CN105985926A - Serum-free culture medium for CHO cell culture - Google Patents
Serum-free culture medium for CHO cell culture Download PDFInfo
- Publication number
- CN105985926A CN105985926A CN201510098842.XA CN201510098842A CN105985926A CN 105985926 A CN105985926 A CN 105985926A CN 201510098842 A CN201510098842 A CN 201510098842A CN 105985926 A CN105985926 A CN 105985926A
- Authority
- CN
- China
- Prior art keywords
- serum
- culture medium
- free medium
- chloride
- acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a serum-free culture medium for CHO cell culture. The serum-free culture medium is mainly prepared from amino acid, inorganic salt, vitamins, trace elements, a carbon source, a nitrogen source and a buffer solution. The content of various nutrients of the culture medium is optimized through a large number of orthogonal experiments, and the serum-free culture medium is beneficial to the growth of CHO cells, improvement of the expression amount and reduction of the expression costs of recombinant proteins.
Description
Technical field
The present invention relates to a kind of serum-free medium cultivated for Chinese hamster ovary celI, belong to animal cell culture technology field.
Background technology
Cell culture technology is to realize cell injuring model by analog cell tumor growth environment.Culture medium provides nutrition for cell injuring model and promotes cell growth and propagation.The development experience of cell culture medium Embryo Gallus domesticus juice, natural medium, synthetic medium, and the most conventional low blood serum medium, serum-free medium, protein-free medium and chemical composition culture medium clearly.
Culture medium refers to maintain zooblast, microbial cell, a kind of liquid that plant cell etc. grows in vitro or solid dielectric.The culture medium being currently used for zooblast can be divided into according to the development course of animal-free medium: the culture medium containing serum, low blood serum medium, serum-free medium (mends the culture medium determined) containing compositions such as some plant hydrolyzed things, culture medium (chemical composition determines, but may contain some recombiant proteins) that chemical composition determines and do not contain the culture medium that the chemical composition of recombiant protein determines.Generally, the growth of zooblast all depends on the existence of serum.In ordinary culture medium, such as not increase serum, overwhelming majority cells can not be bred.But the major drawbacks using serum is to there is potential polluter, expensive, differences between batches are big, and production and scientific research are brought inconvenience.Additionally, for utilizing cell producer gene engineered protein product, the existence of serum will affect the purification of recombination expression product, reduce product purity.Therefore scientist is through researching and developing various culture medium without serum, secrete recombinant protein medicine efficiently so that can grow in the medium through genetic engineering modified cell simultaneously, thus realize expressing at a high level antibody and other recombiant proteins.
Chinese hamster ovary cell (Chinese hamster ovary celI) is a kind of important host cell, and it is because transfection is convenient, and table is the highest, is often used in the various recombiant proteins of expression.In the market, each large-scale culture medium company is all proposed the Chinese hamster ovary celI culture medium of various varying level.Several major companies include Gibco, and Hyclone, Invrine, Millipore, Sartorious etc. are all proposed corresponding CHO culture medium.The culture medium product that each company is top at present is the culture medium that chemical composition determines.Relative to the serum-free medium containing uncertain composition, the culture medium that composition determines has quality controllable, definite ingredients, and the protein drug lot stability of production is good.Chemically defined culture medium has been the only selection that bio-pharmaceuticals is following.
Summary of the invention
Present invention aim at providing a kind of and can be used for the serum-free medium that Chinese hamster ovary celI is cultivated, in order to it expresses the purification of recombiant protein.
The invention discloses the serum-free medium cultivated for Chinese hamster ovary celI, including various aminoacid, inorganic salt, vitamin, trace element, carbon source, nitrogen source, and buffer.
