Shearing force coupling beta-sitosterol NK cell large-scale culture method
By adding β-sitosterol and appropriate shear force to NK cell culture, the problem of low amplification fold in traditional methods was solved, and a large number of highly pure NK cells with strong tumor-killing activity were efficiently cultured.
Patent Information
- Application Number
- CN202511127298.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-13
- Publication Date
- 2025-11-18
AI Technical Summary
Existing technologies make it difficult to obtain large quantities of high-purity NK cells with strong tumor-killing activity. Traditional cytokine combination stimulation methods result in small amplification folds and pose safety risks.
NK cell culture was performed using a β-sitosterol and shear force coupling method, which involved adding IL-2, IL-15 and β-sitosterol to serum-free medium and applying appropriate shear force in a bioreactor for large-scale culture.
It significantly improved the expansion rate and purity of NK cells, with a proliferation rate of more than 800 times. The proportion of CD16+CD56+ double positive cells was 95.60%, and the killing rate of tumor cells reached 85.65%, thus achieving the acquisition of a large number of high-purity NK cells with strong tumor-killing activity.
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Figure CN120966752A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of NK cell culture, in particular to a shear force coupled beta-sitosterol NK cell large-scale culture method. BACKGROUND
[0002] NK cells account for a small proportion in peripheral blood, generally no more than 10% of the total number of lymphocytes, and it is difficult to obtain a large number of high-purity NK cells in a short period of time.
[0003] In order to achieve the purpose of in vitro expansion, researchers culture NK cells in peripheral blood mononuclear cells by the method of feeder cells, which can obtain a large number of NK cells, but the introduction of human cells may cause certain safety hazards. At present, the commonly used method is to stimulate the expansion of NK cells by cytokines. However, the traditional cytokine (IL-2, IL-15) combination stimulation method for expanding NK cells has a small expansion multiple, which cannot meet the requirements of a large number and high purity.
[0004] Researchers have also tried to use other cell stimulating factors to improve the expansion multiple and purity of NK cells, for example, Chen Wei, Yang Ji, Lin Di, et al. IL-15 and IL-21 cytokine combination optimization of clinical grade human NK cell culture system [J]. Clinical Military Medical Journal, 2017, 45(1): 6. DOI: 10.16680 / j.1671-3826.2017.01.18. The results show that although IL-15 and IL-21 promote the proliferation of NK cells, the tumor killing activity is not improved. Therefore, how to obtain a large number of high-purity NK cells with strong tumor killing activity is a problem currently faced.
[0005] Based on this, the present application is proposed. SUMMARY
[0006] In order to solve the above technical problems, the present application provides the following technical solutions:
[0007] The present application provides a shear force coupled beta-sitosterol NK cell large-scale culture method, comprising the following steps:
[0008] (1) isolating NK cells from peripheral blood;
[0009] (2) inoculating the NK cells into a serum-free medium containing beta-sitosterol for culture to obtain an NK cell seed solution;
[0010] (3) inoculating the NK cell seed solution into a bioreactor for large-scale culture, coupling shear force culture in the large-scale culture stage, and collecting NK cells after the culture ends.
[0011] Preferably, the serum-free medium is X-VIVO 15 or KBM 581.
[0012] Preferably, IL-2 and IL-15 are further added in the serum-free medium, the concentration of IL-2 is 800-1200 IU / mL, and the concentration of IL-15 is 80-120 IU / ml.
[0013] Preferably, the addition amount of β-sitosterol is 0.8-1.2 mg / L of the volume of the medium.
[0014] Preferably, the medium in the bioreactor is a serum-free medium containing IL-2, IL-15 and β-sitosterol; the concentration of IL-2 is 800-1200 IU / mL, the concentration of IL-15 is 80-120 IU / ml, and the addition amount of β-sitosterol is 0.8-1.2 mg / L.
[0015] Preferably, the shear force for shear culture is 40-60 rpm.
[0016] Preferably, the time for ending the culture is 14 days.
[0017] The present application improves the expansion multiple, purity and activity of NK cells by adding β-sitosterol in the NK cell culture medium, the proliferation multiple of NK cells reaches more than 800 times, the proportion of CD16+CD56+ double positive cells is 95.60%, the killing rate of tumor cells reaches 85.65%, and the purpose of obtaining a large number of NK cells with high purity and strong tumor killing activity is initially achieved. Further, we have explored the influence of shear force on cell expansion, and the results prove that shear force coupled with β-sitosterol can further improve the expansion multiple of NK cells during large-scale culture of NK cells, so that the number of NK cells reaches the level of hundreds of billions. BRIEF DESCRIPTION OF DRAWINGS
[0018] Figure 1 To culture the cell proliferation multiple at different times;
[0019] Figure 2 To flow cytometry graph;
[0020] Figure 3 To NK cell killing rate under different effector target ratios;
[0021] Figure 4 To the influence of coupled shear force on the expansion multiple of NK cell seed solution;
[0022] Figure 5 To the influence of coupled shear force on large-scale culture of NK cells. DETAILED DESCRIPTION
[0023] The technical solutions provided by the present application will be described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.
