CN112048463A - Serum substitute for cell culture - Google Patents
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Abstract
The invention discloses a serum substitute for cell culture, wherein each liter of serum substitute comprises the following components: amino acid 140-165mg, asparagine 15-20mg, VC15-25 μ M, vitamin H70-110 μ M, tocopherol acetate 150-200 μ M, tocopherol 80-120 μ M, vitamin A10-15 μ M, bovine pituitary extract 15-25g, insulin-like growth factor I80-120 μ g, catalase 250-350 μ g, human recombinant insulin 450-550 μ g, human transferrin 5000-10000 μ g, superoxide dismutase 5000000U, corticosterone 5mM-20mM, D-galactose 150000-250000mM, ethanolamine hydrochloride 100-150mM, glutathione 49-165mM, carnitine L80-120 mM, inorganic salt 140.901-161.403mg and Pluronic F-685-15 g.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a serum substitute for cell culture.
Background
Animal serum is the most abundant natural culture medium used in cell culture, contains abundant nutrients necessary for cell growth, and is commonly used for in vitro culture of animal cells. The serum mainly comprises fetal calf serum, adult calf serum, horse serum, sheep serum, chicken serum, rabbit serum and the like, wherein the fetal calf serum is most commonly used. Animal serum provides growth factors, hormones, transfer proteins, factors adherent to and spreading on the culture substrate, protease inhibitors and other nutrients required for cell proliferation in cell culture. The advantage of using serum is that serum contains most of the factors that promote cell proliferation and maintenance, it is almost a universal growth supplement, and it is suitable for cell culture of animal, human, insect cells, and the like. The use of serum thus eliminates the need to optimize the medium for each cell line, saving a lot of time and effort.
However, studies have shown that improper use of animal serum can inhibit cell growth and cause toxicity, and the components of animal serum are not well defined, and may influence experimental studies, which is not favorable for basic studies of cell metabolism and studies of cell action of specific components. The above problems are effectively avoided if an artificial cell culture medium can be provided in place of animal serum.
Therefore, how to provide an animal serum substitute is a technical problem that needs to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of this, the invention replaces serum with artificially configured culture medium, improves the vitality of cells and the density of cells, reduces the content of endotoxin in cells, and reduces the production cost.
A serum replacement for cell culture, comprising the following components per liter of serum replacement: amino acid 140-165mg, asparagine 15-20mg, VC15-25 μ M, vitamin H70-110 μ M, tocopherol acetate 150-200 μ M, tocopherol 80-120 μ M, vitamin A10-15 μ M, bovine pituitary extract 15-25g, insulin-like growth factor I80-120 μ g, catalase 250-350 μ g, human recombinant insulin 450-550 μ g, human transferrin 5000-10000 μ g, superoxide dismutase 5000000U, corticosterone 5mM-20mM, D-galactose 150000-250000mM, ethanolamine hydrochloride 100-150mM, glutathione 49-165mM, L-carnitine hydrochloride 80-120mM, inorganic salt 140.901-161.403mg and Pluronic F-685-15 g.
