A culture kit for nk cells, a culture method thereof, and an application thereof
By optimizing the NK cell culture kit and culture method, the problem of long-term NK cell expansion was solved, and efficient and stable NK cell culture was achieved, which is suitable for the preparation of drugs for treating solid tumors.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG XIANKANGDA BIOTECH CO LTD
- Filing Date
- 2026-04-01
- Publication Date
- 2026-06-30
AI Technical Summary
Existing technologies cannot maintain the expanded state of NK cells for a long time, leading to a decline in cell activity and function, which limits their effectiveness in clinical applications.
Using specific NK cell culture kits and methods, including amplification medium, high-efficiency induction medium, NK-A coating solution, NK-B mixture and NK-C mixture, the high amplification capacity and immune function of NK cells are maintained by optimizing the culture medium components and cytokine combinations.
It achieves long-term expansion of NK cells (more than 21 days), maintains high expansion capacity and immune function, overcomes the problems of decreased cell activity and functional decline, and is suitable for the preparation of drugs for treating solid tumors.
Smart Images
Figure CN122303144A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture, and more particularly to a kit for NK cell culture and the application of NK cells cultured by the kit in the preparation of drugs for treating solid tumors. Background Technology
[0002] Natural killer (NK) cells are important immune cells in the human immune system, possessing broad immune surveillance capabilities against tumor cells, virus-infected cells, and other pathogens. Due to their unique immune functions, NK cells are widely used in tumor immunotherapy, immunotherapy for viral infections, and the treatment of autoimmune diseases. However, in clinical applications, effectively expanding peripheral blood NK cells and maintaining their long-term activity remains a crucial research topic in immunotherapy.
[0003] Traditional NK cell expansion methods rely heavily on cytokine support, such as IL-2 and IL-15. While these methods can expand NK cells in a short time, they typically cannot maintain this expanded state long-term and suffer from problems such as decreased cell viability and loss of function. In most existing methods, the expansion cycle of NK cells is usually between 13 and 16 days. Beyond this time, cell proliferation and immune function often decline significantly, limiting their clinical application, especially in immunotherapy where long-term maintenance of NK cell function is required.
[0004] In recent years, with in-depth research into immune cell biology, researchers have gradually realized that the key to maintaining long-term NK cell expansion lies in optimizing the culture medium, adding specific proliferation factors, and adjusting the microscopic factors of the cellular environment. For example, introducing a three-dimensional culture system that supports cell survival and proliferation, optimizing the combination of cytokines, and using specific gene modification methods can significantly improve the expansion effect and activity of NK cells. Nevertheless, existing long-term expansion methods still have certain limitations and are generally difficult to achieve long-term stable NK cell expansion in clinical practice. Summary of the Invention
[0005] Based on the above problems, the present invention aims to provide a long-term NK cell expansion protocol using peripheral blood, including an NK cell culture kit and a culture method; NK cells can be cultured for more than 21 days and can maintain high NK cell expansion capacity and immune function, effectively overcoming the problems of decreased cell activity and functional decline, and providing stable and efficient NK cell expansion capacity.
[0006] The technical solution of the present invention is as follows:
[0007] An NK cell culture kit, comprising amplification medium, high-efficiency induction medium, NK-A coating solution, NK-B mixture, and NK-C mixture; wherein:
[0008] The amplification medium includes a basal medium, to which are added and contain the following components at final concentrations: 10-50 mg / L NAD+, 1-3 g / L human serum albumin, 2-10 mg / L transferrin, 50-200 mg / L β-glucan, 1-5 mg / L glutathione, 1-5 μg / L β-mercaptoethanol, and 1-5 mg / L linoleic acid.
[0009] The high-efficiency induction medium contains the aforementioned basal medium, to which are added and contain the following components at final concentrations: 10-50 mg / L NAD+, 1-3 g / L human serum albumin, 2-10 mg / L transferrin, 50-200 mg / L β-glucan, 1-5 mg / L glutathione, 1-5 μg / L β-mercaptoethanol, 1-5 mg / L linoleic acid, 1-2 g / L soybean peptides, 100-300 mg / L nicotinamide, 50-200 mg / L inulin, and 1-5 mM N-acetyl-L-cysteine.
[0010] The NK-A coating solution contains 20-80 mg of heparin sodium, 5-20 μg of CD16 monoclonal antibody, and 5-20 μg of 4-1BB antibody;
[0011] The NK-B mixture contains 10-30 μg of Inbakicept and 2-10 × 10⁻⁶ mg of NK-B solution. 6 UI IL-2, 200~600ng IL-15;
[0012] NK-C mixture contains 1~5ug of Linoan and 2~10×10 6 UI IL-2, 200~600ng IL-18.
[0013] Preferably, in the NK cell culture kit, the amplification medium and the high-efficiency induction medium are each dispensed into 1L reagent bottles, and the ratio of the number of bottles containing the amplification medium to the number of bottles containing the high-efficiency induction medium is 1:2.
[0014] Preferably, in the NK cell culture kit, the NK-A coating solution, NK-B mixture and NK-C mixture are dispensed into 1mL reagent bottles, and the ratio of the number of bottles containing the NK-A coating solution, NK-B mixture and NK-C mixture is 1:1:2.
[0015] Preferably, in the NK cell culture kit, the basal culture medium is one of DMEM / F12, IMDM, or RPMI-1640.
[0016] This invention also provides a method for culturing NK cells using the above-described kit, comprising the following steps:
[0017] On day 0, T75 culture flasks were coated with NK-A coating solution from the above kit; NK-B mixture from the above kit was mixed with high-efficiency induction medium to obtain high-efficiency induction mixed culture medium for later use; NK-C mixture from the above kit was mixed with amplification medium to obtain amplification mixed culture medium for later use.
