Improved NK cell culture medium and preparation method thereof
By improving the design of NK cell culture medium supplements and complex sustained-release formulations, the problems of insufficient cytokine supply and limited microenvironment regulation in NK cell culture medium have been solved, achieving efficient expansion and functional maintenance of NK cells, which is suitable for the reproductive health of breeding livestock.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-13
- Publication Date
- 2026-04-10
AI Technical Summary
Existing NK cell culture media suffer from insufficient continuous supply of cytokines and limited microenvironment regulation methods, resulting in low NK cell expansion efficiency and affecting the reproductive health of breeding livestock.
Using a modified NK cell culture medium, a combination of supplements and a complex sustained-release formulation is employed, including Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, GlutaMAX™ and ionomycin, as well as trehalose and interleukin encapsulated by recombinant human serum albumin. These are combined with photocurable polymers containing acrylate prepolymers and photoinitiators to form a core-shell structure, enabling the controlled release of interleukin.
It significantly enhances the proliferation rate and killing activity of NK cells, maintains their long-term activated state, reduces the risk of contamination, is suitable for industrial production, and optimizes cell expansion efficiency and functional activity.
Smart Images
Figure CN121825875A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of livestock breeding technology, and in particular to an improved NK cell culture medium and its preparation method. Background Technology
[0002] NK cells are the core cells of the animal's innate immune system. Their main functions include: clearing pathogens (viruses, bacteria, etc.) and reducing reproductive system infections; regulating the immune microenvironment to avoid maternal immune rejection of the embryo (especially in terms of embryo transfer success rate); clearing abnormal cells (such as cancerous cells and apoptotic cells) to ensure the normal physiological function of reproductive organs; and secreting cytokines to regulate the balance of reproductive hormones.
[0003] Reproductive system infections in breeding livestock (such as pigs, cattle, and sheep) are a key factor restricting the propagation of superior breeds. The use of traditional antibiotics easily leads to drug resistance and affects the reproductive function of breeding livestock. Therefore, NK cell culture media can be used to expand the NK cells of breeding livestock in vitro, and then reinfuse them to eliminate pathogens in their reproductive system. However, existing NK cell culture media often suffer from insufficient continuous supply of cytokines and limited microenvironment regulation methods. Therefore, there is an urgent need to develop an improved NK cell culture medium to achieve efficient expansion and functional maintenance of NK cells, providing a better technical solution in the field of livestock breeding. Summary of the Invention
[0004] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an improved NK cell culture medium and its preparation method, which achieves efficient expansion and functional maintenance of NK cells through the optimized combination of supplements and the synergistic effect of a complex sustained-release formulation.
[0005] To achieve the above objectives, this application provides the following technical solution:
[0006] In one aspect, this application provides a modified NK cell culture medium, comprising a basal culture medium, a supplement, and a complex sustained-release formulation;
[0007] The supplement includes: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM and ionomycin;
[0008] The inner layer of the composite sustained-release medium includes a protective agent and interleukin, while the outer layer is a photocurable polymer comprising an acrylate prepolymer and a photoinitiator.
[0009] The supplements include functional ingredients such as Polygonatum polysaccharide and α-linolenic acid, which optimize the nutritional metabolism and activation signals of NK cells. The complex sustained-release formulation achieves the controlled release of interleukins through a core-shell structure, solving the problems of poor stability of interleukins and single nutrient composition in traditional culture media, and ensuring the optimized effect of the culture medium on NK cell proliferation and activity maintenance.
[0010] Specifically, the basal culture medium includes any one of X-VIVO15, KBM581, RPMI-1640, DMEM and IMDM; the mass ratio of the basal culture medium, supplement and compound sustained release is (85-95):(3-10):(1-5).
[0011] Specifically, the supplement contains Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is (20-40):(10-25):(15-25):(10-15):(10-15):(10-15).
[0012] Polysaccharides from Polygonatum can enhance antioxidant capacity, α-linolenic acid combined with methyl-β-cyclodextrin optimizes membrane structure, L-carnitine can regulate energy metabolism, and ionomycin activates the calcium signaling pathway. The components work synergistically within the specified concentration range to avoid cytotoxicity or functional inhibition caused by improper concentration, and significantly improve the proliferation rate, survival rate and killing activity of NK cells.
