CN1800368A - Embryo cattle serum substitute without serum for animal cell culture - Google Patents
Embryo cattle serum substitute without serum for animal cell culture Download PDFInfo
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- CN1800368A CN1800368A CNA2005100231187A CN200510023118A CN1800368A CN 1800368 A CN1800368 A CN 1800368A CN A2005100231187 A CNA2005100231187 A CN A2005100231187A CN 200510023118 A CN200510023118 A CN 200510023118A CN 1800368 A CN1800368 A CN 1800368A
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Abstract
The invention relates to a non-serum animal cell culture used fetus cattle serum displace agent, which is formed by recombination human insulin growth factor-1:0.1g-0.5g/l, recombination human platelet sourced growth factor-B:0.1g-0.6g/l, recombination human alkali desmocyte growth factor: 0.05-0.4g/l, cattle transferring: 1g-5g/l, cattle serum protein (V component): 2g-15g/l, and mercaptoethanol 0.3-0.5mol/l.
Description
Technical field
The present invention relates to the animal cell culture foetal calf serum substituting agent of a kind of serum-free prescription, relate in particular to a kind of foetal calf serum substituting agent (serum-free prescription) that can use with the animal cell culture that multiple basic medium is arranged in pairs or groups.
Background technology
The vitro culture of the zooblast substratum that contain serum that adopt are cultivated more, the cost height, serum composition is indeterminate, be difficult for the downstream product purifying, the more important thing is, contain potential virus (as mad cow disease poison etc.) in the serum, shortcomings such as prion or mycoplasma pollution, biological effect for the growth of serum sustenticular cell has obtained proof, but it is not clear fully as yet so far to the complicated ingredient in the serum. not only there is the promoting growth of cell factor in the serum, to also having cell growth inhibitory factor or virulence factor, the biological characteristics that therefore cultured cells reflected in containing blood serum medium is the combined reaction of cell and complicated serum factor together.
In research fields such as somatomedin, protein engineering, gene expression regulations, press for and use the serum free medium culturing cell.Therefore, the substratum of developing alternative serum then becomes a focus of cell engineering research.
The substratum that in cell cultivation process, is mixed with serum substitute, adopted the substratum of known molecular structure and configuration component fully, its composition is clear and definite, uniform quality, help improving the stability of cellular product production and make cellular product be easy to purifying, and avoided because of containing problem such as potential toxicity in the serum.Thereby, adopt serum free medium to carry out the international trend that cell cultures is produced has become the vitro culture of utilizing zooblast.And in some developed countries, the medicine management department vaccine of having laid down hard and fast rule, recombinant protein and monoclonal antibody manufacturer must not use and contain blood serum medium, in order to avoid product is by potential pathogen contamination in the animal serum.
Animal cell culture is becoming a new industry at home, and the serum-free culture technology is one of gordian technique of this industry development.At present, the external existing commodity serum free medium that Chinese hamster ovary celI is cultivated that is used for, domestic also have the scholar to develop a series of serum free mediums that are used for myelomatosis, hybridoma, CHO, BHK and Vero cell, mainly be by adding Regular Insulin, materials such as Transferrins,iron complexes promote cell attachment and growth and breeding etc., and the specific often clone of research object.
[Advances in Animal Cell Biology and Technology forBioprocesses.1989,263-269] such as Merten OW carries out the serum-free culture preliminary study to hybridoma.Zhang Yuanxing etc. [application number: 99113498] substratum is based on substratum RPM1640, added a kind of new making that materials such as Transferrins,iron complexes, steroid hormone, trace element, VITAMIN, thanomin, beta-mercaptoethanol and P-hydroxybenzoic acid form hybridoma can have been grown vigorously, cell density and monoclonal antibody production can with the low albumen serum free medium that has blood serum medium to compare favourably, the substratum that can substitute serum is used for the cultivation of WuT3 hybridoma.Zhang Shuxiang etc. [the journal .2003 of East China University of Science, 29 (4) 372-375] have realized the low albumen cultivation of external serum-free of hybridoma by adding Regular Insulin, Transferrins,iron complexes, bovine serum albumin, Sodium Selenite etc.
