Component-limited insect culture medium containing nanoparticles

Abstract

The invention discloses a component-limited insect culture medium containing nanoparticles, and relates to the technical field of culture medium preparation, and the component-limited insect culture medium is composed of saccharides, amino acids, inorganic salts, vitamins, organic acids and trace elements. The chemical component limited culture medium comprises 10-15 g / L of saccharides, 15-25 g / L of amino acid, 6.5-15.0 g / L of inorganic salt, 0.01-0.5 g / L of vitamins, 0.2-2.0 g / L of organic acid, 0.001-0.01 g / L of trace elements and nano-particles, and the mass of the nano-particles accounts for 0.01%-0.1% of the total mass of the culture medium. The culture medium is free of serum and animal source components, the nano-particles are added into the serum-free culture medium, higher batch stability is achieved, and the culture medium is suitable for large-scale production. The recovery and passage requirements of various insect cells can be met.

Description

Technical Field

[0001] The invention relates to the technical field of culture medium preparation, and in particular to a component-limited insect culture medium containing nanoparticles. Background Art

[0002] The annual demand for insect culture medium in China is very large, and it has great potential in the field of animal vaccine production. At present, it is known that publicly available insect cell culture medium usually needs to be added with serum or hydrolyzate when used (such as Thermo's Grace Insect Medium), which not only increases the production and use costs of the culture medium, but also may cause instability between culture medium production batches.

[0003] Currently, the market for commercial insect culture media with defined ingredients is mainly occupied by foreign culture media, which are expensive and have unstable delivery time. Therefore, there is an urgent need for a relatively low-priced, chemically defined culture medium that is stable between batches.

[0004] For example, the Chinese patent for the preparation method of titanium dioxide nanoparticle culture medium with application number CN202110296135.7 discloses a method for preparing titanium dioxide nanoparticle culture medium, comprising the following steps: 1) mixing titanium dioxide nanoparticles with Tween 20, and ultrasonically treating the obtained mixed solution under ice bath conditions; 2) adding the titanium dioxide-Tween 20 mixed solution obtained in step 1) to a cell culture medium with a mass fraction of serum of 1%-10%; 3) ultrasonically treating the mixed solution obtained in step 2) to obtain a titanium dioxide nanoparticle culture medium. The invention combines a surfactant and an ultrasonic dispersion method to enable titanium dioxide nanoparticles to be uniformly and stably dispersed in the culture medium, and the prepared culture medium can be stably maintained for at least 2 days, thereby facilitating in vitro studies of the biological effects of titanium dioxide nanoparticles; the preparation method of the present invention is simple, easy to operate, and highly repeatable, and can provide a good object for the study of the biological effects of nanoparticles.

[0005] The above patent introduces titanium dioxide nanoparticles into the culture medium so that the titanium dioxide nanoparticles can be evenly and stably dispersed in the culture medium. However, the nanoparticles have no promoting effect on cell culture and are not easy to manipulate and separate in cell culture. Summary of the invention

[0006] The embodiments of the present application solve the problems of high cost and instability between production batches in the prior art by providing a component-limited insect culture medium containing nanoparticles. By providing a serum-free culture medium, adding nanoparticles to the serum-free culture medium, and combining with an ultraviolet light source, the stability of the culture medium and the convenience of the culture process operation are improved, the cell density and cell viability of cell culture are improved, and support is provided for the research and large-scale production of insect cell culture.

[0007] The present application provides a nanoparticle-containing insect culture medium with defined ingredients, wherein the culture medium specifically includes the following components: Sugars: 5-12g / L glucose, 3-6g / L sucrose, 3-7g / L fructose; Amino Acids: 1-3g / L histidine, 0.5-3.0g / L aspartic acid, 0.5-1.2g / L serine, 1.0-1.5g / L glycine, 0.5-2.6g / L arginine, 0.5-1.8g / L asparagine, 0.1-1.0g / L isoleucine, 0.1-1.0g / L leucine, 1.0-3.0g / L glutamic acid, 0.1-1.0g / L valine, 0.5-1.0g / L glutamine, 0.5-1.0g / L lysine, 0.1-0.6g / L methionine, 0.1-0.2g / L phenylalanine, 0.2-0.4g / L proline, 0.2-0.8g / L threonine, 0.01-0.5g / L tyrosine, 0.1-0.5g / L alanine; Inorganic salts: 2.0-4.0g / L potassium chloride, 0.5-1.2g / L calcium chloride, 0.8-1.5g / L sodium dihydrogen phosphate, 1.0-5.0g / L sodium chloride, 1.0-2.0g / L magnesium sulfate, 0.8-2.0g / L magnesium chloride; Vitamins: 0.001-0.01g / L biotin, 0.001-0.1g / L calcium pantothenate, 0.001-0.1g / L folic acid, 0.001-0.1g / L niacin, 0.01-0.1g / L pyridoxine hydrochloride; Organic acids: 0.0001-0.5g / L ketoglutaric acid, 0.001-0.1g / L fumaric acid, 0.02-0.8g / L malic acid, 0.001-0.01g / L succinic acid; Other trace elements: 0.001-0.1g / L zinc chloride, 0.01-0.05g / L ferric chloride, 0.01-0.1g / L aluminum chloride, 0.1-0.5g / L manganese chloride; Nanoparticles: The mass of nanoparticles accounts for 0.01% - 0.1% of the total mass of the culture medium.

