NK cell culture kit and NK cell culture method applying same
By optimizing the pretreatment solution and cytokine combination culture media at different stages in the NK cell culture kit, the problems of limited NK cell expansion and low killing activity in the existing technology have been solved, achieving efficient induction, activation and rapid expansion of NK cells, and obtaining functionally mature NK cells with high killing activity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GUANGDONG PROCAPZOOM BIOSCIENCES CO LTD
- Filing Date
- 2026-02-04
- Publication Date
- 2026-05-12
AI Technical Summary
Existing high-concentration IL-2 single culture systems have problems such as limited expansion fold and low killing activity during NK cell expansion, and it is difficult to simulate the natural development process of NK cells from activation to functional maturity in vivo.
An NK cell culture kit is provided, comprising a pretreatment solution, a first culture medium, a second culture medium, and a third culture medium. By using a specific pretreatment solution and a combination of cytokines and culture media for different stages, the kit targets the activation, invigoration, and expansion stages of NK cells. By utilizing specific cytokines and a basal culture medium, the kit optimizes the concentration of cytokines and the amount of nutrients added at each stage, thereby achieving efficient induction, activation, and rapid expansion of NK cells.
This study achieved efficient induction, activation, and rapid expansion of NK cells, increasing the expansion rate, cell viability, and cytotoxic activity of NK cells against tumor cells, resulting in functionally mature NK cells with high cytotoxic activity.
Smart Images
Figure CN122012392A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of cell culture technology, specifically to an NK cell culture kit and a method for NK cell culture using the same. Background Technology
[0002] Natural killer cells (NK cells), as core effector cells of the innate immune system, play an irreplaceable role in tumor immune surveillance and clearance. The ability of NK cells to recognize and kill malignant transformed cells and virus-infected cells without relying on the major histocompatibility complex (MHC) makes them highly promising candidate cells in the field of adoptive cell immunotherapy. In recent years, immunotherapies based on in vitro expanded NK cells have demonstrated good safety and preliminary efficacy in clinical studies of hematological malignancies and some solid tumors, driving the rapid development of NK cell preparation technology.
[0003] Currently, the in vitro culture and expansion of NK cells mainly uses mononuclear cells derived from peripheral blood or umbilical cord blood as starting cells, and stimulates and expands them by adding various cytokines. Interleukin-2 (IL-2) has long been considered a fundamental cytokine for maintaining and expanding NK cells. However, traditional monoculture systems based on high-dose IL-2 have significant limitations: First, while IL-2 strongly stimulates NK cell proliferation, it also non-specifically expands a large number of coexisting T cells (especially regulatory T cells) in PBMCs, leading to low purity of the final cell product (CD56). + CD3 - The low proportion of NK cells and the potential for T-cell contamination can lead to graft-versus-host disease (GvHD). Furthermore, continuous high-intensity IL-2 stimulation can easily lead to NK cell depletion or senescence, manifested as decreased cytotoxic activity and weakened effector secretion. Moreover, the lack of a phased, regulated culture strategy makes it difficult to mimic the natural developmental process of NK cells in vivo from activation and expansion to functional maturity, resulting in limited NK cell expansion. Therefore, using peripheral blood or umbilical cord blood mononuclear cells as starting cells and inducing their culture using a single high-concentration IL-2 culture system presents challenges in terms of limited NK cell expansion and low cytotoxic activity, significantly limiting the clinical translation and application of NK cell immunotherapy. Summary of the Invention
[0004] To address the limitations of existing single-culture systems containing high concentrations of IL-2 in inducing mononuclear cells to obtain NK cells, which result in limited NK cell expansion and low NK cell cytotoxic activity, this invention provides an NK cell culture kit and a method for NK cell culture using the kit.
[0005] According to a first aspect of the present invention, an NK cell culture kit is provided, the kit comprising a pretreatment solution, a first culture medium, a second culture medium, and a third culture medium; The pretreatment solution includes at least one of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody; The first culture medium includes a first cytokine and a basal culture medium. The first cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, stem cell growth factor (SCF), anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The second culture medium includes a second cytokine and a basal culture medium. The second cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, stem cell growth factor (SCF), and anti-human CD56 monoclonal antibody. The third culture medium includes a third cytokine and a basal culture medium, wherein the third cytokine includes at least one of IL-1α, IL-2, IL-7, IL-15, and IL-21.
[0006] This invention combines a pretreatment solution containing the above-mentioned components, a first culture medium, a second culture medium, and a third culture medium as an NK cell culture kit. By employing the aforementioned specific pretreatment solution and three culture media with different functional and cytokine combinations, the first, second, and third culture media are respectively targeted at different cell growth stages. The first culture medium is mainly used for the NK cell activation stage, the second culture medium is mainly used for the NK cell amplification stage, and the third culture medium is mainly used for the NK cell expansion stage. When using this NK cell culture kit to culture peripheral blood mononuclear cells (PBMCs) or umbilical cord blood mononuclear cells (CBMCs), firstly, the first culture medium... The first, second, and third culture media can adapt to the needs of cells at different growth stages, enabling cells to maintain high cell viability and extremely high expansion rates at each stage. This allows for the efficient induction and differentiation of PBMCs or CBMCs into NK cells. Secondly, the combination of cytokines in the first and second culture media can effectively enrich and directionally induce NK precursor cells, while the pretreatment solution can improve the positive induction rate of NK cells, which is beneficial for better inducing mononuclear cells to differentiate into NK cells. Thirdly, the cytokines in the third culture medium can enhance the function of NK cells, improve the cytotoxicity of the obtained NK cells, and give NK cells high killing activity against tumor cells. Therefore, the NK cell culture kit provided by this invention can achieve efficient induction, activation, and rapid expansion of NK cells to obtain functionally mature NK cells with high killing activity against tumor cells.
