT cells, the purpose of which is to solve the problems raised in the above-mentioned background technology.

[0005] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is: An in vitro efficient expansion and culture method for NKT cells comprises the following steps: S1: coating and cell activation bottle processing; S2: Cell isolation, culture and expansion; S3: Cell collection.

[0006] Further, the coating and cell activation bottle treatment comprises the following steps: S11: Day 0: Use a pipette to take CD16 antibody (concentration 5-50ng / ml) and add it to a T75 culture flask, tighten the cap, and slowly invert it to allow the liquid to fully disperse at the bottom; S12: Seal the bag with sealing film and then place it in a 4°C refrigerator overnight; S13: Gently shake the culture flask when placing it in the refrigerator to ensure that the coating solution is evenly spread on the bottom of the culture flask.

[0007] Furthermore, the cell separation, culture and expansion comprises the following steps: S21: Day 1: S211, draw 50 ml of whole blood, put it into a custom sterile sodium heparin blood collection tube, and mix it by inverting several times; S212, centrifuge the whole blood for 15 minutes at room temperature, 1800rpm / (757g), 9 up and 7 down; after the centrifugation, transfer the upper plasma into a centrifuge tube with a pipette; put the plasma into a water bath and inactivate it at 56 degrees for half an hour; S213, place the inactivated plasma in a refrigerator at 4 degrees for half an hour to reduce the effect of platelets on cell activation; take out the plasma from the refrigerator, centrifuge it for 5 minutes at 3000 rpm (1057g), and take the supernatant for later use; S214, resuspend the blood cell pellet after centrifugation with an equal volume of physiological saline and mix well; slowly add the mixed blood cells to 10 ml / tube of lymphocyte separation solution; place the centrifuge tubes symmetrically in a centrifuge, centrifuge at room temperature for 20 minutes, 2000 rpm / min (939g), 9 for rise and 0 for fall; S215. After centrifugation, aspirate the middle PBMC layer (white film layer) and the cell layer at the junction of the two liquid surfaces; put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 ml, centrifuge at 2000 rpm for 5 minutes, and discard the supernatant; cover the centrifuge tube lid, pat it vigorously in the palm of your hand to fully resuspend the cells, repeat the wash once, and discard the supernatant; S216. Add gentamicin (1x) solution to 1L of GIBCO culture medium and mix well to prepare complete culture medium; S217, add 3 ml of GIBCO complete culture medium, 3 ml of plasma, IL-2 (100 U / ml), IL-15 (concentration 5-50 ng / ml), and INF-Y (concentration 5-50 ng / ml) into the centrifuge tube using a Pasteur pipette, and resuspend the cells; S218. Use a pipette to draw 23 ml of GIBCO complete culture medium into the lymphocyte centrifuge tube, mix well, and transfer to a 75 ml culture flask; S219. Before each subsequent cell transfer, tap the culture flask vigorously to fully disperse the cells; S22: On day 3, add 30 to 60 ml of Gibco complete medium to the 75 ml culture flask; S23: On day 5, transfer the cells to a T175 culture flask and add 20 ml of plasma and Gibco complete medium to 200 ml; S24: Day 7: S241. Divide the cells equally into two T175 culture flasks, adding slightly more volume to the new flask; S242, add 100 ml of Gibco complete culture solution to each culture bottle until the final volume is about 200 ml, and the total volume is 400 ml; S243, taking 6 ml of the cell suspension in the culture bottle and performing quality inspection; S25: Day 9: S251. Divide the two bottles of cells equally into four T175 culture flasks, add complete medium to a volume of 200 ml per flask, and a total volume of 800 ml; S252, use a 5ml syringe to draw the sample from the sampling port for sterility test; S26: Day 11: S261. Add 1 vial of solution B to 1 L of cell culture medium and mix well to prepare complete culture medium; S262. Place the four bottles of cells into two 2 L culture bags respectively and add complete culture medium to a volume of 900 ml / bag; S27: On the 13th day, add 400 ml to 1300 ml of complete culture medium to the cell culture bag, respectively, to a total volume of 2600 ml.

