CN109234223A - Low albumen serum-free cell culture medium - Google Patents
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Abstract
The present invention relates to a kind of low albumen serum-free cell culture medium, the culture medium includes basic physiology buffer mixture and protein ingredient；The protein ingredient includes bovine serum albumin(BSA), transferrins, insulin, and the total addition level of the protein ingredient is no more than 30mg/L.Three kinds of proteinaceous components contents that the culture medium contains only are very low, are conducive to isolating and purifying for product, improve the quality of product；Definite ingredients are prepared conveniently, and large-scale production application is suitable for；Energy good support hybridoma, improves the highest cell density and antibody production of hybridoma.
Description
Technical field
The present invention relates to technical field of cell culture, in particular to a kind of low albumen serum-free cell culture medium.
Background technique
Cell culture is to realize cell injuring model, the in vitro culture of zooblast by simulation cell growth in vivo environment
The physiology, biochemistry and pharmacology activity research field and antibody, hormone and vaccine etc. that technology is widely used in cell and tissue are raw
The field of industrial production of Tetramune.Wherein cell culture medium is the vital a part of cell injuring model technology.Tradition contains
Although blood serum medium is required containing a large amount of cell growth increment institute such as various growth factors, plasma protein, polypeptide and hormone
Ingredient, but that there are differences between batches is big, potential pollution risk, ingredient is indefinite, is unfavorable for purpose product isolates and purifies, holds for serum
Easily by virus and the problems such as mycoplasma infection, and serum free medium is since its constituent is relatively unambiguous, and preparation process is simple,
It can be widely applied in modern biotechnology field.
The ingredient of serum free medium is relatively unambiguous, uniform quality and protein content are low, thus is conducive to improve cell
The stability of production simultaneously makes cell products be easy to purify, it is often more important that can be by this basis to free serum culture
The optimization of based component enables its each comfortable most advantageous growth of different cells or most beneficial for holding in the environment of expression purpose product
Continuous High Density Cultivation.
The ingredient of cell growth is promoted also there are many relevant reports about serum composition substitute.Albumin is much without blood
The main addition factor of clear culture medium, the effects of having adjusting osmotic pressure, and protect cells from mechanical damage.Li Ping etc. (Li Ping,
Jiang Lin, Huang are thought to raise, research (J) the microbiology immunology progress 2004 of Chinese Hamster Ovary engineering cell free serum culture,
32 (4): 46~50) experiment shows that albumin stablizes cell growth there are also apparent, increases the effect of cell strain product expression amount.
The serum free medium of general suspension culture all contains bovine serum albumin(BSA).(Hu Xuemei, Zhang Yuanxing, the amino acid such as Hu Xuemei
To effect (J) the East China University of Science's journal 1996,22 (6) of Chinese hamster ovary cell in serum free medium: 283~
285) influence of 15 kinds of amino acid and its concentration versus cell culture in Chinese hamster ovary celI free serum culture is investigated using orthogonal experiment,
The result shows that arginine, leucine, proline and the methionine of addition have apparent facilitation to cell growth, and it is highly concentrated
Serine, the tryptophan of degree have apparent inhibiting effect to cell growth.Insulin can promote RNA, protein and fatty acid
Synthesis, inhibit Apoptosis, be important liability factor.(Jan L, the Haggstrorm L.Specific such as Jan
growth rate as a parameter for tracing growth-limiting substances in animal
Cell cultures (J) .Journal of Biotechnology1995,42:163~165) think the pancreas in batch experiments
Island element exhausts rapidly the main reason for being specific cell growth rate decline.Selenium has bright in the growth course of mammalian cell
Aobvious effect, trace elements of selenium participate in the mechanism of glutathione peroxidase and superoxide dismutase, eliminate oxide
The injury of enzyme and oxygen radical to cell.(Xue Qingshan is compiled Xue Qingshan, the Beijing philosophy and technique (M) science and technology of in vitro culture
Publishing house, 2001,162~164) point out that the appropriate addition of selenous acid can promote cell to grow, but if excessive concentration,
Toxic effect can be generated to cell.