[0008] Furthermore, the nanoparticles have an inner layer of TiO2 and Fe3O4 composite nanoparticles and an outer layer of Tween 20.

[0009] Furthermore, the mass ratio of TiO2 and Fe3O4 in the inner layer of the nanoparticles is 1:1; The ratio of the mass of TiO2 and Fe3O4 composite nanoparticles to the volume of Tween 20 is 5mg:1mL-40mg:1mL.

[0010] Furthermore, the nanoparticles include two types, one of which has a mass ratio of TiO2 to Fe3O4 of 3:1, and the other of which has a mass ratio of TiO2 to Fe3O4 of 1:3.

[0011] Furthermore, different nanoparticles are used at different stages of cell culture, specifically: In the early stage of cell culture, i.e. 0-24 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 3:1 are used; in the middle stage of cell growth, i.e. 24-72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:1 are used; in the cell growth stabilizer, i.e. after 72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:3 are used.

[0012] The method for culturing the above-mentioned insect culture medium containing nanoparticles is specifically as follows: S1. suspending the nanoparticles in sterile deionized water to form a uniform dispersion; S2, adding the nanoparticle dispersion into the culture medium, and performing ultrasonic treatment for 1-2 hours using an ultrasonic cleaning apparatus to obtain a nanoparticle culture medium; The content of the nanoparticle dispersion does not exceed 0.5% of the mass of the culture medium; the specific parameters of the ultrasonic treatment of the ultrasonic cleaning instrument are: frequency 10kHZ, power 100w; S3, the nanoparticle culture medium is placed in a culture container, the bottom of the culture container is transparent, an ultraviolet light source is arranged at the bottom of the culture container, a magnet is used to control the nanoparticles to precipitate at the bottom, and at this time, an ultraviolet light source is used to irradiate from the bottom; Among them, the wavelength of the ultraviolet light source is 320-400nm, the power is <0.1W / cm², the irradiation time is 10-20min, 1-2 times a day.

[0013] Furthermore, an ultraviolet light source is arranged at the bottom of the culture container, and a stirring device is arranged above the ultraviolet light source.

[0014] Furthermore, in step S3, the nanoparticles are precipitated at the bottom layer, and when an ultraviolet light source is used to irradiate from the bottom, a stirring device is started, and the stirring speed of the stirring device is 30-60 rpm.

[0015] Furthermore, a moving magnetic field is set outside the culture container, and the moving magnetic field can move up and down; the magnetic strength of the moving magnetic field is 0.1-1.5T; the moving speed is 5-500mm / h; in step S3, the moving magnetic field is used to control the precipitation of nanoparticles at the bottom layer.

[0016] Furthermore, at different stages of cell culture, the magnetic strength and moving speed of the moving magnetic field are different, specifically: In the initial stage of cell culture, i.e. 0-24 hours, the magnetic strength of the moving magnetic field is 0.1-0.5T; the moving speed is 5-50mm / h; in the middle stage of cell growth, i.e. 24-72 hours, the magnetic strength of the moving magnetic field is 0.5-1T; the moving speed is 50-250mm / h; in the stable period of cell growth, i.e. after 72 hours, the magnetic strength of the moving magnetic field is 1-1.5T; the moving speed is 250-500mm / h.

