+

WO1998004680A1 - Milieu ne contenant pas de serum, destine a la culture de cellules mammaliennes dependantes d'un support - Google Patents

Milieu ne contenant pas de serum, destine a la culture de cellules mammaliennes dependantes d'un support Download PDF

Info

Publication number
WO1998004680A1
WO1998004680A1 PCT/US1997/013079 US9713079W WO9804680A1 WO 1998004680 A1 WO1998004680 A1 WO 1998004680A1 US 9713079 W US9713079 W US 9713079W WO 9804680 A1 WO9804680 A1 WO 9804680A1
Authority
WO
WIPO (PCT)
Prior art keywords
serum
set forth
cells
tissue culture
anchorage
Prior art date
Application number
PCT/US1997/013079
Other languages
English (en)
Inventor
Michael Butler
John Michael Berry
Original Assignee
University Of Manitoba
Kohn, Kenneth, I.
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by University Of Manitoba, Kohn, Kenneth, I. filed Critical University Of Manitoba
Priority to AU38135/97A priority Critical patent/AU3813597A/en
Publication of WO1998004680A1 publication Critical patent/WO1998004680A1/fr

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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/395—Thyroid hormones
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention provides a serum-free cell culture medium for anchorage-dependent mammalian cells.
  • Mammalian cells are used in large-scale bioprocesses for the production of health-care products such as vaccines and recombinant proteins that are the basis of the commercial operations of many biotechnology and pharmaceutical companies.
  • the cells are grown in a liquid medium of defined chemicals which is often supplemented with blood serum, typically at 10% v/v.
  • the serum is normally of bovine origin and provides undefined hormones, growth factors and micronutrients required for cell growth.
  • Serum-free media formulations are used routinely for suspension cultures [Butler, 1992].
  • serum-free formulations containing a cocktail of up to five ingredients have been developed and are now used routinely.
  • anchorage-dependent mammalian cells are more fastidious in their growth requirements and serum-free formulations for such cells are not as satisfactory as for suspension cultures. Nevertheless the need for serum-free cultures of anchorage-dependent cells is considerable.
  • Anchorage-dependent mammalian cell cultures are used exclusively for human viral vaccine production and the generation of artificial skin used in the treatment of human injuries. Also, anchorage-dependent cells are used in many processes for the production of veterinary vaccines and recombinant proteins.
  • Anchorage-dependent cell lines widely used in commercial bioprocesses include: MRC-5, WI-38, FS-4 (human fibroblasts), Vero (African green monkey), BHK (Baby hamster kidney) and MDCK (Madin Darby canine kidney).
  • Vero cells are used in microcarrier processes for the production of human polio and rabies vaccines [Montagnon et al, 1984; van Wezel, 1985].
  • Alternative cell lines for such processes include human diploid fibroblasts such as MRC-5 which are also grown in microcarrier cultures [Forestall et al, 1992]. These viral production processes are normally dependent upon the use of bovine serum with the attendant problems discussed herein above.
  • Cinatl et al [1992] recognized the importance of the cell-substratum interaction when they developed a protein-free medium which was suitable for growth of Vero cells only in static cultures on a modified plastic surface based on poly vinyl formal. Growth on polystyrene culture plates in this medium was poor. Although the initial protein-free formulation produced low growth rates of Vero cells, they later published the full formulation of an improved medium (PFEK-1) which contained 97 components [Cinatl et al, 1993]. They reported growth rates equivalent to serum-based medium on a PVF surface and the production of human pathogenic viruses. A more recent attempt to produce a serum-free formulation for Vero cells was published by Merten et al [1994].
  • MDSS2 a proprietary formulation from AXCELL Biotechnologies was described as capable of supporting Vero cell growth in microcarrier cultures.
  • the cells grew in MDSS2 without adaptation and were capable of producing rabies virus.
  • the growth curves shown indicate an extremely high doubling time (80 hours) with maximum cell densities occurring after 12 days.
  • the cultures were reported to require five changes of medium (50%) over this period.
  • a high doubling time (38 hours) and a long lag phase (4 days) were reported for a serum-free medium by Zhaolie et al [1996]. It would be useful to have a serum-free medium formulation with fewer than 97 components that can be used for anchorage-dependent growth of these cells on various surfaces including T-flasks different microcarrier types. Further it would be useful to have a serum-free formulation in which cells do not clump in culture and that have a high cell growth rate with a minimal lag phase.
  • a serum-free medium for growing anchorage-dependent mammalian cells in tissue culture as set forth in Table 1 comprises a basal medium supplemented with chemically defined components (Table 1) which replace the serum.
  • the tissue culture is stationary or agitated using microcarriers.
  • the fibronectin is added to the medium immediately prior to use.
  • the fibronectin is used to precoat tissue culture flasks or the microcarriers.
  • the present invention provides a serum-free medium for growing anchorage-dependent Vero (African green monkey) cells in tissue culture including a basal medium enriched with the components replacing serum at the concentration range as set forth in Table 2.
  • the tissue culture is stationary or agitated using microcarriers.
  • the fibronectin is added to the medium immediately prior to use.
  • the fibronectin is used to precoat tissue culture flasks or the microcarriers.
  • the present invention also provides a method of culturing anchorage- dependent mammalian cells in tissue culture including the step of culturing in a basal medium enriched with the components replacing serum at the concentration range as set forth in Table 1.
  • the tissue culture is stationary or agitated using microcarriers.
  • the fibronectin is added to the medium immediately prior to use.
  • the fibronectin is used to precoat tissue culture flasks or the microcarriers.
