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CN116731951A - Serum-free cell culture medium and application thereof - Google Patents

Serum-free cell culture medium and application thereof Download PDF

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Publication number
CN116731951A
CN116731951A CN202310424864.5A CN202310424864A CN116731951A CN 116731951 A CN116731951 A CN 116731951A CN 202310424864 A CN202310424864 A CN 202310424864A CN 116731951 A CN116731951 A CN 116731951A
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vitamin
components
serum
culture medium
component
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张晓丹
李殿明
田春辉
齐春梅
刘甜甜
张导春
任百亮
党将将
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Qingdao Mingqin Biotechnology Co ltd
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Qingdao Mingqin Biotechnology Co ltd
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Abstract

The invention relates to a serum-free cell culture medium and application thereof, which comprises an amino acid component, a vitamin component, a carbohydrate component, a trace element component, an inorganic salt component and other molecular compound components. The culture medium of the invention contains no serum and clear components, avoids the biosafety risk and batch difference caused by using serum, adopts glucose and D-ribose to combine energy supply, combines the growth promotion regulation of propolis flavone and epimedium flavone, has the effect of improving the cell survival rate and proliferation on the basis of maintaining the normal growth of cells, thereby improving the yield and quality stability of biological products and obtaining greater economic benefit.

Description

Serum-free cell culture medium and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a serum-free cell culture medium and a method for cell culture by adopting the serum-free cell culture medium.
Background
With the rapid development of biotechnology, monoclonal antibodies, cytokines, vaccines and various bioactive proteins and other biological products are increasingly used in diagnosis and therapy, and the demand for such products is greatly increased, so that research on the technology for producing biological products by culturing animal cells in vitro on a large scale is increasingly favored.
The traditional serum culture medium is mainly bovine serum, and human serum, horse serum and the like are also used for culturing certain special cells. However, the use of serum has a plurality of disadvantages, firstly, the serum is a very complex mixture, the clear components, content and action mechanism of the serum are still unclear at present, and in particular, some polypeptide growth factors, hormones, lipids and the like are not fully known, which brings a lot of difficulties to research work; secondly, serum is produced in batches, the difference among batches is very large, the similarity of each batch of serum is extremely difficult to ensure, the standardization and continuity of experiments are limited, the use of serum also makes the standardization of experiments and production difficult, and the separation and purification work in the production of certain transgenic protein biological medicines is difficult to complete due to protein.
The serum-free culture medium is a third type of culture medium after the natural culture medium and the synthetic culture medium, and compared with the traditional culture medium, the serum-free culture medium does not contain animal serum or other animal extraction components, but still can meet the requirement of long-time growth and propagation of cells in vitro, has clear components, and is convenient for subsequent experiments and standardization of production. The main components of the serum-free culture medium in the current market are amino acid, vitamin, carbohydrate, inorganic salt and other auxiliary substances, and most of the serum-free culture medium adopts glucose as an energy source, has uneven effects and high price, and is not convenient to store and use compared with the traditional culture medium. According to the invention, the D-ribose and the glucose are combined for energy supply, so that more continuous and long-term energy supply can be provided for cells during logarithmic growth and rapid proliferation of the cells, and meanwhile, the soybean hormone and the traditional Chinese medicine components are added to promote rapid growth and proliferation of the cells, so that the yield and quality stability of biological products are improved, greater economic benefits are obtained, and the prepared culture medium can be stored at normal temperature and is convenient to transport and store.