NK cell serum-free culture solution and NK cell culture method

Abstract

The invention discloses an NK cell serum-free culture solution and a culture method of NK cells. The NK cell serum-free culture solution comprises a basic matrix, a multi-factor slow-release carrier and a function regulation component, the basic matrix comprises recombinant human serum albumin, recombinant human transferrin, plant-derived lipidosome, 12 core recombinant proteins and 5 micromolecular nutrient substances, and the plant-derived lipidosome is composed of soybean phospholipid and linolenic acid; the multi-factor slow-release carrier is amination modified mesoporous silicon nanoparticles, the amination modified mesoporous silicon nanoparticles load four types of functional factors, the culture solution adopts recombinant human-derived components and plant-derived lipidosome to construct a basic matrix, animal serum dependence is thoroughly abandoned, and the culture solution is a novel culture solution. The serum batch difference, virus pollution and heterogeneous protein sensitization risks are effectively avoided, meanwhile, the concentration of each component is accurate and controllable, and the batch-to-batch consistency and quality stability of the culture solution are remarkably improved.

Description

Technical Field

[0001] This invention belongs to the field of in vitro cell culture technology, and particularly relates to a serum-free culture medium for NK cells and a method for culturing NK cells. Background Technology

[0002] NK cells, as key effector cells in the innate immune system capable of directly killing tumor cells and virus-infected cells, have shown broad application prospects in clinical fields such as tumor immunotherapy and anti-infective immunity. Highly efficient in vitro expansion and functional maintenance technologies are core prerequisites for achieving clinical translation. Currently, NK cell in vitro culture technology is gradually transitioning from traditional serum-containing culture systems to serum-free culture systems. Serum-free culture has become the mainstream research direction due to its ability to avoid issues such as batch-to-batch serum variation and foreign protein contamination. Existing serum-free culture media typically contain basic components such as human or recombinant albumin, transferrin, lipid components, and small molecule nutrients, while also adding cytokines such as IL-2 and IL-15 to regulate cell proliferation. In terms of culture modes, bioreactor suspension culture technology has been developed, and some protocols introduce microcarriers to improve cell adhesion efficiency. By controlling environmental parameters such as rotation speed and oxygen concentration, the cell growth state is optimized, providing fundamental technical support for the large-scale expansion of NK cells and promoting the clinical translation of NK cell therapy.

[0003] Current technologies still have significant shortcomings: First, the sources of serum-free culture media vary, and the concentration control precision of some recombinant proteins or small molecule components is insufficient, making it difficult to guarantee batch-to-batch consistency and failing to fully meet the stringent safety standards for clinical-grade cell preparation. Second, cytokines are mostly added in a single-use manner, making it difficult to achieve precise phased release and synergistic effects, resulting in asynchronous cell proliferation and functional enhancement, and low factor utilization. Third, the combination of functional regulatory components lacks synergistic design, limiting the improvement of key characteristics such as cell damage repair and adaptation to hypoxic environments. Fourth, the degree of automation in the culture process is insufficient, parameter switching relies heavily on manual intervention, the operation process is cumbersome, and it is difficult to achieve standardized control throughout the entire cycle, thus restricting the efficiency of large-scale production. Fifth, carrier materials in some culture systems are difficult to recover efficiently, and the residual amount is difficult to control, further increasing the risks of clinical application of cell products. Summary of the Invention

[0004] To overcome the aforementioned deficiencies in the prior art, this invention provides a serum-free culture medium for NK cells and a method for culturing NK cells, which solves the problems of insufficient safety, consistency, and precise regulation in the existing NK cell culture system.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A serum-free culture medium for NK cells and a method for culturing NK cells, comprising: a basic matrix, a multi-factor sustained-release carrier, and functional regulatory components; The basic matrix contains recombinant human serum albumin, recombinant human transferrin, plant-derived liposomes, insulin, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, nerve growth factor, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor, colony-stimulating factor, interleukin-4, interleukin-6, glucose, glutamine, sodium pyruvate, nucleosides and nucleotides, and amino acids that the human body cannot synthesize. The plant-derived liposomes are composed of soybean lecithin and linolenic acid. The multi-factor sustained-release carrier is aminated mesoporous silica nanoparticles, which are loaded with four types of functional factors: a composite factor of SCF and IL-7, IL-21, IL-12 and IL-37. The functional regulatory components include recombinant human HSP70, betaine, vitamin E succinate, dimethyl oxaloyl glycine, astaxanthin from Haematococcus pluvialis, arginine, and sphingosine-1-phosphate, wherein dimethyl oxaloyl glycine is a HIF-1α stabilizer. The electrolyte concentration of the culture medium is 4-6 mM potassium ions and 1.5-2.0 mM calcium ions, and the osmotic pressure is maintained at 280-300 mOsm / kg.

