Mouse NK cell in-vitro culture method and application
By optimizing the culture method of mouse NK cells and using MEMα medium and multi-factor combination activation medium, the problems of poor species compatibility and low amplification efficiency were solved, achieving efficient amplification and viral transduction, thus meeting experimental requirements.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGZHOU DOUBLLE BIOPRODUCT CO LTD
- Filing Date
- 2025-12-26
- Publication Date
- 2026-05-01
AI Technical Summary
Existing technologies cannot effectively support the in vitro survival and expansion of mouse NK cells. They suffer from poor species compatibility, low expansion efficiency, and insufficient regulation of activation and expansion stages, resulting in high apoptosis rates and insufficient expansion folds, which cannot meet experimental requirements.
A two-step culture method using MEMα medium, FBS, and different concentrations of mIL-2 and other cytokines was employed, including activation culture and expansion culture stages. The culture medium type and cytokine ratio were optimized, and penicillin-streptomycin was used to improve cell activation efficiency and expansion fold.
This study achieved efficient expansion of mouse NK cells, with an expansion factor of approximately 25-fold, improving viral transduction efficiency and optimizing cell transduction methods, thus providing a sufficient and highly active cell source.
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Figure CN121950696A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of cell culture technology, specifically relating to a method and application for in vitro culture of mouse NK cells. Background Technology
[0002] Natural killer cells (NK cells) are key effector cells of the innate immune system, playing a central role in tumor immunity, viral clearance, and immune regulation. Among them, mouse NK cells (mNK cells) are important tool cells for studying tumor models and immune mechanisms. Highly efficient in vitro expansion technology for mNK cells is a core prerequisite for promoting related basic research and translational applications—for example, in scenarios such as mouse tumor transplantation models and CAR-NK cell functional validation, a large number of highly active mNK cells are required to meet experimental needs.
[0003] However, current in vitro culture technology for mNK cells faces significant bottlenecks: On the one hand, existing NK cell culture systems are mostly designed for human NK cells, and their direct application to mNK cells results in poor species compatibility. For example, the commonly used RPMI-1640 culture medium system or activation protocols relying on human interleukin (hIL-2) cannot effectively support the survival and proliferation of mNK cells, often resulting in apoptosis rates exceeding 50% and no effective proliferation. On the other hand, even if some culture systems can maintain the short-term survival of mNK cells, they suffer from extremely low expansion efficiency—the expansion fold of mNK cells cultured in traditional serum-containing systems for 8 days is only about 2-fold, which is far from meeting the high-order cell requirements of experiments such as mouse intravenous infusion and in vitro transduction.
[0004] In addition, existing mNK cell culture protocols lack phased regulation of activation and expansion. Most protocols use a single culture medium for the entire process, resulting in low cell activation efficiency in the early stage and cell proliferation being inhibited in the later stage due to nutrient competition and accumulation of metabolic waste, which further limits the expansion rate.
[0005] To address the aforementioned issues, developing a serum-containing in vitro culture method that adapts to the characteristics of mNK cell species and balances activation efficiency and expansion fold is key to overcoming the current technological bottlenecks in mNK cell research. By optimizing the type of basal culture medium, serum ratio, cytokine combination, and staged culture process, while retaining the supporting role of serum for mNK cell growth, this method solves the problems of "low expansion efficiency, poor applicability, and insufficient stability" in traditional serum-containing systems, providing a sufficient and highly active cell source for mouse NK cell-related research. Summary of the Invention
[0006] To address the aforementioned problems, this invention provides a method and application for in vitro culture of mouse NK cells.
[0007] On one hand, the present invention provides a method for in vitro culture of mNK cells, comprising the following steps: S1. Activation culture: mNK cells are seeded in an activation single-factor activation medium and cultured for 1.5-2.5 days. The activation medium includes MEMα medium, FBS, β-ME and mIL-2. S2. Amplification culture: The activated mNK cells are transferred to the amplification culture medium and cultured for 16.5-17.5 days. The amplification culture medium includes MEMα medium, FBS, β-ME and mIL-2.