[0024] Example 1
[0025] (1) Isolation of NK cells from peripheral blood
[0026] The peripheral blood was diluted 1:1 (v / v) with normal saline, and the diluted blood was slowly added to a centrifuge tube containing lymphocyte separation medium, so that the blood was always on the upper layer of the lymphocyte separation medium, with a clear boundary line, and the volume ratio of the two was 2:1. The up-down speed was adjusted to 800g for centrifugation for 30min. After centrifugation, the mononuclear cell layer was carefully aspirated into another new centrifuge tube with a Pasteur pipette, and RPMI 1640 medium was added to the centrifuge tube to wash the cells, 300g centrifugation for 5min, discard the supernatant, repeat the washing for 2 times, use Stem cell immunomagnetic bead cell sorting kit to sort NK cells, the specific method is: resuspend the cells with freshly prepared NK cell sorting Buffer, adjust the cell density to 1×10 8 cells / ml, and transfer to a 1.5ml EP tube; add mixed antibodies according to the antibody addition amount of 50μl / ml, mix gently after adding mixed antibodies, incubate at room temperature for 10min; blow the magnetic beads evenly, add magnetic beads according to the magnetic bead addition amount of 100μl / ml, mix gently after adding magnetic beads, incubate at room temperature for 5min; transfer the cell suspension to a 5ml round-bottom tube specially for sorting, add NK cell sorting Buffer to 2.5ml, blow evenly after mixing gently, insert into the magnetic pole (without cover), stand for 2.5min; take the round-bottom tube together with the magnetic pole, pour the cell suspension into a new 15ml centrifuge tube, centrifuge at 400g for 5min; discard the supernatant, resuspend the cells with 5ml RPMI 1640 medium to obtain seed NK cell suspension. Take a small amount of seed NK cell suspension for counting by trypan blue staining method.
[0027] (2) Preparation of NK cell seed solution
[0028] The NK cells separated in step (1) were inoculated into serum-free medium containing IL-2, IL-15 and β-sitosterol at a concentration of 1×10 6 cells / mL. KBM 581. Among them, the concentration of IL-2 is 1000 IU / mL, the concentration of IL-15 is 100 IU / mL, and the concentration of β-sitosterol is 1.0 mg / L. Cultured at 37°C, 5% CO2 for 14 days, add IL-2, IL-15 and β-sitosterol to the initial concentration at 3, 5, 7, 9, 11 d of culture, and obtain NK cell seed liquid after culture. At the same time, the experiment without adding β-sitosterol is used as the control group.
[0029] Sample at 7, 9, 11, 14 d of culture respectively to determine the number of cells, and calculate the proliferation fold of NK cells.
[0030] NK cell proliferation fold = total number of NK cells on the nth day / total number of NK cells on the 0th day.
[0031] The results are shown in Figure 1 It can be seen that with the extension of culture time, the cell proliferation fold gradually increases, and the NK cell proliferation fold of the experimental group reaches 841 times, while that of the control group is only 425 times. It shows that the addition of β-sitosterol significantly improves the proliferation ability of NK cells.
[0032] Take the NK cell seed liquid at 14 d of culture for analysis of cell phenotype and detection of cytotoxic activity.
[0033] Cell phenotype detection: mature NK cell molecules can express CD16+CD56+ molecules on the surface at the same time, so as to judge the maturity of NK cells. Take the cells at 0 d and 14 d of culture respectively, and detect their phenotypes by flow cytometry. Use FACS Canto II flow cytometer to obtain cells, read 1×10 4 cells per sample, and use FACSDiva software package to analyze data.
[0034] The results show that Figure 2 The proportion of CD16+CD56+ double positive cells in the experimental group is 95.60%, and that in the control group is 35.89%. It shows that the addition of β-sitosterol promotes the maturity of NK cells, and the purity is higher.
[0035] Cytotoxic activity detection: collect NK cells at 14 d of culture as effector cells. Collect K562 cells in logarithmic growth phase as target cells, and perform cytotoxic activity test according to the effector-target ratio of 5:1, 10:1 and 20:1. In addition, culture effector cell group and target cell group.