[0017] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages: First, the culture medium for culturing insect cells provided in this scheme can support the recovery and subculture requirements of SF9, HF and other insect cells in current scientific research experiments, and has great application prospects; the basic culture medium and foreign culture medium are better than imported culture medium in cell doubling and viability maintenance in insect cell growth and subculture tests; it provides support for the research and large-scale production of insect cell culture; by adding nanoparticles to the culture medium, the inner layer of the nanoparticles is composed of TiO2 and Fe3O4 composites, using the antibacterial and antioxidant properties of TiO2 and the magnetic properties of Fe3O4, and the outer layer is wrapped with Tween 20 to ensure the uniform dispersion and stability of the nanoparticles in the culture medium, ultraviolet light irradiation is used to stimulate the photocatalytic activity of TiO2, and the nanoparticles are irradiated with ultraviolet light when they are precipitated at the bottom layer to avoid direct irradiation of the cells to reduce damage to the cells; Secondly, ultraviolet light can stimulate the photocatalytic activity of TiO2 and generate strong oxidizing free radicals. These free radicals have the ability to kill bacteria and degrade harmful substances. By arranging an ultraviolet light source at the bottom of the culture container, it can be ensured that the ultraviolet light is mainly irradiated on the nanoparticles precipitated at the bottom, thereby stimulating the photocatalytic effect of TiO2; the introduction of the stirring device can promote the uniform dispersion of nanoparticles at the bottom of the culture medium through slight stirring when ultraviolet light is irradiated, avoid the agglomeration of nanoparticles, improve their stability in the culture medium, and further promote TiO2 to produce free radicals; the combination of the photocatalytic effect of TiO2 excited by ultraviolet light and the uniform dispersion of the bottom nanoparticles by the stirring device can more effectively kill or inhibit bacteria and viruses in the culture medium, thereby preventing cell contamination and improving cell viability and proliferation ability; Third, by adjusting the mass ratio of TiO2 to Fe3O4, different photocatalytic activities and magnetic properties can be achieved to meet the needs of different stages of cell culture; by introducing different types of nanoparticles according to different stages of cell culture, the cell culture environment can be precisely regulated and optimized, promoting the research and large-scale production of insect cell culture; Fourth, the magnetic field can affect the movement trajectory and distribution of nanoparticles; by moving the magnetic field, the diffusion rate and direction of nanoparticles in the culture medium can be controlled, thereby achieving directional movement and distribution of particles; the magnetic field can change the ion concentration and potential distribution in the cell culture environment, thereby optimizing the cell growth environment; by precisely controlling the magnetic strength and moving speed of the moving magnetic field, it is possible to avoid excessive mechanical stress and damage to the cells and maintain the integrity and activity of the cells. Adjusting the magnetic strength and moving speed of the moving magnetic field at different stages of cell culture can optimize the cell culture environment, thereby promoting cell growth and division; the magnetic field provides more growth space for cells by affecting the arrangement of the extracellular matrix and the interaction between cells, thereby increasing the cell culture density; by finely controlling the magnetic field parameters, complex cell arrangements and combinations can be achieved under different conditions, improving the flexibility and adaptability of cell culture, and promoting the research and large-scale production of insect cell culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The cell culture VCD viable cell density (×10 6 cells / mL) line graph; Figure 2 A line graph of cell culture VIABILITY (%) according to the first embodiment of the present invention; Figure 3 A schematic diagram of a cell culture container according to embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the structure of a cell culture container according to Embodiment 2 of the present invention; Figure 5 This is a schematic diagram of the structure of a cell culture container according to Embodiment 4 of the present invention. DETAILED DESCRIPTION

[0019] To facilitate the understanding of the present invention, the present application will be described more comprehensively below with reference to the relevant drawings; the drawings show preferred embodiments of the present invention, but the present invention can be implemented in many different forms and is not limited to the embodiments described herein; on the contrary, the purpose of providing these embodiments is to enable a more thorough and comprehensive understanding of the disclosed content of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the term "and / or" used herein includes any and all combinations of one or more related listed items.

[0021] Embodiment 1: The present invention provides a nanoparticle-containing insect culture medium with defined ingredients, the culture medium comprising the following components: Sugars: 5-12g / L glucose, 3-6g / L sucrose, 3-7g / L fructose; Amino Acids: 1-3g / L histidine, 0.5-3.0g / L aspartic acid, 0.5-1.2g / L serine, 1.0-1.5g / L glycine, 0.5-2.6g / L arginine, 0.5-1.8g / L asparagine, 0.1-1.0g / L isoleucine, 0.1-1.0g / L leucine, 1.0-3.0g / L glutamic acid, 0.1-1.0g / L valine, 0.5-1.0g / L glutamine, 0.5-1.0g / L lysine, 0.1-0.6g / L methionine, 0.1-0.2g / L phenylalanine, 0.2-0.4g / L proline, 0.2-0.8g / L threonine, 0.01-0.5g / L tyrosine, 0.1-0.5g / L alanine; Inorganic salts: 2.0-4.0g / L potassium chloride, 0.5-1.2g / L calcium chloride, 0.8-1.5g / L sodium dihydrogen phosphate, 1.0-5.0g / L sodium chloride, 1.0-2.0g / L magnesium sulfate, 0.8-2.0g / L magnesium chloride; Vitamins: 0.001-0.01g / L biotin, 0.001-0.1g / L calcium pantothenate, 0.001-0.1g / L folic acid, 0.001-0.1g / L niacin, 0.01-0.1g / L pyridoxine hydrochloride; Organic acids: 0.0001-0.5g / L ketoglutaric acid, 0.001-0.1g / L fumaric acid, 0.02-0.8g / L malic acid, 0.001-0.01g / L succinic acid; Other trace elements: 0.001-0.1g / L zinc chloride, 0.01-0.05g / L ferric chloride, 0.01-0.1g / L aluminum chloride, 0.1-0.5g / L manganese chloride; The following cell culture experiments were performed on this culture medium, and 10 g / L glucose, 5 g / L sucrose, and 5 g / L fructose; 2 g / L histidine, 2 g / L aspartic acid, 1 g / L serine, 1 g / L glycine, 2 g / L arginine, 1 g / L asparagine, 1 g / L isoleucine, 1 g / L leucine, 2 g / L glutamic acid, 1 g / L valine, 1 g / L glutamine, 1.0 g / L lysine, 0.5 g / L methionine, 0.1 g / L phenylalanine, 0.4 g / L proline, 0.5 g / L threonine, 0.1 g / L tyrosine, and 0.2 g / L Alanine; 3g / L potassium chloride, 1g / L calcium chloride, 1g / L sodium dihydrogen phosphate, 2g / L sodium chloride, 2g / L magnesium sulfate, 2g / L magnesium chloride; 0.01g / L biotin, 0.1g / L calcium pantothenate, 0.1g / L folic acid, 0.1g / L nicotinic acid, 0.1g / L pyridoxine hydrochloride; 0.1g / L ketoglutaric acid, 0.01g / L fumaric acid, 0.5g / L malic acid, 0.01g / L succinic acid; 0.01g / L zinc chloride, 0.05g / L ferric chloride, 0.1g / L aluminum chloride, 0.5g / L manganese chloride as culture medium for cell experiments; The specific experimental steps are: Experiment 1: Preheat 10 mL of culture medium at 27.5°C for at least 30 minutes; Take out a HF cell cryopreservation tube from the liquid nitrogen storage tank and immediately put it into a 37°C water bath. Rotate slowly in the water bath and control the thawing time to 120-240 seconds. Take the cryopreservation tube out of the water bath, wipe it dry, and then put it into the biosafety cabinet. Transfer the cells to a 15 mL centrifuge tube containing 9 mL of pre-warmed culture medium; centrifuge at 1000 rpm for 5 minutes at room temperature and remove the supernatant; Resuspend the cells with pre-warmed culture medium, then transfer to a 125 mL shake flask, count the cells, and continue to add culture medium to adjust the density to 1E6 cells / mL; count the cells every two days and perform subculture operations with a subculture density of 0.5E6 cells / mL.

