CN1908162A - Serum-free culture medium of human embryo kidney (HEK) 293 cell - Google Patents
Serum-free culture medium of human embryo kidney (HEK) 293 cell Download PDFInfo
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- CN1908162A CN1908162A CNA2006100301108A CN200610030110A CN1908162A CN 1908162 A CN1908162 A CN 1908162A CN A2006100301108 A CNA2006100301108 A CN A2006100301108A CN 200610030110 A CN200610030110 A CN 200610030110A CN 1908162 A CN1908162 A CN 1908162A
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Abstract
the invention discloses a non-serum substratum for cell of HEK 293, which is composed of transferring, insulin, Primatone, lipoid compound, amino acid, inorganic salt and vitamin based on basic substratum.
Description
Technical field
The present invention relates to a kind of substratum that is used for human embryo kidney (HEK) 293 cell cultures, specifically, relate to a kind of serum free medium of the HEK293 of being used for cell cultures.
Background technology
Human embryo kidney (HEK) 293 cells are at first set up in 1977 by Graham etc., are the desirable hosts who produces recombinant adenovirus.
Traditional substratum of HEK293 cell is that blood serum medium is arranged.But, there is the use of blood serum medium to be restricted along with people improve constantly the understanding that animal serum may pollute production process even the finished product.Serum free medium, with its have passivity pollute, batch between otherness advantages such as separation and purification little and that help expression product more and more be subjected to people's attention.
The existing at present serum free medium that is fit to the HEK293 cell cultures has: 293-SFMII (Invitrogen), Ex-Cell 525 (JRH), IS293-SF (Irvine Scientific), Pro293s-CDM (Cambrex), CD293 Medium, Freestyle
TM293 and Expression Medium (Gibco) etc., but the price comparison costliness of these serum free mediums.Therefore this area presses for the serum free medium of a kind of low cost of research and development and suitable HEK293 cell cultures.
Summary of the invention
The object of the invention is, a kind of low cost is provided and has been applicable to the serum free medium of human embryo kidney (HEK) 293 cell cultures.
Design of the present invention is such:
In the serum free medium owing to lack serum, the essential blood serum substituting factor of adding, current, the recruitment factor kind of alternative serum has a lot, generally includes somatomedin, hormone, lipid, conjugated protein and trace element etc.In order to develop the serum free medium that is fit to the HEK293 cell, the present invention has added following serum substitute:
(1) Transferrins,iron complexes: a kind of glycoprotein in conjunction with iron, with the single-minded receptor acting of cell surface, transmit iron ion.
(2) Regular Insulin: beta Cell of islet excretory polypeptide hormone, promote the cell growth, regulate the metabolism of glucose and lipid.
(3) Primatone: a kind of peptone, can play stable and the active function of adjusting above-mentioned substance in anteserum-less substrate by combining with VITAMIN, lipid, metal ion, hormone and somatomedin.In the stir culture system, the mechanicalness protection effect that viscosity by influencing substratum and oncotic pressure are brought into play pair cell.
(4) lipid mixture: phosphatide is the integral part of cell, adds lipid mixture and can promote the synthetic of phosphatide, accelerates the synthetic of cytolemma, increases the slipperiness of cytolemma.
Because lack the effect of serum, corresponding variation can take place the metabolism situation of cell when serum-free culture, nutritional needs is corresponding to change, and for this reason, changes the content of composition on the basis of the nutritive ingredient of basic medium that must be when having serum to cultivate.Amino acid is the precursor of protein synthesis, and its utilization is also grown for cell and product production provides energy.Amino acid can also be alleviated the influence of generation, carbonic acid gas and the osmotic pressure of ammonia.The production of adenovirus has bigger demand to amino acid, and therefore, HEK293 cell non-serum culture medium of the present invention contains than the more amino acid of general basic medium.