[0006] Preferably, the concentration range of each component in the base matrix is: recombinant human serum albumin 5-10 g / L, recombinant human transferrin 0.5-1 g / L, soybean lecithin 20-50 μmol / L, and linolenic acid 10-20 μmol / L.

[0007] Preferably, the aminated modified mesoporous silica nanoparticles have a particle size of 40-60 nm, a pore size of 1.5-2.5 nm, and a final concentration of 50-100 μg / mL in the culture medium.

[0008] Preferably, the molar ratio of the four functional factors loaded on the aminated mesoporous silica nanoparticles is 2:3:1:1, where the molar ratio of SCF to IL-7 is 2:1, and the total loading concentration of the four functional factors is 20-30 ng / mL.

[0009] Preferably, the concentration range of each component in the functional regulatory component is as follows: recombinant human HSP70 10-20 ng / mL, betaine 15-25 mM, vitamin E succinate 3-7 μM, dimethyl oxaloyl glycine 15-25 μM, astaxanthin 3-7 μM, arginine 15-25 mM, and sphingosine-1-phosphate 0.5-1.5 μM.

[0010] Preferably, a method for culturing NK cells uses serum-free NK cell culture medium, combined with an automated bioreactor and a microcarrier suspension culture mode, to achieve the full cycle culture of NK cells by continuously adjusting the reactor rotation speed and oxygen concentration, specifically including the following steps: (1) Initial stage of culture: The reactor speed was set to 30-40 rpm to maintain a normal oxygen environment, i.e., oxygen concentration of 21%. Recombinant human HSP70, betaine, and vitamin E succinate in the culture medium were used to repair cell damage. At the same time, the mesoporous silica nanoparticles modified by amylation rapidly released the complex factor of SCF and IL-7 and part of IL-21 to initiate cell proliferation signals. (2) Proliferation enhancement stage: The reactor speed is increased to 40-60 rpm, and a normal oxygen environment is maintained, i.e., oxygen concentration of 21%. IL-21 is continuously released through aminated modified mesoporous silica nanoparticles. The glucose concentration in the culture medium is controlled at 4-6 mM. Cell aggregates of 50-100 μm are formed through microcarriers to promote rapid proliferation of NK cells. (3) Functional adaptation stage: The reactor speed was adjusted to 70-90 rpm, and the oxygen concentration gradient was controlled to 5% on days 8-10 and 2% on days 11-14. IL-12 and IL-37 were slowly released through aminated mesoporous silica nanoparticles. Combined with the synergistic effect of arginine, sphingosine-1-phosphate, dimethyloxaloylglycine and astaxanthin, the killing function of NK cells and their ability to adapt to hypoxia were enhanced. (4) End of culture: NK cells were collected by centrifugation to recover aminated mesoporous silica nanoparticles.

[0011] Preferably, the culture time for the initial culture stage is 1-2 days, the culture time for the proliferation and enhancement stage is 3-7 days, and the culture time for the functional adaptation stage is 8-14 days.

[0012] Preferably, the centrifugation parameters at the end of the culture period are 10,000 rpm for 10 minutes, and the residual amount of aminated modified mesoporous silica nanoparticles after recovery does not exceed 0.01 μg / mL.

[0013] Preferably, no cytokines need to be added during the entire culture process, and the culture medium components and culture parameters are automatically switched through an automated bioreactor.

[0014] The technical effects and advantages of the serum-free NK cell culture medium and NK cell culture method of the present invention are as follows: 1. The culture medium of this invention uses recombinant human components (recombinant human serum albumin, recombinant human transferrin, etc.) and plant-derived liposomes to construct the basic matrix, completely eliminating dependence on animal serum, effectively avoiding the risks of serum batch differences, viral contamination and foreign protein sensitization. At the same time, the concentration of each component is precisely controllable, significantly improving the batch-to-batch consistency and quality stability of the culture medium, providing a safe and reliable basic guarantee for the preparation of clinical-grade NK cells.