[0022] Experiment 2: Preheat 10 mL of culture medium at 27.5°C for at least 30 minutes; Take out a cryopreserved tube of SF9 cells from the liquid nitrogen storage tank and immediately place it in a 37°C water bath; rotate slowly in the water bath, and control the thawing time to 120-240 seconds; take the cryopreserved tube out of the water bath, wipe it dry, and then place it in a biosafety cabinet; Transfer the cells to a 15 mL centrifuge tube containing 9 mL of pre-warmed culture medium; centrifuge at 1000 rpm for 5 minutes at room temperature and remove the supernatant; resuspend the cells with pre-warmed culture medium and transfer to a 125 mL shake flask, count the cells, and continue to add culture medium to adjust the density to 1E6 cells / mL; count the cells every two days and perform subculture operations at a subculture density of 1E6 cells / mL.

[0023] Control group: The HF and SF9 cells were cultured using the same recovery and subculture procedures, using Thermo's Grace medium instead of the culture medium of the example; The cell growth results obtained from the experiment are as follows Figure 1 and Figure 2 shown.

[0024] The culture medium also includes nanoparticles, the mass of which accounts for 0.01% - 0.1% of the total mass of the culture medium.

[0025] The culture method of the culture medium containing nanoparticles is specifically as follows: S1. suspending the nanoparticles in sterile deionized water to form a uniform dispersion; The inner layer of the nanoparticles is a composite nanoparticle of TiO2 and Fe3O4, and the outer layer is Tween 20; the mass ratio of TiO2 and Fe3O4 is 1:1; the ratio of the mass of the composite nanoparticles of TiO2 and Fe3O4 to the volume of Tween 20 is less than 40 mg:1 mL; Preparation of nanoparticles, specifically: Dissolve TiO2 powder and Fe3O4 powder in a mass ratio of 1:1 in deionized water to form a uniform suspension; mix the TiO2 suspension and the Fe3O4 suspension and place them in a reaction kettle; Under magnetic stirring, slowly add sodium hydroxide solution as a precipitant to adjust the solution pH to pH = 9; control the reaction temperature at 60-80 ° C, and continue stirring for 2-4 hours to allow TiO2 and Fe3O4 to fully combine to form composite nanoparticles; After the reaction is completed, the impurity ions are removed by centrifugation and washing to obtain pure TiO2 and Fe3O4 composite nanoparticles; The suspension of TiO2 and Fe3O4 composite nanoparticles and Tween 20 solution are added into a container and mixed, the container is placed in an ice bath, and then ultrasonically treated for 1.5-2 minutes using an ultrasonic cell lyser to make Tween 20 evenly cover the surface of the TiO2 and Fe3O4 composite nanoparticles, and then centrifuged and washed to remove excess Tween 20 to obtain TiO2 and Fe3O4 composite nanoparticles coated with Tween 20, namely nanoparticles; The specific parameters of ultrasonic cell lysis instrument for ultrasonic treatment are: at a frequency of 40kHz, a power of 700-800W, ultrasonic treatment for 1s, an interval of 1s, and 45-60 cycles; the ratio of the mass of TiO2 and Fe3O4 composite nanoparticles to the volume of Tween 20 is 5mg:1mL-40mg:1mL; S2, adding the nanoparticle dispersion into the culture medium, and performing ultrasonic treatment for 1-2 hours using an ultrasonic cleaning apparatus to obtain a nanoparticle culture medium; The content of the nanoparticle dispersion does not exceed 0.5% of the mass of the culture medium; the specific parameters of the ultrasonic treatment of the ultrasonic cleaning instrument are: frequency 10kHZ, power 100w; S3, such as Figure 3 As shown, the nanoparticle culture medium is placed in a culture container, the bottom of the culture container is transparent, an ultraviolet light source is arranged at the bottom of the culture container, a magnet is used to control the nanoparticles to precipitate at the bottom, and at this time, an ultraviolet light source is used to irradiate from the bottom; Among them, the wavelength of the ultraviolet light source is 320-400nm, the power is <0.1W / cm², the irradiation time is 10-20min, 1-2 times a day.

