Immune cell culture medium and application thereof

Abstract

The invention discloses an immune cell culture medium which is formed by adding an immune activator, a signal channel regulator, a metabolism regulator, a glycolysis inhibitor, an insulin deglutide liraglutide injection and transferrin on a basic culture medium, the immune activator is selected from a TLR7 / 8 agonist and an STING agonist; the signal channel regulator is selected from a DNA methyltransferase inhibitor and a histone deacetylase inhibitor; the metabolism regulator is selected from fatty acid oxidation accelerators. The immune cell culture medium is applied to culture of NK cells. According to the immune cell culture medium, the activation-metabolism-appearance three-in-one synergistic regulation effect of immune cells is achieved through multi-target-point small molecule combination, the cell proliferation number can be increased by 50%, and the killing capacity can be improved by 30%. Meanwhile, the dependence on cell factors is reduced, and the vaccine is more suitable for clinical treatment and vaccine production.

Description

Technical Field

[0001] The present invention relates to an immune cell culture medium and its application, belonging to the technical field of cell culture. Background Art

[0002] With the rapid development of biomedical technology, immune cell culture media play an important role in fields such as cell therapy, vaccine research and development, and antibody drug production. The development of immune cell culture media not only needs to meet the basic requirements of cell growth and proliferation, but also needs to consider the maintenance and optimization of cell functions. However, in the actual development process, issues such as the use of animal-derived serum and the selection of cell nutrient components have always been the key points and difficulties in research.

[0003] Animal-derived serum (such as fetal bovine serum FBS) is an indispensable component in traditional immune cell culture media. Its main role is to provide nutrients, hormones, growth factors, and a variety of unknown trace components required for cell growth. However, the use of animal-derived serum also has many problems, which have a profound impact on the development of immune cell culture media.

[0004] In order to overcome the limitations of animal-derived serum, the development of serum-free or low-serum immune cell culture media has become a research hotspot. In this context, the selection of cell nutrient components has become the core issue in the development of culture media. Existing serum-free culture media mostly rely on recombinant proteins (such as cytokines, growth factors), which are costly and may introduce immunogenic components. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides an immune cell culture medium and its application.

[0006] The present invention is achieved by the following technical solutions: An immune cell culture medium is prepared by adding an immune activator, a signal pathway regulator, a metabolic regulator, a glycolysis inhibitor, degludec and liraglutide injection, and transferrin to a basal medium. Among them, the immune activator is selected from TLR7 / 8 agonists or / and STING agonists; the signal pathway regulator is selected from DNA methyltransferase inhibitors or / and histone deacetylase inhibitors; the metabolic regulator is selected from fatty acid oxidation promoters.

[0007] Furthermore, the immune activator is selected from Resiquimod (Chinese name: Ruiquimod), and the concentration is 0.1 - 10 μM.

[0008] Further, the signal pathway regulator is selected from Tofacitinib (Chinese name: tofacitinib) or / and Y-27632 (Chinese name: trans-4-[(R)-1-aminoethyl]-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride), the concentration of Tofacitinib is 10-100 nM, and the concentration of Y-27632 is 5-20 μM.

[0009] Further, the metabolic regulator is selected from Dynasore (Chinese name: 3-hydroxy-2-naphthoic acid [(3,4-dihydroxyphenyl)methylene] hydrazide), and the concentration is 10-100 nM.

[0010] Further, the glycolysis inhibitor is selected from 2-DG (Chinese name: 2-deoxy-D-glucose), and the concentration is 1-5 mM.

[0011] Further, the concentration of the degludec and liraglutide injection is 300-600 IU / L.

[0012] Further, the concentration of the transferrin is 50-200 μg / mL.

[0013] Further, the basal medium is selected from commercialized media such as IMDM medium, DMEM / F12 medium, RPMI 1640 medium, etc.