For commercialization serum free medium mainly based on import, expensive, formula secrecy is unfavorable for subsequent training at present
Process optimization is supported, domestic serum free medium producer is few, and there is a problem of that Market Feedback cell adaptation is not strong, Yi Xieke
It grinds mechanism or research and development unit is also only developed and used by oneself.Therefore independent research can support that hybridoma fast-growth, monoclonal are anti-
The serum free medium of the high expression quantity of body, and related process optimization and amplification are carried out, to meet great expression monoclonal antibody
Demand is most important to target protein industrialization.In addition, not having in general, different cell strains has its unique nutritional need
There is any culture medium that can sufficiently meet the needs of all cell strains or play the maximum expression potentiality of cell, therefore, independently
It researches and develops and the personalized serum free medium optimized for specific cells strain is also pith in scale cell incubation.
In view of this, the present invention is specifically proposed.
Summary of the invention
There are differences between batches for blood serum medium greatly, potential pollution risk, ingredient is indefinite, it is pure to be unfavorable for purpose product separation
Change and be easy the problems such as infected.Chemical component is completely explicitly very strong without albumen serum free medium specific aim, in other words,
It is exactly that versatility is not high, general a kind of culture medium only properly culture of a certain cell, in addition, cell is in complete free serum culture
It is easy to be influenced by physical mechanical factor and chemical factor in base.
It is an object of the invention to overcome deficiency existing for the above culture medium, a kind of low albumen serum-free cell culture is prepared
Base.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
The present invention relates to a kind of low albumen serum-free cell culture medium, the culture medium is mixed comprising basic physiology buffer
Object and protein ingredient；
The protein ingredient includes bovine serum albumin(BSA), transferrins, insulin, and the total addition level of the protein ingredient
No more than 30mg/L.
The culture medium is suitable for zooblast, especially mammaliancellculture.
Compared with prior art, the invention has the benefit that
(1) the three kinds of proteinaceous components contents contained only are very low, are conducive to isolating and purifying for product, improve the quality of product；
(2) definite ingredients, preparation are convenient, are suitable for large-scale production application；
(3) there is stronger versatility；
(4) hybridoma, highest cell density and antibody production can be supported to meet or exceed GIBCO product well
The commercial medium effect of board；
(5) it supports the culture of hybridoma Long Term Passages, can directly be cultivated without adapting to.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the growth curve of serum free medium culture hybridoma 1C3 in one embodiment of the invention；
Fig. 2 is the concentration of glucose of serum free medium culture hybridoma DF92 in one embodiment of the invention, thin
Born of the same parents' density, Cell viability and monoclonal antibody expression quantity with incubation time change curve；
Fig. 3 is dense using the glucose of Gibco serum free medium fermented and cultured hybridoma 1A4 in the prior art
Degree, cell density, Cell viability and monoclonal antibody expression quantity with incubation time change curve；
Fig. 4 be one embodiment of the invention in serum free medium fermented and cultured hybridoma 1A4 concentration of glucose,
Cell density, Cell viability and monoclonal antibody expression quantity with incubation time change curve.
Specific embodiment
Before describing cell culture and method of the invention, it should be understood that the present invention is not limited to the specific methods
And experiment condition, because such methods and condition can change.It should also be understood that its purpose of term used herein is only that
Specific embodiment is described, and is not intended to be restrictive.
The present invention relates to a kind of low albumen serum-free cell culture medium, the culture medium is mixed comprising basic physiology buffer
Object and protein ingredient；
The protein ingredient includes bovine serum albumin(BSA), transferrins, insulin, and the total addition level of the protein ingredient
No more than 30mg/L.