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a serum-free cell culture medium, which has definite chemical components, can obviously improve the cell culture efficiency or the production duration, and provides convenience for the large-scale production of biological products.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a serum-free cell culture medium comprises amino acid component, vitamin component, carbohydrate component, trace element component, inorganic salt component and other molecular compound component,
the amino acid component comprises the following components:
l-histidine 5-20 mg/L
L-leucine 10-30 mg/L
L-isoleucine 15-35 mg/L
L-phenylalanine 5-15 mg/L
L-lysine 20-50 mg/L
L-cysteine 10-20 mg/L
Glycine 10-50 mg/L
L-asparagine 10-50 mg/L
L-glutamic acid 1-10 mg/L
L-alanyl-L-glutamine 1-10 mg/L
L-tryptophan 5-20 mg/L
L-arginine 10-100 mg/L
L-tyrosine 5-20 mg/L
L-aspartic acid 10-30 mg/L
L-cystine 10-30 mg/L
L-methionine 3-10 mg/L
The vitamin component comprises the following components:
vitamin A1-15 mg/L
Vitamin B1 0.5-5 mg/L
Vitamin B2 0.1-1 mg/L
Vitamin B3 0.1-1 mg/L
Vitamin B6 0.5-5 mg/L
Vitamin B7 0.05-2 mg/L
Vitamin B9 2-20 mg/L
Vitamin B15 0.3-5 mg/L
Vitamin C0.5-5 mg/L
Vitamin E1.8-9.4 mg/L
Inositol 5-35 mg/L
1-10 mg/L calcium pantothenate
The carbohydrate component comprises the following ingredients:
glucose 1500-3500 mg/L
D-ribose 1000-3000 mg/L
Putrescine 0.05-5 mg/L
10-20 mg/L of ethanolamine
Linoleic acid 0.05-0.5 mg/L
The trace element component comprises the following components:
copper sulfate pentahydrate 0.05-0.5 mg/L
0.5-5 mg/L zinc sulfate heptahydrate
Ferrous sulfate heptahydrate 0.5-10 mg/L
Cobalt chloride 0.01-0.1 mg/L
Sodium selenite 0.01-1 mg/L
The inorganic salt component comprises the following components:
sodium bicarbonate 1000-3000 mg/L
Magnesium chloride 20-200 mg/L
500-2000 mg/L sodium chloride
100-250 mg/L potassium chloride
The other molecular compound components include the following:
stigmasterol 20-50 mg/L
Flavonol 20-50 mg/L
10-20 mg/L of soybean isoflavone
Insulin 0.1-10 mg/L
Cortisol 0.1-20 mg/L
Epidermal growth factor 0.1-10 mg/L
EDTA disodium 0.1-10 mg/L
Transferrin 10-20 mg/L
Propolis flavone 1-10 mg/L
Epimedium flavone 1-10 mg/L
Preferably, the serum-free cell culture medium comprises:
the amino acid composition is: l-histidine 5mg/L, L-leucine 10mg/L, L-isoleucine 15mg/L, L-phenylalanine 5mg/L, L-lysine 20mg/L, L-cysteine 10mg/L, glycine 10mg/L, L-asparagine 10mg/L, L-glutamic acid 1mg/L, L-alanyl-L-glutamine 1mg/L, L-tryptophan 5mg/L, L-arginine 10mg/L, L-tyrosine 5mg/L, L-aspartic acid 10mg/L, L-cystine 10mg/L, L-methionine 3mg/L.
The vitamin components are as follows: 1mg/L of vitamin A, 0.5mg/L of vitamin B, 0.1mg/L of vitamin B, 0.5mg/L of vitamin B, 0.05mg/L of vitamin B, 9 2mg/L of vitamin B, 0.3mg/L of vitamin B, 0.5mg/L of vitamin C, 1.8mg/L of vitamin E, 5mg/L of inositol and 1mg/L of calcium pantothenate.
The carbohydrate components are: glucose 1500mg/L, D-ribose 1000mg/L, putrescine 0.05mg/L, ethanolamine 10mg/L, and linoleic acid 0.05mg/L.
The trace elements comprise the following components: copper sulfate pentahydrate 0.05mg/L, zinc sulfate heptahydrate 0.5mg/L, ferrous sulfate heptahydrate 0.5mg/L, cobalt chloride 0.01mg/L, and sodium selenite 0.01mg/L.
The inorganic salt comprises the following components: 1000mg/L sodium bicarbonate, 20mg/L magnesium chloride, 500mg/L sodium chloride and 100mg/L potassium chloride.
Other molecular compound components are: 20mg/L stigmasterol, 20mg/L flavonol, 10mg/L soybean isoflavone, 0.1mg/L insulin, 0.1mg/L cortisol, 0.1mg/L epidermal growth factor, 0.1mg/L EDTA disodium, 10mg/L transferrin, 1mg/L propolis flavone, and 1mg/L epimedium flavone.