[0018] On day 1, firstly, PBMCs were resuspended in a high-efficiency induction mixed culture medium to obtain a cell suspension; secondly, the cell suspension was seeded into coated T75 culture flasks, and high-efficiency induction mixed culture medium was added to control the cell density at 1.5 × 10⁻⁶ cells / year. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0019] On days 3 and 5, high-efficiency induction mixed culture medium was added, and the cell density was controlled at 0.5~0.8×10⁶ cells / year. 6 Cells / mL, continue culturing;
[0020] On day 7, firstly, the cells and culture medium in the culture flask were transferred to a 10L culture bag, and expansion mixed culture medium was added to maintain the cell density at 0.8~1.0×10⁶ cells / year. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0021] From day 9 to day 13, amplification mixed culture medium was added every 2-3 days, and the cell density was controlled at 1.0-2.0 × 10⁶ cells / year. 6 Cells / mL, continue culturing;
[0022] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0023] Preferably, in the culture method, the coating process for the T75 culture flask on day 0 is as follows:
[0024] First, add the NK-A coating solution from the above kit to a T75 culture flask and incubate at 2-8°C for 16 hours. After incubation, use a pipette to remove the remaining NK-A coating solution from the T75 culture flask.
[0025] Next, tilt the T75 culture flask from the coated side to the uncoated side, and add 10 mL of PBS to the T75 culture flask for washing. After washing, lay the T75 culture flask flat and gently shake the washed T75 culture flask.
[0026] Finally, stand the T75 culture flask upright and use a pipette to aspirate the PBS washing solution from the bottom of the T75 culture flask to complete the coating process of the T75 culture flask, and set it aside for later use.
[0027] NK cells obtained using the culture method described above in this invention can be used as biological agents or components of biological agents and can be widely applied in the preparation of drugs for the prevention and treatment of solid tumors (such as gastric cancer, pancreatic cancer, rectal cancer, liver cancer, etc.); or NK cells can be directly applied in the preparation of drugs for the prevention or treatment of solid tumors.
[0028] This invention provides an NK cell culture kit and culture formula, which have the following advantages:
[0029] 1) The kit provided by this invention contains components in the highly efficient induction culture medium, such as human serum albumin, which, under the synergistic effect of the addition of soybean peptides, inulin, and N-acetyl-L-cysteine, can activate and induce PBMCs to differentiate into NK cells; the components in the amplification culture medium (such as NAD+, human serum albumin, β-glucan, etc.) can promote the rapid expansion and growth of NK cells (the amplification fold can reach about 987 times after 14 days of culture).
[0030] 2) The Inbakicept factor in the kit can activate NK cells and enhance their toxicity; Linoan can inhibit T cell proliferation without affecting NK cell proliferation, and can improve NK cell purity (up to about 90%), resulting in strong cell efficacy. Attached Figure Description
[0031] Figure 1 This is a graph showing the cell number growth curves after 14 days of NK cell expansion culture in Examples 1 to 5;
[0032] Figure 2 The flow cytometry plots show the CD3-CD56+ purity of NK cells on day 14 of expansion culture in Examples 1 to 5. Detailed Implementation
[0033] The preferred embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.
[0034] This invention efficiently expands NK cells through in vitro culture of peripheral blood mononuclear cells (PBMCs). By utilizing specific combinations of cytokines (such as Inbakicept, Linoan, etc.), optimized culture conditions, and precise cell environment regulation, cell activity and proliferation can be maintained for more than 21 days, and the number of expanded cells is sufficient to meet clinical treatment needs. It can also overcome the shortcomings of traditional expansion methods to a certain extent and improve the therapeutic effect of NK cells.
[0035] Inbakicep is a dimer fusion protein of human IL-15 receptor α (L-15 Ra) Sushl domain / human IgG1 FC, and is an IL-15 superagonist complex. Inbakicep can form complex N-803 (Nogapendekin alla inbakicep) with the IL-15 antibody logapendekin ala in a 1:2 ratio. N-803 can mimic the function of IL-15 and amplify anti-CD20 mAb-mediated NK cell responses and antibody-dependent cytotoxicity (ADCC).
[0036] I. Obtaining PBMCs from Peripheral Blood
[0037] The mononuclear cells used in this invention are obtained from peripheral blood, and the procedure is as follows:
[0038] 1. Take 50-60 mL of peripheral blood sample and place it in a 50 mL centrifuge tube. Centrifuge at 650 g for 15 min at room temperature. After centrifugation, aspirate the lower red cell pellet and dilute the cell pellet to its original volume with physiological saline or PBS to obtain the diluent.
[0039] 2. Take a 50mL centrifuge tube, add 15mL of lymphocyte separation medium, and slowly add the diluent from step 1 to the top layer of the lymphocyte separation medium;
[0040] 3. Centrifuge at 800g for 20 minutes at room temperature (acceleration rate 1-5, deceleration without brake);
[0041] 4. After centrifugation, the sample in the centrifuge tube will be separated into 4 layers from top to bottom: plasma layer - white membrane layer - human lymphocyte separation solution layer - precipitate of red blood cells and granulocytes; carefully aspirate the middle white membrane layer and about half of the liquid below it, transfer them to a new centrifuge tube, add physiological saline or PBS to a volume of 40 mL, mix well, and centrifuge at 300 g for 10 min.
[0042] 5. After centrifugation, discard the supernatant, resuspend the precipitate in 40 mL of physiological saline or PBS, and centrifuge the suspension at 300 g for 10 min.
[0043] 6. After centrifugation, discard the supernatant, resuspend the pellet in a small amount of basal culture medium to obtain mononuclear cells (PBMCs) for later use.
[0044] In other embodiments, mononuclear cells (CBMCs) can also be obtained from umbilical cord blood.
[0045] II. Preparation of culture components for the reagent kit
[0046] The NK cell culture kit provided by this invention comprises reagents obtained by mixing four components: amplification medium, high-efficiency induction medium, NK-A coating solution, NK-B mixture and NK-C mixture; see Table 1 for details.
[0047] Table 1. Reagent Kit Components
[0048]
[0049] In each kit, the amplification medium and high-efficiency induction medium are prepared to a standard 1L volume and dispensed into 1L reagent bottles, while the NK-A coating solution, NK-B mixture, and NK-C mixture can be dispensed into 1mL reagent bottles. The culture group allocation in the kit is as follows:
[0050] 1L of amplification medium: containing basal medium supplemented with NAD+ at final concentrations of 10-50 mg / L, human serum albumin at final concentrations of 1-3 g / L, transferrin at final concentrations of 2-10 mg / L, β-glucan at final concentrations of 50-200 mg / L, glutathione at final concentrations of 1-5 mg / L, β-mercaptoethanol at final concentrations of 1-5 μg / L, and linoleic acid at final concentrations of 1-5 mg / L.