[0008] Furthermore, the cell collection comprises the following steps: S31. Collect all cells from the two culture bags into two 500 ml centrifuge bottles. Centrifuge for 3 times to collect the cell precipitate. Centrifuge at 2000 rpm for 7 minutes. Be careful not to pour out the cell precipitate each time you discard the supernatant. After each centrifugation, tap the bottom of the bottle vigorously to resuspend the cells. S32, add a small amount of physiological saline to one of the centrifuge bottles, transfer the cells to another centrifuge bottle to combine the cells, add physiological saline to 500 ml, invert to mix, and take a small amount of cells for counting. Centrifuge at 2000 rpm for 7 minutes and discard the supernatant; S33, tap the bottom of the bottle vigorously to resuspend the cells, then add 500 ml of physiological saline to resuspend the cells, centrifuge at 2000 rpm for 7 minutes, discard the supernatant, and tap the bottom of the bottle vigorously to resuspend the cells; S34. Take out a bag of 250ml normal saline for use, use a 20ml syringe to draw 10-20ml of normal saline and NKT4 solution and add them into the culture bottle, then draw out the cell suspension together into a 200ml blood bag and seal it with a sealing machine.

[0009] The beneficial effects of the present invention are: 1. Efficient expansion: Through precise cell activation bottle processing and optimized cell separation, culture and expansion steps, this method can significantly improve the expansion efficiency of NKT cells; from initial cell separation to final cell collection, it ensures that NKT cells can obtain optimal growth and expansion conditions in an in vitro environment.

[0010] 2. High purity: The cell separation, culture and expansion step of the present invention adopts advanced lymphocyte separation technology, which effectively removes impurities such as red blood cells and platelets, ensuring the high purity of NKT cells; high-purity NKT cells can not only reduce the side effects that may occur in clinical applications, but also improve the treatment effect.

[0011] 3. High activity: During the culture process, the present invention pays special attention to the protection of cell activity; through reasonable cytokine ratio, temperature control, centrifugation parameter setting and other measures, the activity of NKT cells is retained to the maximum extent; highly active NKT cells can better play their role in immune regulation, anti-tumor and other aspects.

[0012] 4. Easy operation: The in vitro expansion and culture method of NKT cells of the present invention has clear steps and is easy to operate. It does not require complicated equipment and technical support, and is easy to promote and apply in ordinary laboratories. In addition, by optimizing the culture conditions and steps, the method also reduces the difficulty and cost of operation and improves the repeatability and stability of the experiment.

[0013] 5. Reduce the risk of contamination: In the process of cell culture, contamination is an issue that cannot be ignored; the present invention adopts strict aseptic operation technology and quality control measures to effectively reduce the risk of cell contamination. At the same time, regular quality inspection and sterility inspection ensure that the purity and activity of NKT cells are not affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A positive lateral flow diagram of a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 2 A diagram showing the proportion of white blood cells in a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 3 A diagram showing the ratio of CD3+CD56+ and CD3-CD56+ cells in a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 4 A diagram showing the proportion of CD3+CD16+CD56+ cells in a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 5 A diagram showing the proportion of NK cells in a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 6A diagram showing the ratio of CD56+CD16+ cells in a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 7 A diagram showing the ratio of CD3-CD16+ cells in a method for efficiently expanding and culturing NKT cells in vitro according to the present invention; Figure 8 This is a test data diagram of a method for efficiently expanding and culturing NKT cells in vitro according to the present invention. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with the accompanying drawings.

[0016] like Figures 1 to 8 The present invention relates to an in vitro efficient expansion and culture method of NKT cells, comprising the following steps: 1. Coating and Cell Activation Bottle Processing Add NKT1 cells to a T75 culture flask, tighten the cap and slowly invert to ensure that the liquid is fully dispersed at the bottom of the flask.

[0017] Seal the bottle with sealing film and place it in a 4°C refrigerator overnight.

[0018] Gently shake the bottle while placing it in the refrigerator to ensure that the coating solution evenly covers the bottom of the culture bottle.

[0019] 2. Cell Isolation, Culture and Expansion Extract 50 ml of whole blood, put it into a sterile blood collection tube containing sodium heparin, and mix it by inversion.

[0020] The whole blood was centrifuged at room temperature for 15 minutes at 1800 rpm to separate the plasma. After centrifugation, the supernatant plasma was transferred to a new centrifuge tube.

[0021] The plasma was inactivated in a 56°C water bath for 30 minutes and then placed in a 4°C refrigerator for half an hour to reduce the effect of platelets on cell activation.

[0022] The plasma was centrifuged at 3000 rpm for 5 min, the supernatant was removed, and the blood cell pellet was resuspended in physiological saline.

