WO1998006822A1 - Culture medium and use of the same - Google Patents
Culture medium and use of the same Download PDFInfo
- Publication number
- WO1998006822A1 WO1998006822A1 PCT/JP1997/002749 JP9702749W WO9806822A1 WO 1998006822 A1 WO1998006822 A1 WO 1998006822A1 JP 9702749 W JP9702749 W JP 9702749W WO 9806822 A1 WO9806822 A1 WO 9806822A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- culture
- medium
- active substance
- physiologically active
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
Definitions
- the present invention provides a culture medium containing human serum albumin (hereinafter, also referred to as rHSA) obtained by genetic manipulation, a method for culturing animal cells using the medium, or producing a physiologically active substance. Regarding the method of collecting.
- rHSA human serum albumin
- DMEM Dulbecco's modified Eagle's medium
- F12 Natl. Acad. Sci. USA 53, 288 F12 Natl. Acad. Sci. USA 53, 288
- RPMI 164 medium Goding, JW (1980) J. Immunol. Methods 39, 285, JAMA 199 (1957) 519]
- serum must be added to the media. Serum from fetuses, pomas or humans had to be used at concentrations of 1 to 15%.
- Serum itself is expensive, resulting in high costs.
- ⁇ ⁇ ⁇ May be a source of virus and mycoplasma contamination.
- plasma-derived human serum albumin (hereinafter, also referred to as HSA) is used as an additive for maintaining cell proliferation.
- HSA plasma-derived human serum albumin
- the quality of plasma-derived HSA is not always constant.
- contaminating plasma-derived components such as lipoproteins, proteolytic enzyme inhibitors such as 1-antitrypsin, and metal or heme carrier proteins such as transflurin, cell-mouth plasmin, haptoglobin, etc.
- the degree of contamination of fatty acids, calcium ions, tritophan, cysteine, glutathione, trace metal components, etc. differs for each product lot. Therefore, the effect of these components on the culture of animal cells and the like is concerned.
- the present invention has been made in view of the above-described problems, and has as its object to provide a culture medium capable of obtaining sufficient cell proliferation and having a stable quality and a small influence on culture. .
- Another object of the present invention is to provide a method for culturing animal cells that is stable (has good reproducibility).
- Still another object of the present invention is to provide a method for producing and collecting a large amount of a physiologically active substance from animal cells capable of producing the physiologically active substance.
- the present inventors have conducted studies in consideration of the above circumstances, and as a result, have found that cultivation is achieved by using HSA (r HSA) obtained by gene manipulation as an additive to a basic medium.
- HSA HSA
- the present inventors have found that sufficient proliferation of cells can be maintained without changing the effect when using HSA derived from plasma, and completed the present invention. That is, in the medium of the present invention, since rHSA having a constant quality is used, the influence on the culture effect can be further reduced (ie, reproducibility can be ensured).
- the present invention is as follows. (1) A culture medium containing human serum albumin obtained by genetic manipulation.
- the culture medium according to (1) above which contains a DMEM medium, a F12 medium, or an RPM11640 medium as a basic medium.
- Animal cells are human kidney cell lines, Hypridoma, leukocytes, Namalba cells, BALL-1 cells, fibroblasts, lymphocytes, human kidney cells, melanoma cells, Vero cells, He1a cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, HKG cells, mouse myeloma cells, human lymphoblast cells or parent cells for human human hybridoma production, or their dhfr deficiency Culture method of (8) above, which is a strain, HGPRT-deficient strain,
- a bioactive substance characterized by culturing animal cells capable of producing a bioactive substance in the medium of (1) to produce the bioactive substance, and collecting the bioactive substance from the culture. Manufacturing method.
- the physiologically active substance is properokinase, perokinase, antithrombin-III, tissue plasminogen activator, hepatitis B virus surface antigen, pre-S-Hepatitis B virus surface antigen, interferon, colony formation stimulating factor or The method according to the above (11), which is a monoclonal antibody.
- the culture medium of the present invention is not particularly limited as long as it contains human serum albumin (rHSA) obtained by gene manipulation. Specifically, for example, a medium obtained by adding rHSA to a basic medium can be mentioned.
- rHSA human serum albumin
- the basal medium includes a commonly used basal medium, that is, a medium containing a carbon source which can be normally assimilated by animal cells, a digestible nitrogen source, an inorganic salt, and the like, and If necessary, a trace amount of an effective substance such as a nutrient promoting substance or a precursor may be blended.
- a basal medium any known medium for cell culture can be used.
- the aforementioned DMEM medium, F12 medium and RPMI1640 medium are exemplified, and RPMI1640 medium is particularly suitable.
- RHSA contained in the culture medium of the present invention is not particularly limited as long as it is a human serum albumin produced by an HSA producing host prepared through genetic manipulation.