[0051] High-efficiency induction medium 1L: Contains basal medium supplemented with the following components at final concentrations: 10-50 mg / L NAD+, 1-3 g / L human serum albumin, 2-10 mg / L transferrin, 50-200 mg / L β-glucan, 1-5 mg / L glutathione, 1-5 μg / L β-mercaptoethanol, 1-5 mg / L linoleic acid, 1-2 g / L soybean peptides, 100-300 mg / L nicotinamide, 50-200 mg / L inulin, and 1-5 mM N-acetyl-L-cysteine.
[0052] 1 mL NK-A coating solution: contains 20-80 mg heparin sodium, 5-20 μg CD16 monoclonal antibody and 5-20 μg 4-1BB antibody;
[0053] 1 mL NK-B mixture: contains 10-30 μg Inbakicept, 2-10 × 10⁻⁶ mg / mL6 UI IL-2 and 200~600ng IL-15.
[0054] 1mL NK-C mixture: contains 1~5ug of linolenic acid, 2~10×10 6 UI IL-2 and 200~600ng IL-18.
[0055] Preferably, the NK cell culture reagent in the kit includes:
[0056] 1L of amplification medium: The basal medium is supplemented with NAD+ at a final concentration of 15-40 mg / L, human serum albumin at a final concentration of 1.5-2.5 g / L, transferrin at a final concentration of 4-8 mg / L, β-glucan at a final concentration of 80-160 mg / L, glutathione at a final concentration of 2-4 mg / L, β-mercaptoethanol at a final concentration of 2-4 μg / L, and linoleic acid at a final concentration of 2-4 mg / L.
[0057] High-efficiency induction medium 1L: The basal medium is supplemented with the following components: NAD+ at a final concentration of 15-40 mg / L, human serum albumin at a final concentration of 1.5-2.5 g / L, transferrin at a final concentration of 4-8 mg / L, β-glucan at a final concentration of 80-160 mg / L, glutathione at a final concentration of 2-4 mg / L, β-mercaptoethanol at a final concentration of 2-4 μg / L, linoleic acid at a final concentration of 1-2 g / L, soybean peptides at a final concentration of 150-250 mg / L, nicotinamide at a final concentration of 70-160 mg / L, inulin at a final concentration of 2-4 mM, and N-acetyl-L-cysteine.
[0058] 1 mL NK-A coating solution: contains 30-60 mg heparin sodium, 8-16 μg CD16 monoclonal antibody and 8-16 μg 4-1BB antibody;
[0059] 1 mL NK-B mixture: contains 15-25 μg Inbakicept, 3-8 × 10⁻⁶ mg / mL 6 UI IL-2, 300~500ng IL-15;
[0060] 1mL NK-C mixture: contains 2~4ug of linolenic acid, 3~8×10 6 UI IL-2, 300~500ng IL-18.
[0061] The basal culture medium in the kit is any one of DMEM / F12, IMDM or RPMI-1640.
[0062] Specifically, in a preferred embodiment, the culture components in the kit may be formulated including, but not limited to, any of the following groups:
[0063] Group 1)
[0064] 1L of amplification medium: DMEM / F12 basal medium supplemented with NAD+ at a final concentration of 30mg / L, human serum albumin at a final concentration of 3g / L, transferrin at a final concentration of 5mg / L, β-glucan at a final concentration of 120mg / L, glutathione at a final concentration of 3mg / L, β-mercaptoethanol at a final concentration of 2.5ug / L, and linoleic acid at a final concentration of 3mg / L.
[0065] 1L of high-efficiency induction medium: Based on DMEM / F12 medium, supplemented with the following components at final concentrations: NAD+ 30mg / L, human serum albumin 3g / L, transferrin 5mg / L, β-glucan 120mg / L, glutathione 3mg / L, β-mercaptoethanol 2.5ug / L, linoleic acid 3mg / L, soybean peptide 1.5g / L, nicotinamide 200mg / L, inulin 120mg / L, and N-acetyl-L-cysteine 3mM.
[0066] 1mL NK-A coating solution: contains 50mg heparin sodium, 12ug CD16 monoclonal antibody and 13ug 4-1BB antibody;
[0067] 1 mL NK-B mixture: contains 20 μg Inbakicept, 5 × 10 6 UI IL-2 and 400ng IL-15;
[0068] 1mL NK-C mixture: contains 3ug of linolenic acid, 5×10 6 UI IL-2 and 400ng IL-18;
[0069] 2) group
[0070] 1L of amplification medium: Based on IMDM medium, supplemented with NAD+ at a final concentration of 40mg / L, human serum albumin at a final concentration of 2.5g / L, transferrin at a final concentration of 8mg / L, β-glucan at a final concentration of 80mg / L, glutathione at a final concentration of 4mg / L, β-mercaptoethanol at a final concentration of 3ug / L, and linoleic acid at a final concentration of 4mg / L.
[0071] High-efficiency induction medium 1L: Based on IMDM medium, supplemented with the following: final concentration of NAD+ 40mg / L, final concentration of human serum albumin 2.5g / L, final concentration of transferrin 8mg / L, final concentration of β-glucan 80mg / L, final concentration of glutathione 4mg / L, final concentration of β-mercaptoethanol 3ug / L, final concentration of linoleic acid 4mg / L, final concentration of soybean peptide 1g / L, final concentration of nicotinamide 180mg / L, final concentration of inulin 160mg / L, and final concentration of N-acetyl-L-cysteine 4mM.