[0007] Preferably, the concentrations of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody in the pretreatment solution are all 0.5~50 μg / mL.
[0008] Preferably, the first cytokine includes IL-2, IL-12, IL-15, IL-18 and IL-21, wherein the concentration of IL-2 in the first culture medium is 100~2000 U / mL, and the concentrations of IL-12, IL-15, IL-18 and IL-21 in the first culture medium are all 5~50 ng / mL. Preferably, the second cytokine includes IL-2, IL-12, IL-15, IL-18 and IL-21, wherein the concentration of IL-2 in the second culture medium is 100~2000 U / mL, and the concentrations of IL-12, IL-15, IL-18 and IL-21 in the second culture medium are all 5~50 ng / mL. Preferably, the third cytokine includes IL-2, IL-15 and IL-21, with IL-2 having a concentration of 100-2000 U / mL in the third culture medium, and IL-15 and IL-21 having concentrations of 5-50 ng / mL in the third culture medium.
[0009] In the first, second, and third culture media, the above-mentioned cytokines were used and their concentrations were controlled within the above-mentioned ranges. The cytokines could produce a synergistic effect, which could improve the differentiation efficiency of functional NK cell subsets, thereby increasing the NK cell induction positivity rate and enhancing the NK cell expansion, cell viability, and killing activity against tumor cells.
[0010] In the process of culturing and inducing NK cells using umbilical cord blood mononuclear cells (CBMCs) as the starting cells, in addition to IL-2, IL-12, IL-15, IL-18, and IL-21, the first cytokines also include FLT3 Ligand, SCF, and anti-human CD16 monoclonal antibody. The concentrations of FLT3 Ligand and SCF in the first culture medium are both 5–50 ng / mL, with FLT3 Ligand preferably at 20 ng / mL and SCF preferably at 40 ng / mL. The concentration of anti-human CD16 monoclonal antibody in the first culture medium is 1–5 μg / mL, with anti-human CD16 monoclonal antibody preferably at 1.5 μg / mL. In addition to IL-2, IL-12, IL-15, IL-18, and IL-21, the second cytokines also include FLT3 Ligand and SCF. The concentrations of FLT3 Ligand and SCF in the second culture medium are both 5–50 ng / mL, with FLT3 Ligand preferably at 20 ng / mL. The concentration of SCF in the second culture medium is preferably 40 ng / mL; the third culture medium also includes 5-50 ng / mL IL-7, preferably 10 ng / mL IL-7.
[0011] In the process of culturing and inducing NK cells using peripheral blood mononuclear cells (PBMCs) as the starting cells, in addition to IL-2, IL-12, IL-15, IL-18, and IL-21, the first cytokine also includes at least one of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The concentrations of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody in the first culture medium are all 0.5~50 μg / mL. Preferably, the first cytokine also includes anti-human CD16 monoclonal antibody and anti-human CD56 monoclonal antibody, with the concentration of anti-human CD16 monoclonal antibody in the first culture medium being 10~20 μg / mL and the concentration of anti-human CD56 monoclonal antibody in the first culture medium being 5~15 μg / mL. μg / mL; In addition to IL-2, IL-12, IL-15, IL-18 and IL-21, the second cytokine also includes anti-human CD56 monoclonal antibody, the concentration of anti-human CD56 monoclonal antibody in the first culture medium is 0.5~50 μg / mL, preferably, the concentration of anti-human CD56 monoclonal antibody in the first culture medium is 10~20 μg / mL.
[0012] Preferably, the concentration of IL-2 in the first culture medium is 2000 U / mL, the concentration of IL-12 in the first culture medium is 20 ng / mL, the concentration of IL-15 in the first culture medium is 15 ng / mL, the concentration of IL-18 in the first culture medium is 30 ng / mL, and the concentration of IL-21 in the first culture medium is 15 ng / mL.
[0013] Preferably, the concentration of IL-2 in the second culture medium is 2000 U / mL, the concentration of IL-12 in the second culture medium is 20 ng / mL, the concentration of IL-15 in the second culture medium is 15 ng / mL, the concentration of IL-18 in the second culture medium is 30 ng / mL, and the concentration of IL-21 in the second culture medium is 15 ng / mL.
[0014] Preferably, the concentration of IL-2 in the third culture medium is 2000 U / mL, the concentration of IL-7 in the third culture medium is 10 ng / mL, the concentration of IL-15 in the third culture medium is 15 ng / mL, and the concentration of IL-21 in the third culture medium is 50 ng / mL.
[0015] By optimizing the concentrations of various cytokines in the first, second, and third culture media, NK cells can maintain rapid proliferation and greatly reduce the metabolic stress caused by high concentrations of cytokines. This allows the cells to maintain high viability throughout the culture cycle, reduces the proportion of apoptosis, and enables the final NK cells to achieve an optimal balance in various performance indicators (including expansion rate, cell viability, and cytotoxicity).
[0016] Preferably, the pretreatment solution includes anti-human CD16 monoclonal antibody and anti-human CD56 monoclonal antibody, with the concentration of anti-human CD16 monoclonal antibody in the pretreatment solution being 8-12 μg / mL and the concentration of anti-human CD56 monoclonal antibody in the pretreatment solution being 14-16 μg / mL.
[0017] Using anti-human CD16 monoclonal antibody and anti-human CD56 monoclonal antibody as pretreatment solutions and controlling their concentrations within the above-mentioned ranges, the induction culture of starting cells (monocytes) can be improved by first using the pretreatment solution containing the above components for plating.
[0018] Preferably, the pretreatment solution also includes Dulbecco's phosphate buffer (DPBS).