[0008] Specifically, in S1, the activation medium is divided into two types: single-factor activation and multi-factor combination activation; More specifically, the activation medium for the single-factor activation includes MEMα medium, 15-25 v / v% FBS, 0.03-0.08 mM β-ME and 250-1500 ng / mL mIL-2; More specifically, the activation medium for the multi-factor combination activation includes MEMα medium, 15-25 v / v% FBS, 0.03-0.08 mM β-ME, 100-1000 ng / mL mIL-2, mIL-15 and mIL-12; Preferably, the dosage of mIL-15 is 50-500 ng / mL, and the dosage of mIL-12 is 5-100 ng / mL.
[0009] Specifically, the activation medium and amplification medium also include penicillin-streptomycin.
[0010] Specifically, the concentration of mIL-2 in the amplification medium is lower than the concentration of mIL-2 in the activation medium.
[0011] Specifically, the concentration of mIL-2 in the amplification medium is 50 ng / mL.
[0012] Specifically, during the culture process, the cell density does not exceed 2E6 cells / mL.
[0013] On the other hand, the present invention provides mNK cells cultured by any of the above-mentioned in vitro culture methods.
[0014] Specifically, the mNK cells were cultured for 18-19 days and then expanded approximately 25-fold.
[0015] Specifically, when a multi-factor combination activation culture method is used, the viral transduction efficiency of the mNK cells is increased by at least 68% compared to mNK cells cultured in an activation medium containing only mIL-2.
[0016] Furthermore, this invention provides the application of the aforementioned mNK cells in in vitro transduction.
[0017] Specifically, the in vitro transduction uses VSVG enveloped virus as the transduction vector.
[0018] In another aspect, the present invention provides an in vitro transduction method for mNK cells, wherein the in vitro transduction method uses the above-mentioned mNK cells and transduces the amplified mNK cells using VSVG enveloped virus.
[0019] Specifically, the VSVG enveloped virus is a VSVG enveloped lentivirus or a VSVG enveloped retrovirus.
[0020] Specifically, the amino acid sequence of the VSVG envelope protein is shown in SEQ ID NO.1.
[0021] Specifically, when using VSVG enveloped retrovirus transduction, the transduction positivity rate of the mNK cells was increased by at least 172% compared to VSVG enveloped lentivirus transduction.
[0022] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this subject matter pertains. Unless otherwise stated, all patents, patent inventions, and disclosures cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definitions provided in this chapter shall prevail.
[0023] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting. Attached Figure Description
[0024] Figure 1 Flow cytometry data for mNK cell separation using negative selection method; Figure 2 When mNK cells were cultured in different culture media systems and with different species of cytokine IL-2, only the MEMα+mIL-2 culture medium system showed that mNK cells could proliferate in small amounts. Figure 3 The figures show the amplification curves and cell morphology diagrams of the two-step mNK method. In the figure, A is the amplification curve, and B and C are cell morphology diagrams at Day 10 of culture. Figure 4The diagram shows mNK cell transduction. In the diagram, A is a flow cytometry plot of lentivirus transduction of different mNK envelope proteins under different culture methods; B is a statistical graph of the positive rate of transduction corresponding to A; C is a flow cytometry plot of mNK cells cultured by mIL2 / 12 / 15 activation method transduced by VSVG enveloped lentivirus and retrovirus expressing CAR CD19; and D is a statistical graph of the mean fluorescence intensity (MFI) of CAR after transduction. Detailed Implementation
[0025] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.
[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.
[0027] Basic Example: Isolation of NK Cells from Mouse Spleen C57BL6 mouse NK cells were isolated using a negative selection method. The isolation kit used was the EasyIso™ Mouse NK Cell Isolation Kit (catalog number: SN4201L) from Eyenos LifeScience. The specific procedures are as follows: 1) Single-cell suspension isolation from mouse spleen: Prepare FACS buffer (1×PBS+0.02% EDTA+2% FBS) and pre-cool on ice. After euthanizing the mice by cervical dislocation, isolate the spleen and place it in FACS buffer. Use sterile scissors to cut the spleen tissue into small pieces as much as possible. Use a 5ml syringe plunger to further grind and homogenize the tissue. After passing through a 70um sterile nylon filter, obtain a single-cell suspension. Centrifuge at 300g, 4℃ for 10 minutes, wash once with FACS buffer, and finally resuspend in 1ml FACS buffer. Approximately 1E8 single-cell suspensions can be isolated from the spleen of one mouse. 2) Add EasyIso TM Add 100 μl of Mouse NK Cell Isolation Cocktail to the sample and mix thoroughly. Incubate at room temperature for 10 minutes. 3) Shake the Streptavidin Nanobeads for 30 seconds and add them to the sample from the previous step at 100 μl / ml. Mix thoroughly and incubate at room temperature for 5 minutes. 4) Add FACS buffer to the sample from the previous step to a total of 2.5 ml, and then transfer it to a 5 ml flow cytometry tube; Place the flow cytometry tube on a magnetic rack and let it stand at room temperature for 3 minutes. Then, quickly tilt the tube to pour the supernatant into a new sample collection tube to obtain isolated NK cells. Flow cytometry analysis is then performed as follows: Figure 1 As shown, the purity of mNK cells before and after separation was detected. Two markers were used for NK cells: NK1.1 and CD49b. The purity before separation was about 2.5%, and after separation it reached more than 90%. The purity of mNK cells after separation increased from 2.59% to 86.77%, and the content of mNK1.1 was 10.17%.