[0072] 1mL NK-A coating solution: contains 30mg heparin sodium, 10ug CD16 monoclonal antibody and 18ug 4-1BB antibody;
[0073] 1 mL NK-B mixture: contains 25 μg Inbakicept, 8 × 10 6 UI IL-2 and 360ng IL-15;
[0074] 1mL NK-C mixture: contains 4ug of Linoan, 8×10 6 UI IL-2 and 300ng IL-18;
[0075] 3) Group
[0076] 1L of amplification medium: Based on RPMI-1640 medium, supplemented with NAD+ at a final concentration of 20mg / L, human serum albumin at a final concentration of 2g / L, transferrin at a final concentration of 4mg / L, β-glucan at a final concentration of 100mg / L, glutathione at a final concentration of 1.5mg / L, β-mercaptoethanol at a final concentration of 3.5ug / L, and linoleic acid at a final concentration of 1.5mg / L.
[0077] High-efficiency induction medium 1L: Based on RPMI-1640 medium, supplemented with the following: final concentration of NAD+ 20mg / L, final concentration of human serum albumin 2g / L, final concentration of transferrin 4mg / L, final concentration of β-glucan 100mg / L, final concentration of glutathione 1.5mg / L, final concentration of β-mercaptoethanol 3.5ug / L, final concentration of linoleic acid 1.5mg / L, final concentration of soybean peptide 2g / L, final concentration of nicotinamide 110mg / L, final concentration of inulin 80mg / L, and final concentration of N-acetyl-L-cysteine 1.5mM.
[0078] 1mL NK-A coating solution: contains 60mg heparin sodium, 15ug CD16 monoclonal antibody and 15ug 4-1BB antibody;
[0079] 1 mL NK-B mixture: contains 15 μg Inbakicept, 8 × 10 6UI IL-2 and 500ng IL-15.
[0080] 1mL NK-C mixture: contains 1.5ug of linolenic acid, 8×10 6 UI IL-2 and 500ng IL-18.
[0081] III. NK Cell Culture
[0082] Use the culture reagents from any of the kits in the second item above to culture NK cells. The culture steps are as follows:
[0083] On day 0, firstly, add the NK-A coating solution from the kit to the T75 culture flask and incubate at 2-8°C for 16 hours. After incubation, use a pipette to remove the remaining NK-A coating solution from the T75 culture flask. Next, tilt the T75 culture flask from the coated side to the non-coated side, and simultaneously add 10 mL of PBS to the T75 culture flask for washing. After washing, lay the T75 culture flask flat and gently shake the washed T75 culture flask. Finally, stand the T75 culture flask upright and use a pipette to remove the PBS washing solution from the bottom of the T75 culture flask, completing the coating treatment of the T75 culture flask, and set it aside for later use.
[0084] Mix the NK-B mixture in the kit with the high-efficiency induction medium until homogeneous to obtain a high-efficiency induction mixed culture medium for later use.
[0085] Mix the NK-C mixture in the kit with the amplification medium until homogeneous to obtain the amplification mixed culture medium, and set aside for later use;
[0086] On day 1, firstly, resuspend 3.0 × 10⁻⁶ cells in a high-efficiency induction mixed culture medium. 7 PBMCs were collected to obtain a cell suspension; next, the cell suspension was seeded into coated T75 culture flasks, and high-efficiency induction mixed culture medium was added to control the cell density at 1.5 × 10⁻⁶ cells / mL. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0087] On days 3 and 5, high-efficiency induction mixed culture medium was added, and the cell density was controlled at 0.5~0.8×10⁶ cells / year. 6 Cells / mL, continue culturing;
[0088] On day 7, firstly, the cells and culture medium in the culture flask were transferred to a 10L culture bag, and expansion mixed culture medium was added to maintain the cell density at 0.8~1.0×10⁶ cells / year. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0089] From day 9 to day 13, amplification mixed culture medium was added every 2-3 days, and the cell density was controlled at 1.0-2.0 × 10⁶ cells / year. 6 Cells / mL, continue culturing;
[0090] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0091] The technical solution of the present invention will be described in detail below through specific embodiments. In the embodiments, the PBMCs used are derived from the same batch of peripheral blood isolated from the first item of the present invention, and will not be repeated here.
[0092] (I) NK cell culture
[0093] Example 1
[0094] 1.1 Preparation of culture components in the kit
[0095] Amplification medium 1L×2 bottles: Based on IMDM, supplemented with NAD+ at a final concentration of 50mg / L, human serum albumin at a final concentration of 1g / L, transferrin at a final concentration of 2mg / L, β-glucan at a final concentration of 50mg / L, glutathione at a final concentration of 1mg / L, β-mercaptoethanol at a final concentration of 1ug / L, and linoleic acid at a final concentration of 5mg / L;
[0096] High-efficiency induction medium 1L×1 bottle: Based on IMDM, supplemented with the following components: final concentration 50mg / L NAD+, final concentration 1g / L human serum albumin, final concentration 2mg / L transferrin, final concentration 50mg / L β-glucan, final concentration 1mg / L glutathione, final concentration 1ug / L β-mercaptoethanol, final concentration 5mg / L linoleic acid, final concentration 2g / L soybean peptide, final concentration 100mg / L nicotinamide, final concentration 200mg / L inulin, and final concentration 1mM N-acetyl-L-cysteine.
[0097] NK-A coating solution 1mL×1 bottle: 80mg heparin sodium, 5ug CD16 monoclonal antibody and 5ug 4-1BB antibody;
[0098] NK-B mixture 1mL×1 bottle: 10ug Inbakicept, 2×10 6 UI IL-2 and 200ng IL-15;
[0099] NK-C mixture 1mL x 2 bottles: 1ug Linoan, 2 x 10 6 UI IL-2 and 200ng IL-18.
[0100] 1.2 NK cell culture
[0101] On day 0, firstly, the NK-A coating solution prepared in step 1.1 of this embodiment was added to a T75 culture flask and incubated in a refrigerator at 2~8℃ for 16 hours. After incubation, the remaining NK-A coating solution was aspirated from the T75 culture flask using a pipette. Next, the T75 culture flask was tilted from the coated side to the non-coated side, and 10mL of PBS was added to the T75 culture flask for washing. After washing, the T75 culture flask was laid flat and the washed T75 culture flask was gently shaken. Finally, the T75 culture flask was placed upright, and the washing solution after PBS washing was aspirated from the bottom of the T75 culture flask using a pipette. The coating treatment of the T75 culture flask was completed and it was ready for use.