[0019] Preferably, the above-mentioned basic culture medium includes at least one of BC-515NK serum-free medium, AMMS NK serum-free medium, ALyS505NK-EX serum-free medium, Ham's F-12 medium, IMDM medium, alpha MEM medium, RPMI1640 medium, and NK cell serum-free medium. The serum-free NK cell culture medium consisted of the following components: 1 mM L-glutamine, 2 mM alanyl-glutamine, 2 mM alanyl-alanine, 2 mM glycine, 20 mg / L folic acid, 20 mg / L nicotinamide, 0.3 μg / L sodium selenite, 0.3 μg / L manganese chloride, 0.05 mg / L disodium 5'-triphosphate hydrate, 0.5 mg / L taurine, 2 mg / L ethanolamine, 8 mg / L β-mercaptoethanol, 5 g / L human serum albumin, 2.5 mg / L recombinant human transferrin, 10 mg / L recombinant human insulin, 7 mg / L linoleic acid, 8 mg / L lauric acid, 10 mg / L β-sitosterol, 100 mg / L Tween 20, 100 mg / L polyoxyethylene lauryl ether, and basal medium. The basal medium consisted of Ham's F-12 medium, IMDM medium, and alpha... MEM culture medium is prepared by mixing in a volume ratio of 1:1:2.
[0020] Preferably, the first culture medium further includes 5-15 vol% of a first nutrient, which is selected from at least one of umbilical cord blood plasma, human AB serum, fetal bovine serum, and serum substitutes.
[0021] Preferably, the second culture medium further includes 5-15 vol% of a second nutrient, which is selected from at least one of umbilical cord blood plasma, human AB serum, fetal bovine serum, and serum substitutes.
[0022] Preferably, the third culture medium further includes 0-5 vol% of a third nutrient, which is selected from at least one of umbilical cord blood plasma, human AB serum, fetal bovine serum, and serum substitutes.
[0023] By adding nutrients to the first, second, and third culture media and controlling the amount of nutrients within the aforementioned range, these nutrients can play a crucial nutritional support role in serum-free or low-serum systems. High amounts of nutrients in the first and second culture media provide abundant lipids, hormones, and unknown growth factors during the NK cell activation, induction, and expansion stages, which is beneficial for high-density NK cell expansion and prevents premature plateau phase. Conversely, low amounts of nutrients in the third culture media are more conducive to NK cell functional maturation rather than excessive proliferation. Therefore, by introducing nutrients into the first, second, and third culture media and controlling their amounts within the aforementioned range, NK cell viability can be maintained while ensuring the acquisition of functionally mature NK cells.
[0024] Preferably, the first culture medium also includes trophoblast cells.
[0025] Preferably, the second culture medium also includes trophoblast cells.
[0026] Preferably, the third culture medium also includes trophoblast cells.
[0027] By introducing feeder cells into the first, second, and third culture media, NK cell proliferation and functional maturation can be promoted, thereby obtaining a larger number of NK cells with high killing ability against tumor cells.
[0028] According to a second aspect of the present invention, a method for culturing NK cells is provided, comprising the following steps: S1. Resuspend mononuclear cells in the first culture medium to obtain a cell suspension; S2. Add the NK pretreatment solution to the culture flask and let it stand at room temperature for 30-120 min or at 4℃ for 12-72 h. Add cell suspension to the culture flask for culture, then add the second culture medium to the culture system and continue culturing, and then add the third culture medium to the culture system and continue culturing to obtain NK cells. The pretreatment solution includes at least one of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody; The first culture medium includes a first cytokine and a basal culture medium. The first cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The second culture medium includes a second cytokine and a basal culture medium. The second cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, and anti-human CD56 monoclonal antibody. The third culture medium includes a third cytokine and a basal culture medium, wherein the third cytokine includes at least one of IL-1α, IL-2, IL-7, IL-15, and IL-21.
[0029] In the NK cell culture method provided in this scheme, mononuclear cells are first resuspended in a first culture medium to prepare a cell suspension. Then, NK pretreatment solution is added to the culture flask to cover the entire bottom of the flask, and the cell suspension is added and cultured for a period of time. Then, a second culture medium is added to the culture flask and cultured for a period of time. Finally, a third culture medium is added to the culture flask and cultured for a period of time. Firstly, coating the bottom of the culture flask with the pretreatment solution containing the above components before culturing the cells can improve the positive induction rate of NK cells. Secondly, the first, second, and third culture media can meet the needs of different growth stages of cells, so that cells can maintain high cell viability and extremely high expansion fold at each stage, and can efficiently induce PBMCs or CBMCs to differentiate into NK cells. Thirdly, the combination of cytokines in the first and second culture media can effectively enrich and directionally induce NK precursor cells, while the pretreatment solution can improve the positive induction rate of NK cells, which is conducive to better inducing mononuclear cells to differentiate into NK cells. Fourthly, the cytokines in the third culture medium can enhance the function of NK cells, improve the cytotoxicity of the obtained NK cells, and make NK cells have high killing activity against tumor cells. Therefore, the NK cell culture kit provided by this invention can achieve efficient induction, activation, rapid expansion of NK cells and improve the positive induction rate of NK cells, thereby obtaining functionally mature NK cells with high killing activity against tumor cells.
[0030] Preferably, the pretreatment solution comprises the following components: 8-12 μg / mL anti-human CD16 monoclonal antibody, 14-16 μg / mL anti-human CD56 monoclonal antibody, and Dulbecco's phosphate buffer (DPBS). The first culture medium consists of the following components: 100~2000 U / mL IL-2, 5~50 ng / mL IL-12, 5~50 ng / mL IL-15, 5~50 ng / mL IL-18, 5~50 ng / mL IL-21, 5~15 vol% first nutrient, feeder cells, and basal culture; The second culture medium consists of the following components: 100~2000 U / mL IL-2, 5~50 ng / mL IL-12, 5~50 ng / mL IL-15, 5~50 ng / mL IL-18, 5~50 ng / mL IL-21, 5~15 vol% first nutrient, feeder cells, and basal culture medium; The third culture medium consists of the following components: 100~2000 U / mL IL-2, 5~50 ng / mL IL-15, 5~50 ng / mL IL-21, 0~5 vol% third nutrient, trophoblast cells, and basal culture medium.