[0014] Preferably, the immune cell medium is composed of the following components: Resiquimod, 1 μM; Tofacitinib, 50 nM; Y-27632, 5 μM; Dynasore, 10 nM; 2-DG, 2 mM; degludec and liraglutide injection (trademark NovoMix), 300 IU / L; transferrin, 100 μg / mL; and the balance is IMDM medium.

[0015] The application of the immune cell medium in culturing NK cells, and its application in being or preparing the culture solution of NK cells.

[0016] A method for culturing NK cells, comprising the following steps: (I) Coating: Pre-coat the bottom of the culture container with Erb-B2 as the coating solution; (II) Culturing: Resuspend peripheral blood mononuclear cells with activation medium A, place them in the above-mentioned pre-coated culture container, and transfer them to an incubator for culturing; on the 3rd to 5th day of culturing, add activation medium B 1-2 times, add amplification medium C on the 7th to 8th day of culturing, and then add amplification medium C once every 2-3 days; culture for a total of 14-21 days; The activation medium A is composed of the above-mentioned immune cell medium, 4-1BBL, IL-15, and IL-18. The concentrations of 4-1BBL, IL-15, and IL-18 are all 80-120 ng / ml, preferably 100 ng / ml. The activation medium B is composed of the immune cell medium and IL-15. The concentration of IL-15 is 80-120 ng / ml, preferably 100 ng / ml. The amplification medium C is composed of the immune cell medium and IL-2. The concentration of IL-2 is 450-550 u / ml, preferably 500 u / ml.

[0017] The immune cell medium of the present invention uses small molecule compounds to replace serum, avoiding the influence of animal-derived components on cell therapy, ensuring the safety of the product, and significantly improving the cell survival rate, amplification multiple, and clone formation rate. Chemical small molecules play a key role in immune cell culture. Their functions are not limited to simple cell amplification, but also involve multiple aspects such as immune cell function regulation, fate determination, and engineering transformation. Chemical small molecules have developed from traditional auxiliary tools to "molecular switches" for immune cell fate programming. Their precision, reversibility, and low cost provide key support for the standardized production of cell therapy products. With the integration of new technologies such as PROTAC and optogenetic tools, it is expected to achieve "customized on demand" immune cell therapy in the future, breaking through major clinical problems such as solid tumor treatment and autoimmune disease reversal.

[0018] The immune cell medium of the present invention is added with small molecule compounds, such as Resiquimod, Tofacitinib, Y-27632, Dynasore. These small molecules play the following roles: Resiquimod binds to TLR7 (mainly expressed in plasmacytoid dendritic cells) and TLR8 (mainly expressed in monocytes and macrophages), triggers the MyD88-dependent signaling pathway, activates the NF-κB and MAPK pathways, and induces the secretion of pro-inflammatory cytokines (such as IFN-α, IL-12, TNF-α) and chemokines. After TLR7 / 8 activation, the secretion of type I interferon (IFN-α / β) is induced, promoting natural killer (NK) cell activation and T cell anti-tumor responses.

[0019] The core role of Tofacitinib in immune cell culture is to regulate the activation of immune cells such as T cells, B cells, and NK cells and the release of pro-inflammatory factors by inhibiting the JAK / STAT pathway. It is widely used in research fields such as RA, HIV, and transplantation immunity, and the efficacy can be enhanced through dose optimization and combination drug use.

[0020] Y-27632 is a Rho-associated kinase (ROCK) inhibitor that reduces apoptosis induced by cellular stress by blocking the ROCK signaling pathway and can enhance the survival rate of immune cells in in vitro culture. In the culture of stem cells and organoids, Y-27632 promotes cell proliferation by regulating the cell cycle process (such as the G1-S phase), and this mechanism has potential assistance for immune cells that need to be amplified (such as T cells and NK cells).

