Serum-free and heterologous component-free NK cell culture medium as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cell therapy, and relates to a serum-free and heterologous component-free NK cell culture medium as well as a preparation method and application thereof. The culture medium comprises a classical culture medium, a proliferation promoting component, vitamins, trace elements, lipids, transferrin, an energy substance and beta-mercaptoethanol, the proliferation promoting component is prepared from human albumin, resveratrol, insulin and hydroxyproline. On the basis of a serum-free and heterologous component-free NK cell culture medium, IL-12, rapamycin, polyinosinic-polycytidylic acid and human AB plasma are additionally added to obtain an activated culture medium, the activated culture medium and an amplification culture medium are jointly used, the culture effect is better, and amplification components IL-2 and human AB plasma are additionally added to obtain an amplification culture medium. Through precise component design, the NK cell in-vitro proliferation efficiency, motility, purity and killing ability are remarkably improved, and key problems existing in a traditional culture medium are efficiently solved.

Description

Technical Field

[0001] This invention belongs to the field of cell therapy technology, and relates to a serum-free and heterologous component-free NK cell culture medium, its preparation method, and its application. Background Technology

[0002] Natural killer cells (NK cells) are key members of the innate immune system, playing an irreplaceable role in immune defense and immune surveillance. Originating from hematopoietic stem cells in the bone marrow, their development is finely regulated by various cytokines and transcription factors, maturing gradually in the bone marrow and other lymphoid organs. Unlike T cells and B cells, NK cells do not require prior antigen sensitization. Utilizing a precise recognition mechanism composed of surface activating and inhibiting receptors, they can rapidly attack target cells such as tumor cells and virus-infected cells, efficiently inducing apoptosis in target cells through the release of perforin and granzymes, and the Fas / FasL pathway.

[0003] In the field of clinical treatment, NK cells show great promise. In cancer treatment, they can kill hematologic malignancies such as leukemia and lymphoma, as well as solid tumors such as lung cancer and liver cancer. Compared with surgery, chemotherapy, and radiotherapy, they can specifically kill tumor cells with less damage to normal tissues and can also overcome some tumor drug resistance. In the treatment of viral infections, they can clear infected cells and inhibit viral replication and spread in cases of chronic hepatitis B, hepatitis C, and AIDS.

[0004] However, the clinical application of NK cells is severely limited by the difficulty of obtaining sufficient quantities of highly active NK cells in vitro, making in vitro culture technology crucial, with the culture medium being the core component. Traditional NK cell culture often relies on serum-containing or heterologous media, which presents numerous problems. In serum-containing media, the source of serum is unstable, and batch-to-batch variations are significant, making it difficult to guarantee the consistency of NK cell culture efficiency, activity, and function, hindering standardization and large-scale culture. Furthermore, serum may carry exogenous contaminants, increasing the risk of cell contamination, and unknown components can interfere with NK cell growth and metabolism.

[0005] Even if some serum-free culture media can support NK cell growth, if they still contain foreign components (such as animal-derived proteins), there are still safety risks and functional impacts. Foreign components may trigger an immune rejection response, threatening the safety of clinical use; at the same time, the presence of foreign components will also increase the complexity of the culture medium composition, affecting the stability and reproducibility of NK cell culture, which is not conducive to its reliable application in clinical treatment.

[0006] Current NK cell culture media on the market generally suffer from low proliferation efficiency, defective cell viability, and poor component stability. Serum-free and heterologous component-free culture media, through clearly defined homologous or synthetic components, completely avoid the drawbacks of serum and heterologous components. They can precisely provide nutrients such as amino acids, glucose, vitamins, and inorganic salts to meet the metabolic needs of cells, precisely regulate the growth environment such as pH and osmotic pressure, and ensure the safety, stability, and consistency of cell culture. Therefore, developing serum-free and heterologous component-free culture media that can efficiently support large-scale, high-quality expansion of NK cells has become a key direction for overcoming the bottlenecks in the clinical application of NK cells and promoting the safe development of NK cell therapy technology. Summary of the Invention

[0007] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a serum-free and heterologous component-free NK cell culture medium, its preparation method and application.

[0008] To achieve this objective, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a serum-free and heterologous component-free NK cell culture medium, the culture medium comprising classical culture medium, proliferation-promoting components, vitamins, trace elements, lipids, transferrin, energy substances, and β-mercaptoethanol, wherein the proliferation-promoting components include human albumin, resveratrol, insulin, and hydroxyproline.

[0010] This invention aims to address the problems existing in current NK cell culture media, including safety concerns arising from serum or heterologous components, and insufficient NK cell proliferation capacity, low viability, and low cytotoxicity in serum-free media. It provides a serum-free and heterologous component-free culture medium that can efficiently expand NK cells, maintain their high activity and cytotoxicity, thus meeting the needs of clinical immunotherapy. This medium can support efficient NK cell proliferation, activity maintenance, and functional regulation, providing crucial culture support for NK cell-based immunotherapy (such as tumor treatment and antiviral therapy), and is an important tool for cell therapy drug development and clinical applications.