[0031] In the process of culturing and inducing NK cells using umbilical cord blood mononuclear cells (CBMCs) as starting cells, the first culture medium also includes 5-50 ng / mL FLT3 Ligand and 5-50 ng / mL SCF, preferably 15-25 ng / mL FLT3 Ligand and 35-45 ng / mL SCF; the second culture medium also includes 5-50 ng / mL FLT3 Ligand and 5-50 ng / mL SCF, preferably 15-25 ng / mL FLT3 Ligand and 35-45 ng / mL SCF; the third culture medium also includes 5-50 ng / mL IL-7, preferably 5-15 ng / mL IL-7.
[0032] In the process of culturing and inducing NK cells using peripheral blood mononuclear cells (PBMCs) as starting cells, the first culture medium further includes at least one of 0.5-50 μg / mL anti-human CD3 monoclonal antibody, 0.5-50 μg / mL anti-human CD16 monoclonal antibody, and 0.5-50 μg / mL anti-human CD56 monoclonal antibody. Preferably, the first culture medium further includes 10-20 μg / mL anti-human CD16 monoclonal antibody and 5-15 μg / mL anti-human CD56 monoclonal antibody. The second culture medium further includes 0.5-50 μg / mL anti-human CD56 monoclonal antibody. Preferably, the second culture medium further includes 10-20 μg / mL anti-human CD56 monoclonal antibody.
[0033] By optimizing the components of the first, second, and third culture media and controlling the concentration or content of each group within the above-mentioned range, the differentiation efficiency of functional NK cell subsets can be improved, thereby increasing the NK cell induction positivity rate and enhancing the NK cell expansion fold, cell viability, and killing activity against tumor cells.
[0034] Preferably, the basal culture medium includes at least one of BC-515NK serum-free medium, AMMS NK serum-free medium, ALYS505NK-EX serum-free medium, Ham's F-12 medium, IMDM medium, alpha MEM medium, RPMI1640 medium, and NK cell serum-free medium. The serum-free NK cell culture medium consisted of the following components: 1 mM L-glutamine, 2 mM alanyl-glutamine, 2 mM alanyl-alanine, 2 mM glycine, 20 mg / L folic acid, 20 mg / L nicotinamide, 0.3 μg / L sodium selenite, 0.3 μg / L manganese chloride, 0.05 mg / L disodium 5'-triphosphate hydrate, 0.5 mg / L taurine, 2 mg / L ethanolamine, 8 mg / L β-mercaptoethanol, 5 g / L human serum albumin, 2.5 mg / L recombinant human transferrin, 10 mg / L recombinant human insulin, 7 mg / L linoleic acid, 8 mg / L lauric acid, 10 mg / L β-sitosterol, 100 mg / L Tween 20, 100 mg / L polyoxyethylene lauryl ether, and basal medium. The basal medium consisted of Ham's F-12 medium, IMDM medium, and alpha... MEM culture medium is prepared by mixing in a volume ratio of 1:1:2.
[0035] In the process of culturing and inducing NK cells using cord blood mononuclear cells (CBMCs) as starting cells, the basal culture medium used includes at least one of ALyS505NK-EX serum-free medium, Ham's F-12 medium, IMDM medium, alpha MEM medium, RPMI 1640 medium, and serum-free NK cell medium.
[0036] In the process of culturing and inducing NK cells using peripheral blood mononuclear cells (PBMCs) as starting cells, the basal culture medium used includes at least one of BC-515NK serum-free medium, AMMS NK serum-free medium, Ham's F-12 medium, IMDM medium, alpha MEM medium, RPMI 1640 medium, and NK cell serum-free medium.
[0037] Preferably, in S1, the cell density of the cell suspension is (1~3)×10⁻⁶. 6 per mL.
[0038] Preferably, in S2, during the process of adding a second culture medium to the culture system and continuing the culture, the cell density in the culture system is adjusted to (0.6~2)×10 using the second culture medium. 6 The operation is performed per mL.
[0039] By optimizing cell seeding density and density during culture, optimal conditions can be provided for rapid cell proliferation. Reasonable cell density can also effectively prevent metabolic acidosis and waste accumulation, which is conducive to maintaining high cell viability. Attached Figure Description
[0040] Figure 1 To analyze the CD3+ levels in the supernatant of the culture medium after adding a third culture medium and culturing for 15 days in Example 1 using flow cytometry. - CD56 + Flow cytometry results of cells.