[0021] The positive effect of Dynasore in immune cell culture is mainly reflected in the controllable regulation of the endocytosis process to optimize immune signaling, antigen presentation, and anti-infection research. Dynasore inhibits the endocytosis of the T cell receptor (TCR) or B cell receptor (BCR), prolongs their surface residence time, and thus enhances the continuous activation of downstream signaling pathways. For example: in T cell activation experiments, Dynasore treatment can increase the intensity of TCR signals, promote IL-2 secretion and T cell proliferation. In B cell research, delaying BCR endocytosis may enhance the conduction of antigen recognition signals and promote B cell activation. Inhibiting the endocytosis of cytokine receptors such as IL-2 and IL-15 can prolong their binding time with ligands, enhance the activation of downstream signaling pathways, and promote the survival and function of effector T cells or NK cells.

[0022] The present invention realizes the trinity synergistic regulation effect of "activation-metabolism-epigenetics" of immune cells through a combination of multi-target small molecules, which can increase the number of proliferating cells by 50% and the killing ability by 30%. At the same time, it reduces the dependence on cytokines and lowers the production cost (estimated to save 30% of the reagent cost). The present invention can be applied to the process optimization of various cell therapy products such as CAR-T, TIL, and DC vaccines, and has great potential for clinical transformation. Through the design with clear chemical components, the present invention avoids the pollution risk brought by animal-derived serum, significantly improves the batch-to-batch consistency of products, is easy to produce on a large scale, is more suitable for clinical treatment and vaccine production, and is suitable for industrial application.

[0023] All terms and phrases used in the present invention have the general meanings well-known to those skilled in the art. Description of the Drawings

[0024] Figure 1 : Schematic diagram of the comparison of the number of cells in the experimental group and the control group.

[0025] Figure 2 : Flow cytometry result analysis of the experimental group.

[0026] Figure 3 : Flow cytometry result analysis of the control group.

[0027] Figure 4 : Results of the cell killing experiment in the experimental group.

[0028] Figure 5 : Cell killing experiment results of the control group. Detailed implementation methods

[0029] The present invention will be further described below in conjunction with embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art can understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0030] For the instruments, reagents, materials, etc. involved in the following embodiments, unless otherwise specified, they are all conventional instruments, reagents, materials, etc. existing in the prior art and can be obtained through regular commercial channels. For the experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, they are all conventional experimental methods, detection methods, etc. existing in the prior art.

[0031] Example 1 Immune cell culture medium It consists of the following components: Resiquimod, 1 μM; Tofacitinib, 50 nM; Y-27632, 5 μM; Dynasore, 10 nM; 2-DG, 2 mM; Degludec and liraglutide injection (trademark NovoMix), 300 IU / L; Transferrin, 100 μg / mL; The balance is IMDM medium. When preparing, mix each component evenly to obtain it.

[0032] Experiment 1 Cultivation of NK cells Use the immune cell culture medium prepared in Example 1 to culture cells. The method is as follows (refer to another patent of the applicant of the present invention, CN 111944754 A): (I) Extraction of peripheral blood PBMC 1. Collect 150 - 200 ml of peripheral blood with a 200 ml blood collection bag (containing total citric acid 1.916% - 2.118%, g / ml), aliquot it into 50 ml centrifuge tubes, 10 ml per tube, and add an equal volume of PBS for dilution.

[0033] 2. Take another 50 ml centrifuge tube and add 20 ml of lymphocyte separation solution. Place it obliquely and slowly add the above-diluted blood along the side wall.

[0034] 3. Gently stand the centrifuge tube upright and centrifuge at 975 g for 20 minutes at room temperature. The acceleration of the rising and falling speed is 1.

[0035] 4. After centrifugation, the separation solution is divided into four layers. From top to bottom, they are: plasma layer, mononuclear cell layer, separation solution layer, polymorphonuclear leukocyte and red blood cell layer; Carefully suck the middle white film layer, that is, the mononuclear cell layer, with a Pasteur pipette and transfer it to a new 50 ml centrifuge tube.

[0036] 5. Collect 10 - 20 ml of PBMC suspension per tube, add PBS to make up to 40 ml, centrifuge at 300 g for 10 minutes at room temperature, and repeat 2 - 3 times.

