CN109234223B - Low-protein serum-free cell culture medium - Google Patents
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Abstract
The present invention relates to a low-protein serum-free cell culture medium comprising a basic physiological buffer mixture and a protein component; the protein component comprises bovine serum albumin, transferrin and insulin, and the total addition amount of the protein component is not more than 30 mg/L. The culture medium only contains low content of three protein components, which is beneficial to the separation and purification of products and improves the quality of the products; the components are clear, the preparation is convenient, and the method is suitable for large-scale production and application; can well support the hybridoma cells and improve the highest cell density and the antibody yield of the hybridoma cells.
Description
Technical Field
The invention relates to the technical field of cell culture, and particularly relates to a low-protein serum-free cell culture medium.
Background
The cell culture is realized by simulating the growth environment in a cell body, and the in vitro culture technology of animal cells is widely applied to the research fields of physiological, biochemical and pharmacological activities of cells and tissues and the industrial production field of biological products such as antibodies, hormones, vaccines and the like. Wherein the cell culture medium is a vital part of the in vitro cell culture technology. Although the traditional serum-containing culture medium contains a large amount of components necessary for cell growth and proliferation such as various growth factors, plasma proteins, polypeptides, hormones and the like, the serum has the problems of large batch difference, potential pollution risk, undefined components, unfavorable separation and purification of target products, easy infection by viruses and mycoplasma and the like, and the serum-free culture medium has relatively definite components and simple preparation process, and can be widely applied in the field of modern biotechnology.
Serum-free medium is relatively clear in composition, consistent in quality and low in protein content, so that the stability of cell product production is improved, the cell product is easy to purify, and more importantly, different cells can be continuously cultured in high density in an environment which is most beneficial to growth or expression of a target product by optimizing the serum-free medium composition on the basis.
There are also many reports on serum component substitutes that promote cell growth. Albumin is a main additive factor of many serum-free culture media, and has the effects of regulating osmotic pressure, protecting cells from mechanical damage and the like. Experiments of Lemna minor and the like (Lemna minor, Jianjing, Huang Si Yang and the like, research on serum-free culture of Chinese hamster ovary engineering cells [ J ] microbiological immunology progress 2004, 32 (4): 46-50) show that albumin has obvious effects of stabilizing cell growth and increasing expression level of cell strain products. Serum-free media for suspension culture generally contain bovine serum albumin. The influence of 15 amino acids and the concentration thereof on cell culture in CHO cell serum-free culture is examined by an orthogonal test method, and the results show that the added arginine, leucine, proline and methionine have obvious promotion effect on cell growth, and the high-concentration serine and tryptophan have obvious inhibition effect on cell growth. Insulin can promote the synthesis of RNA, protein and fatty acid, and inhibit apoptosis, and is an important cell survival factor. Jan et al (Jan L, Haggstrarm L. specific growth rates as a parameter for tracking growth-limiting substructures in animal cell cultures [ J ] Journal of Biotechnology1995, 42: 163-165) believe that rapid insulin depletion in batch cultures is the primary cause of decreased specific cell growth rate. Selenium has obvious effect in the growth process of mammalian cells, and the trace element selenium participates in the action process of glutathione peroxidase and superoxide dismutase to eliminate the damage of oxidase and oxygen free radical to cells. Xue Qing (Xue Qing Shang Dynasty, theory and technology of in vitro culture [ M ], Beijing scientific and technological Press, 2001, 162-164) indicated that addition of selenious acid in appropriate amount can promote cell growth, but if the concentration is too high, it will produce toxic effect on cells.