[0041] Figure 2 To analyze the CD16 content in the supernatant of the culture medium after adding a third culture medium and culturing for 15 days in Example 1 using flow cytometry + (CD3) - CD56 + Flow cytometry results of cells. Detailed Implementation
[0042] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] Example 1 A method for culturing NK cells includes the following steps: (1) Add the pretreatment solution to the culture flask, shake well so that the pretreatment solution covers the entire bottom of the flask, and let it stand at room temperature for 80 minutes before use. The pretreatment solution consists of the following components: 10 μg / mL anti-human CD16 monoclonal antibody, 15 μg / mL anti-human CD56 monoclonal antibody, and DPBS; (2) Mononuclear cells (CBMCs) were isolated from umbilical cord blood and resuspended in the first culture medium to obtain a cell density of 2 × 10⁻⁶ cells / mL. 6 Cell suspension of cells / mL; The first culture medium consisted of the following components: 2000 U / mL IL-2, 20 ng / mL IL-12, 15 ng / mL IL-15, 30 ng / mL IL-18, 50 ng / mL IL-21, 20 ng / mL FLT3 Ligand, 40 ng / mL SCF, 10 vol% umbilical cord blood plasma, 1.5 μg / mL anti-human CD16 monoclonal antibody, feeder cells (mIL-21 / K562 cells, feeder cells to CBMCs in a 1:1 ratio), and basal culture (Ham's F-12 medium). (3) Remove the coated culture flask, discard the excess pretreatment solution, add the cell suspension containing CBMC to the culture flask, inoculate and culture for 4 days. Then, add the second culture medium every 2 days and continue culturing. After culturing in the second culture medium for 4 days, take samples, stain with trypan blue, count the cells, and then adjust the cell density in the culture system to 1×10⁻⁶ cells using the second culture medium. 6 1 live cells / mL were seeded into a coated culture flask and cultured for 4 days. Then, a third culture medium was added every 2 days and cultured for 15 days. Umbilical cord blood NK cells were harvested. The second culture medium consists of the following components: 2000 U / mL IL-2, 20 ng / mL IL-12, 15 ng / mL IL-15, 30 ng / mL IL-18, 50 ng / mL IL-21, 20 ng / mL FLT3 Ligand, 40 ng / mL SCF, 10 vol% umbilical cord blood plasma, trophoblast cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture medium (Ham's F-12 medium). The third culture medium consists of the following components: 2000 U / mL IL-2, 10 ng / mL IL-7, 15 ng / mL IL-15, 50 ng / mL IL-21, 1 vol% umbilical cord blood plasma, trophoblast cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture medium (Ham's F-12 medium).
[0044] Example 2 A method for culturing NK cells includes the following steps: (1) Add the pretreatment solution to the culture flask, shake well so that the pretreatment solution covers the entire bottom of the flask, and let it stand at room temperature for 80 minutes before use. The pretreatment solution consists of the following components: 10 μg / mL anti-human CD16 monoclonal antibody, 15 μg / mL anti-human CD56 monoclonal antibody, and DPBS; (2) Mononuclear cells (PBMCs) were isolated from peripheral blood and resuspended in the first culture medium to obtain a cell density of 2 × 10⁻⁶ cells / mL. 6 Cell suspension of cells / mL; The first culture medium consisted of the following components: 2000 U / mL IL-2, 20 ng / mL IL-12, 15 ng / mL IL-15, 30 ng / mL IL-18, 50 ng / mL IL-21, 15 μg / mL anti-human CD3 monoclonal antibody, 10 μg / mL anti-human CD16 monoclonal antibody, 10 vol% peripheral blood plasma, feeder cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture (Ham's F-12 medium). (3) Remove the coated culture flask, discard the excess pretreatment solution, add the cell suspension containing CBMC to the culture flask, inoculate and culture for 4 days. Then, add the second culture medium every 2 days and continue culturing. After culturing in the second culture medium for 4 days, take samples, stain with trypan blue, count the cells, and then adjust the cell density in the culture system to 1×10⁻⁶ cells using the second culture medium. 6 The cells were seeded at 100 viable cells / mL and cultured in coated culture flasks for 4 days. Then, a third culture medium was added every 2 days and the cells were cultured for 15 days. Peripheral blood NK cells were then harvested. The second culture medium consists of the following components: 2000 U / mL IL-2, 20 ng / mL IL-12, 15 ng / mL IL-15, 30 ng / mL IL-18, 50 ng / mL IL-21, 15 μg / mL anti-human CD56 monoclonal antibody, 10 vol% peripheral blood plasma, feeder cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture medium (Ham's F-12 medium). The third culture medium consists of the following components: 1000 U / mL IL-2, 10 ng / mL IL-15, 20 ng / mL IL-21, feeder cells (mIL-21 / K562 cells, in a ratio of 1:1), and basal culture medium (Ham's F-12 medium).
[0045] Examples 3-7 Examples 3-7 provide a method for NK cell culture. Compared with Example 1, the difference in composition is that the type or concentration of monoclonal antibody in the pretreatment solution is different. The components and concentrations of the pretreatment solution are shown in Table 1.
[0046] Table 1. Components and concentrations of the pretreatment used in Examples 3-7
[0047] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Examples 3-7 are strictly consistent with those in Example 1.
[0048] Examples 8-13 Examples 8-13 provide an NK cell culture method. Compared with Example 1, the difference in composition is that the types or concentrations of cytokines in the first culture medium are different. The cytokines and concentrations used in the first culture medium are shown in Table 2.
[0049] Table 2. Cytokines and concentrations used in the first culture medium in Examples 8-13
[0050] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Examples 8-13 are strictly consistent with those in Example 1.
[0051] Examples 14-19 Examples 14-19 provide a method for NK cell culture. Compared with Example 1, the difference in composition is that the types or concentrations of cytokines in the second culture medium are different. The cytokines and concentrations used in the second culture medium are shown in Table 3.
[0052] Table 3. Cytokines and concentrations used in the second culture medium in Examples 14-19
[0053] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Examples 14-19 are strictly consistent with those in Example 1.
[0054] Examples 20-23 Examples 20-23 provide an NK cell culture method. Compared with Example 1, the difference is that the types or concentrations of cytokines in the third culture medium are different. The cytokines and concentrations used in the third culture medium are shown in Table 4.
[0055] Table 4. Cytokines and concentrations used in the third culture medium in Examples 20-23
[0056] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in Examples 20-23 are strictly consistent with those in Example 1.
[0057] Example 24 This embodiment provides a method for culturing NK cells. Compared with Embodiment 1, the difference in composition is that neither the second culture medium nor the third culture medium contains feeder cells.