[0037] (II) NK Cell Activation 1. Coating the culture flask with antibodies (1) Take several T-75 cm 2 culture flasks, add 5 ml of Erb-B2 (tyrosine kinase receptor 2) coating solution; (2) Gently shake to spread the solution over the bottom of the culture flask to cover the bottom; (3) Store at 4°C and take out before use; (4) Remove the coating solution, wash the bottom of the flask once with 10 ml of PBS, and use the washed culture flask immediately.

[0038] 2. Prepare the activation medium Experimental group: The basal culture medium for the experimental group is the medium prepared in Example 1.

[0039] Activation medium A: Add 4-1BBL, IL-15, and IL-18 to the basal culture medium, and the concentrations of 4-1BBL, IL-15, and IL-18 after addition are all 100 ng / ml.

[0040] Activation medium B: Add IL-15 to the above basal culture medium, and the concentration of IL-15 after addition is 100 ng / ml.

[0041] Expansion medium C: Add IL-2 to the above basal culture medium, and the concentration of IL-2 after addition is 500 u / ml.

[0042] Control group: The basal culture medium for the control group consists of IMDM medium and human serum albumin. Among them, there is 1 L of IMDM medium and 20 ml of human serum albumin. The preparation of activation medium A, activation medium B, and expansion medium C is the same as above, except that: replace the basal culture medium of the experimental group with the basal culture medium of the control group.

[0043] 3. Inoculate PBMC cells: Take the PBMC cells from step (I), centrifuge at 975 g for 10 minutes, resuspend the cells with activation medium A, and adjust the cell density to 0.6×10 6 - 1.2×10 6 cells / mL, / T75. Add 20 ml of activation medium A to each coated T75 culture flask.

[0044] 4. First fluid replenishment: On the third day of culture, count the cell density, take samples for flow cytometry detection, add activated medium B, and adjust the cell density to 1.0×10 6 ~1.2×10 6 cells / mL.

[0045] 5. Second fluid replenishment: On the fifth day of culture, count the cell density, take samples for flow cytometry detection, add activated culture medium B, and adjust the cell density to 1.0×10 6 ~1.2×10 6 cells / mL.

[0046] 6. When the total volume of the culture medium is close to the maximum value of the container, transfer the whole cells to a larger container.

[0047] 7. Replace the amplification culture medium: On the seventh day of culture, count the cell density, take samples for flow cytometry detection, add amplification medium C, and adjust the cell density to 1.5×10 6 cells / mL, and then replenish the fluid (amplification medium C) every two days for continuous culture for 14 - 21 days.

[0048] (III) Identification of NK cells 1. Transfer 1 ml of the above - cultured cell suspension to a 1.5 ml centrifuge tube, centrifuge at 4°C and 300 g for 5 minutes, and carefully aspirate the supernatant.

[0049] 2. Wash the cells with an appropriate amount of PBS, centrifuge at 4°C and 300 g for 5 minutes, and carefully aspirate the supernatant.

[0050] 3. Resuspend the cells with pre - cooled PBS, and adjust the final cell concentration to 1×10 7 cells / ml, and gently pipette to mix evenly.

[0051] 4. Take 100 μl of the cell suspension as the blank control group, take 100 μl of the cell suspension as the parallel control group and add ISOtype FITC, PerCP, PE - Cy7 and PE antibodies, take 200 μl as the experimental group and add CD45 - PerCP, CD3 - FITC, CD16 - PE - Cy7 and CD56 - PE antibodies, and incubate at 4°C for 30 - 40 minutes.

[0052] 5. Add an appropriate amount of PBS to wash the cells, centrifuge at 4°C and 300 g for 5 minutes, and carefully aspirate the supernatant.

[0053] 6. Resuspend the cells with 500 μl of PBS and perform on - machine detection.

[0054] (IV) Flow cytometry collection 1. Turn on the SA3800 full - spectrum flow cytometer in advance for pre - heating and equipment self - inspection.