[0015] 2. This invention loads different functional factors onto aminated mesoporous silica nanoparticles to achieve phased and differentiated release of the factors—in the initial stage, the rapid release of SCF and IL-7 complex factors initiates proliferation; in the proliferation stage, the continuous release of IL-21 enhances proliferation; and in the functional stage, the slow release of IL-12 and IL-37 enhances the killing function. Combined with the precise molar ratio between the factors, it avoids the problems of concentration fluctuations, short duration of action, and cell overstimulation caused by the traditional one-time addition of factors, and achieves precise adaptation of NK cell growth and functional regulation. The mechanism of phased and differentiated release is that amination modification makes the carrier surface positively charged, which generates differentiated electrostatic / adsorption forces with functional factors with different isoelectric points and molecular properties. The factors bind to the different diffusion rates of the factors in the regular channels with a pore size of 1.5–2.5 nm. Through the loading process, factors that need to be released rapidly are placed near the carrier surface, while factors that need to be released continuously or slowly are placed deep in the channels. Thus, during cell culture, factors are released in sequence according to the order of binding strength from weak to strong and diffusion path from near to far, achieving rapid release of SCF / IL-7, continuous release of IL-21, and slow release of IL-12 / IL-37. At the same time, the dynamic changes in the culture microenvironment further synergistically regulate the release kinetics, ultimately matching the signal requirements of NK cells at each growth stage.

[0016] 3. The recombinant human HSP70 and betaine in the functional regulatory components of this invention can effectively repair cell damage. Dimethyl oxaloyl glycine, astaxanthin and other components work synergistically with cytokines to not only significantly enhance the killing function of NK cells, but also enhance their ability to adapt to hypoxia, thus broadening the applicable scenarios for cells, especially to meet the needs of complex clinical microenvironments.

[0017] 4. This invention combines an automated bioreactor with a microcarrier suspension culture mode. By adjusting parameters such as rotation speed and oxygen concentration in stages, it adapts to the needs of different growth stages of NK cells—the initial stage repairs damage and initiates proliferation; the proliferation stage promotes aggregate formation and rapid proliferation; and the functional stage enhances killing function. The entire culture cycle does not require the addition of cytokines midway, realizing automated and standardized control of the culture process, greatly improving culture efficiency, while ensuring cell uniformity and high activity.

[0018] 5. This invention can efficiently recover aminated mesoporous silica nanoparticles by using specific centrifugation parameters at the end of the culture period, keeping the residual amount at an extremely low level, further reducing the safety risks of cell products and meeting the strict standards for clinical-grade cell preparation.

[0019] 6. This invention eliminates the need for mid-process addition of cytokines during the entire culture process. By automatically switching the culture medium components and culture parameters, it simplifies the operation process, reduces human intervention, lowers operational errors, avoids factor waste, and improves resource utilization efficiency. It is suitable for large-scale, industrialized NK cell preparation. Attached Figure Description

[0020] Figure 1 This is a flowchart of a method for culturing NK cells proposed in this invention. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include," "contain," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "includes..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0023] refer to Figure 1This invention discloses a serum-free culture medium for NK cells and a corresponding culture method. The culture medium consists of a basic matrix containing recombinant human serum albumin and plant-derived liposomes, an aminated mesoporous silica nanoparticle sustained-release carrier loaded with four types of factors including SCF / IL-7 complex factor, and functional regulatory components containing recombinant human HSP70 and dimethyloxaloylglycine, with electrolytes and osmotic pressure maintained within a specific range. The culture method employs an automated bioreactor and microcarrier suspension mode, divided into four stages: initial stage, proliferation enhancement, functional adaptation, and final stage recovery. The entire culture cycle is achieved by precisely controlling the rotation speed and oxygen concentration, eliminating the need for mid-cycle cytokine supplementation. This method can efficiently prepare highly active NK cells, meeting the needs of clinical applications.