At present, commercial serum-free culture media are mainly imported, are high in price and confidential in formula, are not beneficial to optimization of a subsequent culture process, are not provided with lots of domestic serum-free culture medium manufacturers, have the problem of poor market feedback cell adaptability, and are developed by some scientific research institutions or research and development units. Therefore, the serum-free culture medium which can support the rapid growth of hybridoma cells and has high expression of the monoclonal antibody is independently developed, and relevant processes are optimized and amplified to meet the requirement of expressing the monoclonal antibody in large quantities, and the method is of great importance to the industrialization of target proteins. In addition, in general, different cell lines have unique nutritional requirements, and no medium can fully meet the requirements of all cell lines or exert the maximum expression potential of cells, so that the independent development and optimization of personalized serum-free culture mediums aiming at specific cell lines are also an important part in the scale cell culture process.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The serum culture medium has the problems of large batch difference, potential pollution risk, undefined components, unfavorable separation and purification of target products, easy infection and the like. The protein-free and serum-free medium with completely defined chemical components is very specific, in other words, the universality is not high, generally, one medium is only suitable for culturing a certain cell, and in addition, the cell is easily influenced by physical and mechanical factors and chemical factors in the completely serum-free medium.
The invention aims to overcome the defects of the culture medium and prepare a low-protein serum-free cell culture medium.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the present invention relates to a low-protein serum-free cell culture medium comprising a basic physiological buffer mixture and a protein component;
the protein component comprises bovine serum albumin, transferrin and insulin, and the total addition amount of the protein component is not more than 30 mg/L.
The culture medium is suitable for animal cell culture, especially mammalian cell culture.
Compared with the prior art, the invention has the beneficial effects that:
(1) only the three protein components are low in content, which is beneficial to the separation and purification of the product and improves the quality of the product;
(2) the components are clear, the preparation is convenient, and the method is suitable for large-scale production and application;
(3) the universality is stronger;
(4) can well support hybridoma cells, and the highest cell density and antibody yield of the hybridoma cells reach or exceed the effect of a commercial culture medium of a GIBCO brand;
(5) supports long-term subculture of hybridoma cells, and can be directly cultured without adaptation.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing the growth of hybridoma 1C3 in serum-free medium according to one embodiment of the present invention;
FIG. 2 is a graph showing the change of glucose concentration, cell density, cell viability and monoclonal antibody expression of the hybridoma DF92 cultured in a serum-free medium with the lapse of culture time according to an embodiment of the present invention;
FIG. 3 is a graph showing the change of glucose concentration, cell density, cell viability and monoclonal antibody expression level with respect to culture time in the fermentative culture of hybridoma 1A4 using a Gibco serum-free medium of the prior art;
FIG. 4 is a graph showing the glucose concentration, cell density, cell viability rate and monoclonal antibody expression level of hybridoma 1A4 cultured by fermentation in a serum-free medium as a function of culture time in accordance with one embodiment of the present invention.
Detailed Description
Before the present cell cultures and methods are described, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.
The present invention relates to a low-protein serum-free cell culture medium comprising a basic physiological buffer mixture and a protein component;
the protein component comprises bovine serum albumin, transferrin and insulin, and the total addition amount of the protein component is not more than 30 mg/L.
In some embodiments, the total amount of the protein component added is 18mg/L to 30mg/L, and can be selected from 20mg/L, 22mg/L, 24mg/L, 26mg/L and 28 mg/L.
In some embodiments, the protein component comprises bovine serum albumin 10.0-20.0mg/L, transferrin 2.5-5.5mg/L, and insulin 5.0-12.0 mg/L;
in some embodiments, the protein component comprises bovine serum albumin 13.0-17.0mg/L, transferrin 4.5-5.5mg/L, and insulin 9.0-11.0 mg/L;
in some embodiments, the basic physiological buffer mixture is DMEM/F12 based medium with additions of one or more of amino acids, vitamins, inorganic salts, trace elements, lipids;
in some embodiments, the volume ratio of DMEM to F12 in the DMEM/F12 is (0.8-1.2): (0.8 to 1.2); more preferably 1: 1.
In some embodiments, the lipid is selected from the group consisting of:
arachidonic acid 10-50 μ g/L, cholesterol 1500-.
In some embodiments, the lipid is selected from the group consisting of:
arachidonic acid 10-40 μ g/L, cholesterol 2500-.
In some embodiments, the lipid is selected from the group consisting of:
20-30 μ g/L arachidonic acid, 3200 μ g/L cholesterol 2800-, 200 μ g/L linoleic acid 160-, 210 μ g/L linolenic acid 180-, 190 μ g/L oleic acid 170-, 135 μ g/L palmitoleic acid 115-, 80-120 μ g/L stearic acid, and 2900 μ g/L ethanolamine 2500-.
