EP0593539A1 - Additif pour milieu de culture a base de chelate de fer - Google Patents

Additif pour milieu de culture a base de chelate de fer

Info

Publication number
EP0593539A1
EP0593539A1 EP92913614A EP92913614A EP0593539A1 EP 0593539 A1 EP0593539 A1 EP 0593539A1 EP 92913614 A EP92913614 A EP 92913614A EP 92913614 A EP92913614 A EP 92913614A EP 0593539 A1 EP0593539 A1 EP 0593539A1
Authority
EP
European Patent Office
Prior art keywords
citrate
culture medium
fecl
iron
iron salt
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.)
Withdrawn
Application number
EP92913614A
Other languages
German (de)
English (en)
Inventor
Peter Bernt Suhr-Jessen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novo Nordisk AS
Original Assignee
Novo Nordisk AS
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 Novo Nordisk AS filed Critical Novo Nordisk AS
Priority to EP92913614A priority Critical patent/EP0593539A1/fr
Publication of EP0593539A1 publication Critical patent/EP0593539A1/fr
Withdrawn legal-status Critical Current

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
    • C12N5/005—Protein-free medium
    • 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

Definitions

  • Iron c elate culture medium additive Iron c elate culture medium additive.
  • the present invention relates to an iron supplement for culture media, primarily serum-free or protein-free media, for growing mammalian cells, and a culture medium containing said iron supplement.
  • the present invention relates to a culture medium additive comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate.
  • Iron chelates for serum-free media have previously been proposed, e.g. in EP 274 445 describing a culture medium additive containing an iron-EDTA/citric acid chelate and aurin tricarboxylic acid.
  • the iron chelate additive of the present invention has the advantage over the one proposed in EP 274 445 that it is composed of inexpensive constituents, and that it contains fewer constituents which might be a ⁇ ource of contamination.
  • the present invention relates to a culture medium for growing mammalian cells, the medium comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate.
  • the citrate chelator should be mixed with the iron salt so as to generate an equilibrium prior to the addition to the culture medium.
  • This equilibrium may for instance be formed in a concentrated stock solution and, and the process speeded up by stirring, autoclaving, etc.
  • the requisite equilibrium is most conveniently reached when the alkali metal or alkaline earth metal citrate is present in a molar excess relative to the iron salt, in particular a ratio of the citrate to the iron salt of more than 1:1 and less than 500:1.
  • Suitable iron salts for inclusion in the additive of the invention may be selected from the group consisting of FeCl 2 , FeCl 3 , FeS0 4 , Fe 3 (P0 4 ) 2 , Fe(N0 3 ) 3 and Fel 2 .
  • suitable alkali metal or alkaline earth metal citrates for inclusion in the additive of the invention are Na-citrate, K-citrate or Mg- citrate.
  • the iron salt included in the additive is FeCl 2 or FeCl 3
  • the citrate is Na-citrate.
  • a preferred molar ratio of Na-citrate to FeCl 2 /FeCl 3 is between 2:1 and 200:1.
  • the culture medium in which the additive i ⁇ intended to be included is preferably a medium for growing mammalian cells, the additive of the invention constituting an inexpensive iron source which mammalian cells have surprisingly been able to utilise.
  • the medium may for instance be a low-serum medium or, preferably, a serum-free or protein-free medium in which it is important to provide a non-protein iron supplement.
  • mammalian cell ⁇ may al ⁇ o utilise a citrate/iron chloride chelate as the iron source in serum- free media.
  • mammalian cells which exist in an environment enriched in nutrient components and under conditions of considerable osmotic pressure are able to assimilate nutrients in a similar way as a primitive freshwater organism specialized in surviving in a nutrient-poor environment.
  • Adherent BHK cells cultivated in coated T-flask ⁇ containing a serum-free nutrient medium for BHK cells (as described by Maciag et al. 1980, ibid) with transferrin as the only iron source (SFNMT) , were concomitantly inoculated into a series of coated T-flask ⁇ containing serum-free nutrient medium lacking transferrin (SFNM-) but supplemented with a chelated stock solution of Na-citrate and iron chloride.
  • SFNMT transferrin as the only iron source
  • BHK cells were inoculated into spinner flasks containing SFNM- for BHK cells (see example 1) supplemented with a chelated citrate-iron stock solution resulting in 2 mM Citrate and 100 ⁇ M FeCl 3 (final cone.) . Following a few hours where cells were allowed to adhere to coated microcarrier ⁇ , cells spread, propagated and remained es ⁇ entially confluent and healthy for more than two weeks when the experiment was terminated.
  • Adherent CHO cells cultivated in coated T-flasks containing a serum-free nutrient medium for CHO cells (as described by Ham et al. 1979, ibid.) with transferrin as the only iron source (SFNMT) , were concomitantly inoculated into a series of coated T-flask ⁇ containing serum-free nutrient medium lacking transferrin (SFNM-) but supplemented with a chelated stock solution of Na-citrate and iron chloride.
  • SFNMT transferrin as the only iron source
  • Each cell culture was independently treated with respect to replacement of u ⁇ ed medium with fresh serum-free medium of the identical kind or sub-cultivation into new T-flask containing fresh serum-free medium of the identical kind.
  • CHO cells were inoculated into two spinner flasks containing SFNM- for CHO cells (see example 3) supplemented with chelated citrate-iron chloride stock solutions resulting in 2 mM Citrate and 100 and 300 ⁇ M FeCl 3 (final cone), re ⁇ pectively. After a few hour ⁇ where cells were allowed to adhere to coated micro carriers, cells spread, propagated and remained essentially confluent and healthy for more than two weeks when the experiment was terminated.
  • SFNMT transferrin as the only iron source
  • SFNMT transferrin as the only iron source