  • the present invention provides a method of culturing anchorage-dependent Vero (African green monkey) cells in tissue culture including the step of culturing in a basal medium enriched with the components for replacing serum at the concentration range as set forth in Table 2.
  • the tissue culture is stationary or agitated using microcarriers.
  • the fibronectin is added to the medium immediately prior to use.
  • the fibronectin is used to precoat tissue culture flasks or the microcarriers.
  • FIGURE 1 is a graph showing the effect of multiple passages on Vero cell growth in DMEM + 5.0% v/v sCS (open symbols) and VSFM (closed symbols) in T25 culture flasks.
  • the cells were passaged for either 6 (•), 20 ( ⁇ ), 30 (A) or 35 ( ⁇ ) passages in each medium.
  • the cells were inoculated at 0.10 x 10 6 cells/ml in 10 ml of either VSFM or DMEM + 5.0% v/v sCS.
  • FIGURE 2A-B are graphs which show the effect of various serum-free medium formulations on preadapted Vero cell growth in 100 ml spinner flasks on (A) 1.00 g/L Cytodex-1 and (B) 1.70 g/L Cultispher-G.
  • the cells were inoculated at 0.05 x 10 6 cells/ml in either DMEM + 5.0% v/v sCS (•), VSFM ( ⁇ ), Gibco serum-free Vero maintenance media II (A), Gibco serum- free Vero maintenance media HI ( ⁇ ), or Celox TCM supplemented, enriched DMEM ( ⁇ ).
  • the flasks were stirred at 40 rpm continuously (Cytodex-1) or intermittently for 24 hours then continuously and ramped to 60 rpm after 72 hours (Cultispher-G).
  • FIGURE 3 are graphs showing the effects of bead precoating and medium formulation on Vero cell growth in 100 ml spinner flasks on (A) 1.00 g/L Cytodex-1 and (B) 1.70 g/L Cultispher-G. The cells were inoculated at
  • the flasks were stirred at 40 rpm continuously (Cytodex-1) or intermittently for 24 hours then continuously and ramped to 60 rpm after 72 hours (Cultispher-G).
  • the present invention provides a serum-free medium (SFM) for growing anchorage-dependent (AD) mammalian cells in tissue culture including a basal medium supplemented with the components at the concentration range as set forth in Table 1.
  • SFM serum-free medium
  • anchorage-dependent cells are meant cells that must attach to a surface during growth. Examples of such cells are MRC-5, WI-38, FS-4 (human fibroblasts), Vero (African green monkey), BHK (Baby hamster kidney) and MDCK (Madin Darby canine kidney) or other anchorage-dependent cells isolated from mammalian tissue for primary culture.
  • tissue culture is meant the culturing/growth of cells or tissue slices in an artificial medium, i.e. ex vivo.
  • the cells are cultured using either stationary tissue culture or agitated using microcarriers in tissue culture as is generally known in the art.
  • the supplements or components for the basal medium of the present invention are listed in Table 1 with the appropriate concentration range for mammalian cells. Any basal medium may be used in the practice of the present invention if it supports growth of anchorage-dependent cells when used with serum, with the components as listed in Table 1 of the present invention substituting for the serum.
  • Dulbecco's modification of Eagle's medium DMEM
  • DMEM Dulbecco's modification of Eagle's medium
  • the supplemented medium can be referred to as AD-SFM.
  • Concentrated stock solutions (xlOO) are prepared individually or as mixed component stocks.
  • the mixed component stocks include: trace element salts of Se, Fe, Cu and Zn; biotin and vitamin B12; insulin, transferrin, hydrocortisone and triiodothyronine; glutathionine and 2-mercaptoethanol. All stock solutions are prepared as indicated in the Merck Index (10th edition). They are stored at either -20°C (vitamin B12, biotin, insulin, transferrin, hydrocortisone, FGF, triiodothyronine and prostaglandin El), -f-4°C (glutathione, fibronectin and fetuin) or room temperature, 20°C (trace elements and mercaptoethanol).
  • the appropriate growth factor is selected based on the cell type being cultured as is known in the art.
  • the GFs can include epidermal growth factor (EGF), platelet-derived growth factor (PDGF) or transforming growth factor (TGF), and fibroblast growth factor (FGF).
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • TGF transforming growth factor
  • FGF fibroblast growth factor
  • the order of addition of the supplements to enriched DMEM from concentrated (xlOO) stock solutions is chosen such that precipitation of any component is avoided. In one embodiment the order is as follows:
  • GF sterile growth factor
  • hormones hydrocortisone and triiodothyronine
  • prostaglandin El sterile growth factor
  • the fibronectin is used to precoat tissue culture flasks or the microcarriers prior to use and fibronectin is not added directly to the medium.
  • a serum-free medium for growing anchorage-dependent Vero (African green monkey) cells in tissue culture including a basal medium enriched with the components and concentration range as set forth in Table 2.
  • the formulation in Table 2 is optimal for the growth of Vero cells.
  • the medium in this embodiment is designated VSFM.
  • the present invention further provides a method of culturing anchorage- dependent mammalian cells in tissue culture including the step of culturing in a basal medium enriched with the components at the concentration range as set forth in Table 1.
  • the cells are Vero cells and the component concentrations are as set forth in Table 2.
  • the present invention therefore provides a combination of individual growth-promoting components in one formulation. These components have previously been reported individually but not in combination. The specific combinations Applicants have found to be particularly important are: A mixture of trace elements with growth factor (GF), insulin, transferrin, bFGF, fibronectin, vitamin B12, biotin and hydrocortisone. Inclusion of the protein, fetuin reduces the lag phase and increases growth rates. Fibronectin and growth factor are required for cell attachment prior to growth.
  • the proteins may be of animal origin or expressed as recombinant proteins. The use of non- animal sources is preferred because this reduces the risk of contamination. Insulin may be substituted by insulin growth factor (IGF) at a concentration range of 1-100 ng/ml. Transferrin may be substituted by an alternative iron delivery system to the cells [Mercalfe et al., 1994].
  • IGF insulin growth factor
  • Transferrin may be substituted by
  • the AD-SFM is chemically defined and has a low content of proteins and has a lower protein content than serum.