[0011] The classic RPMI 1640 culture medium serves as the core matrix, providing a basic nutritional framework to support the fundamental survival of NK cells. Vitamins, as key coenzymes in cell metabolism, participate in energy synthesis and antioxidant processes, reducing reactive oxygen species damage and maintaining NK cell proliferation activity. Trace elements regulate enzyme activity, ensuring the efficient execution of key physiological processes such as glucose metabolism and nucleic acid synthesis, thus enhancing the stability and expansion efficiency of NK cell culture in vitro. Lipids, as core components of the cell membrane, provide structural raw materials for cell proliferation and participate in signaling pathway regulation, maintaining the cytotoxic function of NK cells. Transferrin is responsible for iron ion delivery, preventing proliferation arrest caused by iron deficiency. Energy substances provide energy for NK cell proliferation, supporting the synthesis and metabolic needs during cell division. β-mercaptoethanol maintains the reducing environment of the system, protects the sulfhydryl structure of cytokines and intracellular functional proteins, and reduces oxidative stress damage.

[0012] The proliferation-promoting components include hydroxyproline, which provides nitrogen to NK cells; insulin, which promotes glucose uptake and protein synthesis, and synergistically enhances NK cell proliferation efficiency with cytokines; human albumin, which binds and transports nutrients and lipids; and resveratrol, which scavenges reactive oxygen species (ROS) produced by NK cells in vitro, inhibiting oxidative stress damage to cellular DNA, proteins, and lipids, reducing apoptosis, and improving cell viability and stability during culture. These components work synergistically to promote NK cell proliferation, activity, and functional maintenance.

[0013] Preferably, the culture medium comprises, by concentration, 5-120 mg / L vitamins, 10-50 mg / L transferrin, 1550-5670 mg / L energy substances, 2.5-5 g / L human albumin, 20-50 mg / L hydroxyproline, 2-5 mg / L insulin, 0.035-0.15 mg / mL trace elements, and by molar concentration, 0.6-260 μM lipids, 10-100 μM β-mercaptoethanol, and 10-30 μM resveratrol.

[0014] Vitamin concentrations can be selected at 5 mg / L, 6 mg / L, 7 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, etc. Transferrin concentrations can be selected at 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, etc. Energy concentrations can be selected at 1550 mg / L, 1594 mg / L, 1700 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, 3200 mg / L, 3500 mg / L, 3800 mg / L, 4000 mg / L, 4500 mg / L, 5000 mg / L, 5200 mg / L, 5500 mg / L, 5600 mg / L, etc. For example, the concentrations of trace elements can be selected as 0.0335 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.12 mg / mL, 0.14 mg / mL, 0.15 mg / mL, etc. The molar concentrations of lipids can be selected as 0.6 μM, 1.5 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, 120 μM, 150 μM, 180 μM, 200 μM, 220 μM, 260 μM, etc. The molar concentrations of β-mercaptoethanol can be selected as 10 μM, 20 μM, 50 μM, 80 μM, 100 μM, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0015] The concentrations of human albumin can be selected from 2.5 g / L, 2.8 g / L, 3 g / L, 3.2 g / L, 3.5 g / L, 3.8 g / L, 4 g / L, 4.2 g / L, 4.5 g / L, 4.8 g / L, and 5 g / L, etc. The concentrations of hydroxyproline can be selected from 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, and 50 mg / L, etc. The concentrations of insulin can be selected from 2 mg / L, 2.5 mg / L, 3 mg / L, 3.5 mg / L, 4 mg / L, 4.5 mg / L, and 5 mg / L, etc. The concentrations of resveratrol can be selected from 10 μM, 15 μM, 20 μM, 25 μM, and 30 μM, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0016] Preferably, the vitamin includes any one or a combination of at least two of para-aminobenzoic acid, vitamin B12, thiamine hydrochloride, or biotin.

[0017] Preferably, the lipid comprises any one or a combination of at least two of arachidonic acid, myristic acid, palmitoleic acid, or stearic acid.

[0018] Preferably, the energy substance includes any one or a combination of at least two of glucose, glutamine, or ornithine hydrochloride.

[0019] Preferably, the concentration of para-aminobenzoic acid in the culture medium is 0.05-10 mg / L, the concentration of vitamin B12 is 0.001-0.05 mg / L, the concentration of thiamine hydrochloride is 5-100 mg / L, and the concentration of biotin is 0.05-10 mg / L.

[0020] The concentrations of para-aminobenzoic acid can be selected from 0.05 mg / L, 0.1 mg / L, 0.5 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 8 mg / L, 10 mg / L, etc.; the concentrations of vitamin B12 can be selected from 0.001 mg / L, 0.002 mg / L, 0.005 mg / L, 0.01 mg / L, 0.02 mg / L, 0.05 mg / L, 0.1 mg / L, 0.15 mg / L, 0.2 mg / L, 0.25 mg / L, 0.3 mg / L, 0.35 mg / L, 0.4 mg / L, 0.45 mg / L, 0.5 mg / L, etc.; the concentrations of thiamine hydrochloride can be selected from 5 mg / L, 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, etc.; and the concentrations of biotin can be selected from 0.05 mg / L, 1 mg / L, 2 mg / L, 5 mg / L, 8 mg / L, 10 mg / L, etc. mg / L, 2 mg / L, 5 mg / L, 8 mg / L, 10 mg / L, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0021] Preferably, the molar concentrations of arachidonic acid, myristic acid, palmitoleic acid, and stearic acid in the culture medium are 0.02-20 μM, 0.2-80 μM, 0.2-80 μM, and 0.2-80 μM, respectively.