In some embodiments, the total addition level of the protein ingredient is 18mg/L~30mg/L, it is also an option that
20mg/L、22mg/L、24mg/L、26mg/L、28mg/L。
In some embodiments, the protein ingredient includes bovine serum albumin(BSA) 10.0-20.0mg/L, transferrins
2.5-5.5mg/L and insulin 5.0-12.0mg/L；
In some embodiments, the protein ingredient includes bovine serum albumin(BSA) 13.0-17.0mg/L, transferrins
4.5-5.5mg/L and insulin 9.0-11.0mg/L；
In some embodiments, the basic physiology buffer mixture is basic culture medium with DMEM/F12, and is added
Enter including one of amino acid, vitamin, inorganic salts, microelement, lipid or a variety of；
In some embodiments, the volume ratio of DMEM and F12 is (0.8~1.2) in the DMEM/F12: (0.8~
1.2)；More preferably 1:1.
In some embodiments, it is selected in the group that the lipid is made of the following:
Arachidonic acid 10-50 μ g/L, cholesterol 1500-3500 μ g/L, linoleic acid 50-250 μ g/L, linolenic acid 50-250
μ g/L, oleic acid 50-250 μ g/L, palmitoleic acid 50-250 μ g/L, stearic acid 50-250 μ g/L, ethanol amine 1500-3500 μ g/L.
In some embodiments, it is selected in the group that the lipid is made of the following:
Arachidonic acid 10-40 μ g/L, cholesterol 2500-3500 μ g/L, linoleic acid 130-230 μ g/L, linolenic acid 150-
250 μ g/L, oleic acid 130-230 μ g/L, palmitoleic acid 75-175 μ g/L, stearic acid 50-150 μ g/L, ethanol amine 2200-3200 μ
g/L。
In some embodiments, it is selected in the group that the lipid is made of the following:
Arachidonic acid 20-30 μ g/L, cholesterol 2800-3200 μ g/L, linoleic acid 160-200 μ g/L, linolenic acid 180-
210 μ g/L, oleic acid 170-190 μ g/L, palmitoleic acid 115-135 μ g/L, stearic acid 80-120 μ g/L, ethanol amine 2500-2900 μ
g/L。
In some embodiments, it is selected in the group that the amino acid is made of the following:
Arginine 36-58mg/L, cystine 8-12mg/L, histidine 9-15mg/L, isoleucine 13-23mg/L, bright ammonia
Sour 13-23mg/L, lysine 14-22mg/L, methionine 4-7mg/L, phenylalanine 9-15mg/L, threonine 18-22mg/L,
Tryptophan 3-5mg/L, tyrosine 9-16mg/L, valine 18-22mg/L, glycine 4-7mg/L, alanine 5-9mg/L, asparagus fern
Amide 8-12mg/L, aspartic acid 8-12mg/L, glutamic acid 8-12mg/L, proline 5-9mg/L, serine 5-9mg/L.
In some embodiments, it is selected in the group that the amino acid is made of the following:
Arginine 40-54mg/L, cystine 9-11mg/L, histidine 11-14mg/L, isoleucine 16-20mg/L, bright ammonia
Sour 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, serine 6-8mg/L.
In some embodiments, it is selected in the group being made of in the vitamin the following:
Choline chloride, calcium pantothenate, folic acid, niacinamide, pyridoxal hydrochloride, riboflavin, thiamine, inositol；
In some embodiments, the concentration of said vitamin can be selected from 0.7-1.8mg/L, it is also an option that 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,1.7mg/L；Respectively
The concentration of vitamin can be identical or different.
