WO2001059069A1 - Cell culture media - Google Patents

Cell culture media Download PDF

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Publication number
WO2001059069A1
WO2001059069A1 PCT/EP2001/001262 EP0101262W WO0159069A1 WO 2001059069 A1 WO2001059069 A1 WO 2001059069A1 EP 0101262 W EP0101262 W EP 0101262W WO 0159069 A1 WO0159069 A1 WO 0159069A1
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Prior art keywords
cell culture
culture medium
component concentrate
medium component
weight
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Application number
PCT/EP2001/001262
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French (fr)
Inventor
Alan Juel Baekgaard
Original Assignee
Medi-Cult A/S
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Application filed by Medi-Cult A/S filed Critical Medi-Cult A/S
Priority to AU2001235452A priority Critical patent/AU2001235452A1/en
Publication of WO2001059069A1 publication Critical patent/WO2001059069A1/en

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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
    • C12N2500/30—Organic components
    • C12N2500/36—Lipids
    • 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
    • C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
    • 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

Definitions

  • the present invention relates to cell culture media and to concentrated components for use in making up cell culture media (cell culture media component concentrates) .
  • Cell culture media are used for the culture of a wide range of cell types under varying circumstances and for varying purposes, which may or may not involve the division and multiplication of the cells.
  • the term "cell culture medium” is used herein to refer to any medium in which cells are maintained in vitro and in active and viable state.
  • the culture of cells requires the supply to the cells of the materials which they need for the maintenance of viability and, if desired, for the production of cell products and/or the multiplication of the cells .
  • lipids which it is desired to include have a variety of differing functions. They include long chain fatty acids, fat soluble vitamins and sterols such as cholesterol. The direct addition of lipids is not practical because of their low solubility. Conventionally in serum containing media, the lipids are added to the medium in the serum, wherein the lipids are carried as soluble lipoproteins . In serum free media, lipids can be carried by albumen, but it is of course desirable to avoid the use of albumen also.
  • a number of publications including "Nutritional and Hormonal Requirements of Mammalian Cells in Culture", D. Barnes, Wld. Rev. Nutr. Diet., Vol.
  • lipids in cell culture via the inclusion of liposomes formed using phospholipids such as are found in such sources as cod liver oil ( O-A-8901027 and others) .
  • the cholesterol which is to be included in the medium may be added with other lipids in such liposomes.
  • Ethanol has been used in preparing culture media for the purpose of dissolving fatty materials including cholesterol and other hydrophobic compounds when making a concentrate to be diluted in aqueous media at a later stage, e.g. in WO-A-9204988.
  • Advantageous serum free cell culture media were described in 098/24883. A number of sterol lipids were disclosed there for use in such media.
  • a combination of stigmasterol and beta sitosterol offers particular advantages not predictable from W098/24883, in particular that the inclusion of beta sitosterol enhances the shelf life of component concentrates containing stigmasterol as determined by the time before visible precipitation of sterol or of Pluronic surfactant (if present) or other components occurs.
  • preferred formulations according to the invention provide improved cell morphology, higher cell growth and improved cell viability as well as allowing the culture of a wide range of cell types .
  • a cell culture medium containing stigmasterol and beta-sitosterol, or esters or other derivatives thereof acceptable for use in cell culture is preferably the acetate.
  • the proportions preferred weight proportions of stigmasterol and beta-sitosterol in said medium are from 1:200 to 1:0.005, more preferably from 1:1 to 1:0.005, still more preferably from 1:0.06 to 1:0.01, e.g. approximately 1:0.03.
  • a cell culture medium according to this aspect of the invention preferably contains from 0.001 to 0.015% by weight of said sterols in aggregate, more preferably about 0.01% of said sterols in aggregate .
  • a concentrate for use as a component in making up a ready to use strength cell culture medium was disclosed in W098/24883 as 'Solution Cx' which contained cholesterol, PVP-10 and ethanolamine dissolved in 96% ethanol. Whilst the performance of cell culture media based on this concentrate is good, the ethanol concentration needed to maintain the ingredients in solution poses a problem for transportation of the concentrate around the world, having regard to the flammability of the concentrate.
