Additive for mammalian cell culture medium and application thereof

Abstract

The invention relates to a mammalian cell culture medium additive and application thereof. The culture medium additive comprises the following effective components: pyrroloquinoline quinone, spirulina, daidzein, alfalfa extract, porphyra polysaccharide, papaya extract and lycium barbarum polysaccharide. By adding 3%-5% (v / v) of the culture medium additive into the culture medium, cell proliferation can be effectively promoted, cell aging can be reduced, cells can efficiently produce target products, the culture medium additive is compatible with various culture media, the use method is flexible, the production cost can be effectively reduced, and economic benefits can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and in particular relates to a mammalian cell culture medium additive and application thereof. Background Art

[0002] The preparation of high-quality, high-purity, and high-natural fidelity proteins has always been a key step in many biomedical experimental studies. Currently, the systems used for protein expression mainly include Escherichia coli expression system, yeast expression system, insect expression system, and mammalian expression system. Among them, the mammalian cell expression system has unique advantages in the initiation signal, processing, secretion, glycosylation, and phosphorylation of proteins, and can guide the post-translational modification and correct folding and refolding of proteins. The expression products are closest to natural higher biological protein molecules in terms of molecular structure, physicochemical properties, and biological functions, and their activity is much higher than that of other expression systems. Therefore, they play an irreplaceable role in biomedical research and are widely used in biopharmaceuticals, gene therapy, and other fields. At present, many important proteins and glycoproteins have been prepared and produced in large quantities using mammalian cell expression systems, such as human tissue-type plasminogen activator, coagulation factor VIII, interferon, hepatitis B surface antigen, erythropoietin, human growth hormone, colony stimulating factor, etc.

[0003] However, compared with the prokaryotic expression system, which is simple, rapid, reproduces quickly, has high expression levels, and is economical, the mammalian cell expression system is complex and time-consuming, requires high operating techniques, and generally has the disadvantages of low expression output and low yield, resulting in its production cost being much higher than that of the prokaryotic expression system.

[0004] The present invention aims to provide a mammalian cell culture medium additive, which can be added to the culture medium in proportion to enable rapid cell proliferation, vigorous metabolism, reduce cell aging, and efficiently produce the target product. The additive is compatible with a variety of culture media, has a flexible use method, greatly reduces production costs, and improves economic benefits. Summary of the invention

[0005] Based on the problems of the prior art, the purpose of the present invention is to provide a mammalian cell culture medium additive that can effectively promote cell proliferation and increase the yield of the target product without inducing cytotoxicity, thereby solving the problems existing in the large-scale production of the above-mentioned mammalian cell expression system.

[0006] To achieve the above purpose, the technical solution provided by the present invention is as follows: A mammalian cell culture medium additive comprises pyrroloquinoline quinone, spirulina, daidzein, alfalfa extract, laver polysaccharide, papaya extract and wolfberry polysaccharide, wherein the concentration of pyrroloquinoline quinone is 1-10 mg / L, the concentration of spirulina is 20-50 mg / L, the concentration of daidzein is 50-100 mg / L, the concentration of alfalfa extract is 20-50 mg / L, the concentration of laver polysaccharide is 100-500 mg / L, the concentration of papaya extract is 5-20 mg / L, and the concentration of wolfberry polysaccharide is 10-50 mg / L.

[0007] Preferably, the mammalian cell culture medium additive comprises: Pyrroloquinoline quinone 1mg / L, Spirulina 20mg / L, Soyzein 50mg / L, Alfalfa extract 20mg / L, Porphyra polysaccharide 100mg / L, Papaya extract 5mg / L and Lycium barbarum polysaccharide 10mg / L.

[0008] Preferably, the mammalian cell culture medium additive comprises: Pyrroloquinoline quinone 5mg / L, Spirulina 30mg / L, Soyzein 75mg / L, Alfalfa extract 30mg / L, Porphyra polysaccharide 300mg / L, Papaya extract 15mg / L and Lycium barbarum polysaccharide 30mg / L.

[0009] Preferably, the mammalian cell culture medium additive comprises: Pyrroloquinoline quinone 10mg / L, Spirulina 50mg / L, Soybean 100mg / L, Alfalfa extract 50mg / L, Porphyra polysaccharide 500mg / L, Papaya extract 20mg / L and Lycium barbarum polysaccharide 50mg / L.

[0010] Preferably, the use of any of the above-mentioned mammalian cell culture medium additives in cell culture includes: adding it to the culture medium at a concentration of 3% to 5% (v / v), and culturing under conventional cell culture conditions.

