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WO2004024899A1 - Facteur de croissance pour culture cellulaire - Google Patents

Facteur de croissance pour culture cellulaire Download PDF

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Publication number
WO2004024899A1
WO2004024899A1 PCT/EP2003/009951 EP0309951W WO2004024899A1 WO 2004024899 A1 WO2004024899 A1 WO 2004024899A1 EP 0309951 W EP0309951 W EP 0309951W WO 2004024899 A1 WO2004024899 A1 WO 2004024899A1
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WIPO (PCT)
Prior art keywords
cell culture
culture medium
formula
alkyl
compound
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Ceased
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PCT/EP2003/009951
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English (en)
Inventor
Kjell Bertheussen
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Medi-Cult AS
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Medi-Cult AS
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Publication date
Application filed by Medi-Cult AS filed Critical Medi-Cult AS
Priority to AU2003264279A priority Critical patent/AU2003264279A1/en
Publication of WO2004024899A1 publication Critical patent/WO2004024899A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • 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
    • C12N2500/76—Undefined extracts from plants

Definitions

  • the present invention relates to the use of aloin as a growth factor in cell culture.
  • Aloe plant and various extracts from it including Aloe Vera have been described in popular and academic literature as having a wide range of properties including various therapeutic or cosmetic properties.
  • Aloe has been found to produce a variety of anthraquinone or anthrone related compounds having reported activities. These include aloin (also known as barbaloin: (10-1' , 5' -anhydroglucosyl) -aloe-emodin anthrone), which has the chemical structure shown in formula 1 below:
  • aloesin (4H-l-benzopyran-4-one-8- ⁇ -D-glucopyranosyl-7-hydroxy-5- methyl-2- (2-oxopropyl) ) having the structure shown in Formula 2 below:
  • Aloesin has been reported in WO 95/23604 to have beneficial properties in promoting cell growth, e.g. in cell culture of human hepatoma cells (see also: Lee KY et al, Biochem Mol Biol Int 1997 Feb; 41(2): 285-92).
  • hydroxyanthraquinones such as emodin were also shown to be phytoestrogens with affinity for human estrogen receptors. Aloin itself is not discussed. The estrogenic activity of these compounds was observed at concentrations in the culture medium of 270 ⁇ g/1 to 2,700 ⁇ g/1.
  • Aloin on the other hand has been reported to be an insecticide (US 4602004), and to inhibit protein synthesis when added to a cell culture (Paszkiewicz- Gadek A et al, Pol J Pharmacol Pharm 1988 Mar-Apr; 40(2): 183-90) and to have cytotoxic effects on monolayers of chicken fibroblasts (Avila H et al; Toxicon 1997 Sep; 35 (9) :1423-30) .
  • aloin and closely related compounds have a very useful growth promoting effect when used in cell culture of eukaryotic cells at very low concentrations.
  • the present invention now provides in a first aspect a cell culture medium comprising cell culture ingredients suitable for the maintenance of eukaryotic cells, characterised by the inclusion therein of a growth promoting amount of a compound of the formula
  • R 1 is a glycosylating moiety, or is H, alkyl, substituted alkyl, aryl, substituted aryl, alkoxy, alkoxyalkyl, alkylamine,
  • R 5 is single bond or is alkylene and R is H, alkyl, substituted alkyl, NH 2 , OH or OR 7 , wherein R 7 is alkyl or substituted alkyl and each of R 2 , R 3 and R 4 is alkyl, aryl, substituted alkyl or aryl, or carboxyalkyl, and n is 0 or 1, in the substantial absence of the range of other compounds produced by the aloe plant with which said compound may be associated in nature.
  • Each alkyl or alkylene group is preferably Ci-C ⁇ .
  • Suitable substituents of substituted groups included OH, halogen (e.g. F, Cl, Br) , amine (NH 2 and primary and secondary amine) or aryl.
  • the glycosylation moiety is
  • the most preferred compound of Formula 3 is aloin (Formula 1) .
  • Said compound of Formula 3 is preferably present at a concentration of from 0.1 to 250 ⁇ g/1, more preferably 0.5 to 100 ⁇ g/1, e.g. 1 to 10 ⁇ g/1.
  • the cell culture medium may contain as other ingredients an iron chelate, aurintricarboxylic acid or other metal binders, trace elements, sterol, fatty acid, and detergent maintaining the ingredients in solution.
  • ingredients of the composition may be of the kind described in W098/24883, WO01/59069 or US 5045454.