  • an aqueous culture medium component concentrate for use at a dilution of 400 times comprising: from 0 to 20% ethanol from 0.1 to 10% propylene glycol from 0.1 to 0.4% of a polyoxyethylene sorbitan fatty acid type surfactant from 0.5 to 7.5% an ⁇ -hydro-w-hydroxypoly (oxyethylene)poly (oxypropylene)poly (oxyethylene) block copolymer 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 5,000 to 75, 000
  • the amount of ethanol is preferably from 1 to 10% by weight, more preferably approximately 10% by weight, which is the upper limit permitted for air shipping without special arrangements.
  • the amount of propylene glycol is preferably from 0.2 to 0.7% by weight, more preferably approximately 0.5% by weight. Inclusion of higher or lower amounts than are indicated above to be preferred may cause less than optimal cell growth and/or encourage precipitation of components.
  • the amount of said sorbitan ester surfactant is preferably about 0.25% by weight. Too little of this surfactant may allow precipitation of less soluble components whilst too much may cause damage to cultured cells and precipitation of sterols and the surfactant .
  • the amount of said block copolymer surfactant is preferably from 0.12 to 4% by weight, more preferably approximately 0.15% by weight. Too little of this surfactant may allow precipitation of less soluble components whilst too much may cause damage to cultured cells.
  • the amount of ethanolamine is preferably from 0.005 to 0.05% by weight, more preferably about 0.012%. Not all cells make use of ethanolamine and these amounts have been selected to provide sufficient to be of use to those that do without damaging those that do not . so as to provide a medium suitable for a wide spectrum of cell types.
  • the amount of soluble fatty acid is preferably from 0.02 to 0.4% by weight, more preferably approximately 0.2% by weight.
  • the most preferred fatty acid is the precursor fatty and acetic acid but other soluble fatty acids may be used, e.g. C 2 to C 4 fatty acids .
  • the amount of sterol is preferably about 0.005 to 0.013% by weight, and preferably this is made up of a mixture of sterols in accordance with the first aspect of the invention.
  • said sufactant is PLURONIC F68.
  • said polyoxyethylene sorbitan fatty acid type surfactant is a polysorbate 20 to 90, most preferably a polysorbate 80.
  • the fatty acid component of said polyoxyethylene sorbitan fatty acid type surfacant is synthetic or is vegetable based.
  • the concentrate contains stigmasterol, more preferably in admixture with beta sitosterol, and probably in proportions discussed above.
  • the invention includes a cell culture medium component concentrate comprising in the approximate relative amounts shown below: ethanol 95ml
  • the sterol lipid is preferably not mammal derived. Synthetic or plant derived sterols are preferred. Sterols derived from non- mammalian animal sources will be acceptable, such as sterols extracted from fish.
  • the culture media is preferably serum-free and also protein- free except that it may be advantageous to add a small quantity of recombinant insulin or any non-animal substitute for insulin, as described in US-A-4045454.
  • Media containing insulin at the appropriate concentration for its use may still be regarded as substantially protein free.
  • the culture media based on concentrates of the present invention may be prepared by making up one of a number of standard or modified basal media and adding to it the additives disclosed in US-A-5045454 from one or more concentrate solutions and the further additives provided by the concentrates of the present invention.
  • the basal media may be any of those known in the art including Eagle's MEM, Dulbecco's modified Eagle's MEM, DM110, DMEM/F12, MCDB110, Ham's F10/F12, CMRL, RPMI 1640, 199, L15, Fischer's or Waymouth's MB 752/1 and all combinations of two or more thereof.
  • the basal medium is RPMI 1640.
  • RPMI-X (a modified RPMI 1640 containing 20mM HEPES and pyruvate) is preferred for the culture of cells of the leukocyte and reticulo-endothelial lineages, including lymphocyte hybridomas, while DME/F12 is preferred for use for other types of cells.