  • the lower protein content ensures ease of product purification that is a problem with the prior art serum containing media. Further without serum standard quality control would ensure that there would be no batch to batch variation as is seen for serum containing media. Further, without serum the potential vector for disease is missing.
  • the composition of the medium is of chemicals which are generally available and does not require the 97 components of the prior art. Additionally, the medium of the present invention can be used with several microcarriers as well as differing plastics.
  • This Example presents data on the development of a novel serum-free formulation for the growth of Vero cells in static and agitated microcarrier cultures.
  • MATERIALS AND METHODS Cell line African green monkey (Vero) cells were obtained from the American Type Culture Collection (ATCC) CCL 81. The cells were maintained in T- flasks in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% iron-enriched calf serum (Gibco). Cultures: Stock cultures were maintained in T-flasks and passaged every 72-96 hours by trypsinization followed by inoculation into fresh medium (1: 10 dilution). Experimental cultures were established in T-25 flasks by inoculation at 10 5 cells/ml in 10 ml medium.
  • DMEM Dulbecco's modified Eagle's medium
  • Febco iron-enriched calf serum
  • Microcarrier cultures (100 ml) were established in glass spinner flasks (Bellco) with Cytodex-1 (Pharmacia) or Cultispher-G (Hyclone) microcarriers.
  • the Cytodex cultures were stirred continuously at 40 ⁇ m.
  • the Cultispher cultures were stirred intermittently for 24 hours then continuously at 40 ⁇ m for 72 hours at which point the stirring speed was increased to 60 ⁇ m.
  • the stirring regimen of the microcarrier cultures was based upon a previous study [Ng et al, 1996]. All cultures were incubated at 37°C with a 10% CO 2 overlay.
  • Serum-free medium The serum-free medium for Vero cells (VSFM) of the present invention was prepared by adding supplements to DMEM as a basal medium (Table 2).
  • the DMEM contained 25 mM glucose, 4 mM glutamine, 36 mM sodium bicarbonate. All supplements were prepared in stock concentrate solutions (xlOO).
  • Control commercial serum-free media was obtained as TCM concentrate (Celox) which replaced serum in enriched DMEM.
  • Vero maintenance medium formulations (II and III) were generously provided by Gibco.
  • Adaptation of stock cells Cells were transferred directly from serum- containing medium (SCM) to VSFM or were gradually adapted. Gradual adaptation involved sub-culturing every 48 hours into a mixture of SCM and VSFM. The ratio of SCM/VSFM was altered from 5: 1 to 1: 10 in 9 stages prior to inoculation into complete VSFM.
  • SCM serum- containing medium
  • the growth profile of Vero cells in T-flasks was determined by daily cell counts from replicate cultures containing either SCM or VSFM (Fig. 1). This showed a maximum cell density of 1.79 x 10 6 cells/ml in SCM as opposed to 1.13 x 10 6 cells/ml in VSFM. The specific growth rate was slightly lower in VSFM and the lag phase was extended to 24 hours (Table 3). The growth profile of cells in VSFM was maintained for 10 passages but over subsequent passages the maximum cell density decreased, so that after 35 passages the cells grew with a reduced growth rate and attained a maximum density of ⁇ 30% of the original value.
  • FIG. 2 shows that cell growth occurred in all media tested. Maximum cell densities were significantly higher in Cultispher-G cultures compared to those of Cytodex-1. The maximum cell densities attained in SCM was significantly higher than any of the serum-free media. Of the serum-free media tested, cell yields were significantly (44%) higher in VSFM compared to the other formulations, which gave progressively lower yields in the order TCM > GibcoIII > GibcoII.
  • the cell attachment was significantly faster in SCM compared to any of the serum-free media. This accounts for the shorter observed lag phase and also there were fewer free cells observed throughout the culturing period which indicates that the cells were more firmly attached to the microcarriers. Fewer free cells were observed in the VSFM cultures compared to the other serum- free cultures. The characteristics of these cultures are identified in Table 4.
  • microcarriers were pre-coated by suspension of the beads in DMEM supplemented with 5% serum or with 5 mg/L fibronectin for 6 hours. The beads were subsequently washed in PBS prior to inco ⁇ oration into cultures.
  • Figure 3 shows the effect of pre-coating the microcarrier beads on cell growth in VSFM prepared with or without fibronectin.
  • a low growth rate of cells occurred in the absence of fibronectin or with beads that were not pre- coated.
  • the addition of fibronectin to VSFM or pre-coating the beads with serum containing medium was equally effective in increasing the growth rate in the serum-free cultures which attained a maximum cell density of 65- 70% of the values attained in SCM.
  • This Example shows that the medium of the present invention can be used for anchorage-dependent growth of cells in stationary T-flask cultures and agitated cultures using 2 microcarrier types.
  • cell yields above 10 6 /ml were attained from inocula of 0.5-1 x 10 5 cells/ml in 5 days.
  • the observed doubling times in the growth phase was 26 hours which is normal for Vero cells.
  • the cell yields were significantly greater in VSFM compared to three other serum-free formulations tested.
  • Vero cell growth in VSFM cultures are superior to other published reports of serum- free cultures, which are restricted to clump cultures [Litwin, 1992], to a single surface type [Cinatl et al, 1993] or offer extremely low growth rates [Merten et al, 1994] or have a long lag phase [Zhaolie et al., 1996].
  • VSFM Vero cell serum-free medium
  • Preadapted cells were inoculated at 0.05 x 10 6 cells/ml on 1.00 g/L Cytodex-1 or 1.70 g/L Cultispher-G in 100 ml spinner flasks.
  • the microcarriers were precoated (18 hours) at 20°C.
  • the cells were grown in VSFM, Gibco Vero cell maintenance media II or III or in Celox TCM supplemented, enriched DMEM.
  • Cinatl, et al. 1992. Polyvinyl formal surface promotes continuous growth of Vero cells in protein-free medium. Biologicals 20, 59-65.
  • MDSS2 new serum-free medium
  • MDCK Madin-Darby canine kidney epithelial cell

