Method for improving cell growth and expression by changing iron form in culture medium

Abstract

The invention discloses a method for improving cell growth and expression by changing an iron form in a culture medium, and relates to the technical field of cell culture, the culture medium comprises a basic culture medium, a supplemented culture medium and an iron source; wherein the iron source is FeCl3, the basic culture medium and the supplementary culture medium are commercially available culture media, the iron source is not added or removed, and the culture media further contain a three-dimensional culture material; the redox balance of the culture medium can be maintained, oxidative stress damage of cells is prevented, hydrolysis or precipitation is not prone to occurring, the preparation process of the culture medium is simplified, and the requirements of cell growth and expression are met.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a method for changing the iron form in a culture medium to improve cell growth and expression. Background Art

[0002] Monoclonal antibodies (abbreviated as MAbs) are generally widely used in the field of medical diagnosis and treatment, and have extremely important social and economic value. Due to the large demand for therapeutic MAbs and low expression levels, optimizing their production process and increasing antibody production are particularly urgent for current large-scale production.

[0003] Serum-free culture medium generally contains carbon source (glucose), amino acids, inorganic salts, vitamins, trace elements and growth factors. Trace elements are also an indispensable part of cell culture. Among them, iron is an important trace element that plays an important role in cell growth and metabolism.

[0004] Iron is a component of many cellular enzymes, proteins, and other biomolecules. It plays an important role in key biological processes such as oxygen transport, redox reactions, and DNA synthesis. Iron is essential for cellular respiration and metabolism. Without iron, cells stop growing and eventually die. Iron can undergo univalent redox reactions. Its redox forms are ferric and ferrous. The main functions of iron in cell culture systems are as follows: Hemoglobin is a heme-rich protein in mammals, and its main function is to bind, transport, and release molecular oxygen; Cytochromes are a group of heme-containing proteins located in mitochondria; As an enzyme cofactor, iron can protect cells from oxidative damage; Iron is one of the important components in the synthesis of DNA and proteins.

[0005] Understanding the chemistry of iron is essential to designing a stable and adaptable culture medium. Iron management is a critical success factor for cell culture. Iron exists in both ferrous and ferric states in physiological solutions. It is often added to cell culture media as nitrate or sulfate. In solution, iron undergoes redox cycling depending on the composition of oxidants and reductants in the culture medium and its exposure to these agents. The sensitivity of iron to the redox potential of the culture medium depends on the molecules with which it is complexed. Iron complexes can be complexed with biomolecules such as amino acids, nucleotides, physiological chelators, proteins, etc. The specific complexes are important because they will determine whether the iron is available to participate in cell growth, catalyze toxic reactions, or become unavailable to the system. Free or ineffectively chelated iron can be very toxic to cells. Properly complexed iron is available to support cell growth and is critical to the cell culture system. Therefore, choosing the right iron form plays an important role in enhancing cell growth and expression. Summary of the invention

[0006] The embodiments of the present application provide a method for improving cell growth and expression by changing the form of iron in the culture medium, thereby solving the problem of iron toxicity to cells in the prior art and achieving the enhancement of cell growth and expression.

[0007] The embodiment of the present application provides a method for changing the form of iron in a culture medium to improve cell growth and expression, wherein the culture medium includes: a basal culture medium, a feed culture medium, and an iron source; wherein the iron source is FeCl3.

[0008] Furthermore, the basal medium and the feed medium are commercially available medium, and no iron source is added or the iron source is removed.

[0009] Furthermore, the culture medium also contains three-dimensional culture materials.

[0010] Furthermore, the preparation method of the three-dimensional cell culture material comprises the following steps: S1. Add 6% polyvinyl alcohol by mass to the basal culture medium, stir for 2 hours at 60°C to obtain a basal culture medium solution containing a gel aqueous solution; the molecular weight of the polyvinyl alcohol is 1750±50; S2. Add glycerol to the basic culture medium solution containing the gel aqueous solution at a volume ratio of 1.5:1 to obtain a mixed solution, and then stir it at a temperature of 60°C for 2 hours to obtain a liquid three-dimensional cell culture material. Add transferrin to the liquid three-dimensional cell culture material, and let it stand at room temperature for 3 hours to obtain a solid three-dimensional cell culture material containing transferrin.

[0011] Furthermore, the concentration of transferrin is 50 mg / L.