[0026] For the nanoparticle culture medium in this scheme, Experiment 3 was performed on the basis of Experiment 2. Experiment 3 and Experiment 2 used the same recovery and subculture operations. The difference between Experiment 3 and Experiment 2 was that nanoparticles were added to the culture medium in Experiment 3, and the mass of the nanoparticles accounted for 0.05% of the total mass of the culture medium. The culture medium was irradiated with ultraviolet light for 20 minutes, twice a day.

[0027] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: The culture medium for culturing insect cells provided in this scheme can support the recovery and subculture needs of SF9, HF and other insect cells in current scientific research experiments, and has great application prospects; this basic culture medium and foreign culture medium are better than imported culture medium in cell doubling and viability maintenance in insect cell growth and subculture tests; it provides support for the research and large-scale production of insect cell culture; Nanoparticles are added to the culture medium. The inner layer of the nanoparticles is composed of TiO2 and Fe3O4. The antibacterial and antioxidant properties of TiO2 and the magnetic properties of Fe3O4 are used. The outer layer is wrapped with Tween 20 to ensure the uniform dispersion and stability of the nanoparticles in the culture medium. Ultraviolet light is used to stimulate the photocatalytic activity of TiO2. When the nanoparticles are precipitated at the bottom, they are irradiated with ultraviolet light to avoid direct irradiation of cells to reduce damage to cells. This reduces dependence on traditional antibiotics and antimicrobial agents and reduces pollution to the environment. TiO2 has excellent photocatalytic properties. It can generate electron-hole pairs under ultraviolet light, and then generate strong oxidizing free radicals. These free radicals can kill bacteria and degrade harmful substances. They can effectively kill or inhibit bacteria and viruses in the culture medium, prevent cell contamination, and improve the success rate of cell culture. Fe3O4 has good magnetism and biocompatibility. It can be used as a magnetic carrier. These composite nanoparticles can be easily manipulated and recovered through an external magnetic field, which is convenient for subsequent treatment and reuse. Tween 20 is a non-ionic surfactant. The encapsulation effect of Tween 20 can effectively prevent the agglomeration of nanoparticles and make them evenly dispersed in the culture medium, thereby improving the long-term stability of the culture medium. At the same time, Tween 20 can also improve the biocompatibility of nanoparticles and reduce toxicity to cells. Ultraviolet light can stimulate the photocatalytic activity of TiO2 and enhance its antibacterial, antiviral and harmful substance degradation capabilities.

[0028] Example 2: The above-mentioned Example 1 improves the stability of the culture medium and the convenience of the culture process by providing a culture medium with limited ingredients and adding nanoparticles to the culture medium, thereby improving the cell density and cell viability of cell culture. In order to further improve the density and cell viability of cell culture, further improvements are made on the basis of Example 1.

[0029] like Figure 4 As shown, an ultraviolet light source is arranged at the bottom of the culture container, and a stirring device is arranged above the ultraviolet light source; In step S3, the nanoparticles are precipitated on the bottom layer, and when the ultraviolet light source is used to irradiate from the bottom, the stirring device is started; Wherein, the stirring speed of the stirring device is 30-60rpm.

[0030] According to the technical solution of this embodiment, Experiment 4 was performed on the basis of Experiment 3. Experiment 4 and Experiment 3 adopted the same recovery and subculture operations. The difference between Experiment 4 and Experiment 3 was that in Experiment 4, an ultraviolet light source was set at the bottom of the culture container, and a stirring device was set above the ultraviolet light source, and the stirring speed was 40 rpm.