  • the recommended concentration of bicarbonate is between 1.0 to 4.8 g/1 and antibiotics such as penicillin or streptomycin should preferably not exceed 50 U/ml and 50 ⁇ g/ml respectively.
  • PLURONIC F68 e.g. a "solution A" (US 5045454, Col. 5) containing 20 mg/ml PLURONIC F68, about 4 mM EDTA, about 3 mM Fe, about 40 mM sodium citrate/citric acid, about 3 mM aurintricarboxylic acid and optionally about 1% trace elements.
  • concentration A US 5045454, Col. 5
  • trace elements may comprise Mn (about 1 ⁇ M) , Cr
  • the basal culture medium may be further supplemented with an appropriate quantity of a further concentrate solution of the kind referred to as "Solution B" in US-A-5045454 comprising about 0.5 to 10 mg/ml insulin (lOOOx) .
  • the cell culture medium may be further supplemented using a concentrate "Solution Dx" as described in W098/24883) containing ethanol and PVP-10, e.g. a lOOOx solution containing: ethanol 96% 500 ml water 500 ml
  • PLURONIC F68 lOOg (Serva 35724 or Sigma P-1300) .
  • Example 1 The invention will be illustrated by the following example.
  • Example 1 The invention will be illustrated by the following example.
  • a preferred cell culture medium component concentrate (x400) has the composition:
  • The, EtOH, propylene glycol Tween 80, and Pluronic F68 are mixed together .
  • Pluronic F68 is dissolved by stirring.
  • Pluronic F68 is used as an anti-foam agent and surfactant and is therefore of importance for the morphology of the cells in the culture.
  • Tween also acts as an anti-foam agent in the medium.
  • Ethanolamine is added to the solution and stirred to achieve good mixing .
  • lipids are then added and are fully dissolved in the mixture .
  • Acetic acid is added as a lipid precursor.
  • the product is non-milky and not cloudy but appears as clear as water without any visible micelles making the product easy to produce, workable and unique in only using very small amounts of Pluronic F68 and Tween 80.
  • the lack of micelles also makes it more easy for the cells in the culture to use the lipids.
  • beta sitosterol is to extend the shelf-life of the concentrate, i.e. to defer the commencement of precipitation of the less soluble components.
  • Figure 1 shows a comparison of the growth rate of Vero cells in basal medium supplemented with either the composition of Example 1 (Cx2-B) or the Cx concentrate disclosed in W098/24883.
  • the experiment was performed seeding 100000 Vero cells/ml at day 0 and then cells were harvested and counted using blue staining each second day.
  • FIG. 3 shows the growth obtained with CHO-K1 cells and with Vero cells using a basal medium supplemented with various single sterols and combinations of sterols .
  • Figure 4 shows the morphology of the cells in the cell cultures of Figure 3.
  • Figure 5 shows growth obtained with mouse hybridoma cells in basal medium supplemented in a similar way as in Figure 3.
  • the cells were cultured in an adherent monolayer in 25 cm 3 Falcon T-flasks containing 5ml medium, without change of medium during the test . There were two subsequent passages.
  • the initial seeding density was 40xl0 3 cells/ml.
  • the basal medium was DME/F12 supplemented with the SSR3x concentrate of W098/24883 (but without cholesterol) containing polyethylene glycol and supplemented with the sterol or sterol mixture shown below.
  • List of cholesterol analogue mixtures :
  • the cells were cultured in suspension in 25ml Corning T-flasks containing 5 ml medium, without change of medium through 3 passages at an initial seeding density of 40xl0 3 cells/ml in basal medium RPMI-X supplemented with the SSR4x concentrate of W098/24883 (but without cholesterol) containing polyethylene glycol and supplemented with the sterol or sterol mixture shown above.
  • the figure shows the number of viable cells of good morphology counted by microscopy.

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Abstract

A cell culture medium comprises stigmasterol and β-sitosterol or esters or other derivatives thereof, with ethanol, propylene glycol, Pluronic F68, Polysorbate 80, ethanolamine and acetic acid.