[0055] 2. New experiment: Select the "Preparation" wizard tab, enter the experiment preparation interface, click the "Experiment Template" button, select "Blank Template", enter the naming information in the Name text box, and click "Create Experiment" to create a new experiment template.

[0056] 3. Place the Sample-S sample tube, click "Preview", set the value of "Fluorescence PMT Voltage" so that the highest value of Intensity_H is around 10 5 and click "Stop" to unload the Sample-S sample tube.

[0057] 4. Place the Unstained sample tube, click "Preview", set other parameters, and do not use the FSC to set the threshold when performing absolute counting! 5. Collect cells from each group: Place the sample tube to be tested, click "Preview", and click "Acquire" to collect when the Flow condition status becomes Stable. After the collection starts, the same parameters must be used for the same group of experiments. If there are any changes, all collections must be redone.

[0058] (V) Flow cytometry result analysis 1. Add the used markers and their corresponding fluorescence signals.

[0059] 2. Import the corresponding fluorescence curves in the database. For new fluorescence signals, a positive control tube should be established separately to create the curve.

[0060] 3. Select the Unstained group, circle the main cell population, and set it as Gate A. Set FSC-H / FSC-A to display the cells in Gate A, and circle the single-cell population B.

[0061] 4. Set side scatter SSC-H / CD45 to display the cells in Gate B, and set Gate C: including all CD45 non-negative cells.

[0062] 5. Set FSC-H / FSC-A to display the cells in Gate C, and set Gate D: including all single cells.

[0063] 6. Set SSC-H / CD3 to display the cells in Gate D, and set R3: including all CD3 non-negative cells.

[0064] 7. Set SSC-H / CD56 to display the cells in Gate D, and set R56: including all CD56 non-negative cells.

[0065] 8. Set CD3 / CD56 to display the cells within the D gate. Set the cross-quadrant gate W: Determine the X-axis and Y-axis positions with reference to Steps 5 and 7. The first quadrant contains CD3−, CD56+ cells.

[0066] 9. Save the data, clean, and turn off the instrument.

[0067] (VI) Cell Killing Experiment 1. Collect tumor cells, count them, resuspend, and adjust the cell density to 1×10 6 ~5×10 6 cells / tube.

[0068] 2. Resuspend the cells in 500 μl of buffer, add 1 μl of CFSE stock solution, and mix well.

[0069] 3. Incubate at 37 °C for 30 min.

[0070] 4. Add 5 volumes of pre-warmed culture medium or PBS containing 10% FBS to wash the cells and remove the staining solution.

[0071] 5. Use the basal medium (IMDM medium) to adjust the cell density to 2×10 6 cells / ml, 1 ml per well.

[0072] 6. Count the number of immune cells, centrifuge, and use the basal medium to adjust the cell density to 2×10 7 cells / ml, 1 ml per well.

[0073] 7. Co-culture the tumor cells and immune cells for 24 hours, and then perform flow cytometry analysis.

[0074] (VII) Experimental Results Comparison of cell expansion numbers: The schematic diagram of the cell number comparison between the experimental group and the control group is as Figure 1 shown (in the figure, "the culture medium of the present invention" is the experimental group). It can be Figure 1 seen that after 10 days of culture, the number of cells cultured with the culture medium of the present invention is significantly better than that of the control group medium, and the cell proliferation number has increased by at least 50%.

[0075] 2. Comparison of cell purity: The flow cytometry results analysis of the experimental group is as Figure 2 shown, and the flow cytometry results analysis of the control group is as Figure 3 shown. In this example, the target cells to be cultured are NK cells, which are characterized by CD3−CD56+, that is, the proportion of the second quadrant in the result graph. The proportion of NK cells cultured with the control medium is only 0.94%, while the proportion of NK cells obtained with the culture medium of the present invention is 57.21%, and the purity is significantly better than that of the control group.