Preferably, the serum-free cell culture medium comprises:
the amino acid composition is: 15mg/L of L-histidine, 20mg/L of L-leucine, 25mg/L of L-isoleucine, 10mg/L of L-phenylalanine, 35mg/L of L-lysine, 15mg/L of L-cysteine, 15mg/L of glycine, 30mg/L of L-asparagine, 5mg/L of L-glutamic acid, 5mg/L of L-alanyl-L-glutamine, 10mg/L of L-tryptophan, 50mg/L of L-arginine, 12mg/L of L-tyrosine, 20mg/L of L-aspartic acid, 20mg/L of L-cystine and 6mg/L of L-methionine.
The vitamin components are as follows: vitamin A8 mg/L, vitamin B1.5 mg/L, vitamin B2.5 mg/L, vitamin B3.5 mg/L, vitamin B6.5 mg/L, vitamin B7 1mg/L, vitamin B9 10mg/L, vitamin B15 2.5mg/L, vitamin C2.5 mg/L, vitamin E5.6 mg/L, inositol 20mg/L, calcium pantothenate 5mg/L.
The carbohydrate components are: 2500mg/L glucose, 2000mg/L D-ribose, 2.5mg/L putrescine, 15mg/L ethanolamine and 0.25mg/L linoleic acid.
The trace elements comprise the following components: copper sulfate pentahydrate 0.25mg/L, zinc sulfate heptahydrate 2.5mg/L, ferrous sulfate heptahydrate 5mg/L, cobalt chloride 0.05mg/L, sodium selenite 0.5mg/L.
The inorganic salt comprises the following components: 2000mg/L sodium bicarbonate, 100mg/L magnesium chloride, 1200mg/L sodium chloride and 175mg/L potassium chloride.
Other molecular compound components are: 35mg/L stigmasterol, 35mg/L flavonol, 15mg/L soybean isoflavone, 5mg/L insulin, 10mg/L cortisol, 5mg/L epidermal growth factor, 5mg/L EDTA disodium, 15mg/L transferrin, 5mg/L propolis flavone and 5mg/L epimedium flavone.
Preferably, the serum-free cell culture medium comprises:
the amino acid composition is: 20mg/L of L-histidine, 30mg/L of L-leucine, 35mg/L of L-isoleucine, 15mg/L of L-phenylalanine, 50mg/L of L-lysine, 20mg/L of L-cysteine, 50mg/L of glycine, 50mg/L of L-asparagine, 10mg/L of L-glutamic acid, 10mg/L of L-alanyl-L-glutamine, 20mg/L of L-tryptophan, 100mg/L of L-arginine, 20mg/L of L-tyrosine, 30mg/L of L-aspartic acid, 30mg/L of L-cystine and 10mg/L of L-methionine.
The vitamin components are as follows: vitamin A15 mg/L, vitamin B15 mg/L, vitamin B2 1mg/L, vitamin B3 1mg/L, vitamin B6 5mg/L, vitamin B7 2mg/L, vitamin B9 20mg/L, vitamin B15 mg/L, vitamin C5 mg/L, vitamin E9.4 mg/L, inositol 35mg/L, and calcium pantothenate 10mg/L.
The carbohydrate components are: 3500mg/L glucose, 3000mg/L D-ribose, 5mg/L putrescine, 20mg/L ethanolamine and 0.5mg/L linoleic acid.
The trace elements comprise the following components: copper sulfate pentahydrate 0.5mg/L, zinc sulfate heptahydrate 5mg/L, ferrous sulfate heptahydrate 10mg/L, cobalt chloride 0.1mg/L, sodium selenite 1mg/L.
The inorganic salt comprises the following components: 3000mg/L sodium bicarbonate, 200mg/L magnesium chloride, 2000mg/L sodium chloride and 250mg/L potassium chloride.
Other molecular compound components are: 50mg/L stigmasterol, 50mg/L flavonol, 20mg/L soybean isoflavone, 10mg/L insulin, 20mg/L cortisol, 10mg/L epidermal growth factor, 10mg/L disodium EDTA, 20mg/L transferrin, 10mg/L propolis flavone and 10mg/L epimedium flavone.