[0012] Furthermore, the three-dimensional culture material has two layers, including a transferrin layer and a ferric chloride solution layer.

[0013] Furthermore, the volume ratio of glycerol to the basic culture medium solution containing the gel aqueous solution in the ferric chloride solution layer material is 4:1, and it is solidified at -20°C, and 50 μM ferric chloride is added to the liquid three-dimensional culture material.

[0014] Furthermore, the volume ratio of glycerol to the basic culture medium solution containing the gel aqueous solution in the three-dimensional culture material in the transferrin layer material is 2:1, 50 mg / L transferrin is added to the liquid three-dimensional culture material, and it is wrapped with the ferric chloride solution layer material at low temperature and left to solidify at room temperature.

[0015] Furthermore, there is a transferrin layer loaded with ferric chloride outside the transferrin layer.

[0016] Furthermore, specifically, 30 μM ferric chloride and 50 mg / L transferrin were added to the liquid three-dimensional culture material loaded with the transferrin layer of ferric chloride, and the double-layer three-dimensional culture material was wrapped after stirring, and allowed to stand at room temperature for 3 hours to obtain a three-layer three-dimensional culture material.

[0017] One or more technical solutions provided in the embodiments of the present application have at least the following technical effects or advantages: First, ferric chloride is acidic when dissolved in water, making the overall environment of the culture medium acidic, which is beneficial to the stability of iron ions. Ferric chloride can also affect the expression of genes related to cell apoptosis to increase cell survival rate and cell proliferation rate. Ferric chloride can also be used as a redox potential indicator to help maintain the redox balance of the culture medium and prevent cell oxidative stress damage. FeCl3 can remain stable in a wide pH range and is not prone to hydrolysis or precipitation. This makes it unnecessary to strictly control the pH value when adding FeCl3 to the culture medium, thereby simplifying the preparation process of the culture medium. FeCl3 has a high solubility in water and is not easy to form precipitation. This makes it possible to maintain a high iron ion concentration when adding FeCl3 to the culture medium, thereby meeting the needs of cell growth and expression.

[0018] Its two- and three-dimensional culture materials provide cells with a growth space that is closer to the in vivo environment, which can better simulate the growth state of cells in the body and promote interaction and signal transduction between cells.

[0019] Third, the two-layer structure of the three-dimensional culture material provides cells with a culture condition that is closer to the in vivo environment, which helps to improve the effect and success rate of cell culture.

[0020] Fourth, by designing a three-layer structure with different porosities, a dynamic iron ion supply system is formed. The high porosity of the outer layer is conducive to the rapid penetration and binding of iron ions, while the low porosity of the middle layer may play a buffering and regulatory role, ensuring a continuous and stable supply of iron ions, optimizing the cell microenvironment, and providing cells with a culture condition that is closer to the in vivo environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The graph of the change of viable cell density over time in a 125 mL shake flask in medium with different concentrations of iron-based substances (10 6 cells / mL); Figure 2 The cell viability of cells in 125 mL shaking flask in different concentrations of iron-based medium changes over time (%); Figure 3is the graph of the change of cell diameter over time in a 125 mL shake flask in a medium with different concentrations of iron forms (μm); Figure 4 This is the antibody protein concentration (mg / L) detected by Cedex method in 125mL shaking flasks with cells in different concentrations of iron-based medium; Figure 5 This is a graph to verify the change of viable cell density over time in 125mL shake flasks in various iron-based medium (10 6 cells / mL); Figure 6 It is a graph to verify the change of cell viability over time in 125mL shake flask in various iron-based medium (%); Figure 7 It verifies the change of cell diameter over time in 125mL shake flask in various iron-based medium (μm); Figure 8 This is a graph of antibody protein concentration (mg / L) detected by Cedex method in 125mL shake flasks to verify cells in various iron-based culture media. DETAILED DESCRIPTION

[0022] 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.