[0011] The present invention provides a mammalian cell culture medium additive and application thereof, which has the following beneficial effects: Compared with the existing cell culture medium, after adding the mammalian cell culture medium additive of the present invention, the cell proliferation is more vigorous, the survival period is prolonged, and the expression amount of the target product is significantly improved. The culture medium additive provided by the present invention can be transported and stored at room temperature, is compatible with a variety of culture media, has a flexible use method, greatly saves production costs, improves economic benefits, and has high application value.

[0012] According to the above contents of the present invention, various other modifications, replacements or changes may be made according to the basic technical knowledge and common means in this field without departing from the basic technical idea of ​​the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a curve diagram of live cell density between the control group and different experimental groups; Figure 2 Cell viability curves of the control group and different experimental groups; Figure 3 Comparison of protein expression levels between the control group and different experimental groups; Figure 4 Comparison of transient protein expression between the control group and different experimental groups. DETAILED DESCRIPTION Example

[0014] The present embodiment provides a mammalian cell culture medium additive, comprising pyrroloquinoline quinone, spirulina, daidzein, alfalfa extract, laver polysaccharide, papaya extract and wolfberry polysaccharide, wherein the concentration of pyrroloquinoline quinone is 1 mg / L, the concentration of spirulina is 20 mg / L, the concentration of daidzein is 50 mg / L, the concentration of alfalfa extract is 20 mg / L, the concentration of laver polysaccharide is 100 mg / L, the concentration of papaya extract is 5 mg / L, and the concentration of wolfberry polysaccharide is 10 mg / L. Example

[0015] The present embodiment provides a mammalian cell culture medium additive, comprising pyrroloquinoline quinone, spirulina, daidzein, alfalfa extract, laver polysaccharide, papaya extract and wolfberry polysaccharide, wherein the concentration of pyrroloquinoline quinone is 5 mg / L, the concentration of spirulina is 30 mg / L, the concentration of daidzein is 75 mg / L, the concentration of alfalfa extract is 30 mg / L, the concentration of laver polysaccharide is 300 mg / L, the concentration of papaya extract is 15 mg / L, and the concentration of wolfberry polysaccharide is 30 mg / L. Example

[0016] The present embodiment provides a mammalian cell culture medium additive, comprising pyrroloquinoline quinone, spirulina, daidzein, alfalfa extract, laver polysaccharide, papaya extract and wolfberry polysaccharide, wherein the concentration of pyrroloquinoline quinone is 10 mg / L, the concentration of spirulina is 50 mg / L, the concentration of daidzein is 100 mg / L, the concentration of alfalfa extract is 50 mg / L, the concentration of laver polysaccharide is 500 mg / L, the concentration of papaya extract is 20 mg / L, and the concentration of wolfberry polysaccharide is 50 mg / L.

[0017] Example 4 Preparation of experimental culture medium additives Add 1L of each component of the culture medium additive to 700ml of ultrapure water and stir at room temperature for 30min until all components are dissolved. Adjust the pH value to 7.0-7.3 with freshly prepared 1M hydrochloric acid or sodium hydroxide solution. After constant volume, filter sterilize with a 0.22μm sterile filter membrane under positive pressure and store at room temperature for later use.

[0018] The preparation of the culture medium additives for specific experiments is as follows: Add 1 L of each component of the culture medium supplement in Example 1 to 700 ml of ultrapure water, stir at room temperature for 30 min until all components are dissolved, adjust the pH value to 7.1 with freshly prepared 1 M hydrochloric acid or sodium hydroxide solution, and after constant volume, filter sterilize with a 0.22 μm sterile filter membrane under positive pressure, store at room temperature for later use, and mark as Supplement 1.

[0019] According to the same preparation method, the culture medium supplement of Example 2 was prepared and marked as Supplement 2.

[0020] According to the same preparation method, the culture medium supplement of Example 3 was prepared and marked as Supplement 3. Example

[0021] In order to better illustrate the beneficial effects of the present invention, this example uses CHO cells expressing t-PA as the research object, and evaluates the effect of the culture medium additive provided by the present invention by detecting the viable cell density, cell viability and t-PA protein expression. The specific culture method is as follows: 5.1 Count the CHO cells cultured in CD CHO medium (Thermo Fisher Scientific, 10743029) at 0.6×10 6 The cells were transferred to four 125 ml cell culture flasks at a density of cells / ml and marked 1 to 4, respectively. 3% (v / v) of Supplement 1 was added to the culture medium in flask 1, 3% (v / v) of Supplement 2 was added to the culture medium in flask 2, and 3% (v / v) of Supplement 3 was added to the culture medium in flask 3. All culture flasks were placed in a shaker containing 5% CO2 and cultured at 37°C for 7 days. Samples were taken every day, stained with trypan blue, and cells were counted. The number of live cells and cell viability were recorded. After 7 days, the cell supernatant was collected and the protein expression was detected by Elisa.