Preferably, the use of any of the serum-free cell culture media described above in cell culture comprises: adding any of the above culture media into a culture container filled with cells, and culturing at 37deg.C.
Compared with the prior art, the invention has the following beneficial effects:
compared with the traditional culture medium, the serum-free cell culture medium provided by the invention has the advantages that the serum-free cell culture medium does not contain serum, the components are clear, the cost is low, and the biosafety risk and batch difference caused by using the serum are avoided; and secondly, the culture medium is powered by combining the D-ribose and the glucose, so that more continuous and long-term energy supply can be provided for cells during logarithmic growth and rapid proliferation of the cells, the growth speed of the cells is faster, the metabolism is vigorous, and the activity of the cells is strong. Compared with the traditional culture medium, the cell protein expression amount in the culture medium is higher, and the intracellular toxin is lower, so that greater economic benefit can be realized. In addition, the culture medium provided by the invention can be stored at room temperature, and is convenient to transport and store.
Drawings
FIG. 1 is a graph showing the comparison of viable cell densities of CHO cells cultured by perfusion culture in media 1, 2, and 3 and by batch feed culture in control media 1, 2;
FIG. 2 is a graph showing cell viability comparison of CHO cells perfusion cultured using media 1, 2, 3 and batch feed cultured using control media 1, 2;
description of the embodiments
Examples
This example provides a serum-free cell culture medium comprising an amino acid component, a vitamin component, a carbohydrate component, a trace element component, an inorganic salt component, and other molecular compound components, wherein,
the amino acid composition is: l-histidine 5mg/L, L-leucine 10mg/L, L-isoleucine 15mg/L, L-phenylalanine 5mg/L, L-lysine 20mg/L, L-cysteine 10mg/L, glycine 10mg/L, L-asparagine 10mg/L, L-glutamic acid 1mg/L, L-alanyl-L-glutamine 1mg/L, L-tryptophan 5mg/L, L-arginine 10mg/L, L-tyrosine 5mg/L, L-aspartic acid 10mg/L, L-cystine 10mg/L, L-methionine 3mg/L.
The vitamin components are as follows: 1mg/L of vitamin A, 0.5mg/L of vitamin B, 0.1mg/L of vitamin B, 0.5mg/L of vitamin B, 0.05mg/L of vitamin B, 9 2mg/L of vitamin B, 0.3mg/L of vitamin B, 0.5mg/L of vitamin C, 1.8mg/L of vitamin E, 5mg/L of inositol and 1mg/L of calcium pantothenate.
The carbohydrate components are: glucose 1500mg/L, D-ribose 1000mg/L, putrescine 0.05mg/L, ethanolamine 10mg/L, and linoleic acid 0.05mg/L.
The trace elements comprise the following components: copper sulfate pentahydrate 0.05mg/L, zinc sulfate heptahydrate 0.5mg/L, ferrous sulfate heptahydrate 0.5mg/L, cobalt chloride 0.01mg/L, and sodium selenite 0.01mg/L.
The inorganic salt comprises the following components: 1000mg/L sodium bicarbonate, 20mg/L magnesium chloride, 500mg/L sodium chloride and 100mg/L potassium chloride.
Other molecular compound components are: 20mg/L stigmasterol, 20mg/L flavonol, 10mg/L soybean isoflavone, 0.1mg/L insulin, 0.1mg/L cortisol, 0.1mg/L epidermal growth factor, 0.1mg/L EDTA disodium, 10mg/L transferrin, 1mg/L propolis flavone, and 1mg/L epimedium flavone.
Examples
This example provides a serum-free cell culture medium comprising an amino acid component, a vitamin component, a carbohydrate component, a trace element component, an inorganic salt component, and other molecular compound components, wherein,
the amino acid composition is: 15mg/L of L-histidine, 20mg/L of L-leucine, 25mg/L of L-isoleucine, 10mg/L of L-phenylalanine, 35mg/L of L-lysine, 15mg/L of L-cysteine, 15mg/L of glycine, 30mg/L of L-asparagine, 5mg/L of L-glutamic acid, 5mg/L of L-alanyl-L-glutamine, 10mg/L of L-tryptophan, 50mg/L of L-arginine, 12mg/L of L-tyrosine, 20mg/L of L-aspartic acid, 20mg/L of L-cystine and 6mg/L of L-methionine.