[0024] General experimental materials: 1. Cell source: NK cells (CD3+) isolated from peripheral blood of healthy volunteers. - CD56 + Purity ≥ 85%); the separation method used was density gradient centrifugation (Ficoll-PaquePlus separation solution). 2. Core Reagents: Recombinant human serum albumin (Catalog No. A7223), recombinant human transferrin (Catalog No. T3309), soybean lecithin (Catalog No. P5638), linolenic acid (Catalog No. L2376), amino-modified mesoporous silica nanoparticles (custom-made, particle size 40-60nm, pore size 1.5-2.5nm), SCF (Catalog No. 255-SC), IL-7 (Catalog No. 207-IL), IL-21 (Catalog No. 318-21). IL-12 (Catalog No. 219-IL), IL-37 (Catalog No. 7118-IL), Recombinant Human HSP70 (Catalog No. ab21685), Betaine (Catalog No. B3883), Vitamin E Succinate (Catalog No. V3250), Dimethyl Oxaloyl Glycine (Catalog No. D3695), Astaxanthin from Haematococcus pluvialis (Catalog No. A4544), Arginine (Catalog No. A8094), Sphingosine-1-phosphate (Catalog No. S9666); 3. Instruments and equipment: Automated bioreactor (BiostatSTR), microcarriers (C3275, particle size 100-200μm), flow cytometer (CytoFLEX), microplate reader (InfiniteM200), high-speed centrifuge (Centrifuge5810R). 4. Detection kits: NK cell activity detection kit (catalog number KTB175), apoptosis detection kit (catalog number KGA107), and nanoparticle residue detection kit (catalog number E-EL-0198). Example 1 This example provides a serum-free culture medium for NK cells and a method for culturing NK cells, specifically for use with a baseline parameter group. The specific implementation details include: Purpose of implementation: To verify the effects of a serum-free culture system with no animal-derived components and synergistic multi-factor sustained release on NK cell proliferation, activity, and hypoxia adaptation under basic parameter conditions, and to determine the basic feasibility of the core culture system.

[0025] Culture medium: It consists of a basic matrix, a multi-factor sustained-release carrier, and functional regulatory components. The specific preparation method is as follows: Preparation of the basic matrix: 8 g / L of recombinant human serum albumin, 0.8 g / L of recombinant human transferrin, 35 μmol / L of soybean lecithin, 15 μmol / L of linolenic acid, 12 core recombinant proteins (insulin, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, nerve growth factor, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor, colony-stimulating factor, interleukin-4, and interleukin-6, with a final concentration of 10 ng / mL for each protein) and 5 small molecule nutrients (0.1 mmol / L of essential amino acids, 0.05 mmol / L of nucleosides, 5 mM of glucose, 2 mM of glutamine, and 1 mM of sodium pyruvate) were dissolved in serum-free basal buffer (PBS, pH 7.2), stirred and dissolved, and then filtered sterile (0.22 μm filter membrane). Preparation of multi-factor sustained-release carrier: Aminated mesoporous silica nanoparticles (50 nm in diameter and 2 nm in pore size) were loaded with four functional factors in a molar ratio of SCF to IL-7 composite factor (SCF:IL-7=2:1):IL-21:IL-12:IL-37=2:3:1:1, with a total loading concentration of 25 ng / mL. The loading method was to incubate at room temperature for 2 h, centrifuge and wash 3 times (8000 rpm, 5 min), and then resuspend in the above-mentioned basic matrix to make the final concentration of nanoparticles 80 μg / mL. Addition of functional regulating components: Add recombinant human HSP 70 15 ng / mL, betaine 20 mM, vitamin E succinate 5 μM, dimethyl oxaloyl glycine 20 μM, astaxanthin from Haematococcus pluvialis 5 μM, arginine 20 mM, and sphingosine-1-phosphate 1 μM to the above system, adjust the electrolyte concentration to potassium ion 5 mM and calcium ion 1.8 mM, and adjust the osmotic pressure to 290 mOsm / kg to obtain the final product.

[0026] Implementation steps: Initial culture phase (1-2 days): Frozen and thawed NK cells (thawing method: rapid thawing in a 37°C water bath, washing twice with PBS) were seeded into an automated bioreactor, and the serum-free culture medium described above was added at a seeding density of 1×10⁶ cells / day. 6The reactor was set at 35 rpm, maintaining a normal oxygen environment (oxygen concentration 21%), with an incubation temperature of 37℃ and a CO2 concentration of 5%. (2) Proliferation enhancement stage (3-7 days): Keep the culture temperature and CO2 concentration constant, increase the reactor speed to 50 rpm, maintain a normal oxygen environment, control the glucose concentration in the culture medium to 5 mM, and form 50-100 μm cell aggregates through microcarriers; (3) Functional adaptation stage (8-14 days): Keep the culture temperature and CO2 concentration constant, adjust the reactor speed to 80 rpm, and adjust the oxygen concentration gradient to 5% O2 on days 8-10 and 2% O2 on days 11-14; (4) End of culture (day 14): Aminated mesoporous silica nanoparticles were recovered by centrifugation at 10,000 rpm for 10 minutes, and NK cells were collected for subsequent detection.

