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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 PDF

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Publication number
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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Prior art keywords
acid
vitamin
serum
free medium
hek
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CNA2006100301108A
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CN100569940C (en
Inventor
易小萍
陈春艳
张元兴
孙祥明
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East China University of Science and Technology
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East China University of Science and Technology
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Abstract

本发明涉及一种用于人胚胎肾(HEK)293细胞培养的无血清培养基，其是在现有基础培养基的基础上添加了转铁蛋白、胰岛素、Primatone、类脂混合物、氨基酸、无机盐和维生素等物质构成。该培养基能使(HEK)293细胞旺盛生长且成本低，是一种具有商业价值的细胞培养。The invention relates to a serum-free medium for culturing human embryonic kidney (HEK) 293 cells, which adds transferrin, insulin, Primatone, lipid mixture, amino acid, inorganic Composition of substances such as salt and vitamins. The culture medium can make (HEK)293 cells grow vigorously and has low cost, and is a kind of cell culture with commercial value.

Description

Human embryo kidney (HEK) 293 cell non-serum culture mediums
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
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
Thioctic Acid 0.1～0.5mg/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
MgSO 4 30～50mg/L
ZnSO 4.7H 2O 0.2～0.5mg/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
Xanthoglobulin 4～10mg/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.
Embodiment 1
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
Embodiment 2
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
Embodiment 3
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.
CNB2006100301108A 2006-08-16 2006-08-16 Serum-free medium for human embryonic kidney (HEK) 293 cells Expired - Fee Related CN100569940C (en)

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CN104357379A (en) * 2014-09-30 2015-02-18 刘兴宇 Stem cell culture medium
CN104651297A (en) * 2013-11-19 2015-05-27 华东师范大学 Culture medium used in high-density large-scale suspended cultivation of human embryo nephrocyte, preparation method and application thereof
CN105647852A (en) * 2016-03-30 2016-06-08 北京普瑞金科技有限公司 Method for serum-free culturing of 293T cells for lentivirus package
CN104450607B (en) * 2014-11-14 2017-07-11 武汉金开瑞生物工程有限公司 For the full chemistry culture medium and cultural method of HEK293 cell suspension growths
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CN114438131A (en) * 2022-02-25 2022-05-06 上海健士拜生物科技有限公司 293 cell transfection method
CN114736871A (en) * 2022-04-27 2022-07-12 中山康晟生物技术有限公司 Adenovirus packaging WAYNE293LVPRO animal cell culture method
CN114836367A (en) * 2022-04-28 2022-08-02 上海东富龙生物试剂有限公司 Chemical component limited culture medium for HEK293 cell culture and adenovirus and adeno-associated virus replication and amplification
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JP2018183154A (en) * 2012-04-24 2018-11-22 ジェネンテック， インコーポレイテッド Cell culture compositions and methods for polypeptide production
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JP2021104023A (en) * 2012-04-24 2021-07-26 ジェネンテック， インコーポレイテッド Cell culture composition and method for polypeptide production
CN104651297A (en) * 2013-11-19 2015-05-27 华东师范大学 Culture medium used in high-density large-scale suspended cultivation of human embryo nephrocyte, preparation method and application thereof
CN104357379B (en) * 2014-09-30 2017-08-08 刘兴宇 Stem cell media
CN104357379A (en) * 2014-09-30 2015-02-18 刘兴宇 Stem cell culture medium
CN104450607B (en) * 2014-11-14 2017-07-11 武汉金开瑞生物工程有限公司 For the full chemistry culture medium and cultural method of HEK293 cell suspension growths
CN105647852A (en) * 2016-03-30 2016-06-08 北京普瑞金科技有限公司 Method for serum-free culturing of 293T cells for lentivirus package
CN111793595A (en) * 2020-07-23 2020-10-20 上海奥浦迈生物科技有限公司 Serum-free culture medium for HEK293 cells
CN111826341A (en) * 2020-07-23 2020-10-27 上海奥浦迈生物科技有限公司 Method for in vitro culture of HEK293 cells for transient expression of proteins
CN112501113A (en) * 2021-02-03 2021-03-16 上海奥浦迈生物科技股份有限公司 HEK293 cell combined gradient culture medium and application thereof
CN113061584A (en) * 2021-05-14 2021-07-02 赵峻岭 Culture medium additive capable of promoting growth of adenovirus
CN114438131A (en) * 2022-02-25 2022-05-06 上海健士拜生物科技有限公司 293 cell transfection method
CN114736871A (en) * 2022-04-27 2022-07-12 中山康晟生物技术有限公司 Adenovirus packaging WAYNE293LVPRO animal cell culture method
CN114836367A (en) * 2022-04-28 2022-08-02 上海东富龙生物试剂有限公司 Chemical component limited culture medium for HEK293 cell culture and adenovirus and adeno-associated virus replication and amplification
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