The said serum free medium that is used for human embryo kidney (HEK) 293 cell cultures of the present invention is made up of amino acid, VITAMIN, inorganic salt and other parts, and it comprises following component and content:
Amino acid moiety:
L-L-Ala 0~30mg/L
L-arginine 50~300mg/L
L-L-glutamic acid 10~150mg/L
Altheine 10~200mg/L
L-aspartic acid 20~300mg/L
L-halfcystine 20~300mg/L
L-Gelucystine 50~600mg/L
L-glycine 0~50mg/L
L-Histidine 40~700mg/L
L-Isoleucine 50~700mg/L
L-leucine 80~1200mg/L
L-Methionin 90~1500mg/L
L-methionine(Met) 20~400mg/L
L-phenylalanine-3,4-quinone 0~500mg/L
L-proline(Pro) 0~50mg/L
L-Threonine 50~600mg/L
L-Serine 50~200mg/L
L-tryptophane 10~200mg/L
L-tyrosinase 15 0~800mg/L
L-Xie Ansuan 50~700mg/L
L-glutaminate 100~9000mg/L
The VITAMIN part:
Vitamin H 0.001~0.01mg/L
Choline chloride 60 5~20mg/L
VITMAIN B1 2~10mg/L
Wei ShengsuB2 0.1~0.5mg/L
Vitamin B12 0.2~1.0mg/L
Thioctic Acid 0.1~0.5mg/L
The inorganic salt part:
Fe(NO
3)
3.9H
2O 2~10mg/L
FeSO
4.7H
2O 5~20mg/L
KCl 200~500mg/L
MgCl
2 20~50mg/L
CuSO
4.5H
2O 0.001~0.01mg/L
Na
2HPO
4.2H
2O 50~100mg/L
NaH
2PO
4.2H
2O 50~100mg/L
Sodium Selenite 0.005~0.05mg/L
NaHCO
3 2000mg/L
NaCl 6999.5mg/L
MgSO
4 30~50mg/L
ZnSO
4.7H
2O 0.2~0.5mg/L
Other parts:
Glucose 2000~5000mg/L
Transferrins,iron complexes 2~20mg/L
Primatone 1000~5000mg/L
Lipid mixture 0.1v/v%~0.5v/v%
Hydrocortisone 0.05~0.5mg/L
HEPES 1000~5000mg/L
Pluronic F-68 1~10g/L
Dextran 10~100mg/L
Reduced glutathion 0~3mg/L
Thymus pyrimidine 0.3~0.5mg/L
Wherein: the lipid that the chemical ingredients that said lipid mixture is produced for GIBCO company is determined, it is composed as follows
Arachidonic acid 2mg/L
1
Cholesterol 220mg/L
1
DL-alpha-tocopherol acetate 70mg/L
1
Linolic acid 10mg/L
1
Linolenic acid 10mg/L
1
Myristic acid 10mg/L
1
Oleic acid 10mg/L
1
Physetoleic acid 10mg/L
1
Palmitinic acid 10mg/L
1
Stearic acid 10mg/L
1
Tween 80 2200mg/L
1
Pluronic F-68 100000mg/L
1
L is the cumulative volume of said serum free medium, L
1Be the overall integrating of said lipid mixture, the molecular weight of used polyoxyethylene glycol is 20000.
Above-mentioned related raw material is commercially available product, and wherein Primatone, HEPES and Pluronic F-68 are all available from Sigma company.
The key step of preparation the present invention said serum free medium is: with above-mentioned raw materials according to its dissolution characteristics classification dissolving separately, then with gained solution in 20~30 ℃ of mixing, make each component concentration as listed above, regulate the pH value to 7.2 of gained mixed solution, promptly get target compound behind the constant volume.
The using method of the said serum free medium of the present invention is identical with the using method of existing serum free medium.
Description of drawings
Fig. 1 is the test-results diagram of embodiment 2 and Comparative Examples 1.
Wherein: the cell density of the substratum of ◇-employing DMEM+10% foetal calf serum;
◆-the adopt cell density of serum free medium.
Embodiment
Below by embodiment content of the present invention is further described, its purpose only is better to understand content of the present invention and unrestricted protection scope of the present invention.
The said preparation that is used for the serum free medium of human embryo kidney (HEK) 293 cell cultures of the present invention:
With following raw materials according according to its dissolution characteristics classification dissolving (being undertaken) separately by the operation steps that supplier provides, then with gained solution in 20~30 ℃ of mixing, make each component concentration following listed, regulate the pH value to 7.2 of gained mixed solution, promptly get target compound behind the constant volume.