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)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Additif pour milieu de culture comprenant un chélate de fer d'un sel de fer soluble, ainsi qu'un citrate de métal alcalin ou de métal alcalino-terreux. L'additif est une source de fer approprié pour des milieux de culture ne contenant ni sérum ni protéine.
EP92913614A 1991-06-21 1992-06-18 Additif pour milieu de culture a base de chelate de fer Withdrawn EP0593539A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP92913614A EP0593539A1 (fr) 1991-06-21 1992-06-18 Additif pour milieu de culture a base de chelate de fer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP91610054 1991-06-21
EP91610054 1991-06-21
PCT/DK1992/000190 WO1993000423A1 (fr) 1991-06-21 1992-06-18 Additif pour milieu de culture a base de chelate de fer
EP92913614A EP0593539A1 (fr) 1991-06-21 1992-06-18 Additif pour milieu de culture a base de chelate de fer

Publications (1)

Publication Number Publication Date
EP0593539A1 true EP0593539A1 (fr) 1994-04-27

Family

ID=8208780

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92913614A Withdrawn EP0593539A1 (fr) 1991-06-21 1992-06-18 Additif pour milieu de culture a base de chelate de fer

Country Status (5)

Country Link
EP (1) EP0593539A1 (fr)
JP (1) JPH06508523A (fr)
AU (1) AU2196892A (fr)
CA (1) CA2111984A1 (fr)
WO (1) WO1993000423A1 (fr)

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2243827B2 (fr) 1996-08-30 2017-11-22 Life Technologies Corporation Milieu de culture de cellules de mammiferes exempt de serum
CA2383460C (fr) * 1999-08-27 2015-10-13 Invitrogen Corporation Composes de liaison metallique et leur utilisation dans des compositions de milieu de culture cellulaire
US6767741B1 (en) 1999-08-27 2004-07-27 Invitrogen Corporation Metal binding compounds and their use in cell culture medium compositions
US20030096414A1 (en) * 2001-03-27 2003-05-22 Invitrogen Corporation Culture medium for cell growth and transfection
CA2417689C (fr) 2002-03-05 2006-05-09 F. Hoffmann-La Roche Ag Methodes ameliorees de culture in vitro de cellules de mammiferes
DE602004005868T2 (de) * 2003-08-08 2007-12-20 Cambridge Antibody Technology Ltd. Myeloma zellkultur in einem transferrin-freien medium mit niedrigem eisengehalt
GB2404665B (en) 2003-08-08 2005-07-06 Cambridge Antibody Tech Cell culture
WO2006050050A2 (fr) 2004-10-29 2006-05-11 Centocor, Inc. Compositions de milieux chimiquement definis
DK2250251T3 (en) 2008-01-09 2018-01-22 Sartorius Stedim Cellca Gmbh Improved culture medium additive and method of using it
AU2012324495B2 (en) * 2011-10-21 2016-04-21 Pfizer Inc. Addition of iron to improve cell culture
EP3778868B1 (fr) * 2019-08-16 2022-01-26 UGA Biopharma GmbH Milieu de culture cellulaire permettant de cultiver des cellules, procédé de culture des cellules et procédé d'expression d'au moins une protéine recombinant dans une culture cellulaire

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CS262822B1 (en) * 1986-10-03 1989-04-14 Kovar Jan Synthetic medium for the cultivation of myelomic cells
NO162160C (no) * 1987-01-09 1989-11-15 Medi Cult As Serumfritt vekstmedium, samt anvendelse derav.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9300423A1 *

Also Published As

Publication number Publication date
WO1993000423A1 (fr) 1993-01-07
JPH06508523A (ja) 1994-09-29
AU2196892A (en) 1993-01-25
CA2111984A1 (fr) 1993-01-07

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