[0023] In order to ensure that the technical means, optimization direction, and expected effects adopted by the present invention are easy to understand, the embodiments of the present invention will be specifically described below. The following embodiments are used in the present invention, but are not used to limit the protection scope of the present invention. The experimental methods in the embodiments are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following embodiments are obtained from commercial channels unless otherwise specified. The various material components of the cell culture medium can be purchased from Sigma, GIBCO, etc. Other reagents or instruments without indicating the manufacturer are conventional reagents or instruments. Embodiment 1

[0024] The present application discloses a method for changing the iron form in a culture medium to improve cell growth and expression, comprising a basal culture medium, a feed culture medium and an iron element. The basal culture medium and the feed culture medium of the present invention are chemically defined culture mediums, and do not contain an iron element or the iron element is removed, and the components and contents are as follows: Amino acid: 400-5000mg / L; Vitamins: 30-1000mg / L; Trace elements & inorganic salts: 2500-25000mg / L; Lipids: 1-20 mg / L; Carbohydrates: 3000-15000 mg / L; Additives: 50-2000mg / L; Add 30 mL of recovery medium to a 125 mL shake flask in advance, preheat it in a 37 °C shaker, then take out a CHOK1 cell cryotube from the liquid nitrogen tank and quickly thaw it in a 37 °C constant temperature water bath, mix it thoroughly to melt, wipe off the surface moisture, wipe the cryotube with 75% alcohol, and quickly add it to the preheated 125 mL shake flask. After sampling and counting, place the cells in a 110-130 rpm, 5-8% CO2, 37 ± 0.5 °C shaker for culture; perform a subculture operation every 2-3 days to ensure that the cells are in the logarithmic growth phase, subculture more than three times, and start inoculation after ensuring that the cells are in a stable doubling state. The culture start time is D0, and the inoculation density is 0.4-0.8×10 6 cells / mL, inoculated into 125mL shake flasks. The specific shaker parameters were set as follows: culture temperature was 37±0.5℃, CO2 was 8%, humidity was 80%, and speed was 110-130rpm.

[0025] During the whole culture process, samples were taken and fed every other day. Countstar counter was used to count to avoid counting errors. Each sample was counted three times, and the cell survival rate was calculated by the plate blue staining method. Glucose and lactate were tested by M100 biochemical analyzer, and the feeding strategy was 3-5%. The culture was harvested at D13, and the samples were collected by centrifugation. The antibody protein IgG concentration was tested using Cedex Bio.