[0027] Test results: After 14 days of culture, the total number of NK cells reached 1.2 × 10⁻⁶. 11 The number of cells increased 240-fold; CD3 was detected by flow cytometry. - CD56 + Cell purity was 97%; NK cell activity assay (effect-to-target ratio 10:1) showed a 92% killing rate against K562 cells, an 86% killing rate against hypoxic MCF-7 cells, and an 89% killing rate against HeLa cells; apoptosis rate was 3.5%; cell viability under hypoxic conditions (2% O2) was 94%; nanoparticle residue assay showed a residue level of 0.008 μg / mL; batch-to-batch CV value was 5%.

[0028] Example 2 This example provides a serum-free culture medium for NK cells and a method for culturing NK cells, used for culturing NK cells from mesoporous silica nanoparticles of different sizes modified with amino groups. Specific implementation details include: Purpose of implementation: The effects of aminated modified mesoporous silica nanoparticles with different particle sizes, pore sizes and final concentrations on the sustained release of multiple factors and the culture effect of NK cells were verified, and the appropriate range of nanoparticle specifications was determined.

[0029] Culture medium: The culture medium formulation was the same as in Example 1, except that the size and final concentration of the aminated mesoporous silica nanoparticles were adjusted. Specifically, the particle size of the aminated mesoporous silica nanoparticles was 40 nm, the pore size was 1.5 nm, and the final concentration in the culture medium was 50 μg / mL.

[0030] Culture medium: Same as in Example 1, only the nanoparticle parameters were adjusted: particle size 40 nm, pore size 1.5 nm, final concentration 50 μg / mL.

[0031] Implementation steps: The implementation steps are completely consistent with those in Example 1.

[0032] Detection results: After 14 days of culture, the total number of NK cells reached 1.0 × 10⁻⁶. 11 One, multiplication factor 200-fold; CD3 - CD56 + Cell purity 96%; kill rate of K562 cells 90%, kill rate of hypoxic MCF-7 cells 85%, kill rate of HeLa cells 88%; apoptosis rate 4%; cell viability under hypoxic conditions 93%; residual nanoparticle content 0.007 μg / mL; batch-to-batch variability CV value 5%.

[0033] Example 3 This example provides a serum-free NK cell culture medium and a method for culturing NK cells, used for NK cell culture with different functional factor loading ratios. Specific implementation details include: Objective: To verify the effects of different functional factor loading ratios on NK cell proliferation and activity, and to determine the optimal factor loading ratio.

[0034] Culture medium: The culture medium formulation is the same as that in Example 1, except that the proportion and total concentration of functional factors loaded on the aminated mesoporous silica nanoparticles are adjusted. Specifically, the molar ratio of the four functional factors is the composite factor of SCF and IL-7 (SCF:IL-7=2:1):IL-21:IL-12:IL-37=2:2:1:1, and the total loading concentration is 20 ng / mL.

[0035] Implementation steps: The implementation steps are completely consistent with those in Example 1.

[0036] Test results: After 14 days of culture, the total number of NK cells was 1.1 × 10⁻⁶. 11 One, multiplication factor 220-fold; CD3 - CD56 + Purity 96%; killing rates against K562, hypoxic MCF-7, and HeLa cells were 91%, 85%, and 88%, respectively; apoptosis rate 3.8%; hypoxic survival rate 93%; nanoparticle residue 0.008 μg / mL; batch-to-batch CV value 5%.

[0037] Example 4 This example provides a serum-free culture medium for NK cells and a method for culturing NK cells, used for NK cell culture with different culture rotation speed parameters. The specific implementation includes: Objective: To verify the effects of different culture rotation speeds on cell aggregate formation, nutrient delivery, and NK cell culture efficacy, and to determine the appropriate rotation speed range for each culture stage.