[0028] Example 1: In vitro expansion of mouse NK cells 1. Preliminary experiment on the effects of different culture media and IL-2 sources on mNK cell expansion. Experimental Groups: Group 1: MEMα medium system + 150 ng / ml mIL-2, wherein the MEMα medium system is MEMα medium + 20% FBS + 1×PS + 0.05mM β-ME; Group 2: MEMα culture medium + 150 ng / ml hIL-2; Group 3: 1640 medium system + 150 ng / ml mIL-2, wherein the 1640 medium system is 1640 medium + 10% FBS + 1×PS + 1.6mM β-ME; Group 4: 1640 culture medium system + 150 ng / ml hIL-2.
[0029] Culture method: The mNK cells isolated in Example 1 were cultured at a ratio of 1×10⁻⁶. 5 The cells were seeded at a density of 1 cell / ml into the above 4 groups of culture media and cultured in a 37℃, 5% CO2 incubator. The cell status was observed every 2 days. After 8 days of culture, the number of cells was counted and the expansion fold was calculated.
[0030] The experimental results are shown in Figure 2Only group 1 (MEMα culture medium system + 150 ng / ml mIL-2) could maintain mNK cells from apoptosis, but the expansion fold after 8 days of culture was only about 2-fold, which is far from meeting the needs of mouse NK cell intravenous reinfusion (usually requiring a higher order of cells); the apoptosis rate of mNK cells in group 2 (MEMα + hIL-2), group 3 (1640 + mIL-2), and group 4 (1640 + hIL-2) all exceeded 50%, and no effective proliferation was observed, indicating that hIL-2 and 1640 culture medium systems are not suitable for basic expansion of mNK cells in this scenario, and the culture protocol needs to be further optimized.
[0031] 2. Two-step in vitro expansion of mNK cells Experimental groups (different activation media, same amplification media): Activation group A (single factor activation): The activation medium was MEMα medium system + 500 (250-1500) ng / ml mIL-2 (MEMα medium system: MEMα medium + 20% FBS + 1×PS + 0.05mM β-ME). Activation group B (multi-factor combination activation): The activation medium is MEMα medium system + 500 (100-1000) ng / ml mIL-2 + 150 (50-500) ng / ml mIL-15 + 20 (5-100) ng / ml mIL-12; (MEMα medium system: MEMα medium + 20% FBS + 1×PS + 0.05mM β-ME); Amplification medium: MEMα medium system + 50 ng / ml mIL-2.
[0032] Cultivation methods: Activation culture phase: The mNK cells isolated in Example 1 were cultured at a rate of 1×10⁻⁶. 6 The cells were inoculated into the activation medium of activation groups A and B at a density of 1 cell / ml and cultured in an incubator at 37°C and 5% CO2 for 2 days. Amplification and Culture Phase: After 2 days of activation culture, collect two groups of cells, centrifuge at 300g for 5 minutes at room temperature, discard the supernatant, resuspend the cells in amplification medium, and adjust the cell density to 1×10⁻⁶. 6 Count / ml, continue to incubate in an incubator; Fluid resuscitation and passage: Observe cell density every 4 days. When the cell density exceeds 2×10⁻⁶, [the cell density is considered positive]. 6 When the cell count is 1 / ml, dilute the cells to 1×10⁻⁶ with amplification medium. 6 Cells / ml, maintain culture until day 19; Cell morphology observation: On day 7 of culture, the morphology of the two groups of cells was observed under an inverted microscope, and the cell morphology characteristics were recorded.