[0013] Specifically, the protective agent includes trehalose and recombinant human serum albumin; the mass ratio of trehalose to recombinant human serum albumin is (1-1.5):1; the interleukin includes any one of IL-2, IL-12, IL-15, IL-18 and IL-21, and the mass ratio of interleukin to protective agent is 1:(5-15).
[0014] By combining trehalose with recombinant human serum albumin for protection, the vitrification effect of trehalose and the steric hindrance effect of recombinant human serum albumin are utilized to prevent the denaturation of interleukins during freeze-drying and sustained release, significantly improving the retention rate of interleukin activity. The limited mass ratio of interleukin to protectant of 1:(5-15) effectively avoids carrier overload or insufficient protection, ensuring precise protection of interleukins by the inner microenvironment, and providing a key guarantee for the sustained and stable release of the sustained-release formulation.
[0015] Specifically, the photoinitiator is an ethanol solution of a mixture of camphorquinone and triethanolamine, wherein the mass ratio of the camphorquinone and triethanolamine mixture to ethanol is (8-10):(90-92); and the mass ratio of camphorquinone to triethanolamine is 1:(1-2).
[0016] Camphorquinone, acting as a photoinitiator, and triethanolamine, as a co-initiator, are uniformly dispersed in an ethanol solvent. Upon absorbing energy from a light source, they initiate the cross-linking of the acrylate prepolymer, forming a porous sustained-release outer layer. The controlled concentration of the ethanol solution prevents prepolymer hydrolysis, ensuring the activity of the photoinitiator and the mechanical strength of the outer polymer layer. This solves the problems of uneven curing and uncontrollable release in traditional sustained-release carriers, guaranteeing precise control of the interleukin release rate by the composite sustained-release outer layer.
[0017] Specifically, the preparation method of the composite sustained-release compound includes the following steps:
[0018] The protective agent and interleukin were dissolved in an 8-10 mM sodium citrate solution with a pH of 4.0-5.5 to obtain the first solution;
[0019] The first solution was pre-frozen at -20℃ to -80℃ for 24-48 hours, then freeze-dried, ground, and passed through a 40-70μm sieve to obtain freeze-dried particles;
[0020] The acrylate prepolymer and photoinitiator are mixed and desolventized under vacuum at 40-50°C for 1-2 hours. The freeze-dried particles are added at a ratio of 2%-8% of the total mass of the acrylate prepolymer and photoinitiator. After mixing, the mixture is poured into a mold, and the light source is placed 8-12 cm away from the mold. The mixture is irradiated for 10-12 minutes and then demolded to obtain the composite sustained-release body.
[0021] In the preparation method of the composite sustained-release compound, the structural stability and functional consistency of the sustained-release compound are ensured through full-process control of the inner layer freeze-drying and the outer layer photocuring. The protective agent and interleukin are dissolved in a weakly acidic sodium citrate solution, and after freeze-drying, the particles are sieved to obtain uniform particles, avoiding agglomeration that affects release. After vacuum desolvation, photocuring forms a core-shell structure. The setting of light source parameters ensures that the activity of the inner layer interleukin is not impaired and the outer layer polymer is completely cured, solving the problems of active ingredient inactivation and uneven release rate in traditional sustained-release compound preparation, and providing a continuous and stable interleukin environment for NK cell culture.
[0022] Specifically, the mass ratio of the protective agent and interleukin to the sodium citrate solution in the first solution is (10-20):(80-90).
[0023] Specifically, the wavelength of the light source is 450-550nm, and the intensity is 30-50mW / cm². 2 .
[0024] The wavelength of 450-550nm corresponds to the absorption peak of camphorquinone. The intensity parameter ensures that light energy can penetrate the precursor, enabling deep cross-linking of the outer polymer layer. This avoids the phenomenon of surface curing without internal cross-linking, ensuring the consistency of the mechanical strength and porosity of the sustained-release body. It solves the problem of quality fluctuation of the sustained-release body caused by the difference of light source in the traditional photocuring process, and lays the foundation for the large-scale preparation of composite sustained-release bodies.
[0025] Specifically, the composite sustained-release body is a micro-cylinder with a diameter of 100-200 μm and a height-to-diameter ratio of 1:(1-1.5).