[0038] Culture medium: The culture medium formulation is completely consistent with that of Example 1.

[0039] Implementation steps: The implementation steps are the same as in Example 1, except that the reactor speed is adjusted at each stage: 30 rpm for the initial culture stage, 40 rpm for the proliferation enhancement stage, and 70 rpm for the functional adaptation stage.

[0040] Test results: After 14 days of culture, the total number of NK cells reached 1.1 × 10⁻⁶. 11 One, multiplication factor 220-fold; CD3 - CD56 + Cell purity 97%; kill rate of K562 cells 91%, kill rate of hypoxic MCF-7 cells 86%, kill rate of HeLa cells 89%; apoptosis rate 3.6%; cell viability under hypoxic conditions 94%; residual nanoparticle content 0.008 μg / mL; batch-to-batch CV value 5%.

[0041] Example 5 This example provides a serum-free NK cell culture medium and a method for culturing NK cells, used for NK cell culture with different concentrations of functional regulatory components. Specific implementation details include: Objective: To verify the effects of different concentrations of functional regulatory components on the hypoxia adaptation and cytotoxic activity of NK cells, and to determine the appropriate concentration range of functional regulatory components.

[0042] Culture medium: The culture medium formula is the same as that in Example 1, except that the concentration of some components in the functional regulation component is adjusted, specifically: dimethyl oxaloyl glycine 15 μM, astaxanthin 3 μM, and arginine 15 mM.

[0043] Implementation steps: The implementation steps are completely consistent with those of Example 1.

[0044] Test results: After 14 days of culture, the total number of NK cells reached 1.0 × 10⁻⁶. 11 One, multiplication factor 200-fold; CD3 - CD56 + Cell purity 96%; kill rate of K562 cells 90%, kill rate of hypoxic MCF-7 cells 85%, kill rate of HeLa cells 88%; apoptosis rate 4%; cell viability under hypoxic conditions 93%; residual nanoparticle content 0.007 μg / mL; batch-to-batch variability CV value 5%.

[0045] Comparative Example 1 This comparative example provides a conventional serum-free NK cell culture method, including the following: Purpose of implementation: The conventional serum-free NK cell culture method was used as a control to verify the advantages of the culture system of the present invention in terms of cell proliferation, activity, and hypoxia adaptation.

[0046] Culture medium: The standard serum-free culture medium is specifically prepared as follows: containing 5 g / L bovine serum albumin, 0.5 g / L transferrin, IL-250 ng / mL, and IL-1520 ng / mL. The remaining basic components are the same as those in Example 1 (excluding functional regulatory components and multi-factor sustained-release carriers).

[0047] Implementation steps: Static culture mode was used, with an inoculation density of 1×10⁻⁶. 6 Cells / mL, maintaining a normoxic environment (21% O2) throughout the process, culture temperature 37℃, CO2 concentration 5%, IL-2 and IL-15 (at the same concentration as the initial) were added every 3 days during the culture process.

[0048] Test results: After 14 days of culture, the total number of NK cells reached 3×10⁻⁶. 10 One, multiplication factor 60 times; CD3 - CD56 + Cell purity 88%; kill rate of K562 cells 65%, kill rate of hypoxic MCF-7 cells 55%, kill rate of HeLa cells 60%; apoptosis rate 12%; cell viability under hypoxic conditions 65%; batch-to-batch CV value 16%.

[0049] Comparing Examples 1-5 with the comparative examples, it can be seen that the serum-free NK cell culture medium and culture method of the present invention, through the absence of animal-derived basic matrix, the multi-factor sustained-release system mediated by aminated modified mesoporous silica nanoparticles, and the precise three-stage control of rotation speed and oxygen concentration, achieves efficient proliferation of NK cells (proliferation fold of 200-240 times, far exceeding the 60 times of the comparative examples), significantly improves cell purity (96-97% vs 88%) and killing activity (killing rate against various tumor cells 85-92% vs 55-65%), reduces cell apoptosis rate (3.5-4% vs 12%) and batch-to-batch variability (CV value 5% vs 16%), and improves cell hypoxia adaptation ability (survival rate 93-94% vs 65%), and the residual amount of nanoparticles is far below the safety threshold (≤0.01μg / mL). Comparative Examples 1-5 with Comparative Examples: This invention achieves efficient NK cell proliferation (200-240 times vs 60 times) through an animal-free basic matrix, nanoparticle-mediated multi-factor sustained release, and three-stage rotation speed-oxygen concentration regulation. It significantly improves cell purity (96-97% vs 88%) and cytotoxic activity (85-92% vs 55-65%), reduces apoptosis rate (3.5-4% vs 12%) and batch-to-batch variability (5% vs 16%), and enhances hypoxia adaptation ability (93-94% vs 65%). Moreover, the residual amount of nanoparticles is below the safety threshold (≤0.01 μg / mL).