[0022] The molar concentrations of arachidonic acid can be selected from 0.02 μM, 0.1 μM, 0.5 μM, 1 μM, 2 μM, 5 μM, 8 μM, 10 μM, 12 μM, 15 μM, 18 μM, and 20 μM, etc. The molar concentrations of myristic acid can be selected from 0.2 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, and 80 μM, etc. The molar concentrations of palmitoleic acid can be selected from 0.2 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, and 80 μM, etc. The molar concentrations of stearic acid can be selected from 0.2 μM, 2 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 60 μM, etc. μM, 70 μM, 80 μM, etc., and other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0023] Preferably, the concentration of glucose in the culture medium is 1000-4000 mg / L, the concentration of glutamine is 584-1461.5 mg / L, and the concentration of ornithine hydrochloride is 10-200 mg / L.

[0024] The concentrations of glucose can be selected from 1000 mg / L, 1500 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, 3500 mg / L, 4000 mg / L, etc.; the concentrations of glutamine can be selected from 584 mg / L, 600 mg / L, 700 mg / L, 800 mg / L, 900 mg / L, 1000 mg / L, 1100 mg / L, 1200 mg / L, 1300 mg / L, 1400 mg / L, 1461.5 mg / L, etc.; and the concentrations of ornithine hydrochloride can be selected from 10 mg / L, 20 mg / L, 50 mg / L, 80 mg / L, 100 mg / L, 120 mg / L, 150 mg / L, 180 mg / L, 200 mg / L, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0025] Preferably, the classical culture medium includes RPMI 1640.

[0026] Preferably, the trace elements include a first trace element and a second trace element.

[0027] Preferably, the first trace element includes any one or a combination of at least two of ferric citrate trihydrate, manganese chloride tetrahydrate, cobalt chloride hexahydrate, or nickel sulfate hexahydrate.

[0028] Preferably, the second trace element includes any one or a combination of at least two of the following: ammonium heptamolybdate tetrahydrate, ammonium metavanadate, potassium iodide, potassium bromide, sodium fluoride, aluminum chloride hexahydrate, rubidium chloride, barium acetate, or germanium dioxide.

[0029] Preferably, the concentration of ferric citrate trihydrate in the culture medium is 25-100 μg / L, the concentration of manganese chloride tetrahydrate is 2-8 μg / L, the concentration of cobalt chloride hexahydrate is 0.5-1.5 μg / L, and the concentration of nickel sulfate hexahydrate is 0.3-0.9 μg / L.

[0030] The concentrations of ferric citrate trihydrate can be selected from 25 μg / L, 30 μg / L, 50 μg / L, 80 μg / L, 100 μg / L, etc.; the concentrations of manganese chloride tetrahydrate can be selected from 2 μg / L, 3 μg / L, 4 μg / L, 5 μg / L, 6 μg / L, 7 μg / L, 8 μg / L, etc.; the concentrations of cobalt chloride hexahydrate can be selected from 0.5 μg / L, 0.8 μg / L, 1 μg / L, 1.2 μg / L, 1.5 μg / L, etc.; and the concentrations of nickel sulfate hexahydrate can be selected from 0.3 μg / L, 0.5 μg / L, 0.7 μg / L, 0.9 μg / L, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0031] Preferably, the concentrations of ammonium heptamolybdate tetrahydrate, ammonium metavanadate, potassium iodide, potassium bromide, sodium fluoride, aluminum chloride hexahydrate, rubidium chloride, barium acetate, and germanium dioxide in the culture medium are 2-7.5 μg / L, 1.5-4.5 μg / L, 0.5-2 μg / L, 0.5-2 μg / L, 2-6.5 μg / L, 0.1-1 μg / L, 1-4 μg / L, 1-5 μg / L, and 1-3 μg / L, respectively.

[0032] The concentrations of ammonium heptamolybdate tetrahydrate can be selected at 2 μg / L, 3 μg / L, 4 μg / L, 5 μg / L, 6 μg / L, 7 μg / L, and 7.5 μg / L, etc. The concentrations of ammonium metavanadate can be selected at 1.5 μg / L, 2 μg / L, 2.5 μg / L, 3 μg / L, 3.5 μg / L, 4 μg / L, and 4.5 μg / L, etc. The concentrations of potassium iodide can be selected at 0.5 μg / L, 1 μg / L, 1.5 μg / L, and 2 μg / L, etc. The concentrations of potassium bromide can be selected at 0.5 μg / L, 1 μg / L, 1.5 μg / L, and 2 μg / L, etc. The concentrations of sodium fluoride can be selected at 2 μg / L, 2.5 μg / L, 3 μg / L, 3.5 μg / L, 4 μg / L, 4.5 μg / L, 5 μg / L, and 5.5 μg / L, etc. The concentrations of aluminum chloride hexahydrate can be selected from 0.1 μg / L, 0.2 μg / L, 0.5 μg / L, 0.8 μg / L, and 1 μg / L, etc.; the concentrations of rubidium chloride can be selected from 1 μg / L, 1.5 μg / L, 2 μg / L, 2.5 μg / L, 3 μg / L, 3.5 μg / L, and 4 μg / L, etc.; the concentrations of barium acetate can be selected from 1 μg / L, 2 μg / L, 3 μg / L, 4 μg / L, and 5 μg / L, etc.; and the concentrations of germanium dioxide can be selected from 1 μg / L, 1.5 μg / L, 2 μg / L, 2.5 μg / L, and 3 μg / L, etc. Other specific values ​​within the above ranges can be selected, which will not be elaborated here.