[0058] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.
[0059] Example 25 This embodiment provides a method for NK cell culture. Compared with Embodiment 1, the difference in structure is that in step (3), after adding the second culture medium and continuing the culture, the adjustment of the cell density in the culture system to 1×10⁻⁶ is omitted. 6 The procedure involves adding live cells per mL, meaning that after adding the second culture medium and continuing the culture for 4 days, the third culture medium is added.
[0060] Apart from the differences mentioned above, the materials, formulation ratios, and preparation operations used in this embodiment are strictly consistent with those in Example 1.
[0061] Comparative Example 1 A method for culturing NK cells includes the following steps: (1) Mononuclear cells (CBMCs) were isolated from umbilical cord blood and resuspended in the first culture medium to obtain a cell density of 2 × 10⁻⁶ cells / mL. 6 Cell suspension of cells / mL; The first culture medium consisted of the following components: 2000 U / mL IL-2, 20 ng / mL IL-12, 15 ng / mL IL-15, 30 ng / mL IL-18, 50 ng / mL IL-21, 20 ng / mL FLT3 Ligand, 40 ng / mL SCF, 11.5 μg / mL anti-human CD16 monoclonal antibody, 15 μg / mL anti-human CD56 monoclonal antibody, 10 vol% umbilical cord blood plasma, feeder cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture (Ham's F-12 medium). (2) Add the cell suspension containing CBMC to the culture flask, inoculate and culture for 4 days. Then, add the second culture medium every 2 days and continue culturing. After culturing in the second culture medium for 4 days, take samples, stain with trypan blue, count the cells, and then adjust the cell density in the culture system to 1×10⁻⁶ cells using the second culture medium. 6 1 live cells / mL were seeded into a culture flask and cultured for 4 days. Then, a third culture medium was added every 2 days and cultured for 15 days. Umbilical cord blood NK cells were harvested. The second culture medium consists of the following components: 2000 U / mL IL-2, 20 ng / mL IL-12, 15 ng / mL IL-15, 30 ng / mL IL-18, 50 ng / mL IL-21, 20 ng / mL FLT3 Ligand, 40 ng / mL SCF, 10 vol% umbilical cord blood plasma, trophoblast cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture medium (Ham's F-12 medium). The third culture medium consists of the following components: 2000 U / mL IL-2, 10 ng / mL IL-7, 15 ng / mL IL-15, 50 ng / mL IL-21, 1 vol% umbilical cord blood plasma, trophoblast cells (mIL-21 / K562 cells, in a 1:1 ratio), and basal culture medium (Ham's F-12 medium).
[0062] Test case 1. Participants This test case uses NK cells obtained in Examples 1-25 and Comparative Example 1 as test subjects to conduct relevant performance tests.
[0063] 2. Test Content (1) CD3- / CD56+, CD16, NKG2D CD3+ was analyzed in the supernatant of the culture medium after culturing Examples 1-25 and Comparative Example 1 for 15 days with a third culture medium supplemented using flow cytometry. - CD56 +CD16 + (CD3) - CD56 + ) cells were tested, including CD3 - CD56 + (%) refers to the percentage of total NK cells in the analyzed target cell population, CD16 + (CD3) - CD56 + ) refers to CD3 - CD56 + CD16 in the population + The positive rate, i.e., the CD16 positivity rate in the NK cell population. + The percentage of the population (mature NK cells) is determined by the following experimental procedures: a. Cell treatment: The supernatant of the culture medium of Examples 1-25 and Comparative Example 1 after being cultured for 15 days with the addition of the third culture medium was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, 1 mL of washing buffer (physiological saline) was added, the cell pellet was resuspended and mixed, centrifuged at 2000 rpm for 5 minutes, 500 μL of washing buffer was added and the cell pellet was resuspended to obtain a cell suspension; b. Antibody incubation: Add 50 μL of cell suspension to a laminar flow plate. Add 1 μL of each of the three antibodies, Anti-CD3, Anti-CD56, and Anti-CD16, to the cell suspension. Mix well and incubate at 4°C in the dark for 30-60 minutes. After the cells precipitate, wash with a laminar flow cytometer with the washing parameters set to "flow rate 2-8 μL / s, 5-10 wash cycles". Resuspend the washed cell pellet in a flow cytometer with 500 µL of physiological saline to obtain the sample to be tested, which can be used for instrumental detection. c. Flow cytometry: Perform instrument quality control. Specifically, prepare quality control microspheres (BD calibration microspheres CS&T ResearchBeads, manufacturer: Becton, Dickinson and Company, item number: 655050). Take 1 mL of cleaning solution, add 2 µL of quality control microspheres, mix well, load the sample, and run "quality control test" or "CS&T test". After the quality control test is qualified, the above-mentioned sample to be tested is then tested on the instrument.
[0064] (2) Amplification results After adding a third culture medium to Examples 1-25 and Comparative Example 1 and culturing for another 15 days, the supernatant of the culture medium was centrifuged at 300 g for 5 min and the cell pellet was collected. The cells were resuspended in the corresponding third culture medium, and the sample was diluted to obtain a cell suspension. A portion of the cell suspension was taken out and mixed with an equal volume of trypan blue staining solution to obtain a mixture. 10 μL of the mixture was taken into a counting chamber for counting, and the amplification factor was calculated according to the following formula: Amplification factor = Number of harvested cells / Number of inoculated cells.