[0102] On day 1, firstly, the NK-B mixture prepared in step 1.1 of this embodiment was mixed evenly with the high-efficiency culture medium to obtain a high-efficiency induction mixed culture medium; then, 3.0 × 10⁻⁶ units of the high-efficiency induction mixed culture medium were resuspended. 7 1 PBMC was used to obtain a cell suspension; next, the cell suspension was seeded into the coated T75 culture flask, and the cell density was controlled at 1.5 × 10⁻⁶ cells / mL. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0103] On days 3 and 5, high-efficiency induction mixed culture medium was added, and the cell density was controlled at 0.5 × 10⁻⁶. 6 Cells / mL, continue culturing;
[0104] On day 7, firstly, take two bottles of the NK-C mixture prepared in step 1.1 of this embodiment and mix them thoroughly with the amplification culture medium to obtain the amplification mixed culture medium; then, transfer the cells and culture medium from the culture bottles to a 10L culture bag, add more amplification mixed culture medium, and control the cell density to 0.8 × 10⁻⁶. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0105] From day 9 to day 13, amplification mixed culture medium was added every two days, and the cell density was controlled at 1.5 × 10⁻⁶ cells / day. 6 Cells / mL, continue culturing;
[0106] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0107] Example 2
[0108] 2.1 Preparation of culture components in the kit
[0109] Amplification medium 1L×2 bottles: Based on IMDM, supplemented with NAD+ at a final concentration of 25mg / L, human serum albumin at a final concentration of 2g / L, transferrin at a final concentration of 5mg / L, β-glucan at a final concentration of 100mg / L, glutathione at a final concentration of 2mg / L, β-mercaptoethanol at a final concentration of 2ug / L, and linoleic acid at a final concentration of 2mg / L.
[0110] High-efficiency induction medium 1L×1 bottle: Based on IMDM, supplemented with the following components: final concentration 25mg / L NAD+, final concentration 2g / L human serum albumin, final concentration 5mg / L transferrin, final concentration 100mg / L β-glucan, final concentration 2mg / L glutathione, final concentration 2ug / L β-mercaptoethanol, final concentration 2mg / L linoleic acid, final concentration 1.5g / L soybean peptide, final concentration 200mg / L nicotinamide, final concentration 100mg / L inulin, and final concentration 2mM N-acetyl-L-cysteine;
[0111] NK-A coating solution 1mL×1 bottle: 50mg heparin sodium, 10ug CD16 monoclonal antibody and 10ug 4-1BB antibody;
[0112] NK-B mixture 1mL×1 bottle: 20ug Inbakicept, 5×10 6 UI IL-2 and 400ng IL-15;
[0113] NK-C mixture 1mL×2 bottles: 2ug Linoan, 5×10 6 UI IL-2 and 400ng IL-18.
[0114] 2.2 NK cell culture
[0115] On day 0, firstly, the NK-A coating solution prepared in step 2.1 of this embodiment was added to a T75 culture flask and incubated in a refrigerator at 2~8℃ for 16 hours. After incubation, the remaining NK-A coating solution was aspirated from the T75 culture flask using a pipette. Next, the T75 culture flask was tilted from the coated side to the non-coated side, and 10mL of PBS was added to the T75 culture flask for washing. After washing, the T75 culture flask was laid flat and the washed T75 culture flask was gently shaken. Finally, the T75 culture flask was placed upright, and the washing solution after PBS washing was aspirated from the bottom of the T75 culture flask using a pipette. The coating treatment of the T75 culture flask was completed and it was ready for use.
[0116] On day 1, firstly, the NK-B mixture prepared in step 2.1 of this embodiment was mixed evenly with the high-efficiency culture medium to obtain a high-efficiency induction mixed culture medium; then, 3.0 × 10⁻⁶ units of the high-efficiency induction mixed culture medium were resuspended. 7 1 PBMC was used to obtain a cell suspension; next, the cell suspension was seeded into the coated T75 culture flask, and the cell density was controlled at 1.5 × 10⁻⁶ cells / mL. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0117] On days 3 and 5, highly efficient induction mixed culture medium was added, and the cell density was controlled at 0.6 × 10⁶ cells / year. 6 Cells / mL, continue culturing;
[0118] On day 7, firstly, take two bottles of the NK-C mixture prepared in step 2.1 of this embodiment and mix them thoroughly with the amplification culture medium to obtain the amplification mixed culture medium; then, transfer the cells and culture medium from the culture bottles to a 10L culture bag, add more amplification mixed culture medium, and control the cell density to 0.8 × 10⁻⁶. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0119] From day 9 to day 13, amplification mixed culture medium was added every two days, and the cell density was maintained at 1.0 × 10⁶ cells / day. 6 Cells / mL, continue culturing;
[0120] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0121] Example 3
[0122] 3.1 Preparation of culture components in the kit
[0123] Amplification medium 1L×2 bottles: Based on IMDM, add final concentrations of 10mg / L NAD+, 3g / L human serum albumin, 10mg / L transferrin, 200mg / L β-glucan, 5mg / L glutathione, 5ug / L β-mercaptoethanol and 1mg / L linoleic acid;
[0124] High-efficiency induction medium 1L×1 bottle: Based on IMDM, add the following to a final concentration: NAD+ 10mg / L, human serum albumin 3g / L, transferrin 10mg / L, β-glucan 200mg / L, glutathione 5mg / L, β-mercaptoethanol 5ug / L, linoleic acid 1mg / L, soybean peptide 1g / L, nicotinamide 300mg / L, inulin 50mg / L, and N-acetyl-L-cysteine 5mM.
[0125] NK-A coating solution 1mL×1 bottle: 20mg heparin sodium, 20ug CD16 monoclonal antibody and 20ug 4-1BB antibody;
[0126] NK-B mixture 1mL×1 bottle: 30ug Inbakicept, 10×10 6 UI IL-2 and 600ng IL-15;
[0127] NK-C mixture 1mL x 2 bottles: 5ug Linoan, 10 x 10 6 UI IL-2 and 600ng IL-18.