[0026] This size ensures that the composite sustained-release medium has a moderate specific surface area, avoiding excessive deposition or excessive loss, while allowing interleukins to be released evenly from the sides and ends of the cylinder. This reduces the burst release effect of traditional spherical particles. The micro-cylindrical morphology settles stably in the culture system, contacts NK cells evenly, promotes the gradient distribution of interleukins, and ensures the consistency of cell activation signals. This solves the problems of uncontrollable morphology and uneven release of traditional sustained-release carriers.
[0027] Specifically, the release period of the composite sustained-release formulation is 7-10 days.
[0028] The release cycle of 7-10 days matches the 5-7 day logarithmic growth phase of NK cell culture in vitro and its functional maintenance requirements. The release rate is precisely controlled by the degree of cross-linking of the outer polymer layer, enabling interleukin to initiate activation in the early stages of culture, maintain proliferation in the middle stages, and enhance function in the later stages, forming a pulsed release curve that conforms to the cell's metabolic rhythm and avoids overactivation or functional depletion caused by concentration fluctuations. The release cycle is coordinated with the frequency of culture medium replenishment, reducing intermediate operational steps, lowering the risk of contamination, and solving the problem of mismatch between the release cycle and cell culture cycle in traditional sustained-release carriers, significantly improving the stability and practicality of the culture system.
[0029] On the other hand, this application provides a method for preparing an improved NK cell culture medium, comprising:
[0030] Adjust the pH of the basal culture medium to 7.2-7.4, sterilize it by filtration through a 0.22μm filter membrane, and set aside for later use;
[0031] Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, GlutaMAX™ and ionomycin, and stir at a stirring rate of 300-400 rpm / min for 5-10 min.
[0032] Continue adding the prepared complex sustained-release medium to the basal culture medium and stir at a stirring rate of 100-200 rpm / min for 1-3 min to obtain the modified NK cell culture medium.
[0033] Compared with the prior art, the beneficial effects of the present invention are:
[0034] By employing a core-shell structure design for a composite sustained-release formulation—with an inner protective layer encapsulating interleukin and an outer photocurable polymer controlling release—the problem of short half-life and large concentration fluctuations associated with traditional exogenous interleukins is solved. The inner trehalose layer, combined with recombinant human serum albumin, protects interleukin activity, while the outer porous polymer controls the release rate, ensuring continuous and uniform release of key factors. This avoids sudden concentration spikes and drops caused by frequent additions, reducing NK cell overactivation or functional exhaustion and maintaining their long-term activated state and effector function. Furthermore, the release cycle of the composite sustained-release formulation is highly matched to the NK cell culture cycle, eliminating the need to replace the formulation midway or frequently add interleukin, reducing the risk of contamination from human error. Simultaneously, the clearly defined concentration range and standardized culture procedures facilitate industrial-scale production, reducing quality control costs. Finally, among the supplements used in this application, Polygonatum polysaccharide enhances antioxidant capacity and inhibits cell apoptosis; α-linolenic acid combined with methyl-β-cyclodextrin optimizes cell membrane phospholipid composition and maintains signal transduction function; L-carnitine promotes fatty acid oxidation and enhances energy supply; and ionomycin activates the calcium signaling pathway and strengthens the expression of cytotoxic molecules. Compared with traditional culture media, this combination increases the proliferation rate of NK cells and comprehensively optimizes cell expansion efficiency and functional activity. Attached Figure Description
[0035] Figure 1 This is a photograph of the modified NK cell culture medium prepared in Example 1 of this application.
[0036] Figure 2 This is a schematic diagram of the preparation process of the improved NK cell culture medium of this application. Detailed Implementation
[0037] The technical solutions in the embodiments of this application will be described in detail below. The described embodiments are only some embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments in this application are within the scope of protection of this application. The present application will be further described below with reference to specific embodiments.
[0038] Example 1
[0039] like Figure 2 As shown, a method for preparing an improved NK cell culture medium includes the following steps:
[0040] 1. First, prepare the composite sustained-release formulation:
[0041] The protective agent (trehalose and recombinant human serum albumin in a 1:1 mass ratio) and interleukin (IL-2) were dissolved in an 8 mM sodium citrate solution at pH 4.0 to obtain the first solution;
[0042] The mass ratio of interleukin (IL-2) to the protective agent (trehalose and recombinant human serum albumin in a 1:1 mass ratio) in the first solution is 1:5; the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a 1:1 mass ratio) and interleukin (IL-2) to the sodium citrate solution in the first solution is 10:90.