Description

CELL CULTURE MEDIA
The present invention relates to cell culture media and to concentrated components for use in making up cell culture media (cell culture media component concentrates) .
Cell culture media are used for the culture of a wide range of cell types under varying circumstances and for varying purposes, which may or may not involve the division and multiplication of the cells. The term "cell culture medium" is used herein to refer to any medium in which cells are maintained in vitro and in active and viable state.
As is well understood in this art, the culture of cells requires the supply to the cells of the materials which they need for the maintenance of viability and, if desired, for the production of cell products and/or the multiplication of the cells .
The inclusion of lipids in cell culture media is a long standing problem. Lipids which it is desired to include have a variety of differing functions. They include long chain fatty acids, fat soluble vitamins and sterols such as cholesterol. The direct addition of lipids is not practical because of their low solubility. Conventionally in serum containing media, the lipids are added to the medium in the serum, wherein the lipids are carried as soluble lipoproteins . In serum free media, lipids can be carried by albumen, but it is of course desirable to avoid the use of albumen also. A number of publications including "Nutritional and Hormonal Requirements of Mammalian Cells in Culture", D. Barnes, Wld. Rev. Nutr. Diet., Vol. 45 pp 167-197 describe the provision of lipids in cell culture via the inclusion of liposomes formed using phospholipids such as are found in such sources as cod liver oil ( O-A-8901027 and others) . For convenience, the cholesterol which is to be included in the medium may be added with other lipids in such liposomes. Ethanol has been used in preparing culture media for the purpose of dissolving fatty materials including cholesterol and other hydrophobic compounds when making a concentrate to be diluted in aqueous media at a later stage, e.g. in WO-A-9204988. Advantageous serum free cell culture media were described in 098/24883. A number of sterol lipids were disclosed there for use in such media. We have now found that a combination of stigmasterol and beta sitosterol offers particular advantages not predictable from W098/24883, in particular that the inclusion of beta sitosterol enhances the shelf life of component concentrates containing stigmasterol as determined by the time before visible precipitation of sterol or of Pluronic surfactant (if present) or other components occurs. Furthermore, preferred formulations according to the invention provide improved cell morphology, higher cell growth and improved cell viability as well as allowing the culture of a wide range of cell types .
Accordingly, there is now provided according to a first aspect of the invention a cell culture medium containing stigmasterol and beta-sitosterol, or esters or other derivatives thereof acceptable for use in cell culture . Where an ester is used it is preferably the acetate.
The proportions preferred weight proportions of stigmasterol and beta-sitosterol in said medium are from 1:200 to 1:0.005, more preferably from 1:1 to 1:0.005, still more preferably from 1:0.06 to 1:0.01, e.g. approximately 1:0.03.
A cell culture medium according to this aspect of the invention preferably contains from 0.001 to 0.015% by weight of said sterols in aggregate, more preferably about 0.01% of said sterols in aggregate . A concentrate for use as a component in making up a ready to use strength cell culture medium was disclosed in W098/24883 as 'Solution Cx' which contained cholesterol, PVP-10 and ethanolamine dissolved in 96% ethanol. Whilst the performance of cell culture media based on this concentrate is good, the ethanol concentration needed to maintain the ingredients in solution poses a problem for transportation of the concentrate around the world, having regard to the flammability of the concentrate. We have now discovered that by alteration of the proportions of the components of said concentrate and by including further ingredients therein it is possible to form a stable solution using a much reduced ethanol concentration, thus removing the transportation problems. Accordingly, there is provided according to a second aspect of the invention an aqueous culture medium component concentrate for use at a dilution of 400 times comprising: from 0 to 20% ethanol from 0.1 to 10% propylene glycol from 0.1 to 0.4% of a polyoxyethylene sorbitan fatty acid type surfactant from 0.5 to 7.5% an α-hydro-w-hydroxypoly (oxyethylene)poly (oxypropylene)poly (oxyethylene) block copolymer surfactant of the structure HO (CH2CH20) a (CH (CH3) -CH2OH) b (CH2 CH20)cH 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 5,000 to 75, 000 from 0 to 15% ethanolamine from 0 to 0.5% of a soluble carboxylic acid, and from 0.001 to 0.15% of a sterol lipid (all percentages by weight) .