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Cette invention concerne un milieu ne contenant pas de sérum qui est destiné à la culture de cellules mammaliennes dépendantes d'un support dans une culture de tissu contenant un milieu de base complété par des composants de remplacement du sérum, la concentration desdits composants appartenant à une plage de concentrations définies dans la table 1 du descriptif de l'invention. Ladite culture de tissu est stationnaire ou agitée au moyen de billes micro-porteuses. Dans une réalisation, on ajoute de la fibronectine au milieu de culture immédiatement avant son utilisation. Il est également possible d'utiliser la fibronectine pour pré-enduire des récipients destinés à la culture du tissu ou les billes micro-porteuses. Dans une de ses réalisations, la présente invention se rapporte à un milieu ne contenant pas de sérum et destiné à la culture de cellules Véro (de grivet d'Afrique) dépendantes d'un support dans une culture de tissu contenant un milieu de base complété par des composants dont la concentration appartient à une plage de concentrations définies dans la table 2 du descriptif de l'invention. La présente invention se rapporte également à un procédé de culture de cellules mammaliennes dépendantes d'un support de culture, telles que des cellules Véro, dans une culture de tissu, ledit procédé consistant à cultiver dans un milieu de base complété par des composants dont la concentration appartient à une plage de concentrations définies dans la table 1 du descriptif de l'invention.
PCT/US1997/013079 1996-07-26 1997-07-25 Milieu ne contenant pas de serum, destine a la culture de cellules mammaliennes dependantes d'un support WO1998004680A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU38135/97A AU3813597A (en) 1996-07-26 1997-07-25 Serum-free medium for growth of anchorage-dependant mammalian cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2266196P 1996-07-26 1996-07-26
US60/022,661 1996-07-26