[0026] Experimental plan: Six kinds of iron substances were added to the basal medium without iron, and two concentrations were set, high and low. The high concentration was marked as H, the low concentration was marked as L (experiments 1-12), and the control was marked as √. In terms of iron content, H was 75 μM, L was 25 μM, and the iron concentration of the control experiment was 50 μM. The experimental culture medium used the basal culture medium and feed medium of Shanghai Duoning Biotechnology Co., Ltd., specifically: Maxfeed 403A1 basal culture medium + Maxfeed403B1 feed medium. The cell culture scheme and results are shown in Tables 1, 2, 3, 4, and 5. The key combinations are as follows: Figure 1 , Figure 2 , Figure 3 , Figure 4 As shown: Table 1 Specific implementation plan for shake flask culture Shake bottle number Inoculation medium <![CDATA[FeSO4]]> <![CDATA[FeCl2]]> <![CDATA[FeCl3]]> <![CDATA[Fe2(SO4)3]]> <![CDATA[Fe(NO3)2]]> <![CDATA[Fe(NO3)3]]> Control experiment Commercial culture medium √ / / / / / Experiment 1 Commercial culture medium / L / / / / Experiment 2 Commercial culture medium / H / / / / Experiment 3 Commercial culture medium / / L / / / Experiment 4 Commercial culture medium / / H / / / Experiment 5 Commercial culture medium / / / L / / Experiment 6 Commercial culture medium / / / H / / Experiment 7 Commercial culture medium / / / / L / Experiment 8 Commercial culture medium / / / / H / Experiment 9 Commercial culture medium / / / / / L Experiment 10 Commercial culture medium / / / / / H Table 2 Viable cell density of different iron forms in 125 mL shake flask (×10 5 cells / mL) over time Cultivation time / day 0 3 5 7 9 11 13 Control experiment 3.48 47.90 119.00 143.00 201.00 129.00 95.00 Experiment 1 3.48 55.40 124.00 203.00 167.00 104.00 91.40 Experiment 2 3.48 56.00 113.00 178.00 119.00 134.00 113.00 Experiment 3 3.48 48.10 134.00 209.00 191.00 131.00 105.00 Experiment 4 3.48 46.70 124.00 220.00 186.00 156.00 127.00 Experiment 5 3.48 67.90 103.00 157.00 205.00 127.00 95.00 Experiment 6 3.48 55.30 125.00 180.00 144.00 118.00 96.20 Experiment 7 3.48 48.50 152.00 205.00 182.00 136.00 116.00 Experiment 8 3.48 37.10 132.00 155.00 147.00 125.00 112.00 Experiment 9 3.48 74.90 122.00 175.00 131.00 133.00 97.20 Experiment 10 3.48 52.80 115.00 172.00 171.00 134.00 91.50 Table 3 Trend of cell viability (%) of different iron forms in 125 mL shake flask over time Cultivation time / day 0 3 5 7 9 11 13 Comparison Example 98.53 99.14 96.12 97.81 92.23 89.21 84.00 Experiment 1 98.53 98.87 95.68 98.65 93.18 87.63 77.66 Experiment 2 98.53 98.60 95.88 97.03 93.23 91.56 87.20 Experiment 3 98.53 99.09 98.35 97.29 94.28 89.37 84.54 Experiment 4 98.53 99.13 98.34 99.00 95.77 94.37 92.26 Experiment 5 98.53 98.57 94.66 98.85 94.34 86.42 76.39 Experiment 6 98.53 99.18 96.06 98.88 91.76 87.27 80.26 Experiment 7 98.53 99.54 95.45 98.51 92.86 89.36 85.47 Experiment 8 98.53 98.64 94.79 98.09 93.85 91.87 89.44 Experiment 9 98.53 97.25 97.01 97.96 91.34 89.01 78.83 Experiment 10 98.53 99.33 95.38 98.17 94.63 87.33 80.02 Table 4 Trends of cell diameter (μm) over time for different combinations of iron forms in 125 mL shake flasks Cultivation time / day 0 3 5 7 9 11 13 Comparison Example 14.59 14.52 15.24 16.24 17.83 18.77 18.85 Experiment 1 14.59 14.42 15.02 15.76 17.34 18.03 18.43 Experiment 2 14.59 14.42 14.67 15.80 16.96 18.42 19.19 Experiment 3 14.59 14.18 14.67 15.76 17.12 17.95 17.76 Experiment 4 14.59 14.40 14.98 16.08 17.11 18.57 18.52 Experiment 5 14.59 14.21 14.91 15.81 17.19 17.92 18.16 Experiment 6 14.59 14.28 14.82 16.00 17.04 17.82 18.16 Experiment 7 14.59 14.40 14.84 16.18 17.16 18.11 18.45 Experiment 8 14.59 14.31 15.01 15.71 17.39 18.56 19.20 Experiment 9 14.59 14.36 15.59 16.57 18.13 18.82 18.97 Experiment 10 14.59 14.28 14.86 15.86 17.12 18.15 18.14 Table 5 Protein expression of different iron forms in 125 mL shake flasks Cultivation time / day D13 Titer (mg / L) Control experiment 2961.00 Experiment 1 2439.00 Experiment 2 2121.00 Experiment 3 3222.00 Experiment 4 3392.00 Experiment 5 2495.00 Experiment 6 2708.00 Experiment 7 2774.00 Experiment 8 2733.00 Experiment 9 2384.00 Experiment 10 2716.00 To avoid the accidental nature of the experimental results, the six iron forms were repeatedly tested in other CHOK1 cells to determine the optimal concentration range for cell growth.