The technical effects of the components are as follows:
amino acids: amino acids are the components of proteins and precursors for purine and pyrimidine base synthesis, and are involved in the growth and metabolism of cells, and the concentration of amino acids in a culture medium generally limits the maximum density and cell viability state which can be achieved by cell growth;
asparagine: asparagine is usually used for microbial culture and is not generally used as a component of a culture medium, but asparagine is used as a component of a cell culture medium and can be used as a main component for synthesizing aspartic acid by cells;
VC: during the cell growth and metabolism process, the cell growth promoting and oxygen free radical damage protecting effects are provided;
vitamin H: vitamin H can be used as coenzyme of various enzymes in cells and can play an auxiliary role, and the vitamin H can also participate in the metabolism of fatty acid and carbohydrate in the cells;
tocopherol acetate: as the derivative of vitamin E, the vitamin E has the function of preventing the cell membrane and unsaturated fatty acid in the cell from being oxidized easily in the cell metabolism process, thereby protecting the integrity of the cell membrane and preventing aging;
and (3) tocopherol: vitamin E, which can reduce oxygen consumption of cells and prevent aging of cells;
vitamin A: vitamin A also has antioxidant effect, and also has effects of maintaining cell function, improving cell membrane elasticity, and preventing cell rupture;
bovine pituitary extract: the extract prepared from bovine pituitary has the effects of obviously improving the in-vitro serum-free culture condition of human cells and promoting the proliferation of in-vitro cultured human cells, is used as a growth supplement for serum-free cell culture in the invention, can promote the proliferation of epithelial cells, endothelial cells and melanocytes, and has antioxidant capacity, and can resist hydrogen peroxide-induced cell necrosis, protein oxidation, membrane disruption and DNA damage;
insulin-like growth factor: is a broad-spectrum growth promoting factor, can promote the growth of cells, and has important promotion effects in the differentiation and proliferation of the cells and the growth and development of individuals;
catalase: is an enzyme scavenger, also called catalase, which is a conjugated enzyme with ferriporphyrin as a prosthetic group and can promote H2O2Decomposing into molecular oxygen and water, scavenging hydrogen peroxide in cells, and protecting cells from H2O2Poisoning;
human recombinant insulin: the uptake of glucose and amino acid by cells is promoted, and the growth of the cells is promoted;
human transferrin: fe2+Can make hydrogen peroxide (H)2O2) The hydroxyl free radical (OH) is generated by reduction, and is the most active and most powerful oxygen free radical, so that the damage to cells is great; transferrin can be combined with iron ions with high affinity, and free iron ions basically do not exist in the extracellular culture environment after the transferrin is added into a serum-free culture medium, so that free radical reaction can not occur, and the damage of free radicals to cells can be avoided; meanwhile, many important proteins in the cell can exert their activity after binding iron ions, and these proteins are basically involved in the replication and repair of DNA, that is, transferrin largely affects the growth and proliferation of the cell;
superoxide dismutase: preventing cell aging;
corticosterone: is one kind of cortical hormone-type twenty-one-carbon steroid hormone, has antibacterial effect, and can prevent cell from being polluted by bacteria and other microorganisms;
d-galactose: increasing oxidative stress of the cell;
ethanolamine hydrochloride: participate in the synthesis of intracellular phospholipids and phosphatidylethanolamine;
glutathione: the compound is composed of glutamic acid, cysteine and glycine, contains sulfur radicals, plays an important role in maintaining the biological functions of cells, can activate various enzymes so as to promote the metabolism of sugar, fat and protein of the cells, can be combined with free radicals in the cells through the sulfur radicals, and can be converted into easily metabolized acid substances so as to accelerate the excretion of the free radicals;
inorganic salts: the osmotic pressure balance inside and outside the cell can be maintained, the permeability of a cell membrane is kept, and meanwhile, salt ions can be used as prosthetic groups synthesized by a plurality of enzymes such as cytochrome enzyme, catalase and the like in the cell and participate in the synthesis of a mitochondrial respiratory chain;
pluronic F-68: protecting the cells in suspension from damage caused by transfer and agitation; air can be prevented from adhering to cells, foam on the cell surface can be stabilized to improve the resistance of the cell membrane to hydrodynamic shear, and interaction between cells can be enhanced.
The components are combined together for synergy, can promote the proliferation of cells, protect the cells from oxidative damage, improve the activity and density of the cells, reduce the content of endotoxin generated by the cells, and better maintain the morphology of the cells.
As a preferred embodiment of the present invention, the amino acids include: alanine 20-25mg, aspartic acid 25-30mg, glutamic acid 60-70mg and proline 30-40 mg.