The vitamin components are as follows: vitamin A8 mg/L, vitamin B1.5 mg/L, vitamin B2.5 mg/L, vitamin B3.5 mg/L, vitamin B6.5 mg/L, vitamin B7 1mg/L, vitamin B9 10mg/L, vitamin B15 2.5mg/L, vitamin C2.5 mg/L, vitamin E5.6 mg/L, inositol 20mg/L, calcium pantothenate 5mg/L.
The carbohydrate components are: 2500mg/L glucose, 2000mg/L D-ribose, 2.5mg/L putrescine, 15mg/L ethanolamine and 0.25mg/L linoleic acid.
The trace elements comprise the following components: copper sulfate pentahydrate 0.25mg/L, zinc sulfate heptahydrate 2.5mg/L, ferrous sulfate heptahydrate 5mg/L, cobalt chloride 0.05mg/L, sodium selenite 0.5mg/L.
The inorganic salt comprises the following components: 2000mg/L sodium bicarbonate, 100mg/L magnesium chloride, 1200mg/L sodium chloride and 175mg/L potassium chloride.
Other molecular compound components are: 35mg/L stigmasterol, 35mg/L flavonol, 15mg/L soybean isoflavone, 5mg/L insulin, 10mg/L cortisol, 5mg/L epidermal growth factor, 5mg/L EDTA disodium, 15mg/L transferrin, 5mg/L propolis flavone and 5mg/L epimedium flavone.
Examples
This example provides a serum-free cell culture medium comprising an amino acid component, a vitamin component, a carbohydrate component, a trace element component, an inorganic salt component, and other molecular compound components, wherein,
the amino acid composition is: 20mg/L of L-histidine, 30mg/L of L-leucine, 35mg/L of L-isoleucine, 15mg/L of L-phenylalanine, 50mg/L of L-lysine, 20mg/L of L-cysteine, 50mg/L of glycine, 50mg/L of L-asparagine, 10mg/L of L-glutamic acid, 10mg/L of L-alanyl-L-glutamine, 20mg/L of L-tryptophan, 100mg/L of L-arginine, 20mg/L of L-tyrosine, 30mg/L of L-aspartic acid, 30mg/L of L-cystine and 10mg/L of L-methionine.
The vitamin components are as follows: vitamin A15 mg/L, vitamin B15 mg/L, vitamin B2 1mg/L, vitamin B3 1mg/L, vitamin B6 5mg/L, vitamin B7 2mg/L, vitamin B9 20mg/L, vitamin B15 mg/L, vitamin C5 mg/L, vitamin E9.4 mg/L, inositol 35mg/L, and calcium pantothenate 10mg/L.
The carbohydrate components are: 3500mg/L glucose, 3000mg/L D-ribose, 5mg/L putrescine, 20mg/L ethanolamine and 0.5mg/L linoleic acid.
The trace elements comprise the following components: copper sulfate pentahydrate 0.5mg/L, zinc sulfate heptahydrate 5mg/L, ferrous sulfate heptahydrate 10mg/L, cobalt chloride 0.1mg/L, sodium selenite 1mg/L.
The inorganic salt comprises the following components: 3000mg/L sodium bicarbonate, 200mg/L magnesium chloride, 2000mg/L sodium chloride and 250mg/L potassium chloride.
Other molecular compound components are: 50mg/L stigmasterol, 50mg/L flavonol, 20mg/L soybean isoflavone, 10mg/L insulin, 20mg/L cortisol, 10mg/L epidermal growth factor, 10mg/L disodium EDTA, 20mg/L transferrin, 10mg/L propolis flavone and 10mg/L epimedium flavone.