Preferably, described serum-free medium includes:
nullMethionine 0.8-1.0mM，Aspartic acid 4.0-5.0mM，Threonine 1.5-2.0mM，Glutamic acid 6.0-8.0mM，Valine 3.0-4.0mM，Cysteine 0.5-1.0mM，Isoleucine 2.5-3.0mM，Leucine 2.5-3.0mM，Tyrosine 0.6-1.0mM，Phenylalanine 0.5-1.0mM，Lysine 2.2-2.5mM，Histidine 0.8-1.0mM，Arginine 1.5-1.8mM，Proline 3 .5-4.0mM，Glycine 0.5-1.0mM，Alanine 0.4-1.0mM，Agedoite 4.0-5.0mM，Insulin 0.001-1.005mM，Cholesterol 0.1-0.2mM，Folic acid 0.01-0.02mM，Nicotinic acid 0.03-0.05mM，Arsenic Lip river alcohol 0.007-0.01mM，Phylloxanthin 0.0005-0.001mM，Nicotiamide 0.007-0.01mM，Vitamin A 0.0003-0.0009mM，Calcium pantothenate 0.01-0.03mM，Sodium Pyruvate 1.0-2.0mM，Calcium chloride 0.2-0.4mM，Magnesium chloride 0.4-0.6mM，Sodium chloride 8.0-10.0mM，Potassium chloride 0.5-1.0mM，Magnesium sulfate 0.5-1.0mM，Sodium bicarbonate 10.0-20.0mM，Potassium sulfate 1.0-2.0mM，. putrescine 0.01mM，Ethanolamine 0.1mM，Palmic acid 0.001mM，Ferrous citrate 10mg/L，Copper sulfate 0.00005-0.0001mM，Sodium selenate 0.0001-0.001mM，Aluminum chloride 0.00005-0.0001mM，Potassium iodide 0.00004-0.00006mM，Glucose 30.0-50.0mM，With PBS 0.5-1M, pH 7.4.
Preferably, described serum-free medium is possibly together with Tween-80, and its content is 1.0-2.0g/L.
The various compositions of the culture medium of the present invention optimize through substantial amounts of orthogonal experiment, may advantageously facilitate the growth of Chinese hamster ovary celI and improve its expression efficiency, reduce the production cost of recombiant protein.
Detailed description of the invention
It should be appreciated by those skilled in the art that following embodiment is merely to illustrate the present invention rather than limits the scope of the present invention.
Embodiment
1
The preparation of serum-free medium
null(1) material of following amount is weighed respectively: methionine 0.8mmol，Aspartic acid 4.0-mmol，Threonine 1.5mmol，Glutamic acid 6.0mmol，Valine 3.0mmol，Cysteine 0.5mmol，Isoleucine 2.5mmol，Leucine 2.5mmol，Tyrosine 0.6mmol，Phenylalanine 0.5mmol，Lysine 2.2mmol，Histidine 0.8mmol，Arginine 1.5mmol，Proline 3 .5mmol，Glycine 0.5mmol，Alanine 0.4mmol，Agedoite 4.0mmol，Insulin 0.001mmol，Cholesterol 0.1mmol，Folic acid 0.01mmol，Nicotinic acid 0.03mmol，Arsenic Lip river alcohol 0.007mmol，Phylloxanthin 0.0005mmol，Nicotiamide 0.007mmol，Vitamin A 0.0003mmol，Calcium pantothenate 0.01mmol，Sodium Pyruvate 1.0mmol，Calcium chloride 0.2mmol，Magnesium chloride 0.4mmol，Sodium chloride 8.0mmol，Potassium chloride 0.5mmol，Magnesium sulfate 0.5mmol，Sodium bicarbonate 10.0mmol，Potassium sulfate 1.0mmol，. putrescine 0.01mmol，Ethanolamine 0.1mmol，Palmic acid 0.001mmol，Ferrous citrate 10mg/L，Copper sulfate 0.00005mmol，Sodium selenate 0.0001mmol，Aluminum chloride 0.00005mmol，Potassium iodide 0.00004mmol，With glucose 30.0mmol；
(2) above-mentioned substance mixing and is dissolved in PBS 0.5M, in pH 7.4, filtration sterilization can prepare the serum-free medium of the present invention.