As a preferred embodiment of the present invention, the inorganic salt includes: 0.8-1.2mg of ferric citrate and CaCl2.2H2O140-160mg、CuSO4.5H20.001-0.003mg of O and FeSO4.7H2O0.1-0.2mg。
The technical effect achieved by the technical scheme is as follows: iron ions are used as prosthetic groups of cytochrome enzyme and catalase and participate in the composition of mitochondrial respiratory chain; the copper ions are used as prosthetic groups of superoxide dismutase and have an antioxidant effect; calcium ions are involved in neurotransmitter synthesis and release, hormone synthesis and secretion.
As a preferred embodiment of the present invention, the method further comprises: linoleic acid 1-10mM, progesterone 0.5-1mM, putrescine hydrochloride 150-250mM and sodium selenite 0.5-1 mM.
The technical effect achieved by the technical scheme is as follows: linoleic acid can repair oxidative damage of cells, and putrescine hydrochloride has the function of promoting synthesis of protein and nucleic acid and can regulate the pH value in the cells; selenium element in sodium selenite is a component of glutathione reductase, and has antioxidant effect.
As a preferred technical scheme of the invention, the serum substitute comprises the following components per liter: alanine 22mg, asparagine 19mg, aspartic acid 26.4mg, glutamic acid 66mg, proline 35.2mg, VC 20. mu.M, vitamin H100. mu.M, tocopherol acetate 185. mu.M, tocopherol l 00. mu.M, vitamin A14. mu.M, bovine pituitary extract 20g, insulin-like growth factor I100. mu.g, catalase 300. mu.g, human recombinant insulin 500. mu.g, human transferrin 6000. mu.g, superoxide dismutase 5000000U, corticosterone 10mM, D-galactose 200000mM, ethanolamine hydrochloride 120mM, glutathione 100mM, levorotatory insulin 10mM, glutathione 100mM, human insulin BCarnitine hydrochloride 100mM, ferric citrate 1mg, CaCl2.2H2O154.4mg、CuSO4.5H2O0.002mg、FeSO4.7H2O0.147mg and Pluronic F-6810 g.
As a preferred embodiment of the present invention, the method further comprises: linoleic acid 5mM, progesterone 0.63mM, putrescine hydrochloride 200mM and sodium selenite 0.52 mM.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The components used in the examples were obtained from sigma
Example 1
A serum replacement for cell culture, comprising the following components per liter of serum replacement:
alanine 20mg, aspartic acid 25mg, glutamic acid 60mg, proline 30mg, asparagine 15mg, VC15 μ M, vitamin H70 μ M, tocopherol acetate 150 μ M, tocopherol 80 μ M, vitamin A10 μ M, bovine pituitary extract 15g, insulin-like growth factor I80 μ g, catalase 250 μ g, human recombinant insulin 450 μ g, human transferrin 5000 μ g, superoxide dismutase 5000000U, corticosterone 5mM, D-galactose 150000mM, ethanolamine hydrochloride 100mM, glutathione 49mM, L-carnitine hydrochloride 80mM, linoleic acid 1mM, progesterone 0.5mM, putrescine hydrochloride 150mM, sodium selenite 0.5mM, ferric citrate 0.8mg, CaCl 0.8 mM2.2H2O140mg、CuSO4.5H2O0.001mg and FeSO4.7H2O0.1mg and Pluronic F-685 g.