Example 4 preparation of culture Medium for experiments
Adding 1L of each component of the culture medium into 800ml of ultrapure water, stirring at room temperature for 30min until all the components are dissolved, regulating the pH value to 7.0-7.3 by using a freshly prepared 1M hydrochloric acid or sodium hydroxide solution, then performing positive pressure filtration sterilization by using a 0.22 mu M sterile filter membrane, and preserving at room temperature for later use.
The preparation of the culture medium for the specific experiment comprises the following steps:
the medium of example 1 was stirred at room temperature for 30min with an amount of 1L of each component added to 800ml of ultrapure water until all components were dissolved, the pH was adjusted to 7.1 with freshly prepared 1M hydrochloric acid or sodium hydroxide solution, and then sterilized by positive pressure filtration through a 0.22 μm sterile filter, and stored at room temperature for later use, labeled medium 1.
The medium of example 2, labeled Medium 2, was prepared in the same manner as the preparation method.
The medium of example 3, labeled Medium 3, was prepared in the same manner as the preparation method.
Commercial medium DMEM (purchased from Gibco) was supplemented with 10% fetal bovine serum as control medium 1.
Commercial Medium SFM (from Gibco) was used as control Medium 2.
Examples
Taking 5T 75 flash cell culture flasks, and respectively adding culture mediums 1, 2 and 3 and control culture mediums 1 and 2; inoculating resuscitated Vero cell strain into the above 5 culture flasks at a density of 1×10 4 Individual/cm 2 ，37℃，5%CO 2 After 4 days of culture, the cells were digested, and 1ml of pancreatin stop solution was used to terminate the digestion and collect the cells. Cell morphology was observed, the average cell density and viability for each medium was counted using a Cedex AS-20 cell density and viability autoanalyzer, and the endotoxin content was measured using an endotoxin detector.
The results show that: the cell density and cell activity of the culture mediums 1, 2 and 3 are obviously superior to those of the control culture mediums 1 and 2, the cell expansion capacity can reach 3 times that of the control culture mediums 1 and 2, the cell growth state is better, and the activity is higher; the endotoxin content of the cells in the culture media 1, 2 and 3 was reduced by 3/5~4/5 compared to the control culture media 1 and 2. See table 1.
TABLE 1
Culture medium Cell density (. Times.10) 6 Personal/ml) Cell viability (%) Endotoxin content (EU/ml)
Culture medium 1 3.87 99 1.17
Culture medium 2 3.94 99 1.05
Culture medium 3 3.85 99 1.21
Control Medium 1 1.23 87 5.24
Control Medium 2 1.04 85 4.96
Examples
In this example, CHO cells expressing t-PA were used as subjects, and the culture medium provided by the present invention and the control medium were evaluated for their effects by detecting the viable cell density, cell viability and the content of expressed t-PA protein, in the following manner:
6.1 CHO cell lines expressing t-PA were grown at 1X 10 6 The density of each/ml was inoculated into a flask containing 30ml of medium 1, 2, 3, respectively, and the flask was placed in a flask containing 5% CO 2 The culture was carried out at 37℃at 120rpm, and then 30% of the culture volume per day, i.e., 9ml of the cell culture solution was obtained, and 300g was centrifuged for 5 minutes, and the supernatant was discarded, and the cells were resuspended in 9ml of each of the new media 1, 2 and 3, and then each of them was added to the original flask and continued to be cultured for 32 days.
6.2 CHO cell lines expressing t-PA were grown at 1X 10 6 The density of each/ml was inoculated into a flask containing 30ml of control medium 1, 2, respectively, and the flask was placed in a flask containing 5% CO 2 In the shaker at 37℃at 120rpm, 3, 5, 7, 9, 11, 13 and 15 days of culture, 5% of the initial culture volume of Celsource CHO cell feed medium was added, respectively.
6.3 Results
6.3.1 FIG. 1 shows the comparison of viable cell densities of CHO cells grown in perfusion culture with media 1, 2, 3 and in fed batch culture with control media 1, 2, showing that media 1, 2, 3 are capable of high density growth (32 days) for longer periods of time than control media fed batch culture, with media 2 being optimally effective.