In some embodiments, it is selected in the group that the vitamin is made of the following:
Choline chloride 0.7-1.8mg/L, calcium pantothenate 0.7-1.8mg/L, folic acid 0.7-1.8mg/L, niacinamide 0.7-
1.8mg/L, pyridoxal hydrochloride 0.7-1.8mg/L, riboflavin 0.7-1.8mg/L, thiamine 0.7-1.8mg/L, inositol 0.7-
1.8mg/L。
In some embodiments, it is selected in the group that the vitamin is made of the following:
Choline chloride 1.0-1.4mg/L, calcium pantothenate 0.8-1.4mg/L, folic acid 1.0-1.4mg/L, niacinamide 1.0-
1.4mg/L, pyridoxal hydrochloride 1.0-1.4mg/L, riboflavin 1.0-1.4mg/L, thiamine 1.0-1.4mg/L, inositol 1.0-
1.4mg/L。
In some embodiments, it is selected in the group that the inorganic salts are made of the following:
Ironic citrate 5-15mg/L, Sodium Pyruvate 40-60mg/L, calcium chloride 1-5mg/L, magnesium sulfate 1-5mg/L, potassium chloride
4-10mg/L, sodium chloride 80-100mg/L, sodium dihydrogen phosphate 100-200mg/L.
In some embodiments, it is selected in the group that the inorganic salts are made of the following:
Ironic citrate 8-12mg/L, Sodium Pyruvate 45-55mg/L, calcium chloride 2-4mg/L, magnesium sulfate 2-4mg/L, potassium chloride
6-8mg/L, sodium chloride 85-100mg/L, sodium dihydrogen phosphate 100-140mg/L.
In some embodiments, the culture medium includes to select in one or more groups being made of the following
Microelement:
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 aluminium；
In some embodiments, the microelement is selenium, is added in the form of selenous acid, and content is 3-8 μ g/L.
In some embodiments, the basic physiology buffer mixture further includes buffer and/or anti-shearing tries hard to keep
Protect agent；
In some embodiments, the buffer is sodium bicarbonate 1.0-1.8g/L and Hepes3.2-4.5mg/L；
In some embodiments, the anti-shearing force protective agent is Pluronic F-68 700-2000mg/L.
According to an aspect of the present invention, the invention further relates to a kind of methods for cultivating cell, comprising: is training as described above
It supports in base and cultivates cell；
In some embodiments, the cell is multipotential stem cell, embryonic stem cell, marrow stromal cell, hematopoiesis ancestral
Cell, lymphoid stem cell, stem cell, T cell, B cell, macrophage, liver cell, pancreatic cell, cancer cell and
Cell line；
In some embodiments, wherein the cell line is selected in the group being made of the following:
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 system, B cell system, 3T3, RIN, A549,
PC12, K562, PER.C6, SP2/0, NS-0, U20S, HT1080, L929, fusion tumor and cancerous cell line.
In a specific embodiment, the cell is hybridoma.Hybridoma is that a kind of energy secretion is single
The unlimited passage cell of one unique antibodies, the monoclonal antibody of secretion is in terms of production of vaccine, in-vitro diagnosis and biological medicine
It has a wide range of applications.The culture medium can make hybridoma normal growth and express monoclonal antibody under floating condition, and one
Aspect meets the needs of external diagnosis reagent industry is to monoclonal antibody, on the other hand can reduce cost.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Embodiment 1
Serum free medium composition selection and optimization.
With six orifice plates, 3mL cultivating system cultivates hybridoma cell strain 1D7, when cultivating 72h in sampling meter cultivating system
Viable cell density.Using L16 (45) orthogonal test method, consults related literatures and determine that 5 classes train cell after screening
Favorable additive is supported, 5 classes are respectively to the favorable additive of cell culture: A, 8 kinds of lipids；B, 8 kinds of dimensions
Raw element；C, 3 kinds of albumen；D, 19 kinds of amino acid；E, 7 kinds of inorganic salts.
Test is basic culture medium with DMEM/F12 (v/v, 1:1), is previously added 6 μ g/L of selenous acid, sodium bicarbonate 1.5g/
L and Hepes 3.8mg/L, Pluronic F-68 1300mg/L.Selected five kinds of additives are carried out using orthogonal test method
Four various concentration levels (1,2,3,4, contained additive concentration is from low to high) are arranged in optimization, each factor (A, B, C, D, E).