[0027] The experimental scheme is to add 6 kinds of iron substances to the basic medium without iron element, set the same concentration of 30μM, the control is marked as √, and the other experiments are marked as +. The cell culture scheme and results are shown in Table 6, Table 7, Table 8, Table 9, Table 10. The key combinations are as follows Figure 5 , Table 6, Table 7, Table 8: Table 6 Specific implementation plan for shake flask culture Shake bottle number Inoculation medium <![CDATA[FeSO4]]> <![CDATA[FeCl2]]> <![CDATA[FeCl3]]> <![CDATA[Fe2(SO4)3]]> <![CDATA[Fe(NO3)2]]> <![CDATA[Fe(NO3)3]]> Control experiment Commercial culture medium √ / / / / / Experiment 1 Commercial culture medium / + / / / / Experiment 2 Commercial culture medium / / + / / / Experiment 3 Commercial culture medium / / / + / / Experiment 4 Commercial culture medium / / / / + / Experiment 5 Commercial culture medium / / / / / + Table 7 Viable cell density of different iron forms in 125 mL shake flask (×10 5 cells / mL) over time Cultivation time / day 0 3 5 7 9 11 13 Control experiment 3.87 40.40 141.00 146.00 110.00 80.70 72.70 Experiment 1 3.87 38.60 130.00 145.00 122.00 92.90 32.00 Experiment 2 3.87 43.00 143.00 160.00 121.00 119.00 98.70 Experiment 3 3.87 39.50 127.00 136.00 112.00 93.40 73.20 Experiment 4 3.87 42.60 137.00 161.00 129.00 86.00 62.10 Experiment 5 3.87 37.90 137.00 142.00 122.00 90.90 69.70 Table 8 Trend of viable cell viability (%) of different iron forms in 125 mL shake flask over time Cultivation time / day 0 3 5 7 9 11 13 Control experiment 98.99 98.29 98.41 96.11 86.28 74.77 55.74 Experiment 1 98.99 97.87 98.28 94.25 90.98 72.14 57.14 Experiment 2 98.99 98.92 98.85 97.15 89.12 91.28 92.11 Experiment 3 98.99 97.41 98.70 95.46 91.48 83.42 83.70 Experiment 4 98.99 99.03 98.99 95.45 91.25 82.98 60.28 Experiment 5 98.99 96.25 96.86 94.67 89.60 83.64 64.46 Table 9 Trends of cell diameter (μm) over time for different combinations of iron forms in 125 mL shake flasks Cultivation time / day 0 3 5 7 9 11 13 Control experiment 14.12 15.02 14.91 16.43 17.23 18.65 18.08 Experiment 1 14.12 14.94 15.01 16.44 17.27 18.61 18.66 Experiment 2 14.12 15.09 15.13 16.65 17.75 18.76 18.96 Experiment 3 14.12 15.52 15.23 17.14 17.89 19.25 19.69 Experiment 4 14.12 14.88 14.80 16.39 17.34 18.92 18.43 Experiment 5 14.12 15.36 14.97 16.63 17.63 18.74 18.88 Table 10 Protein expression of different iron forms in 125 mL shake flasks Cultivation time / day D13 Titer (mg / L) Control experiment 2995.00 Experiment 1 2724.00 Experiment 2 3231.00 Experiment 3 3152.00 Experiment 4 2471.00 Experiment 5 2344.00 The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: Table 1-5 and Figure 1-4The results showed that the cells reached peak density at D7 or D9 in different iron-based medium combinations, and the peak density of the control cells was 20.1×10 6 cells / mL, the optimal combination is Example 4 (FeCl3 form), with a peak density of 22×10 6 cells / mL. As of D13 harvest, the best combination Example 4 had a higher viability than the control, with a D13 viability of 92.26%. After culture to D13, the control cell protein expression was 2961 mg / L. Except for the high and low concentration combinations of FeCl3 (Examples 3 and 4), which were higher than the control group, the other combinations were slightly lower than the control group. This result shows that the growth and expression of cells are significantly improved by replacing the original iron form with FeCl3. Therefore, the subsequent culture medium production and development can consider using this form of iron to replace the original form to achieve the purpose of improving cell protein expression.

[0028] Table 6-10 and Figure 5-8 The results showed that in repeated CHOK1 cell experiments, the cell growth and expression of the FeCl3 form were higher than the control, indicating that the positive effects of the iron form on cell growth and expression have a stable effect in the cells.

[0029] The absorption of iron by cells is mainly through the combination of transferrin and trivalent iron and its entry into cells. Divalent iron needs to be converted into trivalent iron first, or directly enters cells through cell membrane transport proteins. Therefore, generally speaking, the absorption and utilization rate of divalent iron by cells is greater than that of trivalent iron, and its toxicity is lower. However, ferric chloride is acidic when dissolved in water, making the overall environment of the culture medium acidic, which is conducive to the stability of iron ions. In addition, ferric chloride can affect the expression of genes related to cell apoptosis to increase cell survival rate and cell proliferation rate. Ferric chloride can also be used as a redox potential indicator to help maintain the redox balance of the culture medium and prevent cell oxidative stress damage. FeCl3 It can remain stable in a wide pH range and is not prone to hydrolysis or precipitation. This means that when FeCl3 is added to the culture medium, there is no need to strictly control the pH value, thereby simplifying the preparation process of the culture medium. FeCl3 has a high solubility in water and is not easy to form precipitation. This means that when FeCl3 is added to the culture medium, a high iron ion concentration can be maintained, thereby meeting the needs of cell growth and expression. Although iron ions may form complexes with other components, the complexing properties of FeCl3 are relatively weak, and it is not easy to form stable complexes with other components. It has excellent compatibility with other culture medium components, reducing the loss of iron ions and reducing the negative impact on cell growth. Embodiment 2