[0043] The first solution was pre-frozen at -20℃ for 48 hours, then freeze-dried, ground, and passed through a 40μm sieve to obtain freeze-dried particles.
[0044] The acrylate prepolymer was mixed with an ethanol solution of camphor quinone and triethanolamine. After being desolvated under vacuum at 40°C for 2 hours, the freeze-dried particles were added at a ratio of 2% of the total mass of the ethanol solution of the acrylate prepolymer, camphor quinone and triethanolamine mixture. After mixing, the mixture was poured into a mold, and the light source was placed 8 cm away from the mold for 10 minutes. The composite sustained-release body was then demolded.
[0045] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 8:92; the mass ratio of camphorquinone to triethanolamine is 1:1.
[0046] 2. Adjust the pH of the basal culture medium (X-VIVO15) to 7.2, filter it through a 0.22μm filter membrane for sterilization, and set it aside for later use;
[0047] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM Mix with ionomycin at a stirring rate of 300 rpm / min for 10 min;
[0048] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is 20:10:25:15:15:15;
[0049] 4. Continue adding the prepared complex sustained-release medium to the basal culture medium and stir at 100 rpm / min for 3 min to obtain the modified NK cell culture medium; a picture of the modified NK cell culture medium is shown below. Figure 1 As shown;
[0050] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and complex sustained-release formulation was 85:10:5.
[0051] Example 2
[0052] like Figure 2 As shown, a method for preparing an improved NK cell culture medium includes the following steps:
[0053] 1. First, prepare the composite sustained-release formulation:
[0054] The protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.5:1) and interleukin (IL-12) were dissolved in a 10 mM sodium citrate solution at pH 5.5 to obtain the first solution;
[0055] In this solution, the mass ratio of interleukin (IL-12) to the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.5:1) is 1:15; and in the first solution, the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.5:1) and the interleukin (IL-12) to the sodium citrate solution is 20:80.
[0056] The first solution was pre-frozen at -80℃ for 24 hours, then freeze-dried, ground, and passed through a 70μm sieve to obtain freeze-dried particles;
[0057] The acrylate prepolymer was mixed with an ethanol solution of camphor quinone and triethanolamine. After desolvation under vacuum at 50°C for 1 hour, the freeze-dried particles were added at a ratio of 8% of the total mass of the ethanol solution of the acrylate prepolymer, camphor quinone and triethanolamine mixture. The mixture was then poured into a mold, and the light source was placed 12 cm away from the mold for 12 minutes. The composite sustained-release body was then demolded.
[0058] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 10:90; the mass ratio of camphorquinone to triethanolamine is 1:2.
[0059] 2. Adjust the pH of the basal culture medium (KBM581) to 7.4, and sterilize it by filtration through a 0.22μm filter membrane. Set aside for later use.
[0060] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM Mix with ionomycin at a stirring rate of 400 rpm / min for 5 min;
[0061] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is 40:15:15:10:10:10;
[0062] 4. Continue to add the prepared complex sustained-release medium to the basal culture medium and stir at a stirring rate of 100 rpm / min for 3 min to obtain the modified NK cell culture medium.
[0063] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and complex sustained-release formulation was 95:3:2.
[0064] Example 3
[0065] like Figure 2 As shown, a method for preparing an improved NK cell culture medium includes the following steps:
[0066] 1. First, prepare the composite sustained-release formulation:
[0067] The protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.2:1) and interleukin (IL-15) were dissolved in a 9 mM sodium citrate solution at pH 4.8 to obtain the first solution;
[0068] The mass ratio of interleukin (IL-15) to the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.2:1) is 1:8; the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.2:1) and interleukin (IL-15) to the sodium citrate solution in the first solution is 14:86.
[0069] The first solution was pre-frozen at -50℃ for 32 hours, then freeze-dried, ground, and passed through a 50μm sieve to obtain freeze-dried particles.
[0070] The acrylate prepolymer was mixed with an ethanol solution of camphorquinone and triethanolamine. After being desolvated under vacuum at 45°C for 1.2 hours, the freeze-dried particles were added at a ratio of 4% of the total mass of the ethanol solution of the acrylate prepolymer, camphorquinone and triethanolamine mixture. After mixing, the mixture was poured into a mold, and the light source was placed 9 cm away from the mold for 11 minutes. The composite sustained-release body was then demolded.