  • the cell culture medium of the invention is essentially free of material which is not synthetic or derived from plants.
  • it is preferably free of animal derived material, especially animal protein, although a small amount of insulin, preferably recombinant insulin, may be present.
  • Media containing insulin at the appropriate concentration for its use may still be regarded as substantially protein-free.
  • the medium is serum- free .
  • the media of the invention preferably include an iron binding system which may be an iron chelate, aurintricarboxylic acid and an alkali metal EDTA, where the iron chelate preferably comprises a mixture of citrate/citric acid and Fe/EDTA, as in US 5045454.
  • an iron binding system which may be an iron chelate, aurintricarboxylic acid and an alkali metal EDTA, where the iron chelate preferably comprises a mixture of citrate/citric acid and Fe/EDTA, as in US 5045454.
  • Other iron binding systems may be used, as known in the art.
  • EDTA is a preferred chelating agent
  • any biocompatible chelating agent may be used.
  • Citric acid is also added as a chelator rather than as a pH adjusting agent.
  • the medium preferably includes trace elements such as Zn, Cu, Mn, Ni, Al, Cr and Se, initially provided as the ions Zn 2+ , Ca + , Mn 2+ , Ni 2+ , M 3+ and Cr 3+ .
  • the trace elements may comprise Mn (about 20 nM) , Cr (about 20 nM) , Zn (about 20 ⁇ M) , Ni (about 4 nM) , Co (about 4 nM) , Cu (about 0.4 ⁇ M) , Al (about 4 nM) and Se (about 200 nM) .
  • Sterols may be included.
  • Two or more of cholesterol, campesterol, desmosterol, ergosterol, fucosterol, ⁇ - sitosterol, stigmasterol or metabolically acceptable derivatives thereof- may be used.
  • Sterol may be present as a said derivative which is an ester.
  • Other metabolically acceptable derivatives may be used, i.e. derivatives which perform the role of such sterols in cell culture satisfactorily and without toxic effects.
  • estradiol in this context maintain the structure that gives a particular sterol its identity but involve substitutions or reactions with functional groups at specific locations not altering the essential identity of the sterol.
  • the ester is an acetate.
  • other sterols including other methyl cholesterols, various hydroxy-cholesterols, epi-cholesterol, cholestanol, and ⁇ - estradiol may be used.
  • Preferred surfactants for inclusion in the cell culture medium include an -hydro- ⁇ -hydroxypoly (oxyethylene) poly
  • (oxypropylene)poly (oxyethylene) block polymer surfactant of the structure HO (CH 2 CH 2 0) a (CH (CH 3 ) -CH 2 OH) b (CH 2 CH 2 0) C H where a is from 50 to 100, b is from 20 to 40 and c is from 50 to 100, with an average molecular weight of from 7,500 to 10,000.
  • said surfactant is PLURONIC F68.
  • PLURONIC F68 is also known under the names POLOXAMER 188, POLOXALKOL and EXOCORPOL.
  • Other surfactants particularly non-ionic surfactants that do not interfere with the action of the cell culture medium may be included but in general preferably are absent.
  • a polyoxyethylene sorbitan monooleate type surfactant such as MONTANOX 80 preferably is present .
  • the natural surfactant cholic acid may in certain types of cell culture provide a growth stimulating effect.
  • the culture medium contains from 0.035 to 7 mg/1 of sterol, more preferably from 0.07 to 3.5 mg/1, for instance about 0.7 mg/1.
  • the culture medium comprises from 1 to 200 mg/1 of said PLURONIC F68 type surfactant, more preferably 2 to 100 mg/1, more preferably about 20 mg/1.
  • the culture medium comprises from 0.12 to 25 mg/1 of said MONTANOX 80 type surfactant, more preferably 0.25 to 12.5 mg/1, e.g. about 2.5 mg/1.
  • the cell culture medium may further contain a soluble carboxylic acid or metabolically acceptable derivative thereof as a metabolic precursor of lipid fatty acids.
  • Said carboxylic acid is preferably a C 2 to C carboxylic acid which is preferably saturated and preferably contains one carboxylic acid group.
  • the carboxylic acid may be added as the acid as such or in the form of a metabolically acceptable salt or other derivative such as an ester.
  • said carboxylic acid or derivative is present as a concentration of from 0.02 mM to 4 mM, more preferably from 0.04 mM to 2mM, e.g. at about 0.4mM.
  • Said alcohol may be a Ci to C 4 mono-alcohol or diol .
  • Suitable alcohols include methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol or glycerol but ethanol is most preferred.
  • a mixture of ethanol and propylene glycol is particularly preferred, preferably at a ratio of about 1:0.5.