Example 2
A serum replacement for cell culture, comprising the following components per liter of serum replacement: alanine 25mg, aspartic acid 30mg, glutamic acid 70mg, proline 40mg, asparagine 20mg, VC25 μ M, vitamin H110 μ M, tocopherol200 mu M acetate, 120 mu M tocopherol, 15 mu M vitamin A, 25g bovine pituitary extract, 120 mu g insulin-like growth factor I, 350 mu g catalase, 550 mu g human recombinant insulin, 10000 mu g human transferrin, 5000000U superoxide dismutase, 20mM corticosterone, 250000mM D-galactose, 150mM ethanolamine hydrochloride, 165mM glutathione, 120mM L-carnitine hydrochloride, 1.2mg ferric citrate, CaCl2.2H2O160mg、CuSO4.5H2O0.003mg、FeSO4.7H2O0.2mg, linoleic acid 10mM, progesterone 1mM, putrescine hydrochloride 250mM, sodium selenite 1mM and Pluronic F-6815g
Example 3
A serum replacement for cell culture, comprising the following components per liter of serum replacement: alanine 22mg, asparagine 19mg, aspartic acid 26.4mg, glutamic acid 66mg, proline 35.2mg, VC 20. mu.M, vitamin H100. mu.M, tocopherol acetate 185. mu.M, tocopherol l 00. mu.M, vitamin A14. mu.M, bovine pituitary extract 20g, insulin-like growth factor I100. mu.g, catalase 300. mu.g, human recombinant insulin 500. mu.g, human transferrin 6000. mu.g, superoxide dismutase 5000000U, corticosterone 10mM, D-galactose 200000mM, ethanolamine hydrochloride 120mM, glutathione 100mM, L-carnitine hydrochloride 100mM, linoleic acid 5mM, progesterone 0.63mM, putrescine hydrochloride 200mM, sodium selenite 0.52mM, ferric citrate 1mg, CaCl 0.52mM, and the like2.2H2O154.4mg、CuSO4.5H2O0.002mg、FeSO4.7H2O0.147mg and Pluronic F-6810 g.
The preparation method of the serum substitute comprises the following steps:
1) weighing alanine, asparagine, aspartic acid, glutamic acid, proline, bovine pituitary extract, insulin-like growth factor I, catalase, human recombinant insulin, human transferrin, ferric citrate and CaCl according to the proportion2.2H2O、CuSO4.5H2O、FeSO4.7H2O and Pluronic F-68, and uniformly mixing the raw materials to obtain a raw material A;
2) adding water to 1000ml in a container, weighing the rest raw materials, adding the rest raw materials into the solution to a target concentration, mixing the raw materials A, and slightly stirring for dissolving;
3) adding sodium bicarbonate into the container to make the final concentration of the sodium bicarbonate be 2.5g/L, and slightly stirring the sodium bicarbonate to dissolve the sodium bicarbonate to obtain an initial culture medium; adjusting the pH value of the initial culture medium to 7.8 by using 1moL/L sodium hydroxide solution or 1moL/L hydrochloric acid solution;
4) filtering and sterilizing the initial culture medium by using a 0.2 mu m filter membrane under positive pressure to obtain a serum substitute for cell culture;
5) the serum substitute for cell culture is stored under sealed condition and in dark at 2 ℃.
Example 4
Culturing mouse fibroblast with the three serum substitutes, taking DMEM/F12 as basic culture medium, adding serum substitute with concentration of 5%, taking 9 cell culture bottles, correspondingly introducing the three serum substitutes, and making three groups for each substitute, wherein each culture bottle is (1-2) multiplied by 102Inoculating mouse fibroblast at the concentration of (2), culturing in a 5% CO incubator at 37 deg.C for 4h, taking suspension from each cell culture bottle, and culturing at 3 × 104Inoculating 24-well plates at a density of 2ml per well of culture medium, inoculating one 24-well plate per culture medium, placing at 37 deg.C and CO2Culturing in an incubator, after 2 days, adding 0.25(w/v) trypsin solution to each well for digestion, and counting the average cell density and viability of each culture medium by using a Cedex AS-20 cell density and viability automatic analyzer; detecting the content of the intracellular toxins by using a cell endotoxin detector; the results are shown in Table 1;
TABLE 1
Example 5
Comparative example 1: removing the tocopherol acetate and the tocopherol in the example 3 and other components same as the example 3;
comparative example 2: removing the human transferrin from example 3, and other components as in example 3;
comparative example 3: removing the progesterone in example 3 and other components as in example 3;
comparative example 4: removing the corticosterone in the example 3 and other components similar to the example 3;
comparative example 5: removing the L-carnitine hydrochloride in the example 3 and other components which are the same as the components in the example 3;
comparative example 6: the bovine pituitary extract of example 3 was removed and the other components were the same as those of example 3.