10 μ g/L of arachidonic acid, 500 μ g/L of cholesterol, 80 μ g/L of linoleic acid, 80 μ g/L of linolenic acid, 80 μ of oleic acid in A1
G/L, 50 μ g/L of palmitoleic acid, 50 μ g/L of stearic acid, 1000 μ g/L of ethanol amine.
15 μ g/L of arachidonic acid, 1000 μ g/L of cholesterol, 100 μ g/L of linoleic acid, 120 μ g/L of linolenic acid, oleic acid in A2
120 μ g/L, 80 μ g/L of palmitoleic acid, 60 μ g/L of stearic acid, 1500 μ g/L of ethanol amine.
20 μ g/L of arachidonic acid, 2000 μ g/L of cholesterol, 150 μ g/L of linoleic acid, 160 μ g/L of linolenic acid, oleic acid in A3
160 μ g/L, 100 μ g/L of palmitoleic acid, 80 μ g/L of stearic acid, 2000 μ g/L of ethanol amine.
25 μ g/L of arachidonic acid, 3000 μ g/L of cholesterol, 180 μ g/L of linoleic acid, 200 μ g/L of linolenic acid, oleic acid in A4
180 μ g/L, 125 μ g/L of palmitoleic acid, 100 μ g/L of stearic acid, 2700 μ g/L of ethanol amine.
Choline chloride 0.2mg/L, calcium pantothenate 0.2mg/L, folic acid 0.2mg/L, niacinamide 0.2mg/L, hydrochloric acid pyrrole in B1
Tremble aldehyde 0.2mg/L, riboflavin 0.2mg/L, thiamine 0.2mg/L, inositol 0.2mg/L
Choline chloride 0.4mg/L, calcium pantothenate 0.4mg/L, folic acid 0.4mg/L, niacinamide 0.4mg/L, hydrochloric acid pyrrole in B2
Tremble aldehyde 0.4mg/L, riboflavin 0.4mg/L, thiamine 0.4mg/L, inositol 0.4mg/L
Choline chloride 0.8mg/L, calcium pantothenate 0.8mg/L, folic acid 0.8mg/L, niacinamide 0.8mg/L, hydrochloric acid pyrrole in B3
Tremble aldehyde 0.8mg/L, riboflavin 0.8mg/L, thiamine 0.8mg/L, inositol 0.8mg/L
Choline chloride 1.2mg/L, calcium pantothenate 1.2mg/L, folic acid 1.2mg/L, niacinamide 1.2mg/L, hydrochloric acid pyrrole in B4
Tremble aldehyde 1.2mg/L, riboflavin 1.2mg/L, thiamine 1.2mg/L, inositol 1.2mg/L
BSA is 5mg/L, transferrins 2mg/L, insulin 3mg/L in C1；
BSA is 10mg/L, transferrins 2mg/L, insulin 5mg/L in C2；
BSA is 15mg/L, transferrins 5mg/L, insulin 8mg/L in C3；
BSA is 15mg/L, transferrins 5mg/L, insulin 10mg/L in C4.