[0031] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: Ultraviolet light can stimulate the photocatalytic activity of TiO2 and generate strong oxidizing free radicals. These free radicals have the ability to kill bacteria and degrade harmful substances. By setting an ultraviolet light source at the bottom of the culture container, it can be ensured that the ultraviolet light is mainly irradiated on the nanoparticles precipitated at the bottom, thereby stimulating the photocatalytic effect of TiO2. The introduction of the stirring device can promote the uniform dispersion of nanoparticles at the bottom of the culture medium through slight stirring when ultraviolet light is irradiated, avoid the agglomeration of nanoparticles, improve their stability in the culture medium, and further promote the generation of free radicals by TiO2. The combination of the ultraviolet light-excited TiO2 photocatalytic effect and the uniform dispersion of the bottom nanoparticles by the stirring device can more effectively kill or inhibit bacteria and viruses in the culture medium, thereby preventing cell contamination and improving cell viability and proliferation ability; the stirring device can maintain the uniform dispersion of the nanoparticles at the bottom of the culture medium when the nanoparticles are irradiated with ultraviolet light, thereby improving the convenience of operation, simplifying the culture process, improving the culture efficiency, and promoting the research and large-scale production of insect cell culture.

[0032] Embodiment 3: The above-mentioned embodiment 2 improves the stability of the culture medium and the convenience of the culture process operation through the cooperation of the stirring device and the ultraviolet light source, and improves the cell density and cell viability of cell culture. In order to further improve the density and cell viability of cell culture, further improvements are made on the basis of embodiment 2.

[0033] The nanoparticles also include two types, one of which has a mass ratio of TiO2 to Fe3O4 of 3:1, and the other has a mass ratio of TiO2 to Fe3O4 of 1:3; Different nanoparticles are used at different stages of cell culture, specifically: In the early stage of cell culture, i.e. 0-24 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 3:1 are used; in the middle stage of cell growth, i.e. 24-72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:1 are used; in the cell growth stabilizer, i.e. after 72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:3 are used.

[0034] According to the technical scheme of this embodiment, Experiment 5 was carried out on the basis of Experiment 4. Experiment 5 and Experiment 4 adopted the same recovery and subculture operations. The difference between Experiment 5 and Experiment 4 was that in the early stage of cell culture, i.e. 0-24 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 3:1 were used; in the middle stage of cell growth, i.e. 24-72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:1 were used; in the cell growth stabilizer, i.e. after 72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:3 were used.

[0035] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: By adjusting the mass ratio of TiO2 to Fe3O4, different photocatalytic activities and magnetic properties can be achieved to meet the needs of different stages of cell culture; In the early stage of cell culture, the proportion of TiO2 is high and the photocatalytic activity is strong, which can decompose organic pollutants and bacteria in the culture medium and provide a clean growth environment for cells. At the same time, an appropriate amount of Fe3O4 can provide certain magnetic properties, but it will not affect the photocatalytic effect of TiO2; it can improve the cleanliness of the culture medium, promote cell attachment and growth, and increase the starting density of cell culture; In the middle stage of cell growth, composite nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:1 are used. In the middle stage, cells grow rapidly and need more nutrients and growth factors. The equal mass ratio of TiO2 to Fe3O4 can balance the photocatalytic activity and magnetic properties, maintaining a certain cleaning effect while avoiding interference with cell growth, thereby maintaining the cleanliness of the culture medium, promoting rapid cell growth and proliferation, and improving cell viability; During the stable growth period of cells, the composite nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:3 have a higher proportion of Fe3O4 and significantly enhanced magnetic properties. When cell culture is completed or subculture is required, cells can be separated quickly and efficiently through an external magnetic field, thereby improving cell separation efficiency, reducing cell loss and contamination risks, and increasing cell culture density and cell viability. By introducing different types of nanoparticles according to different stages of cell culture, the cell culture environment can be precisely regulated and optimized, promoting the research and large-scale production of insect cell culture.

[0036] Embodiment 4: Embodiment 3 introduces different types of nanoparticles according to different stages of cell culture to improve the density and cell viability of cell culture. To further improve the density and cell viability of cell culture, further improvements are made on the basis of Embodiment 3.

[0037] like Figure 5 As shown, a moving magnetic field is set outside the culture container, and the moving magnetic field can move up and down; the magnetic strength of the moving magnetic field is 0.1-1.5T; the moving speed is 5-500mm / h; In step S3, a moving magnetic field is used to control the nanoparticles to precipitate on the bottom layer; At different stages of cell culture, the magnetic strength and moving speed of the moving magnetic field are different, specifically: In the early stage of cell culture, i.e. 0-24 hours, the magnetic strength of the mobile magnetic field is 0.1-0.5T; the moving speed is 5-50mm / h; in the middle stage of cell growth, i.e. 24-72 hours, the magnetic strength of the mobile magnetic field is 0.5-1T; the moving speed is 50-250mm / h; in the stable period of cell growth, i.e. after 72 hours, the magnetic strength of the mobile magnetic field is 1-1.5T; the moving speed is 250-500mm / h; the technical solution of this embodiment is made on the basis of embodiment 5 Experiment 6 and Experiment 5 used the same recovery and subculture operations. The difference between Experiment 6 and Experiment 5 was that in the early stage of cell culture, i.e. 0-24 hours, the magnetic strength of the moving magnetic field was 0.3 T; the moving speed was 20 mm / h; in the middle stage of cell growth, i.e. 24-72 hours, the magnetic strength of the moving magnetic field was 0.75 T; the moving speed was 150 mm / h; in the cell growth stabilizer, i.e. after 72 hours, the magnetic strength of the moving magnetic field was 1.25 T; the moving speed was 350 mm / h.