[0023] Slowly add the resuspended blood cells into the centrifuge tube containing lymphocyte separation solution and centrifuge at 2000 rpm for 20 minutes at room temperature to separate the PBMC layer (white blood cell layer).

[0024] The PBMC layer was aspirated, washed with physiological saline and fixed to 50 ml, and the cell pellet was resuspended after centrifugation and washing.

[0025] As a patent engineer, you can translate this operation description into the technical implementation steps in the patent application, ensuring that the language is precise and clear, and avoiding the use of non-professional terms or unclear expressions. The following is a description of the original text: In a centrifuge tube, use a Pasteur pipette to sequentially add 3 ml of GIBCO complete medium, 3 ml of plasma, and NKT2 solution, and then resuspend the cells.

[0026] Use a pipette to draw 23 ml of GIBCO complete culture medium into the lymphocyte centrifuge tube, mix thoroughly, and then transfer it to a 75 ml culture flask.

[0027] Before each cell transfer, tap the flask vigorously to ensure that the cells are well dispersed.

[0028] Add 30 ml of GIBCO complete culture medium to a 75 ml culture flask to make the total volume reach 60 ml.

[0029] The cells were transferred to a T175 culture flask and supplemented with 20 ml of plasma and GIBCO complete medium to 200 ml.

[0030] Divide the cells equally between two T175 flasks, adding slightly more volume to the new flasks.

[0031] Add 100 ml of GIBCO complete nutrient solution to each culture bottle to about 200 ml, with a total volume of 400 ml.

[0032] Take 6 ml of the cell suspension in the culture flask for quality inspection.

[0033] Distribute the two flasks of cells evenly into four T175 culture flasks, add complete medium to each flask to 200 ml, and the total volume is 800 ml.

[0034] Use a 5 ml syringe to draw a sample from the sampling port for sterility testing.

[0035] Add 1 vial of Solution B to 1L of cell culture medium and mix well to prepare complete culture medium.

[0036] Place the four bottles of cells into two 2L culture bags respectively, add complete culture medium to each bag to 900ml, and add culture fluid appropriately according to the cell density.

[0037] Add 400ml to 1300ml of complete culture medium to the cell culture bag, with a total volume of 2600ml. Add appropriate amount of culture medium according to the cell density.

[0038] 3. The steps for cell collection are as follows: Collect the cells from both culture bags completely into two 500ml centrifuge bottles. The cell pellet in each centrifuge bottle needs to be centrifuged three times, each time at 2000rpm for 7 minutes. Be careful when discarding the supernatant to ensure that the cell pellet is not poured out. After each centrifugation, tap the bottom of the bottle vigorously to resuspend the cells.

[0039] Add a small amount of saline to one of the centrifuge bottles, transfer the cells to another centrifuge bottle and combine the cells. Then add enough saline to the centrifuge bottle to make the total volume reach 500ml, invert and mix, and extract 10-20ml of the cell suspension for counting. Centrifuge again at 2000rpm for 7 minutes and discard the supernatant.

[0040] Tap the bottom of the bottle vigorously to resuspend the cells, then add 500 ml of physiological saline to the centrifuge bottle to resuspend the cells. Centrifuge again at 2000 rpm for 7 minutes and discard the supernatant. After the centrifugation is completed, tap the bottom of the bottle again to resuspend the cells.