The amount of ethanol is preferably from 1 to 10% by weight, more preferably approximately 10% by weight, which is the upper limit permitted for air shipping without special arrangements. The amount of propylene glycol is preferably from 0.2 to 0.7% by weight, more preferably approximately 0.5% by weight. Inclusion of higher or lower amounts than are indicated above to be preferred may cause less than optimal cell growth and/or encourage precipitation of components.
The amount of said sorbitan ester surfactant is preferably about 0.25% by weight. Too little of this surfactant may allow precipitation of less soluble components whilst too much may cause damage to cultured cells and precipitation of sterols and the surfactant .
The amount of said block copolymer surfactant is preferably from 0.12 to 4% by weight, more preferably approximately 0.15% by weight. Too little of this surfactant may allow precipitation of less soluble components whilst too much may cause damage to cultured cells.
The amount of ethanolamine is preferably from 0.005 to 0.05% by weight, more preferably about 0.012%. Not all cells make use of ethanolamine and these amounts have been selected to provide sufficient to be of use to those that do without damaging those that do not . so as to provide a medium suitable for a wide spectrum of cell types.
The amount of soluble fatty acid is preferably from 0.02 to 0.4% by weight, more preferably approximately 0.2% by weight. The most preferred fatty acid is the precursor fatty and acetic acid but other soluble fatty acids may be used, e.g. C2 to C4 fatty acids .
The amount of sterol is preferably about 0.005 to 0.013% by weight, and preferably this is made up of a mixture of sterols in accordance with the first aspect of the invention.
Preferably, in the said copolymer surfactant a=75, b=30 and c=75 and the molecular weight is about 9350. Most preferably, said sufactant is PLURONIC F68. Preferably, said polyoxyethylene sorbitan fatty acid type surfactant is a polysorbate 20 to 90, most preferably a polysorbate 80. Preferably, the fatty acid component of said polyoxyethylene sorbitan fatty acid type surfacant is synthetic or is vegetable based.
Preferably, as said sterol lipid, the concentrate contains stigmasterol, more preferably in admixture with beta sitosterol, and probably in proportions discussed above.
The invention includes a cell culture medium component concentrate comprising in the approximate relative amounts shown below: ethanol 95ml
1 , 2 propane diol 5ml polysorbate 80 2.7g(3ml)
Pluronic F68 15g
PEG8000 O.lg ethanolamine 0.120ml acetic acid 2.0ml stigmasterol (95%) 0.04 to O.lg beta sitosterol (95%) 0.012 to 0.03g water to 1000ml Culture media as described above can be formulated as a solution of water clear appearance.
The sterol lipid is preferably not mammal derived. Synthetic or plant derived sterols are preferred. Sterols derived from non- mammalian animal sources will be acceptable, such as sterols extracted from fish.
The culture media is preferably serum-free and also protein- free except that it may be advantageous to add a small quantity of recombinant insulin or any non-animal substitute for insulin, as described in US-A-4045454. Media containing insulin at the appropriate concentration for its use may still be regarded as substantially protein free. Analogous to the methods described there, the culture media based on concentrates of the present invention may be prepared by making up one of a number of standard or modified basal media and adding to it the additives disclosed in US-A-5045454 from one or more concentrate solutions and the further additives provided by the concentrates of the present invention.
The basal media may be any of those known in the art including Eagle's MEM, Dulbecco's modified Eagle's MEM, DM110, DMEM/F12, MCDB110, Ham's F10/F12, CMRL, RPMI 1640, 199, L15, Fischer's or Waymouth's MB 752/1 and all combinations of two or more thereof. Preferably however, the basal medium is RPMI 1640. RPMI-X (a modified RPMI 1640 containing 20mM HEPES and pyruvate) is preferred for the culture of cells of the leukocyte and reticulo-endothelial lineages, including lymphocyte hybridomas, while DME/F12 is preferred for use for other types of cells.