[0038] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: The magnetic field can affect the movement trajectory and distribution of nanoparticles; by moving the magnetic field, the diffusion rate and direction of nanoparticles in the culture medium can be controlled, thereby achieving directional movement and distribution of particles, thereby optimizing the growth environment of cells; at the same time, the moving magnetic field can control the nanoparticles to settle at the bottom of the culture container for ultraviolet light irradiation; In the early stage of cell culture, using lower magnetic strength and moving speed can gently affect the cells, promote their uniform distribution and attachment to the surface of the culture container, and lay a good foundation for subsequent cell proliferation; the middle stage of cell growth is the most active stage of cell proliferation. At this time, increasing the magnetic strength and moving speed can stimulate the cells, enhance their metabolic activity and proliferation ability, promote rapid cell growth and division, and thus improve cell viability and density; in the stable period of cell growth, the cell density reaches a higher value, and at this time, efficient separation and collection of cells are required; by increasing the magnetic strength and moving speed, the magnetic properties of Fe3O4 can be more effectively utilized to quickly and efficiently separate cells from the culture medium, reduce cell loss and contamination risks, and improve the final yield and quality of cell culture; By precisely controlling the magnetic strength and speed of the moving magnetic field, we can avoid excessive mechanical stress and damage to cells and maintain the integrity and activity of cells. Adjusting the magnetic strength and speed of the moving magnetic field at different stages of cell culture can optimize the cell culture environment, thereby promoting cell growth and division; the magnetic field affects the arrangement of the extracellular matrix and the interaction between cells, providing more growth space for cells, thereby increasing the cell culture density; The mobile magnetic field can promote the diffusion of nanoparticles in the culture medium, allowing the nanoparticles to be distributed more quickly throughout the culture system, improving the efficiency of nutrient transfer and ensuring that cells can obtain adequate nutritional support; utilizing the magnetic properties of ferroferric oxide, the mobile magnetic field can achieve directional movement and distribution of nanoparticles, prompting a more uniform distribution of nanoparticles during cell culture and avoiding the adverse effects of excessive or low local concentrations on cells; the optimization effect of the magnetic field on the cell culture environment can increase the growth rate and viability of cells, and by adjusting the intensity and movement speed of the magnetic field, it can provide an environment conducive to cell growth, thereby increasing the density and cell viability of cell culture; by increasing the diffusion rate of nanoparticles and optimizing the cell culture environment, the efficiency of cell culture is significantly improved, so that more cell products can be obtained in the same time, reducing production costs and time costs; by finely controlling the magnetic field parameters, complex cell arrangements and combinations under different conditions can be achieved, improving the flexibility and adaptability of cell culture and promoting the research and large-scale production of insect cell culture.

[0039] The cell growth conditions obtained from the experiment are shown in Table 1 and Table 2: Cultivation time (days) 0 2 4 6 8 10 <![CDATA[Experimental 2 VCD (×10 6 cells / mL)]]> 1.0 4.2 3.8 4.6 4.7 5.0 <![CDATA[Experimental 3 VCD (×10 6 cells / mL)]]> 1.0 4.5 4.2 5.0 5.2 5.5 <![CDATA[Experimental 4 VCD (×10 6 cells / mL)]]> 1.0 4.8 4.7 5.4 5.5 5.9 <![CDATA[Experimental viable cell density of 5VCD (×10 6 cells / mL)]]> 1.0 5.1 4.9 5.7 6.1 6.4 <![CDATA[Experimental 6VCD viable cell density (×10 6 cells / mL)]]> 1.0 5.2 5.1 6.0 6.2 6.7 Table 1. VCD viable cell density Cultivation time (days) 0 2 4 6 8 10 Experiment 2VIABILITY (%) 86 91 94 96 97 97 Experiment 3VIABILITY (%) 86 93 95 96 97 97 Experiment 4VIABILITY(%) 86 94 96 97 98 98 Experiment 5VIABILITY (%) 86 95 97 98 98 99 Experiment 6VIABILITY (%) 86 96 97 98 99 99 Table 2. VIABILITY% The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A defined insect culture medium containing nanoparticles, characterized in that: The culture medium specifically includes the following components: Sugars: 5-12g / L glucose, 3-6g / L sucrose, 3-7g / L fructose; Amino Acids: 1-3g / L histidine, 0.5-3.0g / L aspartic acid, 0.5-1.2g / L serine, 1.0-1.5g / L glycine, 0.5-2.6g / L arginine, 0.5-1.8g / L asparagine, 0.1-1.0g / L isoleucine, 0.1-1.0g / L leucine, 1.0-3.0g / L glutamic acid, 0.1-1.0g / L valine, 0.5-1.0g / L glutamine, 0.5-1.0g / L lysine, 0.1-0.6g / L methionine, 0.1-0.2g / L phenylalanine, 0.2-0.4g / L proline, 0.2-0.8g / L threonine, 0.01-0.5g / L tyrosine, 0.1-0.5g / L alanine; Inorganic salts: 2.0-4.0g / L potassium chloride, 0.5-1.2g / L calcium chloride, 0.8-1.5g / L sodium dihydrogen phosphate, 1.0-5.0g / L sodium chloride, 1.0-2.0g / L magnesium sulfate, 0.8-2.0g / L magnesium chloride; Vitamins: 0.001-0.01g / L biotin, 0.001-0.1g / L calcium pantothenate, 0.001-0.1g / L folic acid, 0.001-0.1g / L niacin, 0.01-0.1g / L pyridoxine hydrochloride; Organic acids: 0.0001-0.5g / L ketoglutaric acid, 0.001-0.1g / L fumaric acid, 0.02-0.8g / L malic acid, 0.001-0.01g / L succinic acid; Other trace elements: 0.001-0.1g / L zinc chloride, 0.01-0.05g / L ferric chloride, 0.01-0.1g / L aluminum chloride, 0.1-0.5g / L manganese chloride; Nanoparticles: The mass of nanoparticles accounts for 0.01% - 0.1% of the total mass of the culture medium.