- contaminant components derived from the producing host (E.g., protein-free), more preferably, the rHSA-producing host is cultured by known means, and then collected and purified from the culture filtrate, cells, or cells by known separation means and purification means, respectively. What was done is used.
- the host for obtaining rHSA used in the present invention is not particularly limited as long as it is prepared through genetic manipulation, and may be any of those already described in the known literature and those which will be developed in the future. Can be used. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) that have been rendered rHSA-producing through genetic manipulation, animal cells, and the like.
- yeast as a host, preferably Saccharomyces [e.g. For example, Saccharomyces cerevisiae] or a genus Pichia (eg, Pichia pastoris) is used.
- An auxotrophic strain or an antibiotic-sensitive strain may also be used. More preferably, Saccharomyces cerevisiae AH22 strain (a, his4, leu2, can1) and Pichia pastoris GTS115 strain (his4) are used.
- the method for preparing these rHSA-producing hosts, the method for producing rHSA by culturing the host, and the method for separating and collecting rHSA from cultures should be carried out by using known methods and methods equivalent thereto.
- a method for preparing an rHSA-producing host for example, a method using a normal HSA gene [Japanese Patent Laid-Open No. 58-56684 (corresponding to EP-A-73664)], No. 5 8-9 0 5 15 No. 5 (corresponding to EP—A—7 9 7 39), No. 5 8—15 0 5 17 No.
- an appropriate host preferably Pichia yeast, specifically, the AOX of the GT S115 strain (NRRL accession number Y-15851), and the gene region under the control of AOX and the promoter by a conventional method.
- a transformant is obtained by introducing a plasmid having a transcription unit expressing HSA into the yeast [Japanese Patent Application Laid-Open No. 2-104290 (corresponding to EP-A-344449)] See].
- This transformant has a weak growth ability in a methanol medium. Therefore, the transformant is cultured in a medium containing methanol to cause mutation, and only a viable strain is recovered. In this case, the methanol concentration is, for example, about 0.001 to 5%.
- Medium artificial medium, 1 5 to 4 0 ° C as either good c culture conditions natural medium, 1 is about 1 0 0 0 hours are exemplified.
- the rHSA-producing host can be appropriately fed with high-concentration glucose or methanol by fed-batch culture (half-batch culture).
- a method of obtaining a high concentration of cells and products by avoiding high-concentration substrate inhibition on the producing cells Japanese Patent Application Laid-Open No. 3-83595
- r A method for enhancing the production of HS A Japanese Patent Application Laid-Open No. Hei 9-123495 (corresponding to U.S. Pat. No. 5,334,512, EP-A—5048283)]
- the amount of rHSA to be added to the culture medium of the present invention is, for example, about 0.1 to 5 gZL, preferably about 0.1 to 2 gZL.
- the culture medium of the present invention may contain, if necessary, insulin, peptone, transfrin and the like.
- Insulin is not particularly limited in its origin, and preferably, insulin from insulin or recombinant human insulin is used.
- the origin of peptone is not particularly limited, and peptone derived from beef is preferably used. Plant-derived peptone is also used. There is no particular limitation on the origin of transfusin, and human or human origin is preferably used.
- the insulin content in the culture medium of the present invention is 0.1 to 10 mg ZL, preferably 0.1 to 2 mg / L, and the peptone content is 0.1 to 50 gZL, preferably 1 to 10 gL.
- the content of gZL and transferrin is 0.5 to 20 mg ZL, preferably 1 to 15 mg ZL.
- the culture medium of the present invention may further contain hypoxanthine 0.1 to 100 mgZL, preferably l to 20 mgZL, thymidine 0.01 to 100 mg / L, preferably 1 to 10 mg / L, if necessary.
- selenium 0.0 1 to 1 to 00 ⁇ 2, preferably 1 ⁇ 1 0 g / L, ⁇ - tocopherol (vitamin E) 0. 00 1 ⁇ 1 0mg / / L, preferably 0.0 1-0. 5 mg ZL can be added.
- the medium When a transformed animal cell containing a vector having a resistance gene is to be cultured, the medium may be further adapted to the resistance gene contained in the vector in order to maintain the stability of the plasmid transformation.
- selective agents are added.
- Choice Examples of the agent include those well-known to those skilled in the art, for example, neomycin, hygromycin, mycophenolic acid, hypoxanthine, xanthine, aminobulin, or methotrexite (MTX) and derivatives thereof.
- Animal cells that can be cultured in the culture medium of the present invention may be those that are capable of producing a physiologically active substance by themselves, or those that are transformed by genetic engineering to produce a heterologous physiologically active substance.