[0033] Preferably, the osmotic pressure of the culture medium is 280-320 mOSm / kg.

[0034] Preferably, the pH of the culture medium is 7.0-7.4.

[0035] The osmotic pressure can be selected from 280 mOSm / kg, 290 mOSm / kg, 300 mOSm / kg, 310 mOSm / kg, 320 mOSm / kg, etc., and the pH can be selected from 7.0, 7.1, 7.2, 7.3, 7.4, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0036] In a second aspect, the present invention provides a method for preparing a serum-free and heterologous component-free NK cell culture medium according to the first aspect, the preparation method comprising: physically mixing the raw materials, adjusting the osmotic pressure and pH of the culture medium, and filtering to remove bacteria, thereby obtaining the culture medium.

[0037] Thirdly, the present invention provides an NK cell culture medium combination, the culture medium combination comprising an activation culture medium and an amplification culture medium; the activation culture medium comprising IL-12, rapamycin, polyinosinic-polycytidylic acid, human AB plasma and basal culture medium; the amplification culture medium comprising IL-2, human AB plasma and basal culture medium; the basal culture medium being the culture medium described in the first aspect.

[0038] IL-12 in the culture medium can bind to IL-2 receptors on the surface of NK cells, directly stimulating NK cell proliferation and activation. Rapamycin can both inhibit T cells (blocking the G1 to S phase) and slightly enhance NK cell activity (upregulating perforin / granzyme B). Polyinosinic-Cyclokinase (Poly I:C), as a TLR3 agonist, can directly activate the NF-κB and IRF3 signaling pathways within NK cells; by regulating the immune microenvironment, it inhibits excessive T cell activation and proliferation. Human AB plasma provides nutrients and growth factors, improving expansion efficiency. It reduces cell death during culture, maintains NK cell activity and functional stability, and ensures the killing ability of cells after proliferation. It has no blood type compatibility limitations and high safety, and can replace fetal bovine serum or autologous plasma, meeting the needs of clinical-grade PBMC-NK culture. The high concentration of plasma at this stage provides the cofactors and adhesion matrix required for initial activation.

[0039] IL-2 in the amplification culture medium binds to the IL-2 receptor on the surface of NK cells, directly stimulating NK cell proliferation and activation. Human AB plasma can synergistically activate NK cell proliferation pathways with IL-2 and IL-12, reducing plasma concentration during the amplification phase to maintain cell growth while reducing component complexity.

[0040] The activation medium contains IL-12 at a concentration of 1-20 ng / mL, rapamycin at a molar concentration of 1-20 nM, polyinosinic-polycytidylic acid at a concentration of 1-20 μg / mL, and human AB plasma at a volume percentage of 5%-10%.

[0041] The concentration of IL-12 can be selected from 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 12 ng / mL, 15 ng / mL, 18 ng / mL, 20 ng / mL, etc.; the molar concentration of rapamycin can be selected from 1 nM, 2 nM, 5 nM, 8 nM, 10 nM, 12 nM, 15 nM, 18 nM, 20 nM, etc.; the concentration of polyinosinic-polycytidylic acid (poly I:C) can be selected from 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 12 μg / mL, 15 μg / mL, 18 μg / mL, 20 μg / mL, etc.; and the volume percentage of human AB plasma can be selected from 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0042] Preferably, the concentration of IL-2 in the amplification culture medium is 500-1500 IU / mL, and the volume percentage of human AB plasma is 1%-5%.

[0043] The concentration of IL-2 can be selected from 500 IU / mL, 600 IU / mL, 700 IU / mL, 800 IU / mL, 900 IU / mL, 1000 IU / mL, 1100 IU / mL, 1200 IU / mL, 1300 IU / mL, 1400 IU / mL, 1500 IU / mL, etc. The volume percentage of human AB plasma can be selected from 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc. Other specific values ​​within the above range can be selected, which will not be elaborated here.

[0044] Fourthly, the present invention provides a method for in vitro culture of NK cells, the method comprising: inoculating human peripheral blood mononuclear cells into the activation medium of the NK cell culture medium combination described in the third aspect and then culturing them in the amplification medium.

[0045] Preferably, the culture process includes fluid replenishment during the activation and amplification of the culture medium to maintain a cell density of (0.5-2) × 10⁻⁶ cells / year. 6 cells / mL.

[0046] Cell density can be selected as 0.5 × 10⁻⁶. 6 cells / mL, 0.8×10 6 cells / mL, 1×10 6 cells / mL, 1.2×10 6 cells / mL, 1.5×106 cells / mL, 1.8×10 6 cells / mL, 2×10 6 cells / mL, etc. Other specific point values ​​within the above range can be selected, and will not be elaborated on here.

[0047] Fifthly, the present invention provides the application of the serum-free and heterologous component-free NK cell culture medium according to the first aspect or the NK cell culture medium combination according to the third aspect in NK cell culture.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] Outstanding safety and compliance: The NK cell culture medium of this invention completely eliminates animal serum (such as FBS) and uses only recombinant proteins with clearly defined components, chemically defined small molecules and a small amount of clinical-grade human AB plasma to construct the system, thereby reducing the risk of xenoimmune response and pathogen transmission from the source and fully meeting the regulatory requirements for clinical application.