[0071] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 9:91; the mass ratio of camphorquinone to triethanolamine is 1:1.4.
[0072] 2. Adjust the pH of the basal medium (RPMI-1640) to 7.3, filter it through a 0.22μm filter membrane for sterilization, and set it aside for later use;
[0073] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM Mix with ionomycin at a stirring rate of 350 rpm / min for 7 min;
[0074] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TMThe mass ratio of iodine to ionomycin is 30:25:15:10:10:10;
[0075] 4. Continue to add the prepared complex sustained-release medium to the basal culture medium and stir at a stirring rate of 150 rpm / min for 2 min to obtain the modified NK cell culture medium.
[0076] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and complex sustained-release formulation was 88:7:5.
[0077] Example 4
[0078] like Figure 2 As shown, a method for preparing an improved NK cell culture medium includes the following steps:
[0079] 1. First, prepare the composite sustained-release formulation:
[0080] The protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.3:1) and interleukin (IL-18) were dissolved in an 8.5 mM sodium citrate solution at pH 4.5 to obtain the first solution;
[0081] The mass ratio of interleukin (IL-18) to the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.3:1) in the first solution is 1:12; the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.3:1) and interleukin (IL-18) to the sodium citrate solution in the first solution is 17:83.
[0082] The first solution was pre-frozen at -60℃ for 38 hours, then freeze-dried, ground, and passed through a 60μm sieve to obtain freeze-dried particles;
[0083] The acrylate prepolymer was mixed with an ethanol solution of camphorquinone and triethanolamine. After desolvation under vacuum at 47°C for 1.5 hours, the freeze-dried particles were added at a ratio of 6% of the total mass of the ethanol solution of the acrylate prepolymer, camphorquinone and triethanolamine mixture. After mixing, the mixture was poured into a mold, and the light source was placed 11 cm away from the mold for 11.5 minutes. The mixture was then demolded to obtain the composite sustained-release body.
[0084] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 10:90; the mass ratio of camphorquinone to triethanolamine is 1:1.7.
[0085] 2. Adjust the pH of the basal medium (DMEM) to 7.2, filter it through a 0.22μm filter membrane for sterilization, and set it aside for later use;
[0086] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM Mix with ionomycin at a stirring rate of 350 rpm / min for 8 min;
[0087] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is 24:16:17:13:14:16;
[0088] 4. Continue to add the prepared complex sustained-release medium to the basal medium and stir at a stirring rate of 160 rpm / min for 2.5 min to obtain the modified NK cell culture medium.
[0089] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and complex sustained-release formulation was 92:5:3.
[0090] Example 5
[0091] like Figure 2 As shown, a method for preparing an improved NK cell culture medium includes the following steps:
[0092] 1. First, prepare the composite sustained-release formulation:
[0093] The protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.1:1) and interleukin (IL-21) were dissolved in a 9.5 mM sodium citrate solution at pH 5.0 to obtain the first solution;
[0094] The mass ratio of interleukin (IL-21) to the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.1:1) is 1:7; the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.1:1) and the interleukin (IL-21) to the sodium citrate solution in the first solution is 13:87.
[0095] The first solution was pre-frozen at -30℃ for 30 hours, then freeze-dried, ground, and passed through a 45μm sieve to obtain freeze-dried particles;
[0096] The acrylate prepolymer was mixed with an ethanol solution of camphor quinone and triethanolamine. After vacuum desolventizing at 43°C for 1.2 hours, the freeze-dried particles were added at a ratio of 3% of the total mass of the ethanol solution of the acrylate prepolymer, camphor quinone and triethanolamine mixture. After mixing, the mixture was poured into a mold, and the light source was placed 10 cm away from the mold for 10 minutes. The composite sustained-release body was then demolded.
[0097] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 8:92; the mass ratio of camphorquinone to triethanolamine is 1:1.2.
[0098] 2. Adjust the pH of the basal medium (IMDM) to 7.4, filter it through a 0.22μm filter membrane for sterilization, and set it aside for later use;
[0099] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM Mix with ionomycin at a stirring rate of 320 rpm / min for 6 min;
[0100] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is 25:24:17:11:13:10;
[0101] 4. Continue to add the prepared complex sustained-release medium to the basal culture medium and stir at a stirring rate of 120 rpm / min for 2 min to obtain the modified NK cell culture medium.