Amino acid moiety:
The name of an article | mg/L |
The L-tryptophane | 20 |
The L-halfcystine | 30 |
The L-Gelucystine | 60 |
L-tyrosine | 70 |
The L-Isoleucine | 90 |
The L-leucine | 120 |
The L-Xie Ansuan | 92 |
L-L-glutamic acid | 14.5 |
Altheine | 15 |
The L-glycine | 18.75 |
The L-proline(Pro) | 17.25 |
The L-L-Ala | 4.45 |
The L-arginine | 14.75 |
The L-aspartic acid | 30 |
The L-Histidine | 62.75 |
L-Methionin | 275 |
The L-methionine(Met) | 26.68 |
The L-phenylalanine | 54.8 |
The L-Serine | 87.5 |
L-glutaminate | 584 |
The L-Threonine | 54.5 |
The inorganic salt part:
The name of an article | mg/L |
KCl | 311.8 |
MgCl 2 | 28.61 |
MgSO 4 | 48.84 |
ZnSO 4.7H 2O | 0.432 |
CuSO 4.5H 2O | 0.001 |
CaCl 2 | 2.220 |
Na 2HPO 4.2H 2O | 89.025 |
NaH 2PO 4.2H 2O | 70.65 |
Ironic citrate | 6.1225 |
Fe(NO 3) 3.9H 2O | 2.050 |
FeSO 4.7H 2O | 3.417 |
NaF | 4.2 |
NaHCO 3 | 2000 |
NaCl | 6999.5 |
Sodium Selenite | 0.005 |
The VITAMIN part:
The name of an article | mg/L |
Calcium pantothenate | 2.24 |
Choline chloride 60 | 8.89 |
Folic acid | 2.65 |
Inositol | 12.6 |
Niacinamide | 2.0 |
Vitamin B6 | 2.0 |
VITMAIN B1 | 4.17 |
Wei ShengsuB2 | 0.219 |
Vitamin B12 | 0.68 |
Vitamin H | 0.0037 |
Thioctic Acid | 0.1 |
Other parts:
The name of an article | mg/L |
Reduced glutathion | 0.75 |
Xanthoglobulin | 4.39 |
Thymus pyrimidine | 0.365 |
Regular Insulin | 20 |
Transferrins,iron complexes | 20 |
Macrogol 2000 0 | 2 |
Primatone | 4000 |
Lipid mixture | 0.2%(v/v) |
Hydrocortisone | 0.05 |
Glucose | 4500 |
HEPES | 4000 |
Pluronic F-68 | 1000 |
Dextran | 100 |
Phenol red | 10 |
By making the serum free medium that another kind is used for human embryo kidney (HEK) 293 cell cultures with embodiment 1 described method: its composition and content are as follows:
L-L-Ala 30mg/L
L-arginine 300mg/L
L-L-glutamic acid 150mg/L
Altheine 200mg/L
L-aspartic acid 300mg/L
L-halfcystine 300mg/L
L-Gelucystine 600mg/L
L-glycine 50mg/L
L-Histidine 700mg/L
L-Isoleucine 700mg/L
L-leucine 1200mgL
L-Methionin 1500mg/L
L-methionine(Met) 400mg/L
L-phenylalanine 500mg/L
L-proline(Pro) 50mg/L
L-Threonine 600mg/L
L-Serine 200mg/L
L-tryptophane 200mg/L
L-tyrosine 800mg/L
L-Xie Ansuan 700mg/L
L-glutaminate 9000mg/L
The VITAMIN part:
Vitamin H 0.01mg/L
Choline chloride 60 20mg/L
Calcium pantothenate 10mg/L
Folic acid 5mg/L
Inositol 20mg/L
VITMAIN B1 10mg/L
Wei ShengsuB2 0.5mg/L
Vitamin B6 10mg/L
Vitamin B12 1.0mg/L
Niacinamide 5mg/L
Thioctic Acid 0.5mg/L
The inorganic salt part:
Ironic citrate 20mg/L
NaF 20mg/L
Fe(NO
3)
3.9H
2O 10mg/L
FeSO
4.7H
2O 20mg/L
KCl 500mg/L
MgCl
2 50mg/L
CuSO
4.5H
2O 0.01mg/L
CaCl
2 200mg/L
Na
2HPO
4.2H
2O 100mg/L
NaH
2PO
4.2H
2O 100mg/L
Sodium Selenite 0.05mg/L
NaHCO
3 2000mg/L
NaCl 6999.5mg/L
MgSO
4 50mg/L
ZnSO
4.7H
2O 0.5mg/L
Other parts:
Glucose 5000mg/L
Regular Insulin 20mg/L
Transferrins,iron complexes 20mg/L
Polyoxyethylene glycol (20000) 10mg/L
Primatone 5000mg/L
Lipid mixture 0.5v/v%
Hydrocortisone 0.5mg/L
HEPES 5000mg/L
Pluronic F-68 10g/L
Dextran 100mg/L
Reduced glutathion 3mg/L
Xanthoglobulin 10mg/L
Thymus pyrimidine 0.5mg/L
Cultivate the HEK293 cell that has adapted to the serum-free suspension culture in the square vase of 125ml, inoculum density is 3 * 10
5Cells/ml, liquid amount are 20ml, and the square vase that will connect seed cell again places shaking table, and (model: TZ-03, (shaking table is placed on 36.5 ℃, 5%CO SHENERGYBIOCOLOR) to go up cultivation
2In the incubator), rotating speed is 120r/min, cultivates 5 days, maximum cell density reaches 3.8 * 10
6Cells/ml.Result such as Fig. 1.The serum free medium that present embodiment uses is made by embodiment 1.