Arginase 12 4mg/L in D1, 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, paddy
Propylhomoserin 5mg/L, proline 3 mg/L, serine 3mg/L
Arginine 48mg/L, cystine 10mg/L, histidine 12mg/L, isoleucine 19mg/L, leucine 18mg/ in D2
L, lysine 18mg/L, methionine 7mg/L, phenylalanine 11mg/L, threonine 19mg/L, tryptophan 4mg/L, tyrosine
14mg/L, valine 20mg/L, glycine 5mg/L, alanine 7mg/L, asparagine 10mg/L, aspartic acid 10mg/L, paddy
Propylhomoserin 10mg/L, proline 6mg/L, serine 6mg/L
Arginine 96mg/L, cystine 20mg/L, histidine 24mg/L, isoleucine 38mg/L, leucine 36mg/ in D3
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
Arginine 192mg/L, cystine 40mg/L, histidine 48mg/L, isoleucine 76mg/L, leucine in D4
72mg/L, lysine 72mg/L, methionine 28mg/L, phenylalanine 44mg/L, threonine 76mg/L, tryptophan 16mg/L,
Tyrosinase 15 6mg/L, valine 80mg/L, glycine 20mg/L, alanine 28mg/L, asparagine 40mg/L, aspartic acid
40mg/L, glutamic acid 40mg/L, proline 24mg/L, serine 24mg/L
Ironic citrate 12mg/L, Sodium Pyruvate 50mg/L, calcium chloride 4mg/L, magnesium sulfate 3mg/L, potassium chloride 7mg/ in E1
L, sodium chloride 100mg/L, sodium dihydrogen phosphate 120mg/L
Ironic citrate 18mg/L, Sodium Pyruvate 75mg/L, calcium chloride 6mg/L, magnesium sulfate 4.5mg/L, potassium chloride in E2
7mg/L, sodium chloride 150mg/L, sodium dihydrogen phosphate 150mg/L
Ironic citrate 27mg/L, Sodium Pyruvate 112.5mg/L, calcium chloride 9mg/L, magnesium sulfate 6.75mg/L, chlorination in E3
Potassium 7mg/L, sodium chloride 200mg/L, sodium dihydrogen phosphate 200mg/L
Ironic citrate 40.5mg/L in E4, Sodium Pyruvate 168.75mg/L, calcium chloride 13.5mg/L, magnesium sulfate 10mg/L,
Potassium chloride 7mg/L, sodium chloride 250mg/L, sodium dihydrogen phosphate 250mg/L
The results are shown in Table 1.
1 optimization of orthogonal test of table
The average value of viable cell density is compared in cultivating system when to culture to 72h, and orthogonal experiments show 16
In the group various combination addition factor, A4B4C4D2E1 combination promotes viable cell density maximum.According to very poor R size judge it is each because
Element influences size to viable cell density, and showing that each factor influences primary and secondary sequence is C > A > D > B > E.Determine this in 5 on this basis
Difference addition factor optimal level are as follows: A4B4C4D2E1.The additive concentration combined using optimal level is carried out as culture medium
Preferred concentration, the subsequent cell culture for carrying out embodiment 2.
Embodiment 2
Serum free medium preparation and cell culture
1. prepared by culture medium
It is being to add following substance in basic culture medium with DMEM/F12 (v/v, 1:1):
Bovine serum albumin(BSA) 15.0mg/L, transferrins 5mg/L, insulin 10mg/L, arginine 48mg/L, cystine
10mg/L, histidine 12mg/L, isoleucine 19mg/L, leucine 18mg/L, lysine 18mg/L, methionine 7mg/L, benzene
Alanine 11mg/L, threonine 19mg/L, tryptophan 4mg/L, tyrosine 14mg/L, valine 20mg/L, glycine 5mg/L,
Alanine 7mg/L, asparagine 10mg/L, aspartic acid 10mg/L, glutamic acid 10mg/L, proline 6mg/L, serine 6mg/
L, choline chloride 1.2mg/L, calcium pantothenate 1.2mg/L, folic acid 1.2mg/L, niacinamide 1.2mg/L, pyridoxal hydrochloride 1.2mg/
L, riboflavin 1.2mg/L, thiamine 1.2mg/L, inositol 1.2mg/L, ironic citrate 12mg/L, Sodium Pyruvate 50mg/L, chlorination
Calcium 4mg/L, magnesium sulfate 3mg/L, potassium chloride 7mg/L, sodium chloride 100mg/L, sodium dihydrogen phosphate 120mg/L, 25 μ of arachidonic acid
It is g/L, 3000 μ g/L of cholesterol, 180 μ g/L of linoleic acid, 200 μ g/L of linolenic acid, 180 μ g/L of oleic acid, 125 μ g/L of palmitoleic acid, hard
100 μ g/L of resin acid, 2700 μ g/L of ethanol amine, 6 μ g/L of selenous acid, sodium bicarbonate 1.5g/L and Hepes 3.8mg/L, Pluronic
F-68 1300mg/L.Culture medium conventionally prepare after with 0.22 μm of millipore filter filtration sterilization.