Embodiment
2
The serum-free medium of the present invention compares with commercialization culture medium
The Chinese hamster ovary celI strain of EPO will be expressed according to 5x10 simultaneously5The serum-free medium (preparing as described in embodiment 1) of the concentration inoculation present invention of cells/ml and the serum-free medium FreeStyleCHO Expression of commercialization
Medium (Gibco Products), cultivates by identical condition.At 37 DEG C, 110 revs/min, gas concentration lwevel is to cultivate under the conditions of 6.0%, when concentration of glucose is less than 2 grams per liters, supplements glucose to 8 grams per liters.Cultivating 48 and as a child collected cell conditioned medium, measure EPO concentration, the EPO expression utilizing culture medium of the present invention is 750mg/l, and the EPO expression of commercially available culture medium is 380mg/L.As can be seen here, the serum-free culture based formulas of the present invention is better than the culture medium of current commercialization in terms of promoting sufficient protein expression.
The scope of the present invention is not limited by the specific embodiments described, and described embodiment is only used as illustrating the single example of various aspects of the present invention, also includes method and the component of functional equivalent in the scope of the invention.It practice, in addition to content as herein described, those skilled in the art can easily grasp the multiple improvement to the present invention with reference to described above.Within described improvement also falls into the scope of the appended claims.Every list of references mentioned above is listed in herein all in full as reference.
Claims (3)
1. the serum-free medium cultivated for Chinese hamster ovary celI, including various aminoacid, inorganic salt, vitamin, trace element, carbon source, nitrogen source, and buffer.
2. serum-free medium as claimed in claim 1, it is characterised in that described serum-free medium includes:
nullMethionine 0.8-1.0mM，Aspartic acid 4.0-5.0mM，Threonine 1.5-2.0mM，Glutamic acid 6.0-8.0mM，Valine 3.0-4.0mM，Cysteine 0.5-1.0mM，Isoleucine 2.5-3.0mM，Leucine 2.5-3.0mM，Tyrosine 0.6-1.0mM，Phenylalanine 0.5-1.0mM，Lysine 2.2-2.5mM，Histidine 0.8-1.0mM，Arginine 1.5-1.8mM，Proline 3 .5-4.0mM，Glycine 0.5-1.0mM，Alanine 0.4-1.0mM，Agedoite 4.0-5.0mM，Insulin 0.001-1.005mM，Cholesterol 0.1-0.2mM，Folic acid 0.01-0.02mM，Nicotinic acid 0.03-0.05mM，Arsenic Lip river alcohol 0.007-0.01mM，Phylloxanthin 0.0005-0.001mM，Nicotiamide 0.007-0.01mM，Vitamin A 0.0003-0.0009mM，Calcium pantothenate 0.01-0.03mM，Sodium Pyruvate 1.0-2.0mM，Calcium chloride 0.2-0.4mM，Magnesium chloride 0.4-0.6mM，Sodium chloride 8.0-10.0mM，Potassium chloride 0.5-1.0mM，Magnesium sulfate 0.5-1.0mM，Sodium bicarbonate 10.0-20.0mM，Potassium sulfate 1.0-2.0mM，Putrescine 0.01mM，Ethanolamine 0.1mM，Palmic acid 0.001mM，Ferrous citrate 10mg/L，Copper sulfate 0.00005-0.0001mM，Sodium selenate 0.0001-0.001mM，Aluminum chloride 0.00005-0.0001mM，Potassium iodide 0.00004-0.00006mM，Glucose 30.0-50.0mM，With PBS 0.5-1M, pH 7.4.