[0033] amplification curves and cell morphology, such as Figure 3 As shown, Figure 3 This study demonstrates two-step culture methods for expanding mNK cells using different cytokine systems. One method involves activation culture with only a high concentration of mIL-2 for two days, followed by expansion culture with a low concentration of mIL-2. The other method involves activation culture with a mixture of different cytokines for two days, followed by expansion culture with a low concentration of mIL-2. The proliferation curves of the two groups of mNK cells show "stage differences." Figure 3 Figure A shows the amplification curves, revealing differences in cell proliferation kinetics. The high-concentration IL-2 method resulted in slow proliferation in the early stages and rapid proliferation in the later stages, while the mixed-factor method resulted in rapid proliferation in the early stages and slow proliferation in the later stages. Ultimately, by Day 18, the amplification folds were comparable, approximately 25-fold. In the initial culture period (days 1-9), the activation group B (multi-factor activation) proliferated faster than the activation group A (single-factor activation), with the total number of cells in activation group B being approximately 1.5 times that of activation group A on day 9. These results indicate that multi-factor combined activation can accelerate the initial proliferation of mNK cells.
[0034] On day 7 of culture, the mNK cells in both activation group A and activation group B were typical spindle-shaped with clear cell boundaries and good refractive properties, showing no obvious morphological differences. This indicates that both activation systems can maintain the normal growth state of mNK cells without any abnormal cell differentiation or apoptosis.
[0035] Example 3 Packaging and titer determination of lentiviruses and retroviruses with different envelope proteins 3.1 Lentiviral Packaging with Different Envelope Proteins Experimental materials: Viral packaging cell lines: HEK293T (ATCC, CRL-3216); high-glucose DMEM medium (Gibco, 11965092); newborn calf serum (NCBS, Daxi, DX2001); opti-MEM (Gibco, 51985034); packaging plasmids: expression vector FUGW (addgene, #14883), lentiviral expression vector 164 (pCDH-CMV vector, addgene #72265, expressing CAR CD19, SEQ ID NO.2), and retroviral vector 217 (pBMNZ vector, addgene #1734, expressing CAR CD19 gene, SEQ ID NO.2), packaging plasmids pLp1 (addgene, #209988), pLp2 (addgene, #209989), envelope protein plasmids pVSVG (SEQ ID NO.1), pBaEVless (SEQ ID NO.3), pGaLV (SEQ ID NO.1). NO.4); Transfection reagent PEI (1ug / ml, Polysciences, 24765-1).
[0036] Table 1. DNA Sequences
[0037] The virus packaging method is as follows: 1) HEK293T was cultured in high-sugar DMEM + 10% NCBS. After reaching about 90% confluence, it was normally passaged every 2 days. 2) Day -2, lay 6-hole boards 48 hours in advance, 5E5 / hole; 3) Day 0: Observe the cell state under a microscope. At this time, the cell confluence is 80%-90%, and plasmid transfection can be performed. Prepare transfection mixture 1: 100ul optiMEM + plasmid (FUGW / 164:pLp1:pLp2:pVSVG / pBaEVless / pGaLV=2.4:1.2:0.6:0.6(ug)), and transfection mixture 2: 100ul optiMEM + 16.5ug PEI (PEI:total DNA=3.5:1). Mix well and let stand at room temperature for 5 min. Then mix mixture 1 and 2 and let stand at room temperature for 15 min. After that, add the mixture evenly to a 6-well plate, gently shake to mix, and then transfer the plate to a 37℃ incubator. 4) After transfection for 6-8 hours, remove the supernatant, gently add 2 ml of serum-free DMEM high glucose medium along the wall, and then transfer the plate to a 37°C incubator. 5) After transfection for 48 hours, collect the supernatant, centrifuge at 2000g for 10 min, and carefully transfer the supernatant to a new EP tube for titer determination; 3.2 Retroviral Packaging with Different Envelope Proteins Experimental materials: Virus packaging cell line: HEK293T (ATCC, CRL-3216); high-glucose DMEM medium (Gibco, 11965092); newborn calf serum (NCBS, Daxi, DX-2001); opti-MEM (Gibco, 51985034); packaging plasmid: 217, gag / pol; envelope protein plasmid pVSVG; packaging method as follows: 1) HEK293T was cultured in high-sugar DMEM + 10% NCBS. After reaching about 90% confluence, it was normally passaged every 2 days. 2) Day -2, lay 6-hole boards 48 hours in advance, 5E5 / hole; 3) Day 0: Observe the cell state under a microscope. At this time, the cell confluence is 80%-90%, and plasmid transfection can be performed. Prepare transfection mixture 1: 100ul optiMEM + plasmid (217:gag / pol:pVSVG=2.4:1.2:0.6(ug)), and transfection mixture 2: 100ul optiMEM + 14.7ug PEI (PEI:total DNA=3.5:1). Mix well and let stand at room temperature for 5 min. Then mix mixture 1 and 2 and let stand at room temperature for 15 min. After that, add the mixture evenly to a 6-well plate, gently shake to mix, and then transfer the plate to a 37°C incubator. 