[0050] Precise optimization of amplification efficiency: Specific types and concentrations of key components such as amino acids, vitamins, fatty acids, and antioxidants (resveratrol) are determined to precisely match the metabolic needs of NK cells during their rapid proliferation phase; at the same time, a sequential culture strategy of activation (IL-12 + T cell inhibitor) followed by amplification (IL-2) is adopted to achieve refined regulation of the NK cell growth cycle and significantly improve proliferation efficiency.

[0051] Dual guarantee of purity and function: The innovative introduction of rapamycin and polyinosinic-polycytidylic acid (poly I:C) during the activation phase specifically inhibits T cell proliferation in PBMCs, resulting in a high-purity NK cell population (CD3+) in the later stages of expansion. - CD56 + The optimized nutrition system not only ensures cell proliferation but also maintains the highly activated state of NK cells and their powerful cytotoxic function, ensuring their immune-killing activity.

[0052] Stable and easy-to-standardize process: All additive components are clearly defined and the concentration is fixed, which completely avoids the batch-to-batch variation problem caused by traditional serum culture media, making the cell culture process controllable and the results reproducible, laying a solid foundation for the large-scale and standardized production of clinical-grade NK cell products. Attached Figure Description

[0053] Figure 1 This refers to the purity of NK cells cultured on day 14 of Example 1.

[0054] Figure 2 This is the killing efficiency of NK cells cultured for 14 days in Example 1 against K562 cells under different effector-target ratios.

[0055] Figure 3 This refers to the NK cell viability on day 14 of culture in Example 1. Detailed Implementation

[0056] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0057] The insulin was purchased from Tonghua Anruit Biopharmaceutical Co., Ltd., and its product is called recombinant human insulin.

[0058] The transferrin was purchased from Tonghua Anruit Biopharmaceutical Co., Ltd., whose product name is Recombinant Human Transferrin (Total Iron, Solid).

[0059] The human albumin was purchased from Tonghua Anruit Biopharmaceutical Co., Ltd., under the product name "Recombinant Human Albumin (Solid)".

[0060] The IL-2 was purchased from Bio-Techne under the product name Recombinant Human IL-2;

[0061] Human Male AB plasma was purchased from Access Biologicals under the product name Human Male AB Serum;

[0062] The IL-12 was purchased from BD Biosciences under the brand name BD Pharmingen™ Recombinant Human IL-12 (p70);

[0063] Poly I:C was purchased from Shanghai Yuanmu Biotechnology Co., Ltd. under the trade name Yuanmu Poly I:C.

[0064] Rapamycin was a product called Rapamycin purchased from MedChemExpress.

[0065] Preparation Example 1

[0066] This preparation example provides a 1000× first trace element mother liquor, the composition of which and its preparation method are as follows:

[0067]

[0068] Preparation Example 2

[0069] This preparation example provides a 1000× first trace element mother liquor, the composition of which and its preparation method are as follows:

[0070]

[0071] Preparation Example 3

[0072] This preparation example provides a 1000× first trace element mother liquor, the composition of which and its preparation method are as follows:

[0073]

[0074] Preparation Example 4

[0075] This preparation example provides a 1000× second trace element mother liquor, the composition of which and its preparation method are as follows:

[0076]

[0077] Preparation Example 5

[0078] This preparation example provides a 1000× second trace element mother liquor, the composition of which and its preparation method are as follows:

[0079]

[0080] Preparation Example 6

[0081] This preparation example provides a 1000× second trace element mother liquor, the composition of which and its preparation method are as follows:

[0082]

[0083] Example 1

[0084] This embodiment provides a serum-free and heterologous component-free NK cell culture medium, the composition of which is as follows:

[0085]

[0086] The preparation method is as follows: the raw materials are physically mixed, the osmotic pressure is adjusted to 280-320 mOSm / kg, the pH is adjusted to 7.0-7.4, and sterilization is achieved by filtration using a 0.22 µm PES filter membrane.

[0087] Example 2

[0088] This embodiment provides a serum-free and heterologous component-free NK cell culture medium, the composition of which is as follows:

[0089]

[0090] The preparation method is the same as in Example 1.

[0091] Example 3

[0092] This embodiment provides a serum-free and heterologous component-free NK cell culture medium, the composition of which is as follows:

[0093]

[0094] The preparation method is the same as in Example 1.

[0095] Comparative Example 1

[0096] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain human albumin, the concentration of hydroxyproline is adjusted from 20 mg / L to 40 mg / L, the concentration of resveratrol is adjusted from 10 μM to 20 μM, and the concentration of insulin is adjusted from 2 mg / L to 4 mg / L, while other components and contents remain unchanged.

[0097] The preparation method is the same as in Example 1.

[0098] Comparative Example 2

[0099] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain resveratrol, and the concentration of resveratrol is proportionally allocated to the concentrations of human albumin, insulin, and hydroxyproline, while other components and contents remain unchanged.

[0100] The preparation method is the same as in Example 1.