[Performer:Minnesota Univ. such as Jenkin HM, Austin.Hormel Inst., 1983,44] study the binding substances that has added VITAMIN, amino acid, somatomedin, hormone and other organic compound and inorganic salt and come alternative serum, cultured cells mainly comprises macaque kidney cell, human lung and sexual organ cell.
Stockinger H[US1989000295813] worked out serum free medium by adding materials such as Transferrins,iron complexes and Regular Insulin non-adhesive cell.
Mariani E[Journal of Immunological Methods.1991,145 (1-2): 175-183] introduced the serum free medium that can promote that hybridoma growth and monoclonal antibody are produced.This products substitution the somatomedin in the serum originally, hormone and protein etc.
Wear and breed [Chinese patent application number: 02157709] and disclose a kind of serum free medium and concentrated solution and preparation method thereof, it is to be raw material with bovine serum albumin solution, insulin solutions, purifying human transferrin solution, cholesterol solution, hydrogen peroxide enzyme solution, form by certain prepared, be applicable to the growth of polytype tissue and cell.
The special Mark Lewis-Francis Pu Laodi in Wal [Chinese patent application number: application number: 90100961] is with arginyl-A-O Regular Insulin, a kind of by the proinsulin human with the byproduct in the synthetic insulin human's process of biotechnology, add in the serum-free tissue culture medium (TCM), to stimulate the growth of cells of mamma animals, described cells of mamma animals is positive to Regular Insulin in tissue culture.
Chen Guoan etc. [Chinese patent application number: 03118491] have invented a kind of cell culture medium and cultural method and application, especially a kind of human epidermal cell serum free medium and application thereof.This serum-free culture body base is to adopt based on the IMDM cultivation, add the serum free medium serum substitute of development voluntarily, comprise catalase, bovine serum albumin, human transferrin, cholesterol, Regular Insulin, hydrocortisone etc., restock epithelical cell growth factor, Niu Chuiti extract, thanomin etc. are formed the human epidermal cell serum free medium.The serum free culture system of this invention can be used for going down to posterity and the external serum-free culture of former generation human epidermal cell.
Studies show that, cell can long-term stability go down to posterity in serum free medium, it is extremely narrow that yet the serum free medium of present research mainly shows as the suitable spectrum of cell, cell more is subject to the influence of some mechanical factor and chemical factor in serum free medium, the preservation of substratum and application are convenient not as good as traditional synthetic medium.
How working out a kind of can be the pendulum problem that needs solve in face of the science and technology personnel with the Methods of Serum-Free Medium for Animal Cells with handiness highly to be fit to the different cell of cultivation that multiple basic medium is arranged in pairs or groups.
Summary of the invention
The technical issues that need to address of the present invention have provided a kind of animal cell culture foetal calf serum substituting agent of serum-free prescription, are intended to solve above-mentioned defective.
In order to solve the problems of the technologies described above, the present invention is achieved by the following technical solutions:
The present invention is by recombinant human insulin-like growth factor-1:0.1g～0.5g/L (concentration), recombinant human platelet-derived growth factor-B:0.1g～0.6g/L, recombination human basic fibroblast growth factor: 0.05～0.4g/L, ox Transferrins,iron complexes: 1g～5g/L, bovine serum albumin (V component): 2g～15g/L, mercaptoethanol 0.3～0.5mol/L forms.
Compared with prior art, the invention has the beneficial effects as follows: by adding the major ingredient such as cytokine of reorganization, the substratum that can be mixed with in varing proportions with basic medium, successful cultivation make different clones such as Chinese hamster ovary celI, bhk cell, L929 cell, reached the purpose of alternative serum; Simultaneously, additive is made the independent prescription that is independent of basic medium, can arrange in pairs or groups,, have the handiness of height to be fit to cultivating different cells with multiple basic medium; And can cooperate with the basic medium of homemade cheapness, be used for the mammalian cell serum-free culture, reduce the cost of substratum in production or scientific research greatly, this has also obtained breakthrough progress aspect serum-free culture.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail:
Embodiment 1
The present invention is by recombinant human insulin-like growth factor-1:0.1gL (concentration), recombinant human platelet-derived growth factor-B:0.3g/L, recombination human basic fibroblast growth factor: 0.2g/L, ox Transferrins,iron complexes: 3g/L, bovine serum albumin (V component): 6g/L, mercaptoethanol: 0.5mol/L.