[0128] 3.2 NK cell culture
[0129] On day 0, firstly, the NK-A coating solution prepared in step 3.1 of this embodiment was added to a T75 culture flask and incubated in a refrigerator at 2~8℃ for 16 hours. After incubation, the remaining NK-A coating solution was aspirated from the T75 culture flask using a pipette. Next, the T75 culture flask was tilted from the coated side to the non-coated side, and 10mL of PBS was added to the T75 culture flask for washing. After washing, the T75 culture flask was laid flat and the washed T75 culture flask was gently shaken. Finally, the T75 culture flask was placed upright, and the washing solution after PBS washing was aspirated from the bottom of the T75 culture flask using a pipette. The coating treatment of the T75 culture flask was completed and it was ready for use.
[0130] On day 1, firstly, the NK-B mixture prepared in step 3.1 of this embodiment is mixed evenly with the high-efficiency culture medium to obtain a high-efficiency induction mixed culture medium; then, 3.0 × 10⁻⁶ units of the high-efficiency induction mixed culture medium are resuspended. 7 1 PBMC was used to obtain a cell suspension; next, the cell suspension was seeded into the coated T75 culture flask, and the cell density was controlled at 1.5 × 10⁻⁶ cells / mL. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0131] On days 3 and 5, high-efficiency induction mixed culture medium was added, and the cell density was controlled at 0.8 × 10⁻⁶. 6 Cells / mL, continue culturing;
[0132] On day 7, firstly, take two bottles of the NK-C mixture prepared in step 3.1 of this embodiment and mix them thoroughly with the amplification culture medium to obtain the amplification mixed culture medium; then, transfer the cells and culture medium from the culture bottles to a 10L culture bag, add more amplification mixed culture medium, and control the cell density to 0.8 × 10⁻⁶. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0133] From day 9 to day 13, amplification mixed culture medium was added every two days, and the cell density was maintained at 2.0 × 10⁶ cells / day. 6 Cells / mL, continue culturing;
[0134] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0135] Example 4 (Comparative Example 1)
[0136] 4.1 Preparation of NK cell culture medium (without high-efficiency induction medium and amplification medium)
[0137] Three 1L bottles were prepared using GT-T551 H3 medium as the main culture medium.
[0138] NK-A coating solution 1mL×1 bottle: 80mg heparin sodium, 20ug CD16 monoclonal antibody and 20ug 4-1BB antibody;
[0139] NK-B mixture 1mL×1 bottle: 30ug Inbakicept, 10×10 6 UI IL-2 and 600ng IL-15;
[0140] NK-C mixture 1mL×2 bottles: 5ug Linoan, 10×10 6 UIIL-2 and 600ng IL-18.
[0141] 4.2 NK cell culture
[0142] On day 0, firstly, the NK-A coating solution prepared in step 4.1 of this embodiment was added to a T75 culture flask and incubated in a refrigerator at 2~8℃ for 16 hours. After incubation, the remaining NK-A coating solution was aspirated from the T75 culture flask using a pipette. Next, the T75 culture flask was tilted from the coated side to the non-coated side, and 10mL of PBS was added to the T75 culture flask for washing. After washing, the T75 culture flask was laid flat and the washed T75 culture flask was gently shaken. Finally, the T75 culture flask was placed upright, and the washing solution after PBS washing was aspirated from the bottom of the T75 culture flask using a pipette. The coating treatment of the T75 culture flask was completed and it was ready for use.
[0143] On Day 1, firstly, take one bottle of the NK-B mixture prepared in step 4.1 of this embodiment and mix it evenly with GT-T551 H3 medium to obtain a high-efficiency induction mixed culture medium; then, resuspend 3.0 × 10⁻⁶ tbsp of the high-efficiency induction mixed culture medium. 7 1 PBMC was used to obtain a cell suspension; next, the cell suspension was seeded into the coated T75 culture flask, and the cell density was controlled at 1.5 × 10⁻⁶ cells / mL. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0144] On days 3 and 5, high-efficiency induction mixed culture medium was added every two days, and the cell density was controlled at 0.6 × 10⁶ cells / day. 6 Cells / mL, continue culturing;
[0145] On day 7, firstly, take two bottles of the NK-C mixture prepared in step 4.1 of this embodiment and mix them thoroughly with GT-T551 H3 medium to obtain the amplification mixed culture medium; then, transfer the cells and culture medium from the culture bottles to a 10L culture bag, add more amplification mixed culture medium, and control the cell density to 0.8 × 10⁻⁶. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0146] From day 9 to day 13, amplification mixed culture medium was added every two days, and the cell density was controlled at 1.5 × 10⁻⁶ cells / day. 6 Cells / mL, continue culturing;
[0147] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0148] Example 5 (Comparative Example 2)
[0149] 5.1 Preparation of culture components in the kit (without Inbakicept or Linoan)
[0150] Amplification medium 1L×2 bottles: Based on IMDM, supplemented with NAD+ at a final concentration of 25mg / L, human serum albumin at a final concentration of 2g / L, transferrin at a final concentration of 5mg / L, β-glucan at a final concentration of 100mg / L, glutathione at a final concentration of 2mg / L, β-mercaptoethanol at a final concentration of 2ug / L, and linoleic acid at a final concentration of 2mg / L.
[0151] High-efficiency induction medium 1L×1 bottle: Based on IMDM, supplemented with the following components: final concentration 25mg / L NAD+, final concentration 2g / L human serum albumin, final concentration 5mg / L transferrin, final concentration 100mg / L β-glucan, final concentration 2mg / L glutathione, final concentration 2ug / L β-mercaptoethanol, final concentration 2mg / L linoleic acid, final concentration 1.5g / L soybean peptide, final concentration 200mg / L nicotinamide, final concentration 100mg / L inulin, and final concentration 2mM N-acetyl-L-cysteine;
[0152] NK-A coating solution 1mL×1 bottle: 50mg heparin sodium, 10ug CD16 monoclonal antibody and 10ug 4-1BB antibody;
[0153] NK-B mixture 1mL × 1 bottle: 5 × 10 6 UI IL-2 and 400ng IL-15;
[0154] NK-C mixture 1mL × 2 bottles: 5 × 10 6 UI IL-2 and 400ng IL-18.