[0030] The above embodiment optimizes the growth and expression ability of cells by selecting ferric chloride as the iron source in the culture medium, adds transferrin to the culture medium to increase the utilization of iron by cells, reduce the local accumulation of iron ions, and reduce cell damage, and provides three-dimensional culture materials to construct a three-dimensional culture environment.

[0031] A method for preparing a three-dimensional cell culture material comprises the following steps: S1. Add 6% polyvinyl alcohol by mass to the basal culture medium, stir for 2 hours at 60°C to obtain a basal culture medium solution containing a gel aqueous solution; the molecular weight of the polyvinyl alcohol is 1750±50; S2. Add glycerol to the basic culture medium solution containing the gel aqueous solution at a volume ratio of 1.5:1 to obtain a mixed solution, and then stir the mixed solution at 60°C for 2 hours to obtain a liquid three-dimensional cell culture material. Add transferrin to the liquid three-dimensional cell culture material, and let it stand at room temperature for 3 hours to obtain a solid three-dimensional cell culture material containing transferrin, wherein the concentration of transferrin is 50 mg / L.

[0032] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: Transferrin plays a key role in the absorption and utilization of iron by cells. It combines with trivalent iron to form a stable complex and enters the cell through transport proteins on the cell membrane. Inside the cell, transferrin releases iron ions for the cell to carry out various biosynthetic reactions. Therefore, adding transferrin to the culture medium can increase the utilization rate of iron by cells, reduce the local accumulation of iron ions, and thus reduce the risk of cell damage. Three-dimensional culture materials provide cells with a growth space that is closer to the in vivo environment, which can better simulate the growth state of cells in the body and promote interaction and signal transduction between cells. In addition, three-dimensional culture materials can also increase the attachment area and growth density of cells, thereby increasing cell yield and expression level. By adding transferrin to the three-dimensional culture materials, the growth and expression conditions of cells are further optimized, so that cells can more effectively utilize the iron in the culture medium, and the three-dimensional culture materials provide an environment that is more conducive to cell growth and expression, which not only increases the growth rate and density of cells, but also significantly increases the expression level of cells.

[0033] According to the experimental scheme of Example 1, the experiment was set up. The control experiment was to add only 30 μM ferric chloride. The experiment of this example also added a three-dimensional culture material loaded with transferrin. The volume of the three-dimensional culture material was 10% of the culture medium. The results are shown in Tables 11-13: Table 11 Live cell density in 125 mL shake flask (×10 5 cells / mL) over time Cultivation days / day 0 5 9 13 Controlled trials 3.68 141.00 125.00 99.80 Embodiment 2 3.68 149.00 131.00 108.00 Table 12 Trend of viability of live cells (%) in 125 mL shake flask over time Cultivation time / day 0 5 9 13 Control experiment 98.75 98.35 89.45 90.28 Embodiment 2 98.75 98.44 91.57 91.87 Table 13 Protein expression in shake flask Cultivation time / day D13 Titer (mg / L) Control experiment 3215 Embodiment 2 3425 Embodiment 3

[0034] The above embodiment improves cell growth and expression by using transferrin and three-dimensional culture materials, and is further improved on the basis of the second embodiment in order to improve the iron balance of the culture environment and promote cell growth and proliferation.

[0035] The obtained three-dimensional culture material has two layers, specifically, the three-dimensional culture materials of each layer are prepared separately, the volume ratio of glycerol to the basic culture medium solution containing the gel aqueous solution in the inner layer material is 4:1, so that it does not completely solidify at room temperature and solidifies at -20°C, and then a layer of liquid three-dimensional culture material is wrapped outside, the volume ratio of glycerol to the basic culture medium solution containing the gel aqueous solution in the outer layer three-dimensional culture material is 2:1, and it is left to solidify at room temperature; In the case of liquid three-dimensional culture materials, 50 μM ferric chloride was added to the inner layer material, and 50 mg / L transferrin was added to the outer layer material.