Publications (1)

Publication Number Publication Date
WO1998004680A1 true WO1998004680A1 (fr) 1998-02-05

Family

ID=21810761

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1997/013079 WO1998004680A1 (fr) 1996-07-26 1997-07-25 Milieu ne contenant pas de serum, destine a la culture de cellules mammaliennes dependantes d'un support

Country Status (2)

Country Link
AU (1) AU3813597A (fr)
WO (1) WO1998004680A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029084A2 (fr) * 2000-10-02 2002-04-11 Novo Nordisk A/S Production exempte de serum, a l'echelle industrielle, de facteur vii de recombinaison dans des cellules mammaliennes
WO2004022729A1 (fr) 2002-09-05 2004-03-18 Bavarian Nordic A/S Technique de culture de cellules primaires et d'amplification de virus dans un environnement exempt de serum
GB2394477A (en) * 2002-08-22 2004-04-28 Celltran Ltd Mammalian cell culture methods and apparatus
WO2004078955A1 (fr) * 2003-03-03 2004-09-16 Glaxosmithkline Biologicals S.A. Procede de culture de cellule exempt de substances animales
WO2007003640A1 (fr) * 2005-07-05 2007-01-11 Ares Trading S.A. Milieu de culture sans serum pour la production de gonadotrophines recombinantes
US7189536B2 (en) 2000-11-23 2007-03-13 Bavarian Nordic A/S Modified vaccinia ankara virus variant
US7445924B2 (en) 2000-11-23 2008-11-04 Bavarian Nordic A/S Modified Vaccinia Ankara virus variant and cultivation method
US7947471B2 (en) 2001-10-02 2011-05-24 Novo Nordisk Health Care A/G Method of production of recombinant proteins in eukaryote cells
US20130065309A1 (en) * 2007-07-13 2013-03-14 Medimmune, Llc Preparation of Negative-Stranded RNA Viruses By Electroporation
US20160312180A1 (en) * 2002-12-16 2016-10-27 Technion Research & Development Foundation Limited Medium comprising transforming growth factor beta 1 and basic fibroblast growth factor

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112094802B (zh) * 2020-09-28 2022-10-11 成都柏奥特克生物科技股份有限公司 一种用于培养Vero细胞的无血清培养基