[0041] Take out a bag of 250ml saline for use. Use a 20ml syringe to draw a small amount of saline (about 10-20ml) and add it to the NKT4 solution. Then draw out the cell suspension and inject it into a 200ml blood bag. Finally, seal it with a sealing machine.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for efficiently expanding and culturing NKT cells in vitro, characterized in that: The following steps are involved: S1: coating and cell activation bottle processing; S2: Cell isolation, culture and expansion; S3: cell collection; The coating and cell activation bottle process includes the following steps: S11: Day 0: Use a pipette to take CD16 antibody (concentration 5-50ng / ml) and add it to a T75 culture flask, tighten the cap, and slowly invert it to allow the liquid to fully disperse at the bottom; S12: Seal the bag with sealing film and then place it in a 4°C refrigerator overnight; S13: Gently shake the culture medium when placing it in the refrigerator to ensure that the coating solution is evenly spread on the bottom of the culture bottle; The cell separation, culture and expansion comprises the following steps: S21: Day 1: S211, draw 50 ml of whole blood, put it into a custom sterile sodium heparin blood collection tube, and mix it by inverting several times; S212, centrifuge the whole blood for 15 minutes at room temperature, 1800rpm / (757g), 9 up and 7 down; after the centrifugation, transfer the upper plasma into a centrifuge tube with a pipette; put the plasma into a water bath and inactivate it at 56 degrees for half an hour; S213, place the inactivated plasma in a refrigerator at 4 degrees for half an hour to reduce the effect of platelets on cell activation; take out the plasma from the refrigerator, centrifuge it for 5 minutes at 3000 rpm (1057g), and take the supernatant for later use; S214, resuspend the blood cell pellet after centrifugation with an equal volume of physiological saline and mix well; slowly add the mixed blood cells to 10 ml / tube of lymphocyte separation solution; place the centrifuge tubes symmetrically in a centrifuge, centrifuge at room temperature for 20 minutes, 2000 rpm / min (939g), 9 for rise and 0 for fall; S215. After centrifugation, aspirate the middle PBMC layer (white film layer) and the cell layer at the junction of the two liquid surfaces; put the white film layer into a new centrifuge tube, resuspend and mix with physiological saline to 50 ml, centrifuge at 2000 rpm for 5 min, and discard the supernatant; cover the centrifuge tube lid, pat it vigorously in the palm of your hand to fully resuspend the cells, repeat the wash once, and discard the supernatant; S216. Add gentamicin (1x) solution to 1L of GIBCO culture medium and mix well to prepare complete culture medium; S217, add 3 ml of GIBCO complete culture medium, 3 ml of plasma, IL-2 (100 U / ml), IL-15 (concentration 5-50 ng / ml), and INF-Y (concentration 5-50 ng / ml) into the centrifuge tube using a Pasteur pipette, and resuspend the cells; S218. Use a pipette to draw 23 ml of GIBCO complete culture medium into the lymphocyte centrifuge tube, mix well, and transfer to a 75 ml culture flask; S219. Before each subsequent cell transfer, tap the culture flask vigorously to fully disperse the cells; S22: On day 3, add 30 to 60 ml of Gibco complete medium to the 75 ml culture flask; S23: On day 5, transfer the cells to a T175 culture flask and add 20 ml of plasma and Gibco complete medium to 200 ml; S24: Day 7: S241. Divide the cells equally into two T175 culture flasks, adding slightly more volume to the new flasks; S242. Add 100 ml of Gibco complete medium to each culture bottle until the final volume is about 200 ml, with a total volume of 400 ml. S243, taking 6 ml of the cell suspension in the culture bottle and performing quality inspection; S25: Day 9: S251, divide the two bottles of cells equally into four T175 culture flasks, add complete medium to the volume of 200 ml per flask, and the total volume is 800 ml; S252, use a 5ml syringe to draw the sample from the sampling port for sterility test; S26: Day 11: S261. Add gentamicin (1x) to 1 L of cell culture medium and mix well to prepare complete culture medium. S262. Place the four bottles of cells into two 2 L culture bags respectively and add complete culture medium to a volume of 900 ml / bag; S27: On day 13, add 400 ml to 1300 ml of complete culture medium to the cell culture bag, respectively, to a total volume of 2600 ml; The cell collection comprises the following steps: S31. Collect all cells from the two culture bags into two 500 ml centrifuge bottles. Centrifuge for 3 times to collect the cell precipitate. Centrifuge at 2000 rpm for 7 minutes. Be careful not to pour out the cell precipitate each time you discard the supernatant. After each centrifugation, tap the bottom of the bottle vigorously to resuspend the cells. S32, add a small amount of physiological saline to one of the centrifuge bottles, transfer the cells to another centrifuge bottle to merge the cells, then add physiological saline to 500 ml, invert to mix, aspirate 10-20 ml of cells for counting, centrifuge at 2000 rpm for 7 minutes, and discard the supernatant; S33, tap the bottom of the bottle vigorously to resuspend the cells, then add 500 ml of physiological saline to resuspend the cells, centrifuge at 2000 rpm for 7 minutes, discard the supernatant, and tap the bottom of the bottle vigorously to resuspend the cells; S34. Take out a bag of 250ml normal saline for use, use a 20ml syringe to draw 10-20ml of normal saline and NKT4 solution and add them into the culture bottle, then draw out the cell suspension together into a 200ml blood bag and seal it with a sealing machine.

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