- Cells that themselves produce bioactive substances such as hybridomas that produce monoclonal antibodies, leukocytes that produce interferon (IFN) -1 ⁇ , Namaluba cells, BALL-1 cells, and IFN-3 Fibroblasts, lymphocytes producing IFN-7, human kidney cells producing properokinase or perokinase (pro-UK or UK), and melanoma producing tissue plasminogen activator (TPA).
- Cells for example, Bowes cells are exemplified. ⁇
- the established host cells transformed by gene engineering include Vero cells, He1a cells, CHO cells, WI38 cells, BHK cells, COS-7 cells, MDCK cells, and C127 cells. Cells, HKG cells, human kidney cell lines, and the like. Specifically, CHO— (Chinese hamster ovary cell: ATCC CCL 61), BHK (hamster kidney cell: ATCC CCL 10).
- COS-7 (CV-1 Origin, SV-40 cell: ATCCCRL 16) 51), Vero cells (African green monkey kidney cells: ATCCCCL81) and the like, as well as mouse myeloma cells (X63-Ag8-653; P3U1) and human lymphoblast cells (IM-9.ATCC).
- CCL 159 parent cells for producing human hybridomas, and dhfr-deficient strains, HGPRT-deficient strains, and ⁇ abain-resistant strains.
- the method of culturing animal cells using the culture medium of the present invention is generally performed as follows. That is, for culturing animal cells using the culture medium of the present invention, a dish, flask, roller-bottle, spinner-flask, microcarrier, microcapsule, or hollow fiber well-known for culture is used.
- the culture vessel such as the culture apparatus used can be used, but is not limited thereto.
- the culture method is- In addition to the subculture usually performed in the above-mentioned culture vessel, the old culture solution is continuously or intermittently removed from the culture tank while containing the cells or separated from the cells.
- the culture of animal cells using the culture medium of the present invention can be performed in the form of so-called high-density culture.
- Cell number in particular is preferably carried out in 1 0 7 Zm ⁇ more dense.
- Other culturing conditions include a culturing temperature of 30 to 37 ° (the culturing time is, for example, 1 to 10 days. C) If the medium is appropriately replaced, continuous culturing for a longer period can be performed.
- Production of a physiologically active substance such as a protein by a cell may be performed by a means known per se.
- Examples of such means include, for example, Japanese Patent Application Laid-Open No.
- a physiologically active substance of the present invention for example, antithrombin-III, pro-UK, UK, TP A. hepatitis B virus surface antigen, pre-S—hepatitis B virus surface antigen, IFN, colony formation Bioactive substances such as stimulating factors and monoclonal antibodies can be produced.
- RPMI 1640 medium [Goding, J. W (1980) J. Immunol. Methods 39, 285, JAMA 199 (1957)] 10.2 g was used as a base medium, rHSA 1 g as an additive, insulin Lmg, beef-derived peptone 5g, transferrin 10mg, hypoxanthine 13mg, thymidine 4mg, 0.1 tocopherol 0.13mg and selenium 4tzg 1L of culture medium was prepared. r As HS A
- Example 2 Culture of human kidney cell line The human kidney cell line as a cell number in the culture medium prepared with Example 1 which was attached to microcarrier beads were added to a 1 0 7 Zm 1. 3 7 ° C, 5% C0 was cultured in 2 conditions, 0.6 to 2.5 Production of U / m 1 equivalent pro UK was confirmed in culture medium at 2 days. After that, the culture medium was changed every 2-3 days, and continuous cultivation for a long period (at least one month or more) was possible.
- rHSA human serum albumin
Description
Claims
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97934720A EP0947581A4 (en) | 1996-08-08 | 1997-08-06 | CULTURAL MEDIUM AND ITS USE. |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP8/210004 | 1996-08-08 | ||
JP21000496 | 1996-08-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998006822A1 true WO1998006822A1 (en) | 1998-02-19 |
Family
ID=16582261
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP1997/002749 WO1998006822A1 (en) | 1996-08-08 | 1997-08-06 | Culture medium and use of the same |
Country Status (2)
Country | Link |
---|---|
EP (1) | EP0947581A4 (ja) |