[0050] Comparing Examples 1-5 with Comparative Example 1, the following is a summary of the differences between Examples 1-5 and Comparative Example 1: The core culture protocols of the two are fundamentally different. Examples 1-5 employ a synergistic culture system using an animal-free basic matrix, aminated mesoporous silica nanoparticles for multi-factor sustained release, and three-stage precise control of rotation speed and oxygen concentration, eliminating the need for frequent cytokine supplementation. Comparative Example 1, on the other hand, is a conventional serum-free static culture system containing animal-derived proteins, requiring the addition of IL-2 and IL-15 every 3 days, operating in a constant aerobic environment without multi-factor sustained release or dynamic control design. The differences in culture effects between the two are significant.

[0051] Regarding cell proliferation efficiency, the NK cell proliferation fold of Examples 1-5 remained stable at 200-240 times, with Example 1 achieving the best 240-fold increase, while Comparative Example 1 only reached 60-fold. The proliferation efficiency of the present invention is 3.3-4 times that of conventional methods, enabling efficient large-scale expansion required for clinical use. Regarding cell purity, after culture, CD3+ in Examples 1-5... - CD56 + The purity of NK cells reached 96%-97%, while that of Comparative Example 1 was only 88%, indicating that the system of the present invention can effectively maintain the specific phenotype of NK cells and reduce contamination by other cells.

[0052] Regarding cytotoxic activity and adaptation to the tumor microenvironment, Examples 1-5 showed cytotoxicity rates of 90%-92%, 85%-86%, and 88%-89% against K562, hypoxic MCF-7, and HeLa tumor cells, respectively, compared to only 55%-65% in Comparative Example 1. The cytotoxic activity of the NK cells cultured in this invention was particularly pronounced in the hypoxic tumor microenvironment. In terms of apoptosis rate, Examples 1-5 showed only 3.5%-4%, significantly lower than the 12% in Comparative Example 1. Regarding hypoxia adaptation, the NK cells of Examples 1-5 achieved a survival rate of 93%-94% under 2% O2 conditions, compared to only 65% ​​in Comparative Example 1, confirming that the functional regulatory components of this invention can significantly enhance cell anti-apoptosis and hypoxia adaptation capabilities.

[0053] Regarding batch-to-batch stability and safety, the batch-to-batch CV values ​​of Examples 1-5 were only 5%, lower than the 16% of Comparative Example 1, ensuring standardized production for large-scale culture. The residual amount of aminated modified mesoporous silica nanoparticles in Examples 1-5 was 0.007-0.008 μg / mL, lower than the safety threshold of 0.01 μg / mL, while Comparative Example 1 contained animal-derived protein, posing a potential risk of immunogenicity.

[0054] In summary, this invention, through the synergistic effects of animal-free matrix, multi-factor sustained release, and precise three-stage regulation, comprehensively outperforms conventional culture protocols in terms of NK cell proliferation efficiency, purity, cytotoxic activity, hypoxia adaptability, batch-to-batch stability, and safety. It effectively solves the core defects of traditional NK cell culture technology and provides reliable technical support for the clinical translation of NK cell adoptive immunotherapy.

[0055] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims.

[0056] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A serum-free culture medium for NK cells, characterized in that, include: Basic matrix, multi-factor sustained-release carrier and functional regulatory components; The basic matrix contains recombinant human serum albumin, recombinant human transferrin, plant-derived liposomes, insulin, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, transforming growth factor, nerve growth factor, vascular endothelial growth factor, hepatocyte growth factor, insulin-like growth factor, colony-stimulating factor, interleukin-4, interleukin-6, glucose, glutamine, sodium pyruvate, nucleosides and nucleotides, and amino acids that the human body cannot synthesize. The plant-derived liposomes are composed of soybean lecithin and linolenic acid. The multi-factor sustained-release carrier is aminated mesoporous silica nanoparticles, which are loaded with four types of functional factors: a composite factor of SCF and IL-7, IL-21, IL-12 and IL-37. The functional regulatory components include recombinant human HSP70, betaine, vitamin E succinate, dimethyl oxaloyl glycine, astaxanthin from Haematococcus pluvialis, arginine, and sphingosine-1-phosphate, wherein dimethyl oxaloyl glycine is a HIF-1α stabilizer. The electrolyte concentration of the culture medium is 4-6 mM potassium ions and 1.5-2.0 mM calcium ions, and the osmotic pressure is maintained at 280-300 mOsm / kg.