2. The defined insect culture medium containing nanoparticles according to claim 1, characterized in that: The nanoparticles have an inner layer of TiO2 and Fe3O4 composite nanoparticles and an outer layer of Tween 20.

3. The defined insect culture medium containing nanoparticles according to claim 2, characterized in that: The mass ratio of TiO2 and Fe3O4 in the inner layer of nanoparticles is 1:1; The ratio of the mass of TiO2 and Fe3O4 composite nanoparticles to the volume of Tween 20 is 5mg:1mL-40mg:1mL.

4. The defined insect culture medium containing nanoparticles according to claim 3, characterized in that: The nanoparticles also include two types, one of which has a mass ratio of TiO2 to Fe3O4 of 3:1, and the other has a mass ratio of TiO2 to Fe3O4 of 1:

3.

5. The defined insect culture medium containing nanoparticles according to claim 4, characterized in that: Different nanoparticles are used at different stages of cell culture, specifically: In the early stage of cell culture, i.e. 0-24 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 3:1 are used; in the middle stage of cell growth, i.e. 24-72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:1 are used; in the cell growth stabilizer, i.e. after 72 hours, nanoparticles with a mass ratio of TiO2 to Fe3O4 of 1:3 are used.

6. A nanoparticle-containing defined insect culture medium according to claim 5, characterized in that: The specific cultivation method is: S1. suspending the nanoparticles in sterile deionized water to form a uniform dispersion; S2, adding the nanoparticle dispersion into the culture medium, and performing ultrasonic treatment for 1-2 hours using an ultrasonic cleaning apparatus to obtain a nanoparticle culture medium; The content of the nanoparticle dispersion does not exceed 0.5% of the mass of the culture medium; the specific parameters of the ultrasonic treatment of the ultrasonic cleaning instrument are: frequency 10kHZ, power 100w; S3, the nanoparticle culture medium is placed in a culture container, the bottom of the culture container is transparent, an ultraviolet light source is arranged at the bottom of the culture container, a magnet is used to control the nanoparticles to precipitate at the bottom, and at this time, an ultraviolet light source is used to irradiate from the bottom; Among them, the wavelength of the ultraviolet light source is 320-400nm, the power is <0.1W / cm², the irradiation time is 10-20min, 1-2 times a day.

7. The defined insect culture medium containing nanoparticles according to claim 6, characterized in that: An ultraviolet light source is arranged at the bottom of the culture container, and a stirring device is arranged above the ultraviolet light source.

8. The defined insect culture medium containing nanoparticles according to claim 7, characterized in that: Step S3: When the nanoparticles are precipitated at the bottom layer and irradiated from the bottom with an ultraviolet light source, a stirring device is started with a stirring speed of 30-60 rpm.

9. The defined insect culture medium containing nanoparticles according to claim 6, characterized in that: A moving magnetic field is set outside the culture container, and the moving magnetic field can move up and down; the magnetic strength of the moving magnetic field is 0.1-1.5T; the moving speed is 5-500mm / h; in step S3, the moving magnetic field is used to control the precipitation of nanoparticles at the bottom layer.

10. The defined insect culture medium containing nanoparticles according to claim 9, characterized in that: At different stages of cell culture, the magnetic strength and moving speed of the moving magnetic field are different, specifically: In the initial stage of cell culture, i.e. 0-24 hours, the magnetic strength of the moving magnetic field is 0.1-0.5T; the moving speed is 5-50mm / h; in the middle stage of cell growth, i.e. 24-72 hours, the magnetic strength of the moving magnetic field is 0.5-1T; the moving speed is 50-250mm / h; in the stable period of cell growth, i.e. after 72 hours, the magnetic strength of the moving magnetic field is 1-1.5T; the moving speed is 250-500mm / h.

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