In some embodiments, the amino acid is selected from the group consisting of:
36-58mg/L arginine, 8-12mg/L cystine, 9-15mg/L histidine, 13-23mg/L isoleucine, 13-23mg/L leucine, 14-22mg/L lysine, 4-7mg/L methionine, 9-15mg/L phenylalanine, 18-22mg/L threonine, 3-5mg/L tryptophan, 9-16mg/L tyrosine, 18-22mg/L valine, 4-7mg/L glycine, 5-9mg/L alanine, 8-12mg/L asparagine, 8-12mg/L aspartic acid, 8-12mg/L glutamic acid, 5-9mg/L proline, 9-9 mg/L proline, Serine 5-9 mg/L.
In some embodiments, the amino acid is selected from the group consisting of:
arginine 40-54mg/L, cystine 9-11mg/L, histidine 11-14mg/L, isoleucine 16-20mg/L, leucine 16-20mg/L, lysine 16-20mg/L, methionine 5-6mg/L, phenylalanine 11-13mg/L, threonine 19-21mg/L, tryptophan 3-5mg/L, tyrosine 11-14mg/L, valine 19-21mg/L, glycine 5-6mg/L, alanine 6-8mg/L, asparagine 9-11mg/L, aspartic acid 9-11mg/L, glutamic acid 9-11mg/L, proline 6-8mg/L, arginine, methionine, serine 6-8 mg/L.
In some embodiments, the vitamins are selected from the group consisting of:
choline chloride, calcium pantothenate, folic acid, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine, inositol;
in some embodiments, the concentration of the above vitamins can be selected from 0.7 to 1.8mg/L, and can be selected from 0.8mg/L, 0.9mg/L, 1.0mg/L, 1.1mg/L, 1.2mg/L, 1.3mg/L, 1.4mg/L, 1.5mg/L, 1.6mg/L, 1.7 mg/L; the concentration of each vitamin may be the same or different.
In some embodiments, the vitamin is selected from the group consisting of:
0.7-1.8mg/L of choline chloride, 0.7-1.8mg/L of calcium pantothenate, 0.7-1.8mg/L of folic acid, 0.7-1.8mg/L of nicotinamide, 0.7-1.8mg/L of pyridoxal hydrochloride, 0.7-1.8mg/L of riboflavin, 0.7-1.8mg/L of thiamine and 0.7-1.8mg/L of inositol.
In some embodiments, the vitamin is selected from the group consisting of:
1.0-1.4mg/L of choline chloride, 0.8-1.4mg/L of calcium pantothenate, 1.0-1.4mg/L of folic acid, 1.0-1.4mg/L of nicotinamide, 1.0-1.4mg/L of pyridoxal hydrochloride, 1.0-1.4mg/L of riboflavin, 1.0-1.4mg/L of thiamine and 1.0-1.4mg/L of inositol.
In some embodiments, the inorganic salt is selected from the group consisting of:
5-15mg/L of ferric citrate, 40-60mg/L of sodium pyruvate, 1-5mg/L of calcium chloride, 1-5mg/L of magnesium sulfate, 4-10mg/L of potassium chloride, 80-100mg/L of sodium chloride and 100-200mg/L of sodium dihydrogen phosphate.
In some embodiments, the inorganic salt is selected from the group consisting of:
8-12mg/L of ferric citrate, 45-55mg/L of sodium pyruvate, 2-4mg/L of calcium chloride, 2-4mg/L of magnesium sulfate, 6-8mg/L of potassium chloride, 85-100mg/L of sodium chloride and 140mg/L of 100-one sodium dihydrogen phosphate.
In some embodiments, the medium comprises one or more trace elements selected from the group consisting of:
selenium, molybdate, chromium, cobalt, nickel, zinc, copper, manganese, barium, gallium, lithium, tin, titanium, bromine, iodine, vanadium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, zirconium, cadmium, and aluminum;
in some embodiments, the trace element is selenium, added in the form of selenious acid, in an amount of 3-8 μ g/L.