3. serum-free medium as claimed in claim 2, it is characterised in that described serum-free medium is possibly together with Tween-80, and its content is 1.0-2.0g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510098842.XA CN105985926A (en) | 2015-03-06 | 2015-03-06 | Serum-free culture medium for CHO cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510098842.XA CN105985926A (en) | 2015-03-06 | 2015-03-06 | Serum-free culture medium for CHO cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105985926A true CN105985926A (en) | 2016-10-05 |
Family
ID=57039382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510098842.XA Pending CN105985926A (en) | 2015-03-06 | 2015-03-06 | Serum-free culture medium for CHO cell culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105985926A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106520668A (en) * | 2016-12-31 | 2017-03-22 | 山东金周生物科技有限公司 | Protein serum-free culture medium and preparation method thereof |
| CN106635953A (en) * | 2016-12-13 | 2017-05-10 | 昆明润什生物科技有限公司 | Serum-free protein-free cell culture medium |
| CN109337861A (en) * | 2018-11-12 | 2019-02-15 | 王晓柯 | A kind of highly expressed Chinese hamster ovary celI serum free medium of support product |
| CN110583625A (en) * | 2019-09-17 | 2019-12-20 | 深圳大学 | Umbilical cord storage liquid |
-
2015
- 2015-03-06 CN CN201510098842.XA patent/CN105985926A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635953A (en) * | 2016-12-13 | 2017-05-10 | 昆明润什生物科技有限公司 | Serum-free protein-free cell culture medium |
| CN106635953B (en) * | 2016-12-13 | 2021-02-19 | 昆明润什生物科技有限公司 | Serum-free and protein-free cell culture medium |
| CN106520668A (en) * | 2016-12-31 | 2017-03-22 | 山东金周生物科技有限公司 | Protein serum-free culture medium and preparation method thereof |
| CN109337861A (en) * | 2018-11-12 | 2019-02-15 | 王晓柯 | A kind of highly expressed Chinese hamster ovary celI serum free medium of support product |
| CN109337861B (en) * | 2018-11-12 | 2021-06-25 | 友康恒业生物科技（北京）有限公司 | CHO cell serum-free medium supporting high expression of product |
| CN110583625A (en) * | 2019-09-17 | 2019-12-20 | 深圳大学 | Umbilical cord storage liquid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102827804B (en) | Culture medium and method suitable for the amplification culture of Vero cell microcarrier suspension | |
| CN102858953B (en) | The cell culture medium improved | |
| CN104593316B (en) | Insect cell serum free medium and its application | |
| CN101760442A (en) | Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture | |
| CN101974481A (en) | Serum free culture medium for growing various cells derived from kidney tissue | |
| CN101724600B (en) | Blood serum-free culture medium for supporting suspension culture of CHO cells with large scale and high density | |
| CN101988047B (en) | Insect cell serum-free medium with low cost | |
| CN105985926A (en) | Serum-free culture medium for CHO cell culture | |
| CN104164405A (en) | Serum-free culture system for efficiently culturing human umbilical cord mesenchymal stem cells in vitro | |
| ES2728169T3 (en) | E. coli cell culture procedure for high density | |
| CN103898041B (en) | The cultural method of hybridoma | |
| CN104073463B (en) | A kind of Serum-free and protein-free medium for supporting CHO high density suspension culture | |
| CN102268403A (en) | Serum-free culture medium applicable to large-scale single-cell suspension culture of baby hamster kidney cell | |
| CN102443565B (en) | Medium suitable for cultivating CHO cell and cultivation technology thereof | |
| CN104293729A (en) | Efficient serum-free culture medium | |
| CN103320390A (en) | Large-scale mammal engineering cell cultural method | |
| CN105543163A (en) | Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells | |
| CN104073464A (en) | Serum-free CHO cell culture medium and preparation method thereof | |
| CN104664130A (en) | Polypeptide-containing aquatic microbial feed | |
| CN111748527B (en) | Chemical component limited efficient feeding culture medium and preparation method and application thereof | |
| CN109593801A (en) | A kind of technique of fermenting and producing L-Trp | |
| CN110669730B (en) | Human peripheral blood lymphocyte culture medium | |
| CN108707579A (en) | The serum free medium and preparation method and cultural method of a kind of human T lymphocyte's culture | |
| CN105462911A (en) | Serum-free medium for culturing viruses and preparation method thereof | |
| CN103421736B (en) | Medium additive replacing animal serum in CHO cell culture and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161005 |