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4560655A (en) * 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4786599A (en) * 1983-03-24 1988-11-22 Institut National De La Sante Et De La Recherche Medicale Serum-free animal cell culture medium and methods for the primary culture and production of cell lines using this medium
US5147854A (en) * 1990-05-22 1992-09-15 Hoffmann-La Roche Inc. Tgf-b compositions and method
US5326699A (en) * 1991-02-28 1994-07-05 Kurashiki Boseki Kabushiki Kaisha Serum-free medium for culturing animal cells
US5330911A (en) * 1989-09-28 1994-07-19 Board Of Regents, The University Of Texas System Surfaces having desirable cell adhesive effects
US5405772A (en) * 1993-06-18 1995-04-11 Amgen Inc. Medium for long-term proliferation and development of cells

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4560655A (en) * 1982-12-16 1985-12-24 Immunex Corporation Serum-free cell culture medium and process for making same
US4786599A (en) * 1983-03-24 1988-11-22 Institut National De La Sante Et De La Recherche Medicale Serum-free animal cell culture medium and methods for the primary culture and production of cell lines using this medium
US5330911A (en) * 1989-09-28 1994-07-19 Board Of Regents, The University Of Texas System Surfaces having desirable cell adhesive effects
US5147854A (en) * 1990-05-22 1992-09-15 Hoffmann-La Roche Inc. Tgf-b compositions and method
US5326699A (en) * 1991-02-28 1994-07-05 Kurashiki Boseki Kabushiki Kaisha Serum-free medium for culturing animal cells
US5405772A (en) * 1993-06-18 1995-04-11 Amgen Inc. Medium for long-term proliferation and development of cells