[0102] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and complex sustained-release formulation was 87:8:5.
[0103] Example 6
[0104] like Figure 2 As shown, a method for preparing an improved NK cell culture medium includes the following steps:
[0105] 1. First, prepare the composite sustained-release formulation:
[0106] The protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.4:1) and interleukin (IL-15) were dissolved in a 9 mM sodium citrate solution at pH 5.2 to obtain the first solution;
[0107] The mass ratio of interleukin (IL-15) to the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.4:1) in the first solution is 1:10; the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.4:1) and interleukin (IL-15) to the sodium citrate solution in the first solution is 16:84.
[0108] The first solution was pre-frozen at -70℃ for 42 hours, then freeze-dried, ground, and passed through a 65μm sieve to obtain freeze-dried particles.
[0109] The acrylate prepolymer was mixed with an ethanol solution of camphor quinone and triethanolamine. After being desolvated under vacuum at 48°C for 1.8 hours, the freeze-dried particles were added at a ratio of 3% of the total mass of the ethanol solution of the acrylate prepolymer, camphor quinone and triethanolamine mixture. After mixing, the mixture was poured into a mold, and the light source was placed 10 cm away from the mold for 12 minutes. The composite sustained-release body was then demolded.
[0110] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 10:90; the mass ratio of camphorquinone to triethanolamine is 1:1.8.
[0111] 2. Adjust the pH of the basal medium (DMEM) to 7.3, filter it through a 0.22μm filter membrane for sterilization, and set it aside for later use;
[0112] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM Mix with ionomycin at a stirring rate of 300 rpm / min for 9 min;
[0113] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is 36:14:15:10:15:10;
[0114] 4. Continue to add the prepared complex sustained-release medium to the basal culture medium and stir at a stirring rate of 150 rpm / min for 3 min to obtain the modified NK cell culture medium.
[0115] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and complex sustained-release formulation was 93:4:3.
[0116] Comparative Example 1
[0117] A method for preparing an improved NK cell culture medium includes the following steps:
[0118] 1. Dissolve the protective agent (trehalose and recombinant human serum albumin in a 1:1 mass ratio) and interleukin (IL-2) in an 8 mM sodium citrate solution at pH 4.0 to obtain the first solution;
[0119] 2. Adjust the pH of the basal culture medium (X-VIVO15) to 7.2, filter it through a 0.22μm filter membrane for sterilization, and set it aside for later use;
[0120] 3. Add the following supplement components to the sterilized basal culture medium in the following order: Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TMMix with ionomycin at a stirring rate of 300 rpm / min for 10 min;
[0121] Among them are Polygonatum polysaccharide, α-linolenic acid, L-carnitine, methyl-β-cyclodextrin, and GlutaMAX. TM The mass ratio of iodine to ionomycin is 20:10:25:15:15:15;
[0122] 4. Continue to add the prepared first solution to the basal culture medium and stir at a stirring rate of 100 rpm / min for 3 min to obtain the modified NK cell culture medium.
[0123] Throughout the preparation process, the mass ratio of the basal culture medium, supplement, and first solution was 85:10:5.
[0124] Comparative Example 2
[0125] A method for preparing an improved NK cell culture medium includes the following steps:
[0126] 1. First, prepare the composite sustained-release formulation:
[0127] The protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.2:1) and interleukin (IL-15) were dissolved in a 9 mM sodium citrate solution at pH 4.8 to obtain the first solution;
[0128] The mass ratio of interleukin (IL-15) to the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.2:1) is 1:8; the mass ratio of the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.2:1) and interleukin (IL-15) to the sodium citrate solution in the first solution is 14:86.
[0129] The first solution was pre-frozen at -50℃ for 32 hours, then freeze-dried, ground, and passed through a 50μm sieve to obtain freeze-dried particles.
[0130] The acrylate prepolymer was mixed with an ethanol solution of camphorquinone and triethanolamine. After being desolvated under vacuum at 45°C for 1.2 hours, the freeze-dried particles were added at a ratio of 4% of the total mass of the ethanol solution of the acrylate prepolymer, camphorquinone and triethanolamine mixture. After mixing, the mixture was poured into a mold, and the light source was placed 9 cm away from the mold for 11 minutes. The composite sustained-release body was then demolded.