The recommended concentration of bicarbonate is between 1.0 to 4.8 g/1 and antibiotics such as penicillin or streptomycin should preferably not exceed 50 U/ml and 50μg/ml respectively.
To such basal media may be added an appropriate amount of a "SSR2" as described in US-A-5045454, i.e. a solution containing
EDTA, citrate, iron and aurintricarboxylic acid as well as optionally PLURONIC F68, e.g. a "solution A" (US 5045454, Col. 5) containing 20 mg/ml PLURONIC F68, about 4 mM EDTA, about 3 mM Fe, about 40 mM sodium citrate/citric acid, about 3 mM aurintricarboxylic acid and optionally about 1% trace elements. These amounts are suitable for a 100Ox formulation, i.e. a solution containing 1000 times the concentration of each ingredient desired in the final solution and which is therefore intended to be diluted with 1000 parts of the basal culture medium. The trace elements may comprise Mn (about 1 μM) , Cr
(about 1 μM) , Zn (about 0.1 mM) , Ni (about 0.2 μM) , Co (about 0.2 μm) , Cu (about 20 μM) , Al (about 2 μM) and Se (about 10 μM) . The basal culture medium may be further supplemented with an appropriate quantity of a further concentrate solution of the kind referred to as "Solution B" in US-A-5045454 comprising about 0.5 to 10 mg/ml insulin (lOOOx) . Depending on its intended use, the cell culture medium may be further supplemented using a concentrate "Solution Dx" as described in W098/24883) containing ethanol and PVP-10, e.g. a lOOOx solution containing: ethanol 96% 500 ml water 500 ml
PLURONIC F68 lOOg (Serva 35724 or Sigma P-1300) .
The invention will be illustrated by the following example. Example 1
A preferred cell culture medium component concentrate (x400) has the composition:
a) EtOH 95 ml b) Propylene Glycol (1,2 propane diol) 5 ml c) Tween 80 (Montanox, plant based) 3 ml (2.5g) d) Pluronic F68 15 g e) Ethanolamine 0.120 ml f) Acetic acid 2.0 ml g) Stigmasterol (plant based lipid) 1.0 g h) Beta-Sitosterol (plant based lipid) 0.030 ml i) H20 to 1000 ml
Procedure for -production
The, EtOH, propylene glycol Tween 80, and Pluronic F68 are mixed together . Pluronic F68 is dissolved by stirring. Pluronic F68 is used as an anti-foam agent and surfactant and is therefore of importance for the morphology of the cells in the culture. Tween also acts as an anti-foam agent in the medium. Ethanolamine is added to the solution and stirred to achieve good mixing .
The lipids are then added and are fully dissolved in the mixture . Acetic acid is added as a lipid precursor.
Water is added to final volume
The product is non-milky and not cloudy but appears as clear as water without any visible micelles making the product easy to produce, workable and unique in only using very small amounts of Pluronic F68 and Tween 80. The lack of micelles also makes it more easy for the cells in the culture to use the lipids.
The effect of the inclusion of the small quantity of beta sitosterol is to extend the shelf-life of the concentrate, i.e. to defer the commencement of precipitation of the less soluble components.
The superiority of the media according to the present invention is further demonstrated by the results shown in the accompanying drawings, in which: Figure 1 shows a comparison of the growth rate of Vero cells in basal medium supplemented with either the composition of Example 1 (Cx2-B) or the Cx concentrate disclosed in W098/24883. The experiment was performed seeding 100000 Vero cells/ml at day 0 and then cells were harvested and counted using blue staining each second day. This X-axis = days of harvest and the Y-axis = cell no. X 10000.
Figure 2 shows the results of a similar comparative test using CHO t-PA cells. The experiment was performed using 50000 cells/ml at day (0) and then cells were harvested at day 2-10 and counted using blue staining. The X-axis = days of harvest and the
Y-axis - cell no. X 1000/ml. Figure 3 shows the growth obtained with CHO-K1 cells and with Vero cells using a basal medium supplemented with various single sterols and combinations of sterols .