2. cell culture
Liquid nitrogen takes out the hybridoma cell strain 1C3 cell frozen in filling, and is immediately placed in 37 DEG C of water baths, to dissolve completely
Supernatant is removed in centrifugation afterwards, gently blows and beats cell with a small amount of above-mentioned serum free medium, mixing with cells is made uniformly to be then transferred to square vase
In, it is subsequently placed in 37 DEG C, 5%CO2The CO of moisture-saturated2Secondary culture in incubator.
3. compared with GIBCO brand serum free medium Hybridoma-SFM
Above-mentioned 1C3 cell is expanded culture and is passed in 4 square vases, when phase to be grown to cell log is inoculated with together
It is about 70 × 10 with initial incubation density to 1.5L rolling bottle4Cells/ml is subsequently placed in 37 DEG C, 5%CO2Moisture-saturated incubator
Suspend culture.
As shown in Figure 1, the curve being below is the life of the hybridoma of serum free medium culture of the present invention
Long curve, the curve in top are the Growth of Hybridoma Cell curve of GIBCO Hybridoma-SFM culture medium culture.Entirely
Incubation continues 8 days.Serum free medium of the present invention and GIBCO Hybridoma-SFM culture medium viable cell density are in 120h
Respectively reach 537 × 104Cells/ml and 551 × 104The culture effect of cells/ml, serum free medium of the present invention are several
It is equal to the culture effect of the similar culture medium of external producer's production, while serum free medium cost of the present invention is relatively low
It is honest and clean, and specific chemical components, protein content are extremely low.
Embodiment 3
Fed batch culture hybridoma cell strain prepares monoclonal antibody
Serum free medium of the present invention is used for hybridoma cell strain DF92,1A4 and carries out fermentor suspension culture, training
It is identical in embodiment 2 to support based formulas.DF92 uses Guangzhou Qi Zhi biotech firm 14L bioreactor, and initial incubation volume is
2L, control condition are as follows: 37 ± 0.5 DEG C of Temp, DO50% ± 5%, pH 6.9 ± 0.1, Stir speed 80rpm, 1A4 are used
Applikon 2L bioreactor, initial incubation volume are 2L, control condition are as follows: 37 ± 0.5 DEG C of Temp, DO 50% ±
5%, pH6.9 ± 0.1, Stir speed 80rpm, in suspension incubation, using fed batch culture method, often
Interval about sampling monitoring related data for 24 hours, these data include concentration of glucose in culture solution, cell density, Cell viability and
Monoclonal antibody expression quantity, the cultivation results of two kinds of cells are (Fig. 3 is that the culture medium of the prior art compares) as shown in Figure 2,4, according to culture
Supplemented medium is added in glucose consumption index in base, and from starting feed supplement, feed supplement interval is about for 24 hours.When fermentor inner cell
When motility rate is reduced to 60% or less, stop the fed batch culture operation of fermentor, centrifugation is filtered after collecting supernatant, expressed
Measure fixed and monoclonal antibody purifying.The result shows that serum free medium of the present invention can be normally used for hybridoma
Suspend culture and the preparation of monoclonal antibody, under same culture conditions, the growth conditions and antibody production of cell and outsourcing pair
Quite or it is better than outsourcing culture medium according to culture medium, subsequent experimental is also turned out can be with using monoclonal antibody prepared by the serum free medium
It is normally used for external diagnosis reagent field.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
1. a kind of low albumen serum-free cell culture medium, which is characterized in that the culture medium is mixed comprising basic physiology buffer
Object and protein ingredient；
The protein ingredient includes bovine serum albumin(BSA), transferrins, insulin, and the total addition level of the protein ingredient does not surpass
Cross 30mg/L.