[0131] In an ethanol solution of a mixture of camphorquinone and triethanolamine, the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is 9:91; the mass ratio of camphorquinone to triethanolamine is 1:1.4.
[0132] 2. Adjust the pH of the basal medium (RPMI-1640) to 7.3 and sterilize by filtration through a 0.22μm filter membrane;
[0133] 3. Add the prepared complex sustained-release medium to the sterilized basal culture medium and stir at a stirring rate of 150 rpm / min for 2 min to obtain the modified NK cell culture medium.
[0134] Throughout the preparation process, the mass ratio of the basal culture medium to the complex sustained-release medium was 95:5.
[0135] Comparative Example 3
[0136] A method for preparing an improved NK cell culture medium includes the following steps:
[0137] 1. Dissolve the protective agent (trehalose and recombinant human serum albumin in a mass ratio of 1.4:1) and interleukin (IL-15) in a 9 mM sodium citrate solution at pH 5.2 to obtain the first solution;
[0138] 2. Adjust the pH of the basal medium (DMEM) to 7.3 and sterilize by filtration through a 0.22μm filter membrane;
[0139] 3. Add the prepared first solution to the sterilized basal culture medium and stir at a stirring rate of 150 rpm / min for 3 min to obtain the modified NK cell culture medium.
[0140] Throughout the preparation process, the mass ratio of the basal culture medium to the first solution was 97:3.
[0141] The NK cell culture media prepared in Examples 1-6 and Comparative Examples 1-3 were tested for their killing activity against porcine reproductive and respiratory syndrome virus, the expansion fold of NK cells after 10 days of culture, and the remaining amount of interleukin after 10 days of culture to demonstrate the culture efficiency, activity, and continuous and stable release of interleukin in the modified NK cell culture media prepared in this application.
[0142] The test and statistical results are shown in Table 1 below.
[0143] Table 1. Statistical analysis of test results for NK cell culture media prepared in Examples 1-6 and Comparative Examples 1-3
[0144]
[0145] As can be clearly seen from Table 1, the NK cell culture media prepared in Examples 1-6 have better overall killing activity, expansion fold, and residual amount of interleukin than Comparative Examples 1-3.
[0146] The main reason for this is that this application solves the problems of short half-life and large concentration fluctuations of traditional exogenous interleukins by using a core-shell structure design of a composite sustained-release formulation. This involves an inner protective agent encapsulating interleukin and an outer photocurable polymer controlling the release. The inner trehalose layer, combined with recombinant human serum albumin, protects the activity of interleukin, while the outer porous polymer controls the release rate, ensuring continuous and uniform release of key factors. This avoids sudden increases or decreases in concentration caused by frequent additions, reducing overactivation or functional exhaustion of NK cells and maintaining their long-term activated state and effector function. Furthermore, the release cycle of the composite sustained-release formulation is highly matched with the NK cell culture cycle, eliminating the need to replace the sustained-release formulation or frequently add interleukin, reducing the risk of contamination caused by human error. Simultaneously, the clearly defined concentration range and standardized culture steps facilitate industrial-scale production, reducing quality control costs. Finally, among the supplements used in this application, Polygonatum polysaccharide enhances antioxidant capacity and inhibits cell apoptosis; α-linolenic acid combined with methyl-β-cyclodextrin optimizes cell membrane phospholipid composition and maintains signal transduction function; L-carnitine promotes fatty acid oxidation and enhances energy supply; and ionomycin activates the calcium signaling pathway and strengthens the expression of cytotoxic molecules. Compared with traditional culture media, this combination increases the proliferation rate of NK cells and comprehensively optimizes cell expansion efficiency and functional activity.
[0147] In contrast, no complex sustained-release formulation was prepared in Comparative Example 1, which could not maintain the long-term activated state of NK cells and the interleukin was consumed very quickly.
[0148] In Comparative Example 2, no supplements were added, so the main impact was on the expansion efficiency and functional activity of NK cells, which were reduced to some extent.
[0149] Comparative Example 3 had neither a complex sustained-release formulation nor any supplements added, therefore its cytotoxic activity, amplification fold, and residual interleukin levels were the lowest.