[0155] 5.2 NK cell culture
[0156] On day 0, firstly, the NK-A coating solution prepared in step 5.1 of this embodiment was added to a T75 culture flask and incubated in a refrigerator at 2~8℃ for 16 hours. After incubation, the remaining NK-A coating solution was aspirated from the T75 culture flask using a pipette. Next, the T75 culture flask was tilted from the coated side to the non-coated side, and 10mL of PBS was added to the T75 culture flask for washing. After washing, the T75 culture flask was laid flat and the washed T75 culture flask was gently shaken. Finally, the T75 culture flask was placed upright, and the washing solution after PBS washing was aspirated from the bottom of the T75 culture flask using a pipette. The coating treatment of the T75 culture flask was completed and it was ready for use.
[0157] On day 1, firstly, the NK-B mixture prepared in step 5.1 of this embodiment is mixed evenly with the high-efficiency culture medium to obtain a high-efficiency induction mixed culture medium; then, 3.0 × 10⁻⁶ units of the high-efficiency induction mixed culture medium are resuspended. 7 1 PBMC was used to obtain a cell suspension; next, the cell suspension was seeded into the coated T75 culture flask, and the cell density was controlled at 1.5 × 10⁻⁶ cells / mL. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation.
[0158] On days 3 and 5, highly efficient induction mixed culture medium was added, and the cell density was controlled at 0.6 × 10⁶ cells / year. 6 Cells / mL, continue culturing;
[0159] On day 7, firstly, take two bottles of the NK-C mixture prepared in step 5.1 of this embodiment and mix them thoroughly with the amplification culture medium to obtain the amplification mixed culture medium; then, transfer the cells and culture medium from the culture bottles to a 10L culture bag, add more amplification mixed culture medium, and control the cell density to 0.8 × 10⁻⁶. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation.
[0160] From day 9 to day 13, amplification mixed culture medium was added every two days, and the cell density was controlled at 1.5 × 10⁻⁶ cells / day. 6 Cells / mL, continue culturing;
[0161] On day 14, stop culturing. First, collect the cells and wash them repeatedly with PBS 2-3 times. Second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
[0162] (II) NK cell detection test
[0163] Example 6
[0164] 6.1 NK cell count assay
[0165] In Examples 1 to 5, NK cells cultured in PBMCs were sampled in 200 μL volumes on days 0, 5, 7, 9, 11, and 14, respectively. Cell counts were performed using a cell counter, and the results are shown in Table 2. Figure 1 As shown; where, Figure 1 It was drawn based on the data in Table 2.
[0166] Table 2 NK cell count statistics and expansion folds
[0167]
[0168] From Table 2 and Figure 1 It can be seen that the PBMCs were used to expand and culture NK cells on day 14; the cell expansion folds in Examples 1 to 5 were 819.41, 986.70, 879.14, 613.93 and 461.14 times, respectively.
[0169] 1) Compared with Example 4 (Comparative Example 1), the NK cells cultured in Examples 1 to 3 showed a significantly higher expansion rate; this is because the components of the highly efficient induction medium prepared in the kit of the present invention (such as NAD+, human serum albumin, soybean peptides, inulin, and N-acetyl-L-cysteine) work synergistically to induce and stimulate PBMCs to differentiate into NK cells. At the same time, the components of the expansion medium (such as NAD+, human serum albumin, β-glucan, etc.) can promote the rapid expansion and growth of NK cells.
[0170] 2) The NK cell expansion fold of the cultured cells in Examples 1 to 3 was 1.78 to 2.14 times that of the NK cells cultured in Example 5 (Comparative Example 2). This is because the Inbakicept component of the NK-B mixture and the components of the high-efficiency induction medium, such as NAD+, human serum albumin, soybean peptides, inulin and N-acetyl-L-cysteine, work together to further stimulate and induce PBMCs to differentiate into NK cells.
[0171] 6.2 Flow cytometry detection of NK cell CD3-CD56+ phenotype
[0172] In Examples 1 to 5, NK cells were sampled on day 14 of culture, prepared into cell suspensions, and the final cell concentration was adjusted to 1×10⁻⁶. 6 Add CD3-CD56+ monoclonal antibody marker at a concentration of [number] cells / mL, and incubate in the dark at room temperature for 15 min. After incubation, wash away excess antibody and analyze by flow cytometry. Results are as follows: Figure 2 As shown.
[0173] Depend on Figure 2 It can be seen that the CD3-CD56+ purity of NK cells cultured for 14 days in Examples 1 to 5 were 88.22%, 90.51%, 86.70%, 64.61%, and 56.70%, respectively.
[0174] 1) The CD3-CD56+ purity of NK cells cultured for 14 days in Examples 1 to 3 was much higher than that of NK cells cultured for 14 days in Example 4 (Comparative Example 1). This indicates that the components of the highly efficient induction medium in the NK cell culture medium, such as soybean peptides, inulin and N-acetyl-L-cysteine, can maintain the activity of NK cells, promote the secretion of cytokines and slow down cell senescence, thereby improving cell purity.
[0175] 2) The CD3-CD56+ purity of NK cells cultured for 14 days in Examples 1 to 3 was much higher than that of NK cells cultured for 14 days in Example 5 (Comparative Example 5). This indicates that the linoan component in the NK-C mixture in the NK cell culture medium can inhibit T cell proliferation, thereby achieving the effect of improving NK cell purity.
[0176] It should be understood that the above description of the preferred embodiments of the present invention is quite detailed, but it should not be considered as a limitation on the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be determined by the appended claims.