[0036] The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: The inner layer material is not completely solidified at room temperature, which is conducive to the diffusion and exchange of the inner layer of ferric chloride. Since it still has a certain viscosity, the exchange rate is very slow, which increases its slow release during the culture process, reduces toxicity, and increases cell growth. The outer layer material solidifies at room temperature, providing a stable support structure for the cells. The components such as glycerol and gel aqueous solution in the inner and outer layer materials can provide the cells with the required nutrients and growth factors, which is conducive to cell proliferation and differentiation. The ferric chloride in the inner layer material and the transferrin in the outer layer material can affect the function of cells by regulating the iron metabolism in the cells. The iron combines with the transferrin while being exchanged and utilized through the three-dimensional structure, which reduces the free iron ions in the culture medium and reduces the cytotoxicity. In addition, the ferric chloride increases the stability of the hydrogel, making the structure of the hydrogel more stable in the culture environment and having better adsorption and penetration capabilities. The two-layer three-dimensional culture material provides cells with a culture condition that is closer to the in vivo environment, which helps to improve the effect and success rate of cell culture. By adjusting the composition and proportion of the inner and outer layer materials, ferric chloride is applied to the three-dimensional culture material, providing new possibilities for the application of ferric chloride in fields such as cell culture and tissue engineering.

[0037] According to the experimental scheme of Example 2, the experiment was set up. The control experiment was to add only the three-dimensional culture material. The experiment of this example also added a double-layer three-dimensional culture material, and the volume of the three-dimensional culture material was 10% of the culture medium. The results are shown in Tables 14-16: Table 14 Live cell density in 125 mL shake flask (×10 5 cells / mL) over time Cultivation days / day 0 5 9 13 Controlled trials 3.71 148.00 132.00 108.00 Embodiment 3 3.68 151.00 133.00 111.00 Table 15 Trend of viable cell viability (%) in 125 mL shake flask over time Cultivation time / day 0 5 9 13 Control experiment 98.83 98.30 91.52 91.82 Embodiment 3 98.83 98.51 94.65 93.45 Table 16 Protein expression in shake flask Cultivation time / day D13 Titer (mg / L) Control experiment 3455 Embodiment 3 3581 Embodiment 4

[0038] Example 3 The double-layer three-dimensional culture material is used to slowly release iron ions and reduce local iron ion toxicity. In order to improve the efficiency and quality of cell culture, further improvements are made on the basis of Example 3.

[0039] Design a three-layer three-dimensional culture material, the outer layer contains transferrin loaded with ferric chloride, the middle layer contains transferrin, and the inner layer contains ferric chloride solution; The outer layer contains transferrin loaded with ferric chloride, specifically, 30 μM ferric chloride and 50 mg / L transferrin are added to the liquid three-dimensional culture material of the outer layer, and the double-layer three-dimensional culture material obtained in Example 2 is wrapped after stirring, and the three-layer three-dimensional culture material is left to stand at room temperature for 3 hours to obtain a three-layer three-dimensional culture material. The technical solutions in the above embodiments of the present application have at least the following technical effects or advantages: The ferric chloride solution in the inner layer provides a stable source of iron ions for cell culture. The outer layer is loaded with transferrin containing loaded iron ions, which further ensures the effective supply of iron ions and is completely non-toxic to cells. Transferrin has the ability to bind to iron ions with high affinity and can transport iron ions into cells. The transferrin in the middle layer serves as a buffer and regulatory layer for iron ions, which can adjust the supply rate and amount of iron ions according to the needs of cells. Moreover, ferric chloride affects the porosity of the material, so that the porosity of the material in the outer layer is high, and the porosity of the middle layer is low during the period of low curing temperature because the inner layer is cured at low temperature; the three-dimensional culture materials in the outer and middle layers have different porosities, which affect the diffusion rate of oxygen, nutrients and metabolic waste in the cell growth environment. The layer with a larger porosity can provide a better supply of oxygen and nutrients, while promoting the discharge of metabolic waste, which is beneficial to cell growth and metabolism; transferrin realizes the transport and utilization of iron ions in cell culture materials through reversible binding with iron ions. After transferrin binds to iron ions, it can be endocytosed into the cell through the transferrin receptors on the cell surface, releasing iron ions for cell use; the low porosity of the middle layer makes the ferric chloride in the inner layer more likely to bind to the transferrin on the middle layer when exchanging substances outward, and then the large pores of the outer layer make the combined transferrin easier to contact with cells, so that the sustained and controlled release of iron ions is further enhanced; By designing a three-layer structure with different porosities, a dynamic iron ion supply system is formed. The high porosity of the outer layer is conducive to the rapid penetration and binding of iron ions, while the low porosity of the middle layer may play a buffering and regulatory role, ensuring the continuous and stable supply of iron ions, optimizing the cell microenvironment, and providing cells with a culture condition that is closer to the in vivo environment. By regulating parameters such as iron ion supply and porosity, it promotes cell growth, differentiation, and metabolism. It is suitable for fields such as cell culture and tissue engineering, and may also be expanded to more application fields such as drug screening and disease model establishment. Its unique iron ion supply system and porosity regulation mechanism provide new ideas and methods for research and application in related fields.