Fibroblasts were cultured according to the method of example 4, and the cell density, cell viability, endotoxin content and cell growth morphology are shown in table 2;
TABLE 2
As shown in Table 2, the density and cell viability of fibroblasts were significantly decreased and the endotoxin content was increased by removing any one of the components from the culture medium, indicating that the components had synergistic effects.
Example 6
Comparative example 1: culturing mouse fibroblast cells in DMEM/F12 medium containing 10% fetal bovine serum according to the method of example 4; (ii) a
Comparative example 2: culturing mouse fibroblast cells in DMEM/F12 medium containing 5% fetal bovine serum according to the method of example 4;
the results are shown in Table 3;
TABLE 3
As can be seen from the comparison between Table 3 and Table 1, the medium of the present invention can significantly increase the density and activity of fibroblasts and reduce the endotoxin content of cells, compared with the existing medium.
The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (8)
1. A serum replacement for cell culture, comprising the following components per liter of serum replacement: amino acid 140-165mg, asparagine 15-20mg, VC15-25 μ M, vitamin H70-110 μ M, tocopherol acetate 150-200 μ M, tocopherol 80-120 μ M, vitamin A10-15 μ M, bovine pituitary extract 15-25g, insulin-like growth factor I80-120 μ g, catalase 250-350 μ g, human recombinant insulin 450-550 μ g, human transferrin 5000-10000 μ g, superoxide dismutase 5000000U, corticosterone 5mM-20mM, D-galactose 150000-250000mM, ethanolamine hydrochloride 100-150mM, glutathione 49-165mM, L-carnitine hydrochloride 80-120mM, inorganic salt 140.901-161.403mg and Pluronic F-685-15 g.
2. A serum replacement for cell culture according to claim 1, wherein the amino acids comprise: alanine 20-25mg, aspartic acid 25-30mg, glutamic acid 60-70mg and proline 30-40 mg.
3. A serum replacement for cell culture according to claim 2, wherein the inorganic salts comprise: 0.8-1.2mg of ferric citrate and CaCl2.2H2O140-160mg、CuSO4.5H20.001-0.003mg of O and FeSO4.7H2O0.1-0.2mg。
4. A serum replacement for cell culture according to claim 3, further comprising: linoleic acid 1-10mM, progesterone 0.5-1mM, putrescine hydrochloride 150-250mM and sodium selenite 0.5-1 mM.
5. A serum replacement for cell culture according to claim 3, comprising the following components per litre serum replacement: alanine 22mg, asparagine 19mg, aspartic acid 26.4mg, glutamic acid 66mg, proline 35.2mg, VC 20. mu.M, vitamin H100. mu.M, tocopherol acetate 185. mu.M, tocopherol l 00. mu.M, vitamin A14. mu.M, bovine pituitary extract 20g, insulin-like growth factor I100. mu.g, catalase 300. mu.g, human recombinant insulin 500. mu.g, human transferrin 6000. mu.g, superoxide dismutase 5000000U, corticosterone 10mM, D-galactose 200000mM, ethanolamine hydrochloride 120mM, glutathione 100mM, L-carnitine hydrochloride 100mM, ferric citrate 1mg, CaCl 120mM, glutathione 100mM, L-carnitine hydrochloride 100mM, and the like2.2H2O154.4mg、CuSO4.5H2O0.002mg、FeSO4.7H2O0.147mg and Pluronic F-6810 g.
6. The serum replacement for cell culture according to claim 5, further comprising: linoleic acid 5mM, progesterone 0.63mM, putrescine hydrochloride 200mM and sodium selenite 0.52 mM.
7. The serum replacement for cell culture according to claim 6, wherein the serum replacement is added to the basal medium at a concentration of 5-10% (V/V) during the cell culture.
8. A cell culture one year serum replacement according to claim 7, wherein the basal medium comprises any one of DMEM, MEM or DMEM/F12.
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