Claims
1. An NK cell culture kit, characterized in that, The kit contains amplification medium, high-efficiency induction medium, NK-A coating solution, NK-B mixture, and NK-C mixture; wherein: The amplification medium comprises a basal medium supplemented with and containing the following components at final concentrations: 10-50 mg / L NAD+, 1-3 g / L human serum albumin, 2-10 mg / L transferrin, 50-200 mg / L β-glucan, 1-5 mg / L glutathione, 1-5 μg / L β-mercaptoethanol, and 1-5 mg / L linoleic acid. The high-efficiency induction medium comprises the basal medium, to which are added and contain the following components at final concentrations: 10-50 mg / L NAD+, 1-3 g / L human serum albumin, 2-10 mg / L transferrin, 50-200 mg / L β-glucan, 1-5 mg / L glutathione, 1-5 μg / L β-mercaptoethanol, 1-5 mg / L linoleic acid, 1-2 g / L soybean peptide, 100-300 mg / L nicotinamide, 50-200 mg / L inulin, and 1-5 mM N-acetyl-L-cysteine. The NK-A coating solution contains 20-80 mg of heparin sodium, 5-20 μg of CD16 monoclonal antibody, and 5-20 μg of 4-1BB antibody; The NK-B mixture contains 10~30ug Inbakicept, 2~10×10 6 UI IL-2 and 200~600ng IL-15; The NK-C cocktail comprises 1-5 ug IL-12, 2-10 x 10 6 UI IL-2 and 200-600 ng IL-18.
2. The NK cell culture kit according to claim 1, characterized in that, In the kit: The amplification medium contains, in addition to the basal medium, NAD+ at a final concentration of 25 mg / L, human serum albumin at a final concentration of 2 g / L, transferrin at a final concentration of 5 mg / L, β-glucan at a final concentration of 100 mg / L, glutathione at a final concentration of 2 mg / L, β-mercaptoethanol at a final concentration of 2 μg / L, and linoleic acid at a final concentration of 2 mg / L. The highly efficient induction medium contains, in addition to the basal medium, the following components at final concentrations: NAD+ 25 mg / L, human serum albumin 2 g / L, transferrin 5 mg / L, β-glucan 100 mg / L, glutathione 2 mg / L, β-mercaptoethanol 2 μg / L, linoleic acid 2 mg / L, soybean peptide 1.5 g / L, nicotinamide 200 mg / L, inulin 100 mg / L, and N-acetyl-L-cysteine 2 mM. The NK-A coating solution contains 50 mg of heparin sodium, 10 μg of CD16 monoclonal antibody, and 10 μg of 4-1BB antibody; The NK-B cocktail comprises 20 ug Inbakicept, 5 x 10 6 UI IL-2, 200 ng IL-15; The NK-C cocktail comprises 2 ug linoan, 5 x 10 6 UI IL-2, 200 ng IL-18.
3. The NK cell culture kit according to claim 1 or 2, characterized in that, The amplification medium and the high-efficiency induction medium are each dispensed into 1L reagent bottles, and the ratio of the number of bottles containing the amplification medium to the number of bottles containing the high-efficiency induction medium is 1:
2.
4. The NK cell culture kit according to claim 1 or 2, characterized in that, The NK-A coating solution, the NK-B mixture, and the NK-C mixture are each dispensed into 1mL reagent bottles, and the ratio of the number of NK-A coating solution, the NK-B mixture, and the NK-C mixture in the bottles is 1:1:
2.
5. The NK cell culture kit according to any one of claims 1 to 4, characterized in that, The basal culture medium is one of DMEM / F12, IMDM or RPMI-1640.
6. A method for culturing NK cells, characterized in that, Includes the following steps: On day 0, T75 culture flasks were coated with the NK-A coating solution from the kit as described in claim 1; the NK-B mixture from the kit as described in claim 1 was mixed evenly with high-efficiency induction medium to obtain high-efficiency induction mixed culture medium for later use; the NK-C mixture from the kit as described in claim 1 was mixed evenly with amplification medium to obtain amplification mixed culture medium for later use. On day 1, firstly, PBMCs were resuspended in the highly efficient induction mixed culture medium to obtain a cell suspension; secondly, the cell suspension was seeded into coated T75 culture flasks, and the highly efficient induction mixed culture medium was added to control the cell density at 1.5 × 10⁻⁶ cells / year. 6 The culture flasks were placed in a 37°C, 5% CO2 incubator for static incubation. On days 3 and 5, the highly efficient induction mixed culture medium was added again, and the cell density was controlled at 0.5~0.8×10⁻⁶. 6 Cells / mL, continue culturing; On day 7, firstly, the cells and culture medium in the culture flask were transferred to a 10L culture bag, and the amplification mixed culture medium was added to maintain the cell density at 0.8~1.0×10⁻⁶ cells / mL. 6 The cells / mL were placed in a 37℃, 5% CO2 incubator for static incubation. From day 9 to day 13, the aforementioned amplification mixed culture medium was added every 2-3 days, and the cell density was controlled at 1.0-2.0 × 10⁶ cells / day. 6 Cells / mL, continue culturing; On day 14, stop culturing; first, collect the cells and wash them repeatedly with PBS 2-3 times; second, centrifuge the washed cells at 500g for 10 minutes, remove the supernatant, and collect the cell pellet to obtain high-purity NK cells.
7. The cultivation method according to claim 6, characterized in that, On day 0, the coating process for the T75 culture flask is as follows: First, add the NK-A coating solution from the kit to the T75 culture flask and incubate at 2-8°C for 16 hours. After incubation, use a pipette to remove the remaining NK-A coating solution from the T75 culture flask. Next, tilt the T75 culture flask from the coated side to the uncoated side, and add 10 mL of PBS to the T75 culture flask for washing. After washing, lay the T75 culture flask flat and gently shake the washed T75 culture flask. Finally, stand the T75 culture flask upright and use a pipette to aspirate the washing solution after PBS washing from the bottom of the T75 culture flask to complete the coating treatment of the T75 culture flask, and set it aside for later use.
8. A biological agent, characterized in that, The biological agent comprises NK cells obtained by the culture method as described in claim 6.
9. The use of the biological agent as described in claim 6 in the preparation of drugs for the prevention and treatment of solid tumors.
10. The use of NK cells cultured by the method described in claim 6 in the preparation of drugs for the prevention or treatment of solid tumors.