In some embodiments, the base physiological buffer mixture further comprises a buffer and/or an anti-shear force protectant;
in some embodiments, the buffer is sodium bicarbonate 1.0-1.8g/L and hepes3.2-4.5 mg/L;
in some embodiments, the anti-shear protectant is Pluronic F-68700-.
According to one aspect of the invention, the invention also relates to a method of culturing cells comprising: culturing the cells in a medium as described above;
in some embodiments, the cell is a pluripotent stem cell, an embryonic stem cell, a bone marrow stromal cell, a hematopoietic progenitor cell, a lymphoid stem cell, a bone marrow stem cell, a T cell, a B cell, a macrophage, a liver cell, a pancreatic cell, a cancer cell, and a cell line;
in some embodiments, wherein the cell line is selected from the group consisting of:
CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, T cell line, B cell line, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, HT1080, L929, hybridoma and cancer cell line.
In a specific embodiment, the cell is a hybridoma cell. The hybridoma cell is an unlimited passage cell capable of secreting a single unique antibody, and the secreted monoclonal antibody has wide application in vaccine production, in vitro diagnosis and biomedicine. The culture medium can ensure that hybridoma cells can normally grow and express the monoclonal antibody under the suspension condition, on one hand, the requirement of the in vitro diagnostic reagent industry on the monoclonal antibody is met, and on the other hand, the cost can be reduced.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
And (4) screening and optimizing serum-free medium components.
Hybridoma cell line 1D7 was cultured in a six-well plate, 3mL culture system, and the viable cell density in the culture system was measured by sampling at 72h of culture. By utilizing an L16(45) orthogonal test method, 5 types of additives which play a role in promoting cell culture are determined after relevant literature data are consulted and screened, wherein the 5 types of additives which play a role in promoting cell culture are respectively as follows: a, 8 kinds of lipids; b, 8 vitamins; c, 3 proteins; d, 19 amino acids; and E, 7 inorganic salts.
The test uses DMEM/F12(v/v, 1:1) as basic medium, and selenious acid 6 mug/L, sodium bicarbonate 1.5 mug/L, Hepes 3.8mg/L and Pluronic F-681300 mg/L are added in advance. The five additives selected were optimized using an orthogonal test method, each factor (a, B, C, D, E) setting four different concentration levels (1,2,3,4, with low to high additive concentrations).
In A1, arachidonic acid 10. mu.g/L, cholesterol 500. mu.g/L, linoleic acid 80. mu.g/L, linolenic acid 80. mu.g/L, oleic acid 80. mu.g/L, palmitoleic acid 50. mu.g/L, stearic acid 50. mu.g/L, and ethanolamine 1000. mu.g/L.
In A2, arachidonic acid 15 μ g/L, cholesterol 1000 μ g/L, linoleic acid 100 μ g/L, linolenic acid 120 μ g/L, oleic acid 120 μ g/L, palmitoleic acid 80 μ g/L, stearic acid 60 μ g/L, and ethanolamine 1500 μ g/L.
In A4, arachidonic acid 25 μ g/L, cholesterol 3000 μ g/L, linoleic acid 180 μ g/L, linolenic acid 200 μ g/L, oleic acid 180 μ g/L, palmitoleic acid 125 μ g/L, stearic acid 100 μ g/L, and ethanolamine 2700 μ g/L.