[0040] According to the experimental scheme of Example 3, the experiment was set up. The control experiment was to add only two layers of three-dimensional culture materials. The experiment of this example also added three layers of three-dimensional culture materials. The volume of the three-dimensional culture materials was 10% of the culture medium. The results are shown in Tables 17-19: Table 17 Live cell density in 125 mL shake flask (×10 5 cells / mL) over time Cultivation days / day 0 5 9 13 Controlled trials 3.83 143.00 131.00 108.00 Embodiment 4 3.83 153.00 139.00 120.00 Table 18 Trend of viability of live cells (%) in 125 mL shake flask over time Cultivation time / day 0 5 9 13 Control experiment 97.98 97.64 92.74 91.05 Embodiment 4 97.98 97.86 93.74 93.10 Table 19 Protein expression in shake flask Cultivation time / day D13 Titer (mg / L) Control experiment 3587 Embodiment 4 3692 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 method for changing the form of iron in a culture medium to improve cell growth and expression, characterized in that: The culture medium includes: a basic culture medium, a supplement culture medium, and an iron source; wherein the iron source is FeCl3.

2. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 1, characterized in that: The basal medium and feed medium were commercially available medium without iron source or with iron source removed.

3. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 1, characterized in that: The culture medium also contains three-dimensional culture material.

4. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 3, characterized in that: The method for preparing a three-dimensional cell culture material comprises the following steps: S1. Add 6% polyvinyl alcohol by mass to the basal culture medium, stir for 2 hours at 60°C to obtain a basal culture medium solution containing a gel aqueous solution; the molecular weight of the polyvinyl alcohol is 1750±50; S2. Add glycerol to the basic culture medium solution containing the gel aqueous solution at a volume ratio of 1.5:1 to obtain a mixed solution, and then stir it at a temperature of 60°C for 2 hours to obtain a liquid three-dimensional cell culture material. Add transferrin to the liquid three-dimensional cell culture material, and let it stand at room temperature for 3 hours to obtain a solid three-dimensional cell culture material containing transferrin.

5. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 4, characterized in that: The concentration of transferrin was 50 mg / L.

6. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 5, characterized in that: The three-dimensional culture material has two layers, including a transferrin layer and a ferric chloride solution layer.

7. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 6, characterized in that: The volume ratio of glycerol to the basic culture medium solution containing the gel aqueous solution in the ferric chloride solution layer material was 4:1, and it was solidified at -20°C, and 50 μM ferric chloride was added to the liquid three-dimensional culture material.

8. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 7, characterized in that: The volume ratio of glycerol to the basic culture medium solution containing the gel aqueous solution in the three-dimensional culture material in the transferrin layer material is 2:

1. 50 mg / L transferrin is added to the liquid three-dimensional culture material, and it is wrapped with the ferric chloride solution layer material at low temperature and left to solidify at room temperature.

9. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 8, characterized in that: There is another transferrin layer outside the transferrin layer which is loaded with ferric chloride.

10. A method for changing the form of iron in a culture medium to improve cell growth and expression as claimed in claim 9, characterized in that: Specifically, 30 μM ferric chloride and 50 mg / L transferrin were added to the liquid three-dimensional culture material loaded with the transferrin layer of ferric chloride, and the double-layer three-dimensional culture material was wrapped after stirring, and the three-layer three-dimensional culture material was allowed to stand at room temperature for 3 hours to obtain a three-layer three-dimensional culture material.

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