2. culture medium according to claim 1, which is characterized in that the protein ingredient includes bovine serum albumin(BSA) 10.0-
20.0mg/L, transferrins 2.5-5.5mg/L and insulin 5.0-12.0mg/L；
Preferably, the protein ingredient include bovine serum albumin(BSA) 13.0-17.0mg/L, transferrins 4.5-5.5mg/L and
Insulin 9.0-11.0mg/L.
3. culture medium according to claim 1 or 2, which is characterized in that the basic physiology buffer mixture is with DMEM/
F12 is basic culture medium, and being added includes one of amino acid, vitamin, inorganic salts, microelement, lipid or a variety of；
Preferably, the volume ratio of DMEM and F12 is (0.8~1.2): (0.8~1.2) in the DMEM/F12；More preferably 1:
1。
4. culture medium according to claim 3, which is characterized in that selected in the group that the lipid is made of the following
Out:
Arachidonic acid 10-50 μ g/L, cholesterol 1500-3500 μ g/L, linoleic acid 50-250 μ g/L, linolenic acid 50-250 μ g/
L, oleic acid 50-250 μ g/L, palmitoleic acid 50-250 μ g/L, stearic acid 50-250 μ g/L, ethanol amine 1500-3500 μ g/L.
5. culture medium according to claim 3, which is characterized in that in the group that the amino acid is made of the following
It selects:
Arginine 36-58mg/L, cystine 8-12mg/L, histidine 9-15mg/L, isoleucine 13-23mg/L, leucine 13-
23mg/L, lysine 14-22mg/L, methionine 4-7mg/L, phenylalanine 9-15mg/L, threonine 18-22mg/L, color ammonia
Sour 3-5mg/L, tyrosine 9-16mg/L, valine 18-22mg/L, glycine 4-7mg/L, alanine 5-9mg/L, asparagine
8-12mg/L, aspartic acid 8-12mg/L, glutamic acid 8-12mg/L, proline 5-9mg/L, serine 5-9mg/L.
6. culture medium according to claim 3, which is characterized in that in the group that the vitamin is made of the following
It selects:
Choline chloride 0.7-1.8mg/L, calcium pantothenate 0.7-1.8mg/L, folic acid 0.7-1.8mg/L, niacinamide 0.7-1.8mg/
L, pyridoxal hydrochloride 0.7-1.8mg/L, riboflavin 0.7-1.8mg/L, thiamine 0.7-1.8mg/L, inositol 0.7-1.8mg/L.
7. culture medium according to claim 3, which is characterized in that in the group that the inorganic salts are made of the following
It selects:
Ironic citrate 5-15mg/L, Sodium Pyruvate 40-60mg/L, calcium chloride 1-5mg/L, magnesium sulfate 1-5mg/L, potassium chloride 4-
10mg/L, sodium chloride 80-100mg/L, sodium dihydrogen phosphate 100-200mg/L.
8. culture medium according to claim 3, which is characterized in that the culture medium includes one or more by the following institute
The microelement selected in the group of composition:
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 aluminium；
Preferably, the microelement is selenium, is added in the form of selenous acid, and content is 3-8 μ g/L.
9. culture medium according to claim 3, which is characterized in that the basic physiology buffer mixture further includes buffering
Liquid and/or anti-shearing force protective agent；
Preferably, the buffer is sodium bicarbonate 1.0-1.8g/L and Hepes 3.2-4.5mg/L；
Preferably, the anti-shearing force protective agent is Pluronic F-68 700-2000mg/L.
10. a kind of method for cultivating cell, comprising: cultivate cell in the culture medium described in any one of preceding claims；
Preferably, the cell is that multipotential stem cell, embryonic stem cell, marrow stromal cell, hematopoietic progenitor cells, lymph trunk are thin
Born of the same parents, stem cell, T cell, B cell, macrophage, liver cell, pancreatic cell, cancer cell and cell line；
It is furthermore preferred that wherein the cell line is selected in the group being made of the following:
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 system, B cell system, 3T3, RIN, A549, PC12,
K562, PER.C6, SP2/0, NS-0, U20S, HT1080, L929, fusion tumor and cancerous cell line.
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