B1 contains choline chloride 0.2mg/L, calcium pantothenate 0.2mg/L, folic acid 0.2mg/L, nicotinamide 0.2mg/L, pyridoxal hydrochloride 0.2mg/L, riboflavin 0.2mg/L, thiamine 0.2mg/L, and inositol 0.2mg/L
B2 contains choline chloride 0.4mg/L, calcium pantothenate 0.4mg/L, folic acid 0.4mg/L, nicotinamide 0.4mg/L, pyridoxal hydrochloride 0.4mg/L, riboflavin 0.4mg/L, thiamine 0.4mg/L, and inositol 0.4mg/L
B3 contains choline chloride 0.8mg/L, calcium pantothenate 0.8mg/L, folic acid 0.8mg/L, nicotinamide 0.8mg/L, pyridoxal hydrochloride 0.8mg/L, riboflavin 0.8mg/L, thiamine 0.8mg/L, and inositol 0.8mg/L
B4 contains choline chloride 1.2mg/L, calcium pantothenate 1.2mg/L, folic acid 1.2mg/L, nicotinamide 1.2mg/L, pyridoxal hydrochloride 1.2mg/L, riboflavin 1.2mg/L, thiamine 1.2mg/L, and inositol 1.2mg/L
C1 contains BSA 5mg/L, transferrin 2mg/L, and insulin 3 mg/L;
c2 contains 10mg/L BSA, 2mg/L transferrin and 5mg/L insulin;
c3 contains 15mg/L BSA, 5mg/L transferrin and 8mg/L insulin;
c4 contains 15mg/L BSA, 5mg/L transferrin and 10mg/L insulin.
In D1, arginine 24mg/L, cystine 5mg/L, histidine 6mg/L, isoleucine 9.5mg/L, leucine 9mg/L, lysine 9mg/L, methionine 3.5mg/L, phenylalanine 5.5mg/L, threonine 9.5mg/L, tryptophan 2mg/L, tyrosine 7mg/L, valine 10mg/L, glycine 2.5mg/L, alanine 3.5mg/L, asparagine 5mg/L, aspartic acid 5mg/L, glutamic acid 5mg/L, proline 3mg/L, serine 3mg/L
D2 contains 48mg/L arginine, 10mg/L cystine, 12mg/L histidine, 19mg/L isoleucine, 18mg/L leucine, 18mg/L lysine, 7mg/L methionine, 11mg/L phenylalanine, 19mg/L threonine, 4mg/L tryptophan, 14mg/L tyrosine, 20mg/L valine, 5mg/L glycine, 7mg/L alanine, 10mg/L asparagine, 10mg/L aspartic acid, 10mg/L glutamic acid, 6mg/L proline, 6mg/L serine
D3 contains arginine 96mg/L, cystine 20mg/L, histidine 24mg/L, isoleucine 38mg/L, leucine 36mg/L, lysine 36mg/L, methionine 14mg/L, phenylalanine 22mg/L, threonine 38mg/L, tryptophan 8mg/L, tyrosine 28mg/L, valine 40mg/L, glycine 10mg/L, alanine 14mg/L, asparagine 20mg/L, aspartic acid 20mg/L, glutamic acid 20mg/L, proline 12mg/L, serine 12mg/L
D4 contains 192mg/L arginine, 40mg/L cystine, 48mg/L histidine, 76mg/L isoleucine, 72mg/L leucine, 72mg/L lysine, 28mg/L methionine, 44mg/L phenylalanine, 76mg/L threonine, 16mg/L tryptophan, 56mg/L tyrosine, 80mg/L valine, 20mg/L glycine, 28mg/L alanine, 40mg/L asparagine, 40mg/L aspartic acid, 40mg/L glutamic acid, 24mg/L proline, 24mg/L serine and 24mg/L serine
E1 contains ferric citrate 12mg/L, sodium pyruvate 50mg/L, calcium chloride 4mg/L, magnesium sulfate 3mg/L, potassium chloride 7mg/L, sodium chloride 100mg/L, and sodium dihydrogen phosphate 120mg/L
E2 contains ferric citrate 18mg/L, sodium pyruvate 75mg/L, calcium chloride 6mg/L, magnesium sulfate 4.5mg/L, potassium chloride 7mg/L, sodium chloride 150mg/L, and sodium dihydrogen phosphate 150mg/L
E3 contains ferric citrate 27mg/L, sodium pyruvate 112.5mg/L, calcium chloride 9mg/L, magnesium sulfate 6.75mg/L, potassium chloride 7mg/L, sodium chloride 200mg/L, and sodium dihydrogen phosphate 200mg/L
E4 contains ferric citrate 40.5mg/L, sodium pyruvate 168.75mg/L, calcium chloride 13.5mg/L, magnesium sulfate 10mg/L, potassium chloride 7mg/L, sodium chloride 250mg/L, and sodium dihydrogen phosphate 250mg/L
The results are shown in Table 1.