Embodiment 2
The present invention is by recombinant human insulin-like growth factor-1:0.5g/L (concentration), recombinant human platelet-derived growth factor-B:0.1g/L, recombination human basic fibroblast growth factor: 0.1g/L, ox Transferrins,iron complexes: 2g/L, bovine serum albumin (V component): 8g/L, mercaptoethanol: 0.4mol/L.
Embodiment 3
The present invention is by recombinant human insulin-like growth factor-1:0.3g/L (concentration), recombinant human platelet-derived growth factor-B:0.4g/L, recombination human basic fibroblast growth factor: 0.3g/L, ox Transferrins,iron complexes: 4g/L, bovine serum albumin (V component): 12g/L, mercaptoethanol: 0.3mol/L.
Compound method:
At first prepare and the corresponding to basic medium of cell cultures, for example RPMI1640 or DMEM or F12 or DMEM/F12 etc., compound concentration is 4 times of normal concentration, with 0.2 μ m filter membrane Sterile Filtration;
Respectively with load weighted ox Transferrins,iron complexes and bovine serum albumin, dissolving is back with 0.2 μ m filter membrane Sterile Filtration with above-mentioned basic medium;
The degerming solution of ox Transferrins,iron complexes and bovine serum albumin with under slowly stirring, mixing, add mercaptoethanol in proportion, under slowly stirring, add recombinant human insulin-like growth factor-1, recombinant human platelet-derived growth factor-B and the recombination human basic fibroblast growth factor that has calculated volume then successively, all add 4 times of concentration basic mediums and add ultrapure water to final volume 95% after, adjust pH to 7.2～7.3, use the ultrapure water constant volume to final volume;
Behind 0.1 μ m membrane filtration, divide to install in the glass or plastic containers of having sterilized;
Label, place-20 degree preservations.
The present invention is to have added recombinant human insulin-like growth factor-1, and materials such as recombinant human platelet-derived growth factor, recombination human basic fibroblast growth factor, Transferrins,iron complexes, serum albumin, mercaptoethanol are formed.This formula material adds in the substratum such as RPMI1640 or DMEM, can make Chinese hamster ovary celI, bhk cell, L929 cell etc. adherent fast, grows vigorously.Changed liquid once in 6～12 hours, basic medium adds the cell density that this prescription composition turned out and can reach and the consistent result of use that has 10% blood serum medium to compare favourably, and can alternative serum add the cultivation that is used for Chinese hamster ovary celI, bhk cell, L929 cell etc. in the basic medium to fully.
Claims (4)
1. the animal cell culture foetal calf serum substituting agent of serum-free prescription, it is characterized in that: by recombinant human insulin-like growth factor-1:0.1g～0.5g/L (concentration), recombinant human platelet-derived growth factor-B:0.1g～0.6g/L, recombination human basic fibroblast growth factor: 0.05～0.4g/L, ox Transferrins,iron complexes: 1g～5g/L, bovine serum albumin (V component): 2g～15g/L, mercaptoethanol 0.3～0.5mol/L forms.
2. the animal cell culture foetal calf serum substituting agent of serum-free prescription according to claim 1, it is characterized in that: by recombinant human insulin-like growth factor-1:0.1g/L (concentration), recombinant human platelet-derived growth factor-B:0.3g/L, recombination human basic fibroblast growth factor: 0.2g/L, ox Transferrins,iron complexes: 3g/L, bovine serum albumin (V component): 6g/L, mercaptoethanol 0.5mol/L forms.
3. the animal cell culture foetal calf serum substituting agent of serum-free prescription according to claim 1, it is characterized in that: by recombinant human insulin-like growth factor-1:0.5g/L (concentration), recombinant human platelet-derived growth factor-B:0.1g/L, recombination human basic fibroblast growth factor: 0.1g/L, ox Transferrins,iron complexes: 2g/L, bovine serum albumin (V component): 8g/L, mercaptoethanol 0.4mol/L forms.