Comparative Examples 1
Cultivate adherent HEK293 cell in the square vase of 125ml, inoculum density is 3 * 10
5Cells/ml, square vase are placed on 36.5 ℃, 5%CO
2In the incubator, different is: the substratum of adding is DMEM+10% foetal calf serum (FBS), and liquid amount is 10ml, and static cultivation was cultivated 5 days, and maximum cell density is 2.6 * 10
6Cells/ml.Result such as Fig. 1, serum free medium of the present invention are suitable for HEK293 cell high-density growth.
Claims (1)
1, a kind of serum free medium that is used for human embryo kidney (HEK) 293 cell cultures is made up of amino acid, VITAMIN, inorganic salt and other parts, it is characterized in that said serum free medium comprises following component and content:
Amino acid moiety:
L-L-Ala 0~30mg/L
L-arginine 50~300mg/L
L-L-glutamic acid 10~150mg/L
Altheine 10~200mg/L
L-aspartic acid 20~300mg/L
L-halfcystine 20~300mg/L
L-Gelucystine 50~600mg/L
L-glycine 0~50mg/L
L-Histidine 40~700mg/L
L-Isoleucine 50~700mg/L
L-leucine 80~1200mg/L
L-Methionin 90~1500mg/L
L-methionine(Met) 20~400mg/L
L-phenylalanine-3,4-quinone 0~500mg/L
L-proline(Pro) 0~50mg/L
L-Threonine 50~600mg/L
L-tryptophane 10~200mg/L
L-tyrosinase 15 0~800mg/L
L-Xie Ansuan 50~700mg/L
L-glutaminate 100~9000mg/L
The VITAMIN part:
Vitamin H 0.001~0.01mg/L
Choline chloride 60 5~20mg/L
Calcium pantothenate 2~10mg/L
Folic acid 0~5mg/L
Inositol 5~20mg/L
VITMAIN B1 2~10mg/L
Wei ShengsuB2 0.1~0.5mg/L
Vitamin B6 2~10mg/L
Vitamin B12 0.2~1.0mg/L
Niacinamide 2~5mg/L
The inorganic salt part:
Ironic citrate 5~20mg/L
NaF 2~20mg/L
Fe(NO
3)
3.9H
2O 2~10mg/L
FeSO
4.7H
2O 5~20mg/L
KCl 200~500mg/L
MgCl
2 20~50mg/L
CuSO
4.5H
2O 0.001~0.01mg/L
CaCl
2 2~200mg/L
Na
2HPO
4.2H
2O 50~100mg/L
NaH
2PO
4.2H
2O 50~100mg/L
Sodium Selenite 0.005~0.05mg/L
NaHCO
3 2000mg/L
NaCl 6999.5mg/L
Other parts:
Glucose 2000~5000mg/L
Regular Insulin 2~20mg/L
Transferrins,iron complexes 2~20mg/L
Polyoxyethylene glycol 2~10mg/L
Primatone 1000~5000mg/L
Lipid mixture 0.1v/v%~0.5v/v%
Hydrocortisone 0.05~0.5mg/L
HEPES 1000~5000mg/L
Pluronic F-68 1~10g/L
Dextran 10~100mg/L
Reduced glutathion 0~3mg/L
Wherein: the lipid that the chemical ingredients that said lipid mixture is produced for GIBCO company is determined, it is composed as follows
Arachidonic acid 2mg/L
1
Cholesterol 220mg/L
1
DL-alpha-tocopherol acetate 70mg/L
1
Linolic acid 10mg/L
1
Linolenic acid 10mg/L
1
Myristic acid 10mg/L
1
Oleic acid 10mg/L
1
Physetoleic acid 10mg/L
1
Palmitinic acid 10mg/L
1
Stearic acid 10mg/L
1
Tween 80 2200mg/L
1
Pluronic F-68 100000mg/L
1
L is the cumulative volume of said serum free medium, and L1 is the overall integrating of said lipid mixture, and the molecular weight of used polyoxyethylene glycol is 20000.
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CNB2006100301108A CN100569940C (en) | 2006-08-16 | 2006-08-16 | Human embryo kidney (HEK) 293 cell non-serum culture mediums |
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