4) After transfection for 6-8 hours, remove the supernatant, gently add 2 ml of serum-free DMEM high glucose medium along the wall, and then transfer the plate to a 37°C incubator. 5) After transfection for 48 hours, collect the supernatant, centrifuge at 2000g for 10 min, and carefully transfer the supernatant to a new EP tube for titer determination; 3.3 Determination of Lentiviral and Retroviral Titers Experimental materials: polybrene (Beyotime, C0351-1ml); FMC63 antibody (Heyousheng, FMC-ARAP100) 1) HEK293T was grown to approximately 90% confluence for subculturing and plating, using 2.5E5 / ml / 12-well plate. 2) Add 10 μL of viral supernatant and 8 μg / ml of polybrene, mix thoroughly, and transfer the plate to a 37°C incubator. 3) Flow cytometry was performed after 48 hours. For the FUGW expression vector, the FITC positivity rate N was directly detected. For CARCD19, the positivity rate N was detected using the FMC63 antibody. The titer formula is as follows: 2.5E5×N / 10×1000 (TU / ml).
[0038] Example 4: Mouse NK cell transduction experiment 4.1 Effects of different envelope protein lentiviruses on mNK cell transduction efficiency Experimental materials: mNK cells from activation groups A (mIL-2 activation) and B (mIL-2+mIL-15+mIL-12 activation) in Example 2 were cultured to day 4. The viral vectors were VSVG enveloped lentivirus, BaEVless enveloped lentivirus, and GaLV enveloped lentivirus (all carrying fluorescent reporter genes for detecting transduction positivity rate).
[0039] Transduction methods: (1) Day -1, prepare the retrorection (takara, T100A) with 1×PBS to a concentration of 10 ug / ml. For a 24-well plate, add 0.5 ml. Then transfer the plate to 4°C and place it horizontally overnight. (2) Day 0, Lentiviral plating: Add the corresponding enveloped lentivirus with infection multiplicity MOI=10 to the culture system described in step (1), mix well, centrifuge at 32°C and 3200rpm for 1hr or place in a 37°C CO2 constant temperature incubator for 3hr. (3) Virus transduction: After the virus was plated, the supernatant was removed, and then the Day 4 highly active mNK cells obtained by in vitro culture were selected, and after adjusting the cell density to 2.5E5 / ml with amplification medium, they were gently and evenly seeded into 24-well plates. Then the plates were transferred to a 37℃ CO2 constant temperature incubator. (4) Day 3, Transduction verification: After 3 days of culture, the transduction effect was verified. For FUGW lentivirus, it expresses EGFP. The transduction positivity rate can be detected by flow cytometry detection of FITC signal.
[0040] Results analysis: like Figure 4 As shown in A and B, the transduction efficiency of the three enveloped protein viruses on mNK cells showed significant specific differences: VSVG enveloped lentivirus had the highest transduction efficiency (11.89%), while the other two enveloped protein viruses, BaEVless and GaLV enveloped lentiviruses, could not effectively transduce mNK cells (regardless of the activation mode). This differs from the conventional result that "BaEVless enveloped virus is more effective" in human NK cell transduction, which is speculated to be due to the higher conservation of VSVG viral receptors (such as low-density lipoprotein receptors) on the surface of mouse NK cells, while BaEVless viral receptors show expression differences between human and mouse NK cells. Meanwhile, compared with the transduction efficiency of activation group A (single-factor activation) in Example 2 (7.08%), the transduction efficiency of activation group B (multi-factor activation) was increased by approximately 68%, indicating that the addition of mIL-15 and mIL-12 can improve transduction efficiency by activating mNK cell signaling pathways (such as STAT4 and STAT5 pathways), upregulating viral receptor expression, or enhancing the cell's ability to take up the virus.