2. The serum-free NK cell culture medium as described in claim 1, characterized in that, The concentration ranges of each component in the basic matrix are as follows: recombinant human serum albumin 5-10 g / L, recombinant human transferrin 0.5-1 g / L, soybean lecithin 20-50 μmol / L, and linolenic acid 10-20 μmol / L.

3. The serum-free NK cell culture medium as described in claim 1, characterized in that, The aminated mesoporous silica nanoparticles have a particle size of 40-60 nm and a pore size of 1.5-2.5 nm, and a final concentration of 50-100 μg / mL in the culture medium.

4. The serum-free NK cell culture medium as described in claim 3, characterized in that, The molar ratio of the four functional factors loaded on the aminated mesoporous silica nanoparticles is 2:3:1:1, where the molar ratio of SCF to IL-7 is 2:1, and the total loading concentration of the four functional factors is 20-30 ng / mL.

5. The serum-free NK cell culture medium as described in claim 1, characterized in that, The concentration ranges of each component in the functional regulatory component are as follows: recombinant human HSP70 10-20 ng / mL, betaine 15-25 mM, vitamin E succinate 3-7 μM, dimethyl oxaloyl glycine 15-25 μM, astaxanthin 3-7 μM, arginine 15-25 mM, and sphingosine-1-phosphate 0.5-1.5 μM.

6. A method for culturing NK cells, characterized in that, Using the serum-free NK cell culture medium described in claim 1, combined with an automated bioreactor and microcarrier suspension culture mode, the entire NK cell cycle culture is achieved by continuously adjusting the reactor rotation speed and oxygen concentration, specifically including the following steps: (1) Initial stage of culture: The reactor speed was set to 30-40 rpm to maintain a normal oxygen environment, i.e., oxygen concentration of 21%. Recombinant human HSP70, betaine, and vitamin E succinate in the culture medium were used to repair cell damage. At the same time, the mesoporous silica nanoparticles modified by amylation rapidly released the complex factor of SCF and IL-7 and part of IL-21 to initiate cell proliferation signals. (2) Proliferation enhancement stage: The reactor speed is increased to 40-60 rpm, and a normal oxygen environment is maintained, i.e., oxygen concentration of 21%. IL-21 is continuously released through aminated modified mesoporous silica nanoparticles. The glucose concentration in the culture medium is controlled at 4-6 mM. Cell aggregates of 50-100 μm are formed through microcarriers to promote rapid proliferation of NK cells. (3) Functional adaptation stage: The reactor speed was adjusted to 70-90 rpm, and the oxygen concentration gradient was controlled to 5% on days 8-10 and 2% on days 11-14. IL-12 and IL-37 were slowly released through aminated mesoporous silica nanoparticles. Combined with the synergistic effect of arginine, sphingosine-1-phosphate, dimethyloxaloylglycine and astaxanthin, the killing function of NK cells and their ability to adapt to hypoxia were enhanced. (4) End of culture: NK cells were collected by centrifugation to recover aminated mesoporous silica nanoparticles.

7. The method for culturing NK cells as described in claim 6, characterized in that, The culture time for the initial stage is 1-2 days, the culture time for the proliferation and enhancement stage is 3-7 days, and the culture time for the functional adaptation stage is 8-14 days.

8. The method for culturing NK cells as described in claim 6, characterized in that, The centrifugation parameters at the end of the culture period were 10,000 rpm for 10 minutes, and the residual amount of aminated modified mesoporous silica nanoparticles after recovery did not exceed 0.01 μg / mL.

9. The method for culturing NK cells as described in claim 6, characterized in that, The entire culture process does not require the addition of cytokines midway, and the culture medium components and culture parameters are automatically switched through an automated bioreactor.

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