TABLE 1 orthogonal test optimization
The average value of the viable cell density in the culture system when the culture is carried out for 72 hours is compared, and the results of orthogonal experiments show that the combination A4B4C4D2E1 in 16 different combinations of the added factors has the greatest promotion on the viable cell density. And judging the influence of each factor on the living cell density according to the range R to obtain the influence primary and secondary sequences of each factor as C > A > D > B > E. On the basis, the optimal levels of the different addition factors in the 5 are determined as follows: A4B4C4D2E 1. The preferred concentration as culture medium is performed at an optimal level of combined additive concentration, followed by the cell culture of example 2.
Example 2
Serum-free medium preparation and cell culture
1. Preparation of the Medium
The following were added to a basal medium of DMEM/F12(v/v, 1: 1):
15.0mg/L bovine serum albumin, 5mg/L transferrin, 10mg/L insulin, 48mg/L arginine, 10mg/L cystine, 12mg/L histidine, 19mg/L isoleucine, 18mg/L leucine, 18mg/L lysine, 7mg/L methionine, 11mg/L phenylalanine, 19mg/L threonine, 4mg/L tryptophan, 14mg/L tyrosine, 20mg/L valine, 5mg/L glycine, 7mg/L alanine, 10mg/L asparagine, 10mg/L aspartic acid, 10mg/L glutamic acid, 6mg/L proline, 6mg/L serine, 1.2mg/L choline chloride, 1.2mg/L calcium pantothenate, 1.2mg/L folic acid, 1.2mg/L of nicotinamide, 1.2mg/L of pyridoxal hydrochloride, 1.2mg/L of riboflavin, 1.2mg/L of thiamine, 1.2mg/L of inositol, 12mg/L of ferric citrate, 50mg/L of sodium pyruvate, 4mg/L of calcium chloride, 3mg/L of magnesium sulfate, 7mg/L of potassium chloride, 100mg/L of sodium chloride and 120mg/L of sodium dihydrogen phosphate, 25 mu g/L of arachidonic acid, 3000 mu g/L of cholesterol, 180 mu g/L of linoleic acid, 200 mu g/L of linolenic acid, 180 mu g/L of oleic acid, 125 mu g/L of palmitoleic acid, 100 mu g/L of stearic acid, 2700 mu g/L of ethanolamine, 6 mu g/L of selenious acid, 1.5g/L of sodium bicarbonate, 3.8mg/L of Hepes and Pluronic F-681300 mg/L. The medium was prepared according to a conventional method and then sterilized by filtration through a 0.22 μm microporous filter.
2. Cell culture
Taking out the frozen hybridoma cell strain 1C3 cells from liquid nitrogen tank, rapidly placing in 37 deg.C water bath, centrifuging to remove supernatant after completely melting, gently blowing and beating cells with small amount of the above serum-free culture medium to mix the cells uniformly, transferring to a square bottle, placing in 37 deg.C 5% CO2Humidity saturated CO2Subculturing in an incubator.
3. Comparison with GIBCO brand serum-free Medium Hybridoma-SFM
The above 1C3 cells were expanded and transferred into 4 flasks, and inoculated into 1.5L spinner flasks together at an initial culture density of about 70X 10 when grown to the cell log phase4cells/ml, then placed at 37 ℃ in 5% CO2And (5) carrying out suspension culture in a humidity saturation incubator.
As shown in FIG. 1, the lower curve is the growth curve of the Hybridoma cultured in the serum-free medium according to the present invention, and the upper curve is the growth curve of the Hybridoma cultured in the GIBCO Hybridoma-SFM medium. The whole culture process lasted 8 days. The living cell density of the serum-free culture medium and the GIBCO Hybridoma-SFM culture medium respectively reaches 537 multiplied by 10 within 120 hours4cells/ml and 551X 104cells/ml, the culture effect of the serum-free culture medium is almost equal to that of similar culture media produced by foreign manufacturers, and meanwhile, the serum-free culture medium is relatively low in cost, clear in chemical components and extremely low in protein content.