[0036] Cytotoxic activity = [1-(experimental group OD value-effector cell OD value) / target cell OD value] × 100%].
[0037] The results are shown in Figure 3As shown, the experimental groups all have higher cell killing rates than the control group at different effector target ratios. When the effector target ratio continues to be 20:1, the cell killing rate of the experimental group is 85.65%, while that of the control group is only 60.24%. This shows that the addition of β-sitosterol has the effect of improving the killing activity of NK cells.
[0038] (3) Large-scale culture of NK cells
[0039] The NK cell seed solution of step (2) was inoculated into a 2L bioreactor at a 10(v / v)% inoculation amount, and the culture medium was a serum-free medium containing IL-2, IL-15 and β-sitosterol KBM 581, the concentration of IL-2 was 1000 IU / mL, the concentration of IL-15 was 100 IU / mL, and the concentration of β-sitosterol was 1.0 mg / L. Cultured at 37°C, 50 rpm, 5% CO2 for 14 days, NK cells were obtained.
[0040] The number of NK cell proliferation was calculated to reach 100 billion.
[0041] As can be seen, after large-scale culture of NK cells according to the present embodiment, the number of NK cells reached the level of hundreds of billions, solving the problem of small amplification factor of traditional NK cell culture methods. Moreover, the cells have high purity and also have a high killing rate on tumor cells.
[0042] Comparative Example 1
[0043] This comparative example is the previous experimental process of the present technology. In the process of culturing NK cell seed solution, we tried to couple shear force to culture NK cells, hoping to improve the amplification factor, purity and activity of NK cells. However, the experimental results showed that under different shear forces, the amplification factor of NK cells decreased compared with the experimental group without coupling shear force. See Figure 4 . This shows that it is not suitable to couple shear force during the culture of NK cells. The action of shear force may cause additional damage to NK cells, thus leading to a decrease in the proliferation factor of NK cells.
[0044] Subsequently, we tried to couple shear force in a large-scale bioreactor and found that appropriate shear force (40-60 rpm) could promote the proliferation of NK cells (see Figure 5 ), so we decided to use the technical means of increasing shear force culture in the bioreactor.
[0045] In summary, the large-scale culture method of NK cells coupled with shear force and β-sitosterol according to the present application can make the number of NK cells reach the level of hundreds of billions, the proportion of CD16+CD56+ double positive cells is 95.60%, and the killing rate on tumor cells reaches 85.65%.
[0046] The above merely describes the preferred embodiments of the present application, and it should be pointed out that, for those skilled in the art, several improvements and refinements can be made without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.
Claims
1. A method for large-scale culture of NK cells coupled with shear force and β-sitosterol, characterized in that, Includes the following steps: (1) Isolate NK cells from peripheral blood; (2) The NK cells were seeded into serum-free culture medium containing β-sitosterol and cultured to obtain NK cell seed solution; (3) The seed culture of the NK cells is inoculated into a bioreactor for large-scale culture. The large-scale culture stage is coupled with shear force culture. The NK cells are collected after the culture is completed.
2. The method for large-scale culture of NK cells with shear force coupled to β-sitosterol as described in claim 1, characterized in that, The serum-free culture medium used is X-VIVO15 or KBM 581.
3. The method for large-scale culture of NK cells with shear force coupled to β-sitosterol as described in claim 2, characterized in that, The serum-free culture medium also contains IL-2 and IL-15, with the concentration of IL-2 being 800–1200 IU / mL and the concentration of IL-15 being 80–120 IU / mL.
4. The method for large-scale culture of NK cells with shear force coupled to β-sitosterol as described in claim 3, characterized in that, The amount of β-sitosterol added is 0.8–1.2 mg / L.
5. The method for large-scale culture of NK cells with shear force coupled to β-sitosterol as described in claim 1, characterized in that, The culture medium in the bioreactor is a serum-free culture medium containing IL-2, IL-15 and β-sitosterol; the concentration of IL-2 is 800-1200 IU / mL, the concentration of IL-15 is 80-120 IU / mL, and the amount of β-sitosterol added is 0.8-1.2 mg / L.
6. The method for large-scale culture of NK cells with shear force coupling β-sitosterol as described in claim 5, characterized in that, The shear force used in the shearing culture is 40–60 rpm.
7. The method for large-scale culture of NK cells with shear force coupled to β-sitosterol as described in claim 6, characterized in that, The culture period ended in 14 days.