WO (1) | WO1998006822A1 (ja) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000029554A2 (en) * | 1998-11-19 | 2000-05-25 | Thomas Jefferson University | Compositions for preserving haptenized tumor cells for use in vaccines |
JP2003535585A (ja) * | 2000-06-09 | 2003-12-02 | ビトロライフ・アーベー | 哺乳動物の配偶子および胚の培養基サプリメント、およびその使用法 |
WO2005071064A1 (ja) * | 2004-01-21 | 2005-08-04 | Mitsubishi Pharma Corporation | 造血幹細胞及び造血前駆細胞の増幅方法 |
JP2015502756A (ja) * | 2011-12-22 | 2015-01-29 | モガム バイオテクノロジー リサーチ インスティチュート | ナチュラルキラー細胞の製造方法、その方法により製造されたナチュラルキラー細胞、並びにそれを含む腫瘍及び感染性疾患治療用組成物 |
US11766456B2 (en) | 2014-11-26 | 2023-09-26 | GC Cell Corporation | Method for culturing natural killer cells using T cells |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CA2608058C (en) | 2005-05-12 | 2013-09-10 | Crucell Holland B.V. | Host cell specific binding molecules capable of neutralizing viruses and uses thereof |
DK2230311T3 (da) * | 2005-06-28 | 2014-05-19 | Ventria Bioscience | Bestanddele af celledyrkningsmedier produceret ud fra planteceller |
AU2007255384B2 (en) | 2006-06-06 | 2012-09-27 | Crucell Holland B.V. | Human binding molecules having killing activity against staphylococci and uses thereof |
WO2008009641A1 (en) * | 2006-07-17 | 2008-01-24 | Novozymes A/S | Cell culture media |
CN101768206B (zh) * | 2008-12-31 | 2013-05-15 | 华北制药集团新药研究开发有限责任公司 | 一种重组人血清白蛋白的纯化方法及其应用 |
WO2010096588A2 (en) | 2009-02-20 | 2010-08-26 | Ventria Bioscience | Cell culture media containing combinations of proteins |
CN102470170B (zh) * | 2009-07-13 | 2015-03-18 | 巴拉特生物技术国际有限公司 | 用作轮状病毒疫苗的组合物及其方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6374493A (ja) * | 1986-06-03 | 1988-04-04 | デルタ バイオテクノロジ− リミテツド | 酵母におけるガラクト−ス制御遺伝子発現の誘導 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB8725529D0 (en) * | 1987-10-30 | 1987-12-02 | Delta Biotechnology Ltd | Polypeptides |
JPH0322979A (ja) * | 1989-06-19 | 1991-01-31 | Kyowa Hakko Kogyo Co Ltd | 新規プラスミノーゲン活性化因子 |
WO1992013951A1 (en) * | 1991-02-04 | 1992-08-20 | The Salk Institute Biotechnology/Industrial Associates, Inc. | Production of human serum albumin in methylotrophic yeast cells |
ES2211889T3 (es) * | 1994-08-31 | 2004-07-16 | Mitsubishi Pharma Corporation | Procedimiento para la purificacion de albumina de suero recombinante. |
-
1997
- 1997-08-06 WO PCT/JP1997/002749 patent/WO1998006822A1/ja not_active Application Discontinuation
- 1997-08-06 EP EP97934720A patent/EP0947581A4/en not_active Withdrawn
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS6374493A (ja) * | 1986-06-03 | 1988-04-04 | デルタ バイオテクノロジ− リミテツド | 酵母におけるガラクト−ス制御遺伝子発現の誘導 |
Non-Patent Citations (3)
Title |
---|
CYTOTECHNOLOGY, Vol. 19, No. 1, (1995-1996), CASTRO P.M.L. et al., "CHO Cell Growth and Recombinant Interferon-gamma Production: Effects of BSA, Pluronic and Lipids", p. 27-36. * |
See also references of EP0947581A4 * |
THROMBOSIS RESEARCH, Vol. 31, No. 4, (1983), WILLENS C. et al., "The Proliferation of Human Umbilical Vein Endothelial Cells in Serum-Free Medium", p. 623-634. * |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000029554A2 (en) * | 1998-11-19 | 2000-05-25 | Thomas Jefferson University | Compositions for preserving haptenized tumor cells for use in vaccines |
WO2000029554A3 (en) * | 1998-11-19 | 2000-08-24 | Univ Jefferson | Compositions for preserving haptenized tumor cells for use in vaccines |
US6248585B1 (en) | 1998-11-19 | 2001-06-19 | Thomas Jefferson University | Compositions for preserving haptenized tumor cells for use in vaccines |
JP2003535585A (ja) * | 2000-06-09 | 2003-12-02 | ビトロライフ・アーベー | 哺乳動物の配偶子および胚の培養基サプリメント、およびその使用法 |
WO2005071064A1 (ja) * | 2004-01-21 | 2005-08-04 | Mitsubishi Pharma Corporation | 造血幹細胞及び造血前駆細胞の増幅方法 |
JP2015502756A (ja) * | 2011-12-22 | 2015-01-29 | モガム バイオテクノロジー リサーチ インスティチュート | ナチュラルキラー細胞の製造方法、その方法により製造されたナチュラルキラー細胞、並びにそれを含む腫瘍及び感染性疾患治療用組成物 |
US11766456B2 (en) | 2014-11-26 | 2023-09-26 | GC Cell Corporation | Method for culturing natural killer cells using T cells |
Also Published As
Publication number | Publication date |
---|---|
EP0947581A1 (en) | 1999-10-06 |
EP0947581A4 (en) | 2004-07-28 |
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