6.3.2 FIG. 2 shows the cell viability of CHO cells in perfusion culture using medium 1, 2, 3 compared to control medium 1, 2 in fed batch culture, demonstrating that medium 1, 2, 3 is capable of maintaining high viability growth for longer periods of time, with a cell viability of greater than 90% on day 32, compared to control medium fed batch culture, where the effect of medium 2 is optimal.
6.3.3 Table 2 shows that the perfusion culture of CHO cells using culture media 1, 2 and 3 and the batch feed culture of control culture media 1 and 2 show that the culture media 1, 2 and 3 have higher (3 times) expression level of t-PA protein and more stable yield compared with the batch feed culture of control culture media, wherein the effect of culture media 2 is optimal.
TABLE 2
The technical scheme of the invention is further described through the specific embodiments. It should be apparent to those skilled in the art that the examples are merely provided to aid in understanding the present invention and should not be construed as limiting the invention in any way.

Claims (5)

1. A serum-free cell culture medium comprises an amino acid component, a vitamin component, a carbohydrate component, a trace element component, an inorganic salt component and other molecular compound components, and is characterized in that,
the amino acid component comprises the following components:
the vitamin component comprises the following components:
the carbohydrate component comprises the following ingredients:
the trace element component comprises the following components:
the inorganic salt component comprises the following components:
the other molecular compound components include the following:
2. serum-free cell culture medium according to claim 1, characterized in that the medium is formulated as follows:
the amino acid composition is: l-histidine 5mg/L, L-leucine 10mg/L, L-isoleucine 15mg/L, L-phenylalanine 5mg/L, L-lysine 20mg/L, L-cysteine 10mg/L, glycine 10mg/L, L-asparagine 10mg/L, L-glutamic acid 1mg/L, L-alanyl-L-glutamine 1mg/L, L-tryptophan 5mg/L, L-arginine 10mg/L, L-tyrosine 5mg/L, L-aspartic acid 10mg/L, L-cystine 10mg/L, L-methionine 3mg/L. The vitamin components are as follows: 1mg/L of vitamin A, 0.5mg/L of vitamin B, 0.1mg/L of vitamin B, 0.5mg/L of vitamin B, 0.05mg/L of vitamin B, 9 2mg/L of vitamin B, 0.3mg/L of vitamin B, 0.5mg/L of vitamin C, 1.8mg/L of vitamin E, 5mg/L of inositol and 1mg/L of calcium pantothenate. The carbohydrate components are: glucose 1500mg/L, D-ribose 1000mg/L, putrescine 0.05mg/L, ethanolamine 10mg/L, and linoleic acid 0.05mg/L. The trace elements comprise the following components: copper sulfate pentahydrate 0.05mg/L, zinc sulfate heptahydrate 0.5mg/L, ferrous sulfate heptahydrate 0.5mg/L, cobalt chloride 0.01mg/L, and sodium selenite 0.01mg/L. The inorganic salt comprises the following components: 1000mg/L sodium bicarbonate, 20mg/L magnesium chloride, 500mg/L sodium chloride and 100mg/L potassium chloride. Other molecular compound components are: 20mg/L stigmasterol, 20mg/L flavonol, 10mg/L soybean isoflavone, 0.1mg/L insulin, 0.1mg/L cortisol, 0.1mg/L epidermal growth factor, 0.1mg/L EDTA disodium, 10mg/L transferrin, 1mg/L propolis flavone, and 1mg/L epimedium flavone. Other molecular compound components are: 20mg/L stigmasterol, 20mg/L flavonol, 10mg/L soybean isoflavone, 0.1mg/L insulin, 0.1mg/L cortisol, 0.1mg/L epidermal growth factor, 0.1mg/L EDTA disodium, 10mg/L transferrin, 1mg/L propolis flavone, and 1mg/L epimedium flavone.