[0101] Comparative Example 3

[0102] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain insulin, and the concentration of insulin is proportionally allocated to the concentrations of human albumin, resveratrol, and hydroxyproline, while the other components and contents remain unchanged.

[0103] The preparation method is the same as in Example 1.

[0104] Comparative Example 4

[0105] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain hydroxyproline. The concentration of hydroxyproline is proportionally allocated to the concentrations of human albumin, resveratrol, and insulin, while other components and contents remain unchanged.

[0106] The preparation method is the same as in Example 1.

[0107] Comparative Example 5

[0108] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain resveratrol, while other components and contents remain unchanged.

[0109] The preparation method is the same as in Example 1.

[0110] Comparative Example 6

[0111] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain hydroxyproline, while other components and contents remain unchanged.

[0112] The preparation method is the same as in Example 1.

[0113] Comparative Example 7

[0114] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain human albumin, while other components and contents remain unchanged.

[0115] The preparation method is the same as in Example 1.

[0116] Comparative Example 8

[0117] This comparative example provides a serum-free and heterologous NK cell culture medium, which differs from Example 1 only in that it does not contain insulin, while other components and contents remain unchanged.

[0118] The preparation method is the same as in Example 1.

[0119] Application Example 1

[0120] This application example provides an NK cell culture medium combination, which includes an activation medium and an amplification medium. The activation medium includes IL-12 20 ng / mL, rapamycin 10 nM, polyinosinic-polycytidylic acid 10 μg / mL, human AB plasma 7.5% (v / v), and a basal medium. The amplification medium includes IL-2 500 IU / mL, human AB plasma 2.5% (v / v), and a basal medium. The basal medium is the serum-free and heterologous component-free NK cell culture medium described in Example 1.

[0121] The preparation method is as follows: IL-12, rapamycin, polyinosinic-polycytidylic acid 10, human AB plasma and basal culture medium are mixed to obtain activation culture medium; basal culture medium is mixed with IL-2 and human AB plasma to obtain amplification culture medium.

[0122] Application Example 2

[0123] This application example provides an NK cell culture medium combination, which includes an activation medium and an amplification medium. The activation medium includes IL-12 10 ng / mL, rapamycin 1 nM, polyinosinic-polycytidylic acid 20 μg / mL, human AB plasma 5% (v / v), and a basal medium. The amplification medium includes IL-2 1000 IU / mL, human AB plasma 1% (v / v), and a basal medium. The basal medium is the serum-free and heterologous component-free NK cell culture medium described in Example 2.

[0124] The preparation method is described in Application Example 1.

[0125] Application Example 3

[0126] This application example provides an NK cell culture medium combination, which includes an activation medium and an amplification medium. The activation medium includes IL-12 5 ng / mL, rapamycin 20 nM, polyinosinic-polycytidylic acid 2 μg / mL, human AB plasma 10% (v / v), and a basal medium. The amplification medium includes IL-2 1500 IU / mL, human AB plasma 5% (v / v), and a basal medium. The basal medium is the serum-free and heterologous component-free NK cell culture medium described in Example 3.

[0127] The preparation method is described in Application Example 1.

[0128] Application Example 4

[0129] This application example provides an NK cell culture medium composition that differs from Application Example 1 only in that "7.5% (v / v) human AB plasma" is replaced with "15% (v / v) human AB plasma" in the activation medium, while other components and contents remain unchanged.

[0130] The preparation method is described in Application Example 1.

[0131] Application Example 5

[0132] This application example provides an NK cell culture medium composition that differs from Application Example 1 only in that "7.5% (v / v) human AB plasma" is replaced with "2% (v / v) human AB plasma" in the activation medium, while other components and contents remain unchanged.

[0133] The preparation method is described in Application Example 1.

[0134] Application Example 6

[0135] This application example provides an NK cell culture medium composition that differs from Application Example 1 only in that the activation medium does not contain rapamycin, and its reduced concentration is proportionally allocated to the concentrations of IL-12 and polyinosinic-polycytidylic acid (poly I:C), while other components and contents remain unchanged.

[0136] The preparation method is described in Application Example 1.

[0137] Application Example 7

[0138] This application example provides an NK cell culture medium composition that differs from Application Example 1 only in that the activation medium does not contain polyinosinic-polycytidylic acid (poly I:C), and its reduced concentration is proportionally allocated to the concentrations of rapamycin and IL-12, while other components and contents remain unchanged.

[0139] The preparation method is described in Application Example 1.

[0140] Application Example 8

[0141] This application example provides an NK cell culture medium composition that differs from Application Example 1 only in that the activation medium does not contain IL-12, and its reduced concentration is proportionally allocated to the concentrations of polyinosinic-polycytidylic acid (PII) and rapamycin, while other components and contents remain unchanged.

[0142] The preparation method is described in Application Example 1.

[0143] Compare and contrast examples 1-8

[0144] This comparative application example provides eight combinations of NK cell culture media. The only difference between this example and application example 1 is that the basal culture medium is replaced by the serum-free and heterologous component-free NK cell culture medium described in example 1, while the other components and contents remain unchanged.

[0145] The preparation method is described in Application Example 1.

[0146] Comparative Application Example 9

[0147] This comparative application example provides an NK cell culture medium, which is based on RPMI 1640 with the addition of 10% FBS (v / v) and contains 500 IU mL IL-2.