  • the medium may comprise polyvinylpyrrolidone such as PVP-10, molecular weight 10,000, preferably plant cell tested. Suitably this is present at a concentration of from 12.5 mg/1 to 2.5 g/1, more preferably 25 mg/1 to 1.25 g/1, e.g. about 250 mg/1.
  • the culture medium may in addition contain all or any of the ingredients in the proportions or amounts disclosed in US-A-5,045,454, W098/24883 or WO01/59069 for use in those inventions .
  • the medium preferably comprises one or more chelators preventing precipitation of iron at culture temperatures, typically 37 °C.
  • the most preferred chelators are EDTA (ethylenediaminetetraacetic acid) and citrate buffer.
  • EDTA ethylenediaminetetraacetic acid
  • the molar ratio of citrate buffer to EDTA is at least 3:1 citrate buffer: EDTA by molar concentration, more preferably about 10:1 citrate buffer: EDTA, where citrate buffer represents the sum of citric acid and citrate.
  • Iron is preferably supplied in the medium as the metal cheltate complex defined as Fe-EDTA together with citrate buffer. More particularly, there may be provided about 0.15 to 30 ⁇ M Fe-EDTA (more preferably 0.3-15 ⁇ m, e.g. about 3 ⁇ m) and about 0.05 to 10 ⁇ M Na 2 (or K 2 ) EDTA (more preferably 0.1 - 5 ⁇ M, e.g. about 1 ⁇ M) with about 0.75 to 150 ⁇ M citric acid (more preferably 1.5 to 75 ⁇ M, e.g. about 15 ⁇ M) and about 1.25 to 250 ⁇ M Na 3 Citrate (more preferably 2.5 to 125 ⁇ M, e.g. about 25 ⁇ M) .
  • An agent for presenting iron to the cells is preferably provided which preferably is aurintricarboxylic acid. This is preferably present at a concentration of from 0.15 to 30 ⁇ M, more preferably from 0.3 to 15 ⁇ M, e.g. 3 ⁇ M.
  • the pH of the medium may be between about 7.0 and 7.8, most preferably about 7.4 but the optimum may vary according to the nature of the cells in culture. Standard or modified basal media may be used as the bulk of the cell culture medium.
  • the basal media may be any of those known in the art including Eagle's MEM, Dulbecco's modified Eagle's MEM, Ham's F10/F12, DM110, CMRL, RPMI 1640, 199, L15, Fischer's or Waymouth' s MB 752/1.
  • the basal medium is RPMI 1640.
  • RPMI-X a modified RPMI 1640 containing 20 mM HEPES and pyruvate
  • DME/F12 is preferred for use for other types of cells.
  • Basal medium DM 110 is particularly preferred for adherent and suspension cell culture as it contains ingredients over and above those of older basal media such as RPMI that compensate for the absence of serum when it is used to make up a serum-free medium. It is a development of the DME/F12 medium and consists of al:l mixture of DME (DMEM) and Ham's MCDB 110. Ham's MCDB 110 is itself a further development of Ham's F12 medium. DME/F12 however also works well with the concentrates of the invention. Generally basal media designed for use in serum-free media are preferred.
  • the recommended concentration of bicarbonate is 2.2 g/1 and antibiotics such as penicillin or streptomycin should preferably not exceed 50 U/ml and 50 ⁇ g/ml respectively.
  • basal media may be added an appropriate amount of a concentrate as described herein.
  • the cell culture medium preferably comprises a polyamine such as putrescine or spermidine, preferably at a concentration of from 40 ⁇ g/1 to 0.8 mg/1, more preferably 80 ⁇ g/1 to 0.4 mg/1, e.g. about 0.08 mg/1.
  • a polyamine such as putrescine or spermidine
  • Long chain fatty acids such as lipoic acid and linoleic acid are preferably present at a concentration of from 7 ⁇ g/1 to 1.4 mg/1, more preferably from 14 ⁇ g/1 to 0.7 mg/1, e.g. about 0.14 mg/1.
  • Ethanolamine or a suitable substitute such as phospho- ethanolamine is preferably present at a concentration of 1 ⁇ M to 0.2 mM, more preferably 2 ⁇ M to 0.1 mM, e.g. about 0.02 M.
  • Calciferol (ergocalciferol or vitamin D) or any one of many commercially available analogues is preferably included at a concentration of from 5 ⁇ g/1 to 1 mg/1, more preferably from 10 ⁇ g/1 to 0.5 mg/1, e.g. about 0.1 mg/1.
  • Retinol acetate or one of many acceptable alternatives such as retinoic acid (vitamin A) is preferably present at a concentration of from 7 ⁇ g/1 to 1.4 mg/1, more preferably from 14 ⁇ g/1 to 0.7 mg/1, e.g. 0.14 mg/1.
  • retinoic acid vitamin A
  • the majority of the ingredients of the cell culture concentrate of the invention generally were disclosed in W098/24883.
  • cell culture medium concentrates for use at a dilution of lOOOx and the components were separated into concentrates referred to there as solutions A, B, Cx and Dx.