4. the animal cell culture foetal calf serum substituting agent of serum-free prescription according to claim 1, it is characterized in that: by recombinant human insulin-like growth factor-1:0.3g/L (concentration), recombinant human platelet-derived growth factor-B:0.4g/L, recombination human basic fibroblast growth factor: 0.3g/L, ox Transferrins,iron complexes: 4g/L, bovine serum albumin (V component): 12g/L, mercaptoethanol 0.3mol/L forms.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2005100231187A CN100432218C (en) | 2005-01-05 | 2005-01-05 | Embryo cattle serum substitute without serum for animal cell culture |
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| CNB2005100231187A CN100432218C (en) | 2005-01-05 | 2005-01-05 | Embryo cattle serum substitute without serum for animal cell culture |
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| CN1800368A true CN1800368A (en) | 2006-07-12 |
| CN100432218C CN100432218C (en) | 2008-11-12 |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126077B (en) * | 2007-08-31 | 2010-07-14 | 中国水产科学研究院黑龙江水产研究所 | Blood serum substituting agent and blood serum free rainbow trout embryo cell line culture medium |
| WO2010121465A1 (en) * | 2009-04-23 | 2010-10-28 | 中国科学院广州生物医药与健康研究院 | New serum-free medium for inducing pluripotent stem cells quickly with high efficiency and method using thereof |
| CN104830766A (en) * | 2015-05-22 | 2015-08-12 | 广州和能生物科技有限公司 | Serum-free human peripheral blood lymphocyte culture medium |
| CN105794772A (en) * | 2016-05-16 | 2016-07-27 | 天津市中奥天元科技发展有限公司 | Serum-free cell cryopreservation liquid |
| WO2016201657A1 (en) * | 2015-06-17 | 2016-12-22 | 深圳市达科为生物工程有限公司 | Serum replacement for culturing lymphocytes, and preparation method therefor |
| CN114381428A (en) * | 2022-01-21 | 2022-04-22 | 广东善玺迦纳栗生物科技股份有限公司 | Cell culture medium for subcutaneous or injured tissue injection |
| CN115197894A (en) * | 2022-07-08 | 2022-10-18 | 江苏丰华生物制药有限公司 | Method for culturing CHO (Chinese hamster ovary) cells by adopting serum substitute |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1193091C (en) * | 2002-01-25 | 2005-03-16 | 华东理工大学 | Serumless medium suitable for growth and maintenance of young hamster kidney cell |
-
2005
- 2005-01-05 CN CNB2005100231187A patent/CN100432218C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101126077B (en) * | 2007-08-31 | 2010-07-14 | 中国水产科学研究院黑龙江水产研究所 | Blood serum substituting agent and blood serum free rainbow trout embryo cell line culture medium |
| WO2010121465A1 (en) * | 2009-04-23 | 2010-10-28 | 中国科学院广州生物医药与健康研究院 | New serum-free medium for inducing pluripotent stem cells quickly with high efficiency and method using thereof |
| CN104830766A (en) * | 2015-05-22 | 2015-08-12 | 广州和能生物科技有限公司 | Serum-free human peripheral blood lymphocyte culture medium |
| WO2016201657A1 (en) * | 2015-06-17 | 2016-12-22 | 深圳市达科为生物工程有限公司 | Serum replacement for culturing lymphocytes, and preparation method therefor |
| CN105794772A (en) * | 2016-05-16 | 2016-07-27 | 天津市中奥天元科技发展有限公司 | Serum-free cell cryopreservation liquid |
| CN114381428A (en) * | 2022-01-21 | 2022-04-22 | 广东善玺迦纳栗生物科技股份有限公司 | Cell culture medium for subcutaneous or injured tissue injection |
| CN115197894A (en) * | 2022-07-08 | 2022-10-18 | 江苏丰华生物制药有限公司 | Method for culturing CHO (Chinese hamster ovary) cells by adopting serum substitute |
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| CN100432218C (en) | 2008-11-12 |
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