[0148] Test Example 1

[0149] Cell culture method: Isolated human peripheral blood mononuclear cells were seeded in activation medium and cultured for 7 days, then transferred to expansion medium and cultured for 14 days. During culture, fluid replacement was performed to maintain the cell density at (0.5-2)×10⁻⁶ cells / day. 6 Approximately [number] cells / mL.

[0150] (1) Proliferation capacity test

[0151] To dynamically and accurately track the entire growth process of NK cells, six key sampling time points were set at days 0, 3, 5, 7, 11, and 14. At each time point, aseptic procedures were performed in a biosafety cabinet. 0.5 mL of cell suspension was aspirated from each culture flask, transferred to sterile 1.5 mL centrifuge tubes, and labeled with group labels. Each group had three replicates to ensure data reliability. Trypan blue staining and counting were used: 10 μL of cell suspension was mixed with an equal volume of 0.4% trypan blue staining solution and added to a hemocytometer. The cell concentration at each time point was recorded using a CountStar cell counter. The fold increase was calculated based on the culture volume to comprehensively analyze the effect of serum-free and heterologous component-free culture medium on the proliferation capacity and viability stability of NK cells.

[0152] Multiplication factor = Total number of cells at each time point / Initial total number of cells seeded

[0153] (2) Cell purity test

[0154] NK cell immunophenotype assays were performed on day 14 of culture to verify cell purity. 1.0 × 10⁶ cells were collected under aseptic conditions. 6 Transfer cells to flow cytometry tubes, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and wash once to remove residual culture medium. Resuspend cells in 200 μL PBS, and add fluorescently labeled anti-human CD3-FITC antibody and CD56-PE antibody at the recommended dilution ratio in the antibody manufacturer's instructions. Also, prepare isotype control tubes (containing corresponding fluorescently labeled IgG antibodies) to eliminate non-specific binding interference. Incubate the flow cytometry tubes at 4°C in the dark for 30 minutes, gently inverting and mixing every 10 minutes to ensure sufficient antibody binding to cells. After incubation, add 1 mL PBS, centrifuge at 2000 rpm for 5 minutes, discard the supernatant, and wash twice to remove unbound antibodies. Finally, resuspend cells in 0.2 mL PBS and immediately analyze by flow cytometry to determine the percentage of CD3⁻CD56⁺ cells, accurately assessing NK cell purity.

[0155] (3) Cell viability detection

[0156] Collect the cells to be tested, add FVD780 working solution diluted 1:1000 to the cell pellet, and incubate at 4°C in the dark for 30 min; wash with PBS to stop staining, detect by flow cytometry, and count the percentage of FVD780 negative cells, which is the cell viability.

[0157] (4) Lethality test

[0158] Using CFSE-labeled K562 cells as target cells, the cytotoxic function of NK cells on day 14 was detected. Target cells were first prepared: K562 cells in logarithmic growth phase were washed twice with PBS, then CFSE staining solution was added to a final concentration of 5 μM, and incubated at 37°C in the dark for 15 minutes, mixing every 5 minutes. Five volumes of complete culture medium were added to terminate staining, and the cells were centrifuged and washed twice for later use. Unsorted NK cells (effect cells) and CFSE-labeled K562 cells (target cells) were seeded into 96-well plates at effector-to-target ratios (E / T) of 2:1, 1:1, and 0.5:1, with a total volume of 200 μL per well. Target cell control wells (containing only K562 cells) and effector cell control wells (containing only NK cells) were also included. After co-culturing the 96-well plates at 37°C and 5% CO2 for 4 hours, 100 μL of 1:1000 diluted FVD780 working solution was added to each well. After thorough mixing, the plates were incubated at 4°C in the dark for 30 min. The proportion of FVD780 positive cells in CFSE-positive target cells was detected by flow cytometry, the specific killing rate was calculated, and the functional activity of NK cells was systematically evaluated.

[0159] The results of NK cell purity, killing efficiency against K562 cells at different effector-to-target ratios, and NK cell viability cultured to day 14 using the culture medium combination of Example 1 are as follows: Figure 1-3 As shown in Table 1, the purity, expansion fold, cell viability, and K562 killing efficiency (E:T=2:1) ​​of NK cells cultured to day 14 using the culture medium combinations of Application Examples 1-8 and Comparative Application Examples 1-9 are shown in Table 1.

[0160] Table 1

[0161]

[0162] As shown in Table 1, the culture medium combination prepared based on the NK cell culture medium obtained in this invention, when used to culture NK cells, improves the in vitro proliferation efficiency, purity, cell viability, and killing ability of NK cells. Human albumin, resveratrol, insulin, and hydroxyproline have a certain synergistic effect on the above effects. The activating components (rapamycin, IL-12, and polyinosinic-polycytidylic acid) added to the activating medium combination also have a certain synergistic effect on the above effects. Moreover, the amount of human AB plasma added to the activating medium also affects the expansion of NK cells. If the amount added is too low, the expansion fold will be greatly reduced. Although the amount added is too high, the effect on the expansion effect is not significant, but the production cost will be greatly increased.

[0163] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A serum-free and heterologous component-free NK cell culture medium, characterized in that, The culture medium includes classical culture medium, proliferation-promoting components, vitamins, trace elements, lipids, transferrin, energy substances, and β-mercaptoethanol. The proliferation-promoting components include human albumin, resveratrol, insulin, and hydroxyproline.