Figure 4 shows the morphology of the cells in the cell cultures of Figure 3.
Figure 5 shows growth obtained with mouse hybridoma cells in basal medium supplemented in a similar way as in Figure 3.
In Figures 1 and 2, it can be seen that faster growth is obtained, especially toward the end of the growth period, using the concentrate according to the invention with both cell types tested.
In Figures 3 and 4, the cell lines tested were as follows: CHO-K1 ATTC CCL61 Chinese hamster ovary epithelium Vero ATCC CCL81 Monkey kidney fibroblast
The cells were cultured in an adherent monolayer in 25 cm3 Falcon T-flasks containing 5ml medium, without change of medium during the test . There were two subsequent passages. The initial seeding density was 40xl03 cells/ml. The basal medium was DME/F12 supplemented with the SSR3x concentrate of W098/24883 (but without cholesterol) containing polyethylene glycol and supplemented with the sterol or sterol mixture shown below. List of cholesterol analogue mixtures:
1 Cholesterol 2 μg/ml
2 Stigmasterol 1 μg/ml
3 mix Stigmasterol 1 μg/ml + β-sitosterol 60 % 0,3 μg/ml
4 mix Stigmasterol 1 μg/ml + β-sitosterol 95 % synth. 0,1 μg/ml
5 mix Stigmasterol 1 μg/ml + β-sitosterol 95 % synth. 0,3 μg/ml 6 mix Stigmasterol 1 μg/ml + β-sitosterol 95 % synth. 1 μg/ml
7 = 5 + Campesterol 0 , 3 μg/ml C negative control (no sterol) 8 β-sitosterol 95 % synth 1 μg/ml
It can be seen that both as regards growth (Fig. 3) and morphology (Fig. 4), superior results are obtained using the mixture of sterols according to the first aspect of the invention compared to cholesterol, β-sitosterol or stigmasterol alone. In Figure 5, the cell line tested is 1E6 ECACC 86112001, mouse hybridoma fusion Balb/c with P3Uχ myeloma. The cells were cultured in suspension in 25ml Corning T-flasks containing 5 ml medium, without change of medium through 3 passages at an initial seeding density of 40xl03 cells/ml in basal medium RPMI-X supplemented with the SSR4x concentrate of W098/24883 (but without cholesterol) containing polyethylene glycol and supplemented with the sterol or sterol mixture shown above. The figure shows the number of viable cells of good morphology counted by microscopy.
Once again it can be seen that better results are obtained with the mixture of sterols of the invention than with the controls .
Many variations and modifications of the invention are possible within the scope of the invention.

Claims

1. A cell culture medium containing stigmasterol and beta- sitosterol, or esters or other derivatives thereof acceptable for use in cell culture.
2. A cell culture medium as claimed in Claim 1, wherein the proportions of stigmasterol and beta-sitosterol in said medium are from 1:200 to 1:0.005.
3. A cell culture medium as claimed in Claim 1, wherein the proportions of stigmasterol and beta-sitosterol in said medium are from 1:1 to 1:0.005.
4. A cell culture medium as claimed in Claim 1, wherein the proportions of stigmasterol and beta-sitosterol in said medium are from 1:0.06 to 1:0.01.
5. A cell culture medium as claimed in Claim 1, wherein the proportions of stigmasterol and beta-sitosterol in said medium are approximately 1:0.03.
6. A cell culture medium as claimed in any preceding claim, containing from 0.001 to 0.015% of said sterols in aggregate.
7. A cell culture medium as claimed in Claim 6, containing about 0.01% of said sterols in aggregate.
8. An aqueous cell culture medium component concentrate comprising: from 0 to 20% ethanol from 0.1 to 10% propylene glycol from 0.1 to 0.4% of a polyoxyethylene sorbitan fatty acid type surfactant from 0.5 to 7.5% an α-hydro-w-hydroxypoly (oxyethylene) poly (oxypropylene)poly (oxyethylene) block copolymer surfactant of the structure HO (CH2CH20) a (CH (CH3) -CH2OH) b (CH2 CH20) CH 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 5,000 to 75,000 from 0 to 0.15% ethanolamine from 0 to 0.5% of a soluble carboxylic acid, and from 0.001 to 0.015% of a sterol lipid (all percentages by weight) .