3. Serum-free cell culture medium according to claim 1, characterized in that the medium is formulated as follows:
the amino acid composition is: 15mg/L of L-histidine, 20mg/L of L-leucine, 25mg/L of L-isoleucine, 10mg/L of L-phenylalanine, 35mg/L of L-lysine, 15mg/L of L-cysteine, 15mg/L of glycine, 30mg/L of L-asparagine, 5mg/L of L-glutamic acid, 5mg/L of L-alanyl-L-glutamine, 10mg/L of L-tryptophan, 50mg/L of L-arginine, 12mg/L of L-tyrosine, 20mg/L of L-aspartic acid, 20mg/L of L-cystine and 6mg/L of L-methionine. The vitamin components are as follows: vitamin A8 mg/L, vitamin B1.5 mg/L, vitamin B2.5 mg/L, vitamin B3.5 mg/L, vitamin B6.5 mg/L, vitamin B7 1mg/L, vitamin B9 10mg/L, vitamin B15 2.5mg/L, vitamin C2.5 mg/L, vitamin E5.6 mg/L, inositol 20mg/L, calcium pantothenate 5mg/L. The carbohydrate components are: 2500mg/L glucose, 2000mg/L D-ribose, 2.5mg/L putrescine, 15mg/L ethanolamine and 0.25mg/L linoleic acid. The trace elements comprise the following components: copper sulfate pentahydrate 0.25mg/L, zinc sulfate heptahydrate 2.5mg/L, ferrous sulfate heptahydrate 5mg/L, cobalt chloride 0.05mg/L, sodium selenite 0.5mg/L. The inorganic salt comprises the following components: 2000mg/L sodium bicarbonate, 100mg/L magnesium chloride, 1200mg/L sodium chloride and 175mg/L potassium chloride. Other molecular compound components are: 35mg/L stigmasterol, 35mg/L flavonol, 15mg/L soybean isoflavone, 5mg/L insulin, 10mg/L cortisol, 5mg/L epidermal growth factor, 5mg/L EDTA disodium, 15mg/L transferrin, 5mg/L propolis flavone and 5mg/L epimedium flavone.
4. Serum-free cell culture medium according to claim 1, characterized in that the medium is formulated as follows:
the amino acid composition is: 20mg/L of L-histidine, 30mg/L of L-leucine, 35mg/L of L-isoleucine, 15mg/L of L-phenylalanine, 50mg/L of L-lysine, 20mg/L of L-cysteine, 50mg/L of glycine, 50mg/L of L-asparagine, 10mg/L of L-glutamic acid, 10mg/L of L-alanyl-L-glutamine, 20mg/L of L-tryptophan, 100mg/L of L-arginine, 20mg/L of L-tyrosine, 30mg/L of L-aspartic acid, 30mg/L of L-cystine and 10mg/L of L-methionine. The vitamin components are as follows: vitamin A15 mg/L, vitamin B15 mg/L, vitamin B2 1mg/L, vitamin B3 1mg/L, vitamin B6 5mg/L, vitamin B7 2mg/L, vitamin B9 20mg/L, vitamin B15 mg/L, vitamin C5 mg/L, vitamin E9.4 mg/L, inositol 35mg/L, and calcium pantothenate 10mg/L. The carbohydrate components are: 3500mg/L glucose, 3000mg/L D-ribose, 5mg/L putrescine, 20mg/L ethanolamine and 0.5mg/L linoleic acid. The trace elements comprise the following components: copper sulfate pentahydrate 0.5mg/L, zinc sulfate heptahydrate 5mg/L, ferrous sulfate heptahydrate 10mg/L, cobalt chloride 0.1mg/L, sodium selenite 1mg/L. The inorganic salt comprises the following components: 3000mg/L sodium bicarbonate, 200mg/L magnesium chloride, 2000mg/L sodium chloride and 250mg/L potassium chloride. Other molecular compound components are: 50mg/L stigmasterol, 50mg/L flavonol, 20mg/L soybean isoflavone, 10mg/L insulin, 20mg/L cortisol, 10mg/L epidermal growth factor, 10mg/L disodium EDTA, 20mg/L transferrin, 10mg/L propolis flavone and 10mg/L epimedium flavone.
5. A method of cell culture comprising: the culture medium according to any one of claims 1 to 4 is added into a culture container filled with cells, and the cells are cultured at 37 ℃.
CN202310424864.5A 2023-04-20 2023-04-20 Serum-free cell culture medium and application thereof Pending CN116731951A (en)

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