2. The serum-free and heterologous component-free NK cell culture medium according to claim 1, characterized in that, The culture medium, in terms of concentration, includes vitamins 5-120 mg / L, transferrin 10-50 mg / L, energy substances 1550-5670 mg / L, human albumin 2.5-5 g / L, hydroxyproline 20-50 mg / L, insulin 2-5 mg / L, trace elements 0.035-0.15 mg / mL, and, in terms of molar concentration, lipids 0.6-260 μM, β-mercaptoethanol 10-100 μM, and resveratrol 10-30 μM.

3. The serum-free and heterologous component-free NK cell culture medium according to claim 1 or 2, characterized in that, The vitamins include any one or a combination of at least two of para-aminobenzoic acid, vitamin B12, thiamine hydrochloride or biotin; Preferably, the lipid comprises any one or a combination of at least two of arachidonic acid, myristic acid, palmitoleic acid, or stearic acid; Preferably, the energy substance includes any one or a combination of at least two of glucose, glutamine, or ornithine hydrochloride; Preferably, the concentration of para-aminobenzoic acid in the culture medium is 0.05-10 mg / L, the concentration of vitamin B12 is 0.001-0.05 mg / L, the concentration of thiamine hydrochloride is 5-100 mg / L, and the concentration of biotin is 0.05-10 mg / L. Preferably, the molar concentrations of arachidonic acid, myristic acid, palmitoleic acid, and stearic acid in the culture medium are 0.02-20 μM, 0.2-80 μM, 0.2-80 μM, and 0.2-80 μM, respectively. Preferably, the concentration of glucose in the culture medium is 1000-4000 mg / L, the concentration of glutamine is 584-1461.5 mg / L, and the concentration of ornithine hydrochloride is 10-200 mg / L. Preferably, the classical culture medium includes RPMI 1640.

4. The serum-free and heterologous component-free NK cell culture medium according to any one of claims 1-3, characterized in that, The trace elements include a first trace element and a second trace element; Preferably, the first trace element includes any one or a combination of at least two of ferric citrate trihydrate, manganese chloride tetrahydrate, cobalt chloride hexahydrate, or nickel sulfate hexahydrate; Preferably, the second trace element includes any one or a combination of at least two of the following: ammonium heptamolybdate tetrahydrate, ammonium metavanadate, potassium iodide, potassium bromide, sodium fluoride, aluminum chloride hexahydrate, rubidium chloride, barium acetate, or germanium dioxide. Preferably, the concentration of ferric citrate trihydrate in the culture medium is 25-100 μg / L, the concentration of manganese chloride tetrahydrate is 2-8 μg / L, the concentration of cobalt chloride hexahydrate is 0.5-1.5 μg / L, and the concentration of nickel sulfate hexahydrate is 0.3-0.9 μg / L. Preferably, the concentrations of ammonium heptamolybdate tetrahydrate, ammonium metavanadate, potassium iodide, potassium bromide, sodium fluoride, aluminum chloride hexahydrate, rubidium chloride, barium acetate, and germanium dioxide in the culture medium are 2-7.5 μg / L, 1.5-4.5 μg / L, 0.5-2 μg / L, 0.5-2 μg / L, 2-6.5 μg / L, 0.1-1 μg / L, 1-4 μg / L, 1-5 μg / L, and 1-3 μg / L, respectively. Preferably, the osmotic pressure of the culture medium is 280-320 mOSm / kg; Preferably, the pH of the culture medium is 7.0-7.

4.

5. The method for preparing serum-free and heterologous component-free NK cell culture medium according to any one of claims 1-4, characterized in that, The preparation method includes: physically mixing the raw materials, adjusting the osmotic pressure and pH of the culture medium, and filtering to remove bacteria, thereby obtaining the final product.

6. An NK cell culture medium composition, characterized in that, The culture medium combination includes an activation medium and an amplification medium; the activation medium includes IL-12, rapamycin, polyinosinic-polycytidylic acid, human AB plasma and basal medium; the amplification medium includes IL-2, human AB plasma and basal medium; the basal medium is the serum-free and heterologous component-free NK cell culture medium according to any one of claims 1-4.

7. The NK cell culture medium combination according to claim 6, characterized in that, The activation medium contains IL-12 at a concentration of 1-20 ng / mL, rapamycin at a molar concentration of 1-20 nM, polyinosinic-polycytidylic acid at a concentration of 1-20 μg / mL, and human AB plasma at a volume percentage of 5%-10%. Preferably, the concentration of IL-2 in the amplification culture medium is 500-1500 IU / mL, and the volume percentage of human AB plasma is 1%-5%.

8. A method for in vitro culture of NK cells, characterized in that, The method includes: Human peripheral blood mononuclear cells are seeded into the activation medium of the NK cell culture medium combination according to claim 6 or 7 and cultured, and then cultured in the expansion medium.

9. The method for in vitro culture of NK cells according to claim 8, characterized in that, The culture process, including activation and amplification, also includes fluid replenishment to maintain cell density at (0.5-2) × 10⁻⁶. 6 cells / mL.

10. The use of the serum-free and heterologous component-free NK cell culture medium according to any one of claims 1-4 or the NK cell culture medium combination according to claim 6 or 7 in NK cell culture.

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