9. A cell culture medium component concentrate as claimed in Claim 8, wherein the amount of ethanol is from 1 to 10% by weight .
10. A cell culture medium component concentrate as claimed in Claim 8, wherein the amount of ethanol is approximately 10% by weight .
11. A cell culture medium component concentrate as claimed in any one of Claims 8 to 10, wherein the amount of propylene glycol is from 0.2 to 0.7% by weight.
12. A cell culture medium component concentrate as claimed in Claim 11, wherein the amount of propylene glycol is approximately 0.5% by weight.
13. A cell culture medium component concentrate as claimed in any one of Claims 8 to 12 , wherein the amount of said sorbitan ester surfactant is about 0.25% by weight.
14. A cell culture medium component concentrate as claimed in any one of Claims 8 to 13, wherein the amount of said block copolymer surfactant is from 0.12 to 4% by weight.
15. A cell culture medium component concentrate as claimed in Claim 14, wherein the amount of said block copolymer surfactant is approximately 0.15% by weight.
16. A cell culture medium component concentrate as claimed in any one of Claims 8 to 15, wherein the amount of ethanolamine is from 0.005 to 0.05% by weight.
17. A cell culture medium component concentrate as claimed in Claim 16 wherein said amount of ethanolamine is about 0.012%.
18. A cell culture medium component concentrate as claimed in any one of Claims 8 to 17, wherein the amount of soluble fatty acid is from 0.2 to 0.4% by weight.
19. A cell culture medium component concentrate as claimed in
Claim 18, wherein the amount of soluble fatty acid is approximately 0.2% by weight.
20. A cell culture medium component concentrate as claimed in any one of Claims 8 to 19, wherein the amount of sterol is about
0.013% by weight.
21. A cell culture medium component concentrate as claimed in any one of Claims 8 to 20, wherein, in the said copolymer surfactant a=75, b=30 and c=75 and the molecular weight is about 8350.
22. A cell culture medium component concentrate as claimed in Claim 22, wherein said surfactant is PLURONIC F68.
23. A cell culture medium component concentrate as claimed in any one of Claims 8 to 22, wherein said polyoxyethylene sorbitan fatty acid type surfactant is a polysorbate 20 to 90.
24. A cell culture medium component concentrate as claimed in any one of Claims 8 to 22 , wherein said polyoxyethylene sorbitan fatty acid type surfactant is a polysorbate 80 of non-animal or plant origin.
25. A cell culture medium component concentrate as claimed in any one of Claims 8 to 23, wherein the fatty acid component of said polyoxyethylene sorbitan fatty acid type surfactant is synthetic or is vegetable based.
26. A cell culture medium component concentrate as claimed in any one of Claims 8 to 25, wherein as said sterol lipid, the concentrate contains stigmasterol.
27. A cell culture medium component concentrate as claimed in Claim 26, wherein as said sterol lipid, the concentrate contains stigmasterol in admixture with beta sitosterol.
28. A cell culture medium component concentrate as claimed in Claim 27, wherein the proportions of the said sterols are in accordance with any one of Claims 2 to 5.
29. A cell culture medium component concentrate comprising in the approximate relative amounts shown below: ethanol 95ml
1,2 propane diol 5ml polysorbate 80 2.7g(3ml)
Pluronic F68 15g
PEG8000 O.lg ethanolamine 0.120ml acetic acid 2.0ml stigmasterol (95%) O.lg beta sitosterol (95%) 0.030g water to 1000ml
30. A cell culture medium component concentrate as claimed in any one of Claims 1 to 29 which is a solution of water clear appearance .
PCT/EP2001/001262 2000-02-11 2001-02-07 Cell culture media WO2001059069A1 (en)

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