  • Solution Cx contained a very high concentration of ethanol to keep its sterol component in solution.
  • One aspect of the present invention is based on the discovery that it is possible to prepare a stable concentrate for use at a dilution of 20x as a direct replacement for fetal bovine serum as used conventionally at a 5% dilution in basal medium in cell culture practice.
  • a preferred cell culture medium of the invention comprises : -
  • a still more preferred cell culture medium comprises:-
  • An especially preferred cell culture medium comprises:
  • Said sterol preferably comprises stigmasterol and ⁇ - sitosterol in a ratio of 11:3 parts by weight.
  • Said polyoxyethylene sorbitan monooleate surfactant is preferably Montanox 80.
  • the polyvinylpyrrolidone is preferably PVP-10 (plant cell tested) and said polyethyleneglycol is preferably PEG MW 8000.
  • Said ⁇ -hydro- ⁇ -hydroxypoly (oxyethylene) poly (oxypropylene) poly (oxyethylene) block copolymer surfactant is preferably Pluronic F68.
  • the long chain fatty acid is a mixture of lipoic acid and linoleic acid at a ratio of about 2:0.8 by weight .
  • Such a culture medium preferably further comprises ⁇ M to nM quantities of trace elements.
  • the invention includes a concentration for dilution to form a cell culture medium as described above, preferably for dilution 1:20 to form said cell culture medium.
  • Figure 2 show cell growth results obtained in Example 3;
  • Figure 3 shows in graph form results obtained in Example 4 and
  • An example of a cell culture medium concentrate according to the invention for dilution 1:20 with basal cell culture medium is as follows :-
  • ATA Aurintricarboxylic acid 60 ⁇ M Na 2 -EDTA 20 ⁇ M
  • the resulting cell culture medium is suitable especially for the culture of adhesive cells.
  • Example 2 Vero cells (ATCC CCL 81) from stock cultures in serum- containing medium were cultured as an adherent monolayer in a conventional manner in culture media with and without aloin in 25 cm 2 Falcon Primeria T-flasks containing 5 ml medium, without change of medium.
  • the initial seeding density was 100 x 10 3 cells/ml.
  • the culture medium with aloin was obtained by diluting the adherent cell concentrate of Example 1 1:20 DM110 basal medium.
  • the control medium was the same except for the lack of aloin.
  • Example 3 Vero cells cultured in serum-free medium for four weeks were seeded at 200 x 10 3 cells/ml in 25 cm 2 Falcon Primaria T-flasks containing 5 ml of medium and were cultured as an adherent monolayer without change of medium for seven days. Viable cells were counted daily using an inverted microscope with a calibrated grid. Results from using serum-free medium produced by diluting the concentrate for adherent cells of Example 1 (5%) with DM 110 (95%) are shown in Figure 2.
  • the control here is a medium produced by adding 5% fetal bovine serum (FBS) to DME/F12 basal medium. It can be seen that the cell numbers continue to increase using the medium of the invention but plateau after four days using the control.
  • FBS fetal bovine serum
  • Example 4 Monoclonal IgG antibody was produced in T-flask culture by hybridoma IS7 (a low producer) in suspension culture in a known manner using three different media.
  • Medium 1 was a serum containing medium obtained by supplementing RPMI basal medium with 10 percent specially selected fetal bovine serum (FBS) .
  • FBS fetal bovine serum
  • Two alternative media according to the invention were used. The first was RPMI supplemented with 5 percent of the suspension cell culture concentrate of Example 1 (i.e. containing the extra Pluronic F68 and ethanol) and the second was DM110 similarly supplemented.
  • Antibody production was monitored over 8 to 9 days. The results are shown in Figure 2. Here there is a 130% increase in antibody concentration at Day 9 with the DM 110 based medium of the invention compared to the serum containing medium.
  • Example 4 was repeated but producing monoclonal IgG by the culture of a high producer hybridoma, EOl. The results are show in Figure 4. Here there is a 50% increase in antibody concentration at Day 10 as between the serum containing control and the DM 110 based serum-free medium.
  • Example 1 increase the growth of adherent cells and increase the antibody production of hybridoma cell lines compared to serum containing media.
  • the word . or' is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ⁇ exclusive or' which requires that only one of the conditions is met.
  • the word ⁇ comprising' is used in the sense of ⁇ including' rather than in to mean ⁇ consisting of .