[0065] (3) Cell activity (target cell killing ability) The cell activity (i.e., the killing ability of NK cells against target cells) of the NK cells obtained in Examples 1-25 and Comparative Example 1 were tested respectively. The specific experimental procedures are as follows: a. Target cell plating: Remove Huh7 cells from the incubator and observe cell growth under an inverted biological microscope. Cell confluence should be approximately 80%–90%. Discard the old culture medium, gently wash with DMEM medium, discard the washed medium, add 0.25% Trypsin-EDTA digestion solution, and incubate at 37°C for 1.5–2 minutes. After most cells detach, stop the digestion with 6 mL of complete culture medium (90% DMEM high glucose + 10% FBS) and resuspend the cells to obtain a cell suspension. Transfer the cell suspension to a 50 mL centrifuge tube, add DMEM medium to rinse the culture flask, transfer the washings to the centrifuge tube, and centrifuge at 250 g for 4 minutes. Discard the supernatant after centrifugation, resuspend the cells in complete culture medium (90% DMEM high glucose + 10% FBS), and count the cells using a cell counter at a rate of 1.0–5.0 × 10⁻⁶. 4 Cells were seeded at a density of 1 live cells / well into E-Plate 16 (6 plates) and cultured overnight; b. Effector cell killing: After Huh7 target cells were monitored for 20-24 hours by the RTCA S16 instrument, effector cells (i.e. NK cells) were added to the corresponding cell amount in E-Plate 16, the killing situation was monitored, and the killing ratio of NK cells to Huh7 target cells (i.e., killing rate) was calculated.
[0066] 3. Experimental Results Table 1. Results of flow cytometry detection, amplification fold, and kill rate.
[0067] CD3+ was analyzed in the supernatant of the culture medium after culturing Examples 1-25 and Comparative Example 1 for 15 days with a third culture medium supplemented using flow cytometry. - CD56 + CD16 + (CD3)- CD56 + The cells were tested, and the fold expansion of NK cells obtained in Examples 1-25 and Comparative Example 1 and their killing rate against Huh7 cells were also detected. The results are shown in Table 1. The supernatant of the culture medium after Example 1 was cultured for 15 days with a third culture medium supplemented once was analyzed by flow cytometry. - CD56 + CD16 + (CD3-CD56) + The cell detection results are as follows: Figure 1 and Figure 2 As shown.
[0068] From Table 1, Figure 1 and Figure 2 It can be known that: In the NK cell culture method provided in Comparative Example 1, mononuclear cells were first resuspended and cultured using the first culture medium, then cultured again using the second culture medium, and finally cultured again without the third culture medium to obtain umbilical cord blood NK cells. Compared with Comparative Example 1, in the NK cell culture method provided in Example 1, the culture flasks were pre-coated with a pretreatment solution before culturing mononuclear cells using the first, second, and third culture media. Test results showed that the CD3+ cells obtained from NK cells cultured using the methods provided in Examples 1-25 were significantly higher than those in Comparative Example 1. - CD56 + CD16 + (CD3) - CD56 + The proportion of NK cells was higher, the expansion rate of NK cells was higher throughout the culture process, and the obtained NK cells had a higher killing rate against Huh7 cells.
[0069] Comparison of test data from Examples 1, 3-7 shows that, in the process of culturing CBMCs to obtain umbilical cord blood NK cells, pre-coating the culture flasks with a pretreatment solution containing 8-12 μg / mL anti-human CD16 monoclonal antibody and 14-16 μg / mL anti-human CD56 monoclonal antibody can improve the NK cell induction positivity rate. Specifically, this is reflected in the fact that, using the method provided in Example 1, the CD3+ NK cells obtained after CBMC culture are... - CD56 + CD16 + (CD3) - CD56 + The proportion of cells was higher than in Examples 3-7.
[0070] Comparison of test data from Examples 1, 8-13, 14-19, and 20-23 shows that after coating culture flasks with pretreatment solution, using a first culture medium containing IL-2, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, and anti-human CD16 monoclonal antibody, a second culture medium containing IL-2, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, and SCF, and a third culture medium containing IL-2, IL-15, and IL-21, and regulating the concentration range of cytokines in the first, second, and third culture media, and culturing CBMCs using the above-mentioned first, second, and third culture media can increase the CD3 concentration in the obtained NK cells. - CD56 + CD16 + (CD3) - CD56 + The percentage of NK cells, the fold increase of NK cells during the entire culture process, and the killing rate of the obtained NK cells against Huh7 cells.
[0071] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.
Claims
1. An NK cell culture kit, characterized in that: The kit includes a pretreatment solution, a first culture medium, a second culture medium, and a third culture medium; The pretreatment solution includes at least one of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The first culture medium includes a first cytokine and a basal culture medium. The first cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The second culture medium includes a second cytokine and a basal culture medium. The second cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, and anti-human CD56 monoclonal antibody. The third culture medium includes a third cytokine and a basal culture medium, wherein the third cytokine includes at least one of IL-1α, IL-2, IL-7, IL-15, and IL-21.
2. The NK cell culture kit as described in claim 1, characterized in that: The first cytokine includes IL-2, IL-12, IL-15, IL-18 and IL-21, wherein the concentration of IL-2 in the first culture medium is 100~2000 U / mL, and the concentrations of IL-12, IL-15, IL-18 and IL-21 in the first culture medium are all 5~50 ng / mL; The second cytokine includes IL-2, IL-12, IL-15, IL-18, and IL-21, and an anti-human CD56 monoclonal antibody. The concentration of IL-2 in the second culture medium is 100-2000 U / mL, and the concentrations of IL-12, IL-15, IL-18, and IL-21 in the second culture medium are all 5-50 ng / mL. The third cytokine includes IL-2, IL-15 and IL-21, wherein the concentration of IL-2 in the third culture medium is 100~2000 U / mL, and the concentrations of IL-15 and IL-21 in the third culture medium are both 5~50 ng / mL.