[0076] 3. Comparison of cell killing ability: The results of the cell killing experiment in the experimental group are as shown in Figure 4 , and the results of the cell killing experiment in the control group are as shown in Figure 5 . In this embodiment, the target cell is the human chronic myeloid leukemia cell line - K562 cell, and the cell viability dye is 7-AAD. When the K562 cell membrane is intact, 7-AAD cannot enter and shows negative, that is, the left region of the set gate. When NK cells bind to K562 cells and kill them, the membrane permeability of K562 cells increases, 7-AAD enters and binds to DNA, generating a strong fluorescence signal, showing positive, that is, the right region of the set gate. The value in this region represents the killing ability of the immune cells used. As shown in the figure, the killing ability of the cells cultured with the medium of the present invention reaches 81.83%, which is significantly better than 59.55% of the control group medium, and the killing ability is increased by 37.41%.

[0077] The above embodiments are provided to those skilled in the art to fully disclose and describe how to implement and use the claimed embodiments, rather than to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. An immune cell culture medium, characterized in that: The method is prepared by adding an immune activator, a signal pathway regulator, a metabolic regulator, a glycolysis inhibitor, degludec insulin liraglutide injection and transferrin to a basal culture medium, wherein the immune activator is selected from a TLR7 / 8 agonist and / or a STING agonist; the signal pathway regulator is selected from a DNA methyltransferase inhibitor and / or a histone deacetylase inhibitor; and the metabolic regulator is selected from a fatty acid oxidation promoter.

2. The immune cell culture medium according to claim 1, characterized in that: The immune activator is selected from Resiquimod, and the concentration is 0.1 to 10 μM.

3. The immune cell culture medium according to claim 1, characterized in that: The signal pathway regulator is selected from Tofacitinib and / or Y-27632, the concentration of Tofacitinib is 10 to 100 nM, and the concentration of Y-27632 is 5 to 20 μM.

4. The immune cell culture medium according to claim 1, characterized in that: The metabolic regulator is selected from Dynasore, and the concentration is 10-100 nM.

5. The immune cell culture medium according to claim 1, characterized in that: The glycolysis inhibitor is selected from 2-DG, and the concentration is 1 to 5 mM.

6. The immune cell culture medium according to claim 1, characterized in that: The concentration of the insulin degludec liraglutide injection is 300 to 600 IU / L; Or: the concentration of transferrin is 50-200 μg / mL.

7. The immune cell culture medium according to claim 1, characterized in that: The basal culture medium is selected from IMDM culture medium, DMEM / F12 culture medium or RPMI 1640 culture medium.

8. The immune cell culture medium according to any one of claims 1 to 7, characterized in that The immune cell culture medium is composed of the following components: Resiquimod, 1 μM; Tofacitinib, 50 nM; Y-27632, 5 μM; Dynasore, 10 nM; 2-DG, 2 mM; degludec insulin liraglutide injection, 300 IU / L; transferrin, 100 μg / mL; the remainder is IMDM culture medium.

9. Use of the immune cell culture medium according to any one of claims 1 to 8 in culturing NK cells, or in preparing or preparing a culture medium for NK cells.

10. A method for culturing NK cells, characterized in that: The following steps are involved: (i) Coating: Use Erb-B2 as coating solution to pre-coat the bottom of the culture container; (ii) Cultivation: resuspend the peripheral blood mononuclear cells with activation medium A, place in the above-mentioned pre-coated culture container, and transfer to an incubator for cultivation; on the 3rd to 5th day of cultivation, add activation medium B 1 to 2 times, on the 7th to 8th day of cultivation, add expansion medium C, and then add expansion medium C every 2 to 3 days; culture for a total of 14 to 21 days; The activation medium A is composed of the immune cell culture medium of Example 1, 4-1BBL, IL-15 and IL-18, and the concentrations of 4-1BBL, IL-15 and IL-18 are all 80 to 120 ng / ml; The activation medium B is composed of the immune cell culture medium of Example 1 and IL-15, and the concentration of IL-15 is 80-120 ng / ml; The expansion culture medium C is composed of the immune cell culture medium of Example 1 and IL-2, and the concentration of IL-2 is 450-550u / ml.

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