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  • Wood Science & Technology (AREA)
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Abstract

L'invention concerne un milieu de culture cellulaire comprenant des ingrédients de culture cellulaire pour la maintenance de cellules d'eucaryotes, caractérisé en ce qu'il contient une quantité d'un facteur de croissance, l'aloïne, ou plus généralement un composé de formule: dans laquelle R1 représente un entité glycosylante, ou un atome d'H, un groupe alkyle, aryle, alkyle substitué, aryle substitué, alcoxy, alcoxyalkyle, alkylamine, R5-CO-R6 dans lequel R5 représente une liaison simple ou un groupe alkylène et R6 représente un atome d'H, un groupe alkyle, alkyle substitué, NH2, OH, ou OR7, dans lequel R7 représente un groupe alkyle ou alkyle substitué et chacun des R2, R3 et R4 représentant un alkyle, aryle, alkyle ou aryle substitué, ou carboxyalkyle, et n prenant la valeur 0 ou 1, en l'absence sensible de la gamme d'autres composés produits par la plante aloe avec laquelle ce composé peut être associé en nature.
PCT/EP2003/009951 2002-09-13 2003-09-08 Facteur de croissance pour culture cellulaire Ceased WO2004024899A1 (fr)

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AU2003264279A AU2003264279A1 (en) 2002-09-13 2003-09-08 Growth factor for cell culture

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GB0221330A GB0221330D0 (en) 2002-09-13 2002-09-13 Growth factor for cell culture
GB0221330.4 2002-09-13

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007036291A3 (fr) * 2005-09-28 2007-07-26 Cellca Gmbh Milieu de culture cellulaire ameliore
EP3383894B1 (fr) 2015-12-02 2020-05-06 CSL Behring Lengnau AG Milieux améliorés pour l'expression de protéines recombinantes vitamine k-dépendantes
CN112094802A (zh) * 2020-09-28 2020-12-18 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 (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023604A1 (fr) * 1994-03-03 1995-09-08 Namyang Aloe Co., Ltd. Compositions de stimulation de la croissance cellulaire renfermant de l'aloesine

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023604A1 (fr) * 1994-03-03 1995-09-08 Namyang Aloe Co., Ltd. Compositions de stimulation de la croissance cellulaire renfermant de l'aloesine

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
AVILA H ET AL: "CYTOTOXICITY OF A LOW MOLECULAR WEIGHT FRACTION FROM ALOE VERA (ALOE BARBADENSIS MILLER) GEL", TOXICON, ELMSFORD, NY, US, vol. 35, no. 9, 1997, pages 1423 - 1430, XP001104064, ISSN: 0041-0101 *
CHE Q-M ET AL: "BARBALOIN STIMULATES GROWTH OF EUBACTERIUM-SP STRAIN BAR A BARBALOIN-METABOLIZING BACTERIUM FROM HUMAN FECES", CHEMICAL AND PHARMACEUTICAL BULLETIN (TOKYO), vol. 39, no. 3, 1991, pages 757 - 760, XP002268645, ISSN: 0009-2363 *
MATSUDA HISASHI ET AL: "Phytoestrogens from the roots of Polygonum cuspidatum (Polygonaceae): Structure-requirement of hydroxyanthraquinones for estrogenic activity", BIOORGANIC AND MEDICINAL CHEMISTRY LETTERS, vol. 11, no. 14, 23 July 2001 (2001-07-23), pages 1839 - 1842, XP002268646, ISSN: 0960-894X *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007036291A3 (fr) * 2005-09-28 2007-07-26 Cellca Gmbh Milieu de culture cellulaire ameliore
JP2009509514A (ja) * 2005-09-28 2009-03-12 セルカ ゲーエムベーハー 改良された細胞培地
DE102005046225B4 (de) * 2005-09-28 2012-01-05 Cellca Gmbh Verbessertes Zellkulturmedium
US8426202B2 (en) 2005-09-28 2013-04-23 Cellca Gmbh Cell culture medium
EP3383894B1 (fr) 2015-12-02 2020-05-06 CSL Behring Lengnau AG Milieux améliorés pour l'expression de protéines recombinantes vitamine k-dépendantes
US12209261B2 (en) 2015-12-02 2025-01-28 CSL Behring Lengnau AG Media for the expression of recombinant vitamin K-dependent proteins
CN112094802A (zh) * 2020-09-28 2020-12-18 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

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GB0221330D0 (en) 2002-10-23

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