3. The NK cell culture kit as described in claim 2, characterized in that: The concentration of IL-2 in the first culture medium is 2000 U / mL, the concentration of IL-12 in the first culture medium is 20 ng / mL, the concentration of IL-15 in the first culture medium is 15 ng / mL, the concentration of IL-18 in the first culture medium is 30 ng / mL, and the concentration of IL-21 in the first culture medium is 15 ng / mL. And / or, The concentration of IL-2 in the second culture medium was 2000 U / mL, the concentration of IL-12 in the second culture medium was 20 ng / mL, the concentration of IL-15 in the second culture medium was 15 ng / mL, the concentration of IL-18 in the second culture medium was 30 ng / mL, and the concentration of IL-21 in the second culture medium was 15 ng / mL. And / or, The concentration of IL-2 in the third culture medium was 2000 U / mL, the concentration of IL-15 in the third culture medium was 15 ng / mL, and the concentration of IL-21 in the third culture medium was 50 ng / mL.
4. The NK cell culture kit as described in claim 1, characterized in that: The pretreatment solution includes anti-human CD16 monoclonal antibody and anti-human CD56 monoclonal antibody. The concentration of anti-human CD16 monoclonal antibody in the pretreatment solution is 8~12 μg / mL, and the concentration of anti-human CD56 monoclonal antibody in the pretreatment solution is 14~16 μg / mL.
5. The NK cell culture kit as described in claim 1, characterized in that: The basal culture medium includes at least one of the following: BC-515NK serum-free medium, AMMS NK serum-free medium, ALyS505NK-EX serum-free medium, Ham's F-12 medium, IMDM medium, alpha MEM medium, RPMI 1640 medium, and NK cell serum-free medium. The serum-free NK cell culture medium consisted of the following components: 1 mM L-glutamine, 2 mM alanyl-glutamine, 2 mM alanyl-alanine, 2 mM glycine, 20 mg / L folic acid, 20 mg / L nicotinamide, 0.3 μg / L sodium selenite, 0.3 μg / L manganese chloride, 0.05 mg / L disodium 5'-triphosphate hydrate, 0.5 mg / L taurine, 2 mg / L ethanolamine, 8 mg / L β-mercaptoethanol, 5 g / L human serum albumin, 2.5 mg / L recombinant human transferrin, 10 mg / L recombinant human insulin, 7 mg / L linoleic acid, 8 mg / L lauric acid, 10 mg / L β-sitosterol, 100 mg / L Tween 20, 100 mg / L polyoxyethylene lauryl ether, and basal medium. The basal medium consisted of Ham's F-12 medium, IMDM medium, and alpha... MEM culture medium is prepared by mixing in a volume ratio of 1:1:
2.
6. The NK cell culture kit as described in claim 1, characterized in that: The first culture medium further includes 5-15 vol% of a first nutrient, and / or the second culture medium further includes 5-15 vol% of a second nutrient, and / or the third culture medium further includes 0-4 vol% of a third nutrient; The first nutrient, the second nutrient, and the third nutrient are independently selected from at least one of umbilical cord blood plasma, human AB serum, fetal bovine serum, and serum substitutes.
7. The NK cell culture kit as described in claim 1, characterized in that: The first culture medium further includes trophoblast cells, and / or the second culture medium further includes trophoblast cells, and / or the third culture medium further includes trophoblast cells.
8. A method for culturing NK cells, characterized in that, Includes the following steps: S1. Resuspend mononuclear cells in the first culture medium to obtain a cell suspension; S2. Add the NK pretreatment solution to the culture flask and let it stand at room temperature for 30-120 min or treat it at 4℃ for 12-72 h. Add the cell suspension to the culture flask for culture, then add the second culture medium to the culture system for further culture, and then add the third culture medium to the culture system for further culture to obtain NK cells. The pretreatment solution includes at least one of anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The first culture medium includes a first cytokine and a basal culture medium. The first cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, anti-human CD3 monoclonal antibody, anti-human CD16 monoclonal antibody, and anti-human CD56 monoclonal antibody. The second culture medium includes a second cytokine and a basal culture medium. The second cytokine includes at least one of IL-1α, IL-2, IL-3, IL-6, IL-7, IL-12, IL-15, IL-18, IL-21, FLT3 Ligand, SCF, and anti-human CD56 monoclonal antibody. The third culture medium includes a third cytokine and a basal culture medium, wherein the third cytokine includes at least one of IL-1α, IL-2, IL-7, IL-15, and IL-21.
9. The NK cell culture method as described in claim 8, characterized in that, The pretreatment solution comprises the following components: 8-12 μg / mL anti-human CD16 monoclonal antibody and 14-16 μg / mL anti-human CD56 monoclonal antibody. The first culture medium comprises the following components: 100~2000 U / mL IL-2, 5~50 ng / mL IL-12, 5~50 ng / mL IL-15, 5~50 ng / mL IL-18, 5~50 ng / mL IL-21, 5~15 vol% first nutrient, feeder cells, and basal culture; The second culture medium comprises the following components: 100~2000 U / mL IL-2, 5~50 ng / mL IL-12, 5~50 ng / mL IL-15, 5~50 ng / mL IL-18, 5~50 ng / mL IL-21, 5~15 vol% first nutrient, feeder cells, and basal culture medium; The third culture medium comprises the following components: 100~2000 U / mL IL-2, 5~50 ng / mL IL-15, 5~50 ng / mL IL-21, 0~5 vol% third nutrient, trophoblast cells, and basal culture medium.
10. The NK cell culture method as described in claim 8, characterized in that: In S1, the cell density of the cell suspension is (1~3)×10⁻⁶. 6 cells / mL And / or, In S2, the process of adding a second culture medium to the culture system and continuing the culture includes adjusting the cell density in the culture system to (0.6~2)×10 using the second culture medium. 6 The operation is performed per mL.