[0022] 5.2 Comparative analysis of live cell density and cell viability in the four culture flasks. Figure 1 , Figure 2 , inoculated with the same cell density, the growth rate and the number of living cells in bottles 1 to 3 were significantly higher than those in bottle 4, indicating that the culture medium additive provided by the present invention can significantly promote cell proliferation, prolong cell survival time, increase cell activity, and reduce cell aging and death.

[0023] 5.3 Comparison of the expression levels of the target protein in culture flasks 1 to 4 revealed that the expression level of t-PA protein in culture flasks 1 to 3 was much higher than that in culture flask 4, among which the protein expression level in supplement group 2 was the highest, which was 2.65 times that of the control group. This indicates that under the same culture conditions, the culture medium supplement provided by the present invention has the effect of significantly increasing the expression level of the target protein, which can greatly reduce production costs and improve economic benefits in actual production. Example

[0024] HEK293 cells that had been passaged three times and had a viability of more than 90% were transfected. The experiment was divided into four groups (control group and supplement groups 1 to 3). The control group used OPM-293 CD03 medium (OPM, 81075-001), and supplement groups 1 to 3 added 5% (v / v) of supplements 1 to 3 to the OPM-293 CD03 medium used. Before transfection, the cell density was adjusted to 2×10 6 / ml; dilute 33μg target protein plasmid and 66μg transfection reagent (Hieff Trans® Liposomal Transfection Reagent) with 1.5ml Opti-MEM culture medium (Thermo Fisher, 31985070), and incubate at room temperature for 5min; gently mix the diluted plasmid and transfection reagent, and incubate the mixture at room temperature for 20min to form DNA-liposome complex; directly add 3ml DNA-liposome complex to 30ml suspended HEK293 cell culture medium, shake the culture bottle, gently mix, and transfer to a CO2 incubator for culture without changing the medium. Harvest the protein after 72 hours.

[0025] The experimental results are shown in Figure 4 Under the same transfection conditions, the cells added with the culture medium additive of the present invention had a higher transient expression level, and the expression amount of the target protein could reach 37.8 mg / ml, which was 212% higher than that of the control group OPM-293 CD03 culture medium.

[0026] It should be noted that the specific test method described in the above embodiments is only an exemplary description, which is used to explain the present invention in detail and does not constitute a limitation on the scope of the present invention. For those skilled in the art, they can still modify the technical solutions recorded in the above embodiments, or replace some of the technical features therein by equivalents. All modifications and replacement improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A mammalian cell culture medium additive, characterized in that Each liter of culture medium supplement contains 1-10 mg of pyrroloquinoline quinone, 20-50 mg of spirulina, 50-100 mg of soybean yellow element, 20-50 mg of alfalfa extract, 100-500 mg of laver polysaccharide, 5-20 mg of papaya extract and 10-50 mg of wolfberry polysaccharide.

2. The mammalian cell culture medium additive according to claim 1, characterized in that: Each liter of culture medium supplement contains 1 mg of pyrroloquinoline quinone, 20 mg of spirulina, 50 mg of soybean zein, 20 mg of alfalfa extract, 100 mg of laver polysaccharide, 5 mg of papaya extract and 10 mg of wolfberry polysaccharide.

3. The mammalian cell culture medium supplement according to claim 1, characterized in that: Each liter of culture medium supplement contains 5 mg of pyrroloquinoline quinone, 30 mg of spirulina, 75 mg of soybean zein, 30 mg of alfalfa extract, 300 mg of laver polysaccharide, 15 mg of papaya extract and 30 mg of wolfberry polysaccharide.

4. The mammalian cell culture medium supplement according to claim 1, characterized in that: Each liter of culture medium supplement contains 10 mg of pyrroloquinoline quinone, 50 mg of spirulina, 100 mg of soybean zein, 50 mg of alfalfa extract, 500 mg of laver polysaccharide, 20 mg of papaya extract and 50 mg of wolfberry polysaccharide.

5. The mammalian cell culture medium supplement according to claim 4, characterized in that: During the cell culture process, the culture medium additive is added to the culture medium at a concentration of 3% to 5% (v / v).

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