[0041] 4.2 Comparison of mNK cell transduction efficiency between lentiviruses and retroviruses Experimental materials: mNK cells from activation group B in Example 2, cultured to day 7, were selected. The viral vectors were VSVG enveloped lentivirus (164 / VSVG / LV, carrying the CARCD19 gene, clone number FMC63) and VSVG enveloped retrovirus (217 / VSVG / RV, carrying the same CARCD19 gene).
[0042] The transduction method is the same as in 4.1. For CAR CD19, the detection method is to incubate mNK cells with antibody FMC63-APC (Heyousheng, APAR-100) 3 days after transduction, and then detect the APC signal by flow cytometry to detect the transduction positivity rate and MFI.
[0043] Results analysis: like Figure 4As shown in C and D, the transduction efficiency of the two viral vectors differed significantly: the positive transduction rate of VSVG enveloped retrovirus was 27.60%, significantly higher than that of VSVG enveloped lentivirus (10.16%, an increase of approximately 172%). Simultaneously, the CARCD19 expression intensity (MFI value approximately 3000) in mNK cells transduced with retrovirus was also significantly higher than that with lentivirus (MFI value approximately 1400). This result is consistent with the phenomenon of "high transduction efficiency of transfected viruses in human NK cells." The self-inhibition signal carried by the lentiviral genome 3'LTR can also play a role in mouse NK cells, reducing their transduction efficiency. This finding provides a basis for selecting the optimal viral vector for the preparation of mouse CAR-NK cells.
[0044] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.
Claims
1. A method for in vitro culture of mNK cells, characterized in that, Includes the following steps: S1. Activation culture: Seed mNK cells in activation culture medium and culture for 1.5-2.5 days; S2, Expansion Culture: Transfer the activated mNK cells to expansion culture medium and culture for 16.5-17.5 days; Both the activation medium and the amplification medium are based on MEMα medium and contain FBS, β-ME and mIL-2.
2. The in vitro culture method according to claim 1, characterized in that, The activation medium described in S1 includes single-factor activation medium and multi-factor combined activation medium.
3. The in vitro culture method according to claim 2, characterized in that, The single-factor activation medium includes MEMα medium, 15-25 v / v% FBS, 0.03-0.08 mM β-ME, and 250-1500 ng / mL mIL-2.
4. The in vitro culture method according to claim 2, characterized in that, The multi-factor combination activation medium includes MEMα medium, 15-25 v / v% FBS, 0.03-0.08 mM β-ME, 100-1000 ng / mL mIL-2, mIL-15 and mIL-12.
5. The in vitro culture method according to claim 4, characterized in that, The dosage of mIL-15 is 50-500 ng / mL, and the dosage of mIL-12 is 5-100 ng / mL.
6. The in vitro culture method according to any one of claims 1-5, characterized in that, Both the activation medium and the amplification medium contain penicillin-streptomycin.
7. The in vitro culture method according to any one of claims 1-5, characterized in that, The concentration of mIL-2 in the amplification medium was 50 ng / mL.
8. The in vitro culture method according to any one of claims 1-5, characterized in that, During the culture process, the cell density does not exceed 2E6 cells / mL.
9. mNK cells cultured by the in vitro culture method according to any one of claims 1-8.
10. The mNK cell according to claim 9, characterized in that, After 18-19 days of cultivation, the amplification factor reached approximately 25-fold.
11. The mNK cell according to claim 10, characterized in that, When the culture method described in claim 3 or 4 is used, the viral transduction efficiency of the mNK cells is increased by at least 68% compared to mNK cells cultured in an activation medium containing only mIL-2.
12. The use of the mNK cells according to any one of claims 9-11 in in vitro transduction.
13. The application according to claim 12, characterized in that, The in vitro transduction used VSVG enveloped virus as the transduction vector.
14. The application according to claim 13, characterized in that, The VSVG enveloped virus is a VSVG enveloped lentivirus or a VSVG enveloped retrovirus.
15. The application according to claim 14, characterized in that, The amino acid sequence of the VSVG envelope protein is shown in SEQ ID NO.
1.
16. The application according to claim 14, characterized in that, When transduced with VSVG enveloped retrovirus, the transduction positivity rate of the mNK cells was increased by at least 172% compared with VSVG enveloped lentivirus transduction.