[0150] The foregoing illustrative description of this application and its embodiments is not restrictive and is merely one embodiment of this application; the actual structure is not limited to this. Therefore, if those skilled in the art, inspired by this description, design similar structures and embodiments without departing from the spirit of this application, such designs should fall within the protection scope of this application.
Claims
1. An improved NK cell culture medium, characterized in that, The base medium, the supplement and the composite sustained-release body are included. The supplement comprises: polygonatum polysaccharide, alpha-linolenic acid, L-carnitine, methyl-beta-cyclodextrin, GlutaMAX TM and ionomycin; The inner layer of the composite sustained-release body includes a protective agent and interleukin, and the outer layer is a light-cured polymer including an acrylic ester prepolymer and a photoinitiator.
2. The method for preparing the modified NK cell culture medium according to claim 1, characterized in that, The base medium includes any one of X-VIVO15, KBM581, RPMI-1640, DMEM and IMDM; the mass ratio of the base medium, the supplement and the composite sustained-release body is (85-95):(3-10):(1-5).
3. The improved NK cell culture medium of claim 1, wherein, The supplement, Huangjing polysaccharide, alpha-linolenic acid, L-carnitine, methyl-beta-cyclodextrin, GlutaMAX TM And the mass ratio of ionomycin is (20-40):(10-25):(15-25):(10-15):(10-15):(10-15).
4. The improved NK cell culture medium of claim 1, wherein, The protective agent includes trehalose and human recombinant serum albumin; the mass ratio of the trehalose and the human recombinant serum albumin is (1-1.5):1; the interleukin includes any one of IL-2, IL-12, IL-15, IL-18 and IL-21, and the mass ratio of the interleukin to the protective agent is 1:(5-15).
5. The improved NK cell culture medium of claim 1, wherein, The photoinitiator is an ethanol solution of a mixture of camphorquinone and triethanolamine, wherein the mass ratio of the mixture of camphorquinone and triethanolamine to ethanol is (8-10):(90-92); the mass ratio of the camphorquinone to the triethanolamine is 1:(1-2).
6. The modified NK cell culture medium of claim 1, wherein, The preparation method of the composite sustained-release body includes the following steps: The protective agent and the interleukin are dissolved in a 8-10mM sodium citrate solution with pH 4.0-5.5 to obtain a first solution; The first solution is pre-frozen at-20℃ to-80℃ for 24-48 hours, and then ground after freeze-drying, and then filtered through a 40-70μm sieve to obtain freeze-dried particles; The acrylic ester prepolymer is mixed with the photoinitiator, and then the freeze-dried particles are added in a proportion of 2%-8% of the total mass of the acrylic ester prepolymer and the photoinitiator after vacuum desolventizing at 40-50℃ for 1-2 hours, and then poured into a mold after mixing, and then irradiated for 10-12 minutes at a distance of 8-12cm from the light source, and then demolded to obtain the composite sustained-release body.
7. The improved NK cell culture medium of claim 6, wherein, The light source has a wavelength of 450-550 nm and an intensity of 30-50 mW / cm2 2 .
8. The improved NK cell culture medium of claim 6, wherein, The composite sustained-release body is a micro-cylinder with a diameter of 100-200μm and a height-diameter ratio of 1:(1-1.5).
9. The improved NK cell culture medium of claim 6, wherein, The release period of the composite sustained-release body is 7-10 days.
10. A method of preparing an improved NK cell culture medium, characterized by, The base medium is adjusted to pH 7.2-7.4, filtered with a 0.22μm filter membrane to remove bacteria, and then prepared for use; The prepared composite sustained-release body is continuously added to the base medium, and then stirred at a stirring rate of 100-200rpm / min for 1-3min to obtain the improved NK cell culture medium. To the sterilized basal medium, the various components of the supplement were added in proportion in turn: polygonatum polysaccharide, alpha-linolenic acid, L-carnitine, methyl-beta-cyclodextrin, GlutaMAX TM and ionomycin, at a stirring rate of 300-400 rpm / min for 5-10 min; The base medium is adjusted to pH 7.2-7.4, filtered with a 0.22μm filter membrane to remove bacteria, and then prepared for use; The prepared composite sustained-release body is continuously added to the base medium, and then stirred at a stirring rate of 100-200rpm / min for 1-3min to obtain the improved NK cell culture medium.