Cited By (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002029084A3 (fr) * 2000-10-02 2002-09-26 Novo Nordisk As Production exempte de serum, a l'echelle industrielle, de facteur vii de recombinaison dans des cellules mammaliennes
WO2002029083A3 (fr) * 2000-10-02 2003-08-21 Novo Nordisk As Production exempte de serum, a l'echelle industrielle, de proteines recombinantes dans des cellules mammiferes
WO2002029084A2 (fr) * 2000-10-02 2002-04-11 Novo Nordisk A/S Production exempte de serum, a l'echelle industrielle, de facteur vii de recombinaison dans des cellules mammaliennes
US7459270B2 (en) 2000-11-23 2008-12-02 Bavarian Nordic A/S Modified Vaccinia Ankara virus variant
US8470598B2 (en) 2000-11-23 2013-06-25 Bavarian Nordic A/S Modified Vaccinia Ankara virus variant and cultivation method
US7964395B2 (en) 2000-11-23 2011-06-21 Bavarian Nordic A/S Modified vaccinia ankara virus variant and cultivation method
US7964398B2 (en) 2000-11-23 2011-06-21 Bavarian Nordic A/S Modified vaccinia ankara virus variant and cultivation method
US7964396B2 (en) 2000-11-23 2011-06-21 Bavarian Nordic A/S Modified vaccinia ankara virus variant and cultivation method
US8236560B2 (en) 2000-11-23 2012-08-07 Bavarian Nordic A/S Modified Vaccinia Ankara virus variant and cultivation method
US7189536B2 (en) 2000-11-23 2007-03-13 Bavarian Nordic A/S Modified vaccinia ankara virus variant
US7335364B2 (en) 2000-11-23 2008-02-26 Bavarian Nordic A/S Modified Vaccinia Ankara virus variant
US7445924B2 (en) 2000-11-23 2008-11-04 Bavarian Nordic A/S Modified Vaccinia Ankara virus variant and cultivation method
US7947471B2 (en) 2001-10-02 2011-05-24 Novo Nordisk Health Care A/G Method of production of recombinant proteins in eukaryote cells
GB2394477B (en) * 2002-08-22 2005-03-30 Celltran Ltd Cell culture
GB2394477A (en) * 2002-08-22 2004-04-28 Celltran Ltd Mammalian cell culture methods and apparatus
US7695939B2 (en) 2002-09-05 2010-04-13 Bavarian Nordic A/S Method for the cultivation of primary cells and for the amplification of viruses under serum free conditions
EA009008B1 (ru) * 2002-09-05 2007-10-26 Бавариан Нордик А/С Способ амплификации вируса оспы
US8673318B2 (en) 2002-09-05 2014-03-18 Bavarian Nordic A/S Method for the cultivation of primary cells and for the amplification of viruses under serum free conditions
DE60320520T3 (de) 2002-09-05 2019-05-09 Bavarian Nordic A/S Verfahren zur amplifikation von einem poxvirus in serumfreien verhältnissen
US7964397B2 (en) 2002-09-05 2011-06-21 Bavarian Nordic A/S Method for the cultivation of primary cells and for the amplification of viruses under serum free conditions
WO2004022729A1 (fr) 2002-09-05 2004-03-18 Bavarian Nordic A/S Technique de culture de cellules primaires et d'amplification de virus dans un environnement exempt de serum
US8329466B2 (en) 2002-09-05 2012-12-11 Bavarian Nordic A/S Method for the cultivation of primary cells and for the amplification of viruses under serum free conditions
US10066205B2 (en) * 2002-12-16 2018-09-04 Technion Research & Development Foundation Limited Medium comprising transforming growth factor beta 1 and basic fibroblast growth factor
US20160312180A1 (en) * 2002-12-16 2016-10-27 Technion Research & Development Foundation Limited Medium comprising transforming growth factor beta 1 and basic fibroblast growth factor
US10590382B2 (en) 2002-12-16 2020-03-17 Technion Research & Development Foundation Limited Medium comprising transforming growth factor beta 1 and basic fibroblast growth factor
CN1756837B (zh) * 2003-03-03 2010-05-26 葛兰素史密丝克莱恩生物有限公司 无动物细胞培养方法
US8034617B2 (en) 2003-03-03 2011-10-11 Glaxosmithkline Biologicals S.A. Cell culture in culture media free of components of animal origin
EP2192175A1 (fr) * 2003-03-03 2010-06-02 GlaxoSmithKline Biologicals S.A. Procédé de culture de céllule exempt de substances animales
EP2186885A1 (fr) * 2003-03-03 2010-05-19 GlaxoSmithKline Biologicals S.A. Procédé de culture de céllule exempt de substances animales
WO2004078955A1 (fr) * 2003-03-03 2004-09-16 Glaxosmithkline Biologicals S.A. Procede de culture de cellule exempt de substances animales
US8093052B2 (en) 2005-07-05 2012-01-10 Ares Trading S.A. Serum-free culture medium for the production of recombinant gonadotropins
EA012840B1 (ru) * 2005-07-05 2009-12-30 Арес Трейдинг С.А. Применение бессывороточной культуральной среды, содержащей антиоксидант, для снижения окисления рекомбинантных гонадотропинов
WO2007003640A1 (fr) * 2005-07-05 2007-01-11 Ares Trading S.A. Milieu de culture sans serum pour la production de gonadotrophines recombinantes
US20130065309A1 (en) * 2007-07-13 2013-03-14 Medimmune, Llc Preparation of Negative-Stranded RNA Viruses By Electroporation

Also Published As

Publication number Publication date
AU3813597A (en) 1998-02-20

Similar Documents

Publication Publication Date Title
Sinacore et al. Adaptation of mammalian cells to growth in serum-free media
EP1360314B1 (fr) Milieu chimiquement defini pour cellules mammaliennes cultivees
EP2213725B2 (fr) Milieu exempt de proteines animales pour la culture de cellules
USRE46897E1 (en) Recombinant cell clones having increased stability and methods of making and using the same
EP1891207A2 (fr) Procede de culture de cellules animales comprenant l'apport de peptones derives de plantes
US7598083B2 (en) Chemically defined media compositions
Chun et al. Usability of size-excluded fractions of soy protein hydrolysates for growth and viability of Chinese hamster ovary cells in protein-free suspension culture
WO1998004680A1 (fr) Milieu ne contenant pas de serum, destine a la culture de cellules mammaliennes dependantes d'un support
IL171680A (en) Methods for culturing cells derived from per.c6 cells
Crespi et al. Microcarrier culture: Applications in biological production and cell biology
CN112410280B (zh) 一种用于pk15细胞培养的无血清培养基及其应用
AU2009272857B2 (en) Scalable process for culturing PER.C6 cells and producing products therefrom
AU2012238257B2 (en) Animal Protein-Free Media for Cultivation of Cells
AU2002243824A1 (en) Chemically defined medium for cultured mammalian cells
LEONG Department zyxwvutsrqponm

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH KE LS MW SD SZ UG ZW AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: JP

Ref document number: 1998509000

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载