Example 3
Preparation of monoclonal antibody by culturing hybridoma cell strain in fed-batch mode
The serum-free culture medium is used for suspension culture of hybridoma cell strains DF92 and 1A4 in a fermentation tank, and the formula of the culture medium is the same as that in example 2. DF92 was prepared using a 14L bioreactor from Guangzhou Qizhi Bio Inc, with an initial culture volume of 2L and under the following conditions: temp 37. + -. 0.5 ℃, DO 50% + -5%, pH 6.9. + -. 0.1, Stir speed 80rpm, 1A4 using an Applikon 2L bioreactor, initial culture volume of 2L, control conditions: temp 37 +/-0.5 ℃, DO 50% +/-5%, pH6.9 +/-0.1, and Stir speed 80rpm, wherein a fed batch culture method is adopted during the suspension culture process, relevant data including glucose concentration in a culture solution, cell density, cell viability and monoclonal antibody expression amount are sampled and monitored at intervals of about 24h, the culture results of the two cells are shown in figures 2 and 4 (figure 3 is a culture medium control of the prior art), a fed medium is added according to glucose consumption indexes in the culture medium, and the feeding interval is about 24h from the beginning of feeding. When the cell viability in the fermentation tank is reduced to below 60%, the fed-batch culture operation of the fermentation tank is stopped, and the supernatant is collected by centrifugation and then filtered for expression amount measurement and monoclonal antibody purification. The result shows that the serum-free culture medium can be normally used for hybridoma cell suspension culture and monoclonal antibody preparation, the growth state and the antibody yield of the cells are equivalent to or superior to those of an externally purchased contrast culture medium under the same culture condition, and subsequent experiments also prove that the monoclonal antibody prepared by using the serum-free culture medium can be normally used in the field of in vitro diagnostic reagents.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (2)
1. A low-protein, serum-free cell culture medium comprising a basal physiological buffer mixture and a protein component;
the protein components are bovine serum albumin, transferrin and insulin; the addition amount is as follows: 15.0mg/L of bovine serum albumin, 5mg/L of transferrin and 10mg/L of insulin;
the basal physiological buffer mixture consists of a basal medium and:
48mg/L arginine, 10mg/L cystine, 12mg/L histidine, 19mg/L isoleucine, 18mg/L leucine, 18mg/L lysine, 7mg/L methionine, 11mg/L phenylalanine, 19mg/L threonine, 4mg/L tryptophan, 14mg/L tyrosine, 20mg/L valine, 5mg/L glycine, 7mg/L alanine, 10mg/L asparagine, 10mg/L aspartic acid, 10mg/L glutamic acid, 6mg/L proline, 6mg/L serine, 1.2mg/L choline chloride, 1.2mg/L calcium pantothenate, 1.2mg/L folic acid, 1.2mg/L histidine, Nicotinamide 1.2mg/L, pyridoxal hydrochloride 1.2mg/L, riboflavin 1.2mg/L, thiamine 1.2mg/L, inositol 1.2mg/L, ferric citrate 12mg/L, sodium pyruvate 50mg/L, calcium chloride 4mg/L, magnesium sulfate 3mg/L, potassium chloride 7mg/L, sodium chloride 100mg/L, sodium dihydrogen phosphate 120mg/L, arachidonic acid 25 μ g/L, cholesterol 3000 μ g/L, linoleic acid 180 μ g/L, linolenic acid 200 μ g/L, oleic acid 180 μ g/L, palmitoleic acid 125 μ g/L, stearic acid 100 μ g/L, ethanolamine 2700 μ g/L, selenious acid 6 μ g/L, Sodium bicarbonate 1.5g/L, Hepes 3.8.8 mg/L and Pluronic F-681300 mg/L;
the basic culture medium is DMEM/F12 basic culture medium; the volume ratio of DMEM to F12 in the DMEM/F12 basal medium is 1: 1.
2. A method of culturing cells comprising: culturing cells in the medium of claim 1; the cell is a hybridoma cell.
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