WO1995029231A1 - Serum-free medium supplement - Google Patents

Serum-free medium supplement Download PDF

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Publication number
WO1995029231A1
WO1995029231A1 PCT/US1995/004885 US9504885W WO9529231A1 WO 1995029231 A1 WO1995029231 A1 WO 1995029231A1 US 9504885 W US9504885 W US 9504885W WO 9529231 A1 WO9529231 A1 WO 9529231A1
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Prior art keywords
serum
medium supplement
eukaryotic cell
cell culture
culture medium
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Ceased
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PCT/US1995/004885
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French (fr)
Inventor
Nick C. Wan
Jason C. Goodrick
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Genzyme Corp
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Genzyme Corp
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Publication date
Application filed by Genzyme Corp filed Critical Genzyme Corp
Priority to AU23598/95A priority Critical patent/AU702402B2/en
Priority to JP52777095A priority patent/JPH09512171A/en
Priority to EP95917608A priority patent/EP0763102A4/en
Publication of WO1995029231A1 publication Critical patent/WO1995029231A1/en
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
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/38—Vitamins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • mammalian cell culture provides the only viable production source.
  • Mammalian cells have the capability to synthesize such agents with the proper configuration, correct disulfide bonding, and arrays of sugar side chains, all of which result in the desired activity of the naturally occurring agent. Therefore, many agents derived from mammalian cells are more likely to be efficacious and are less likely to be immunogenic in target mammals if expressed by bacterial or yeast fermentation.
  • Feeding with concentrated nutrients is the preferred in vitro cultivation strategy because the product can be recovered more economically from a smaller volume of liquid, i.e., more concentrated.
  • Methods of supplementation involve either boosting the concentration of nutrients in a basal formulation or feeding the culture with supplements (Jo E.C. et al, UK Patent Application #2251249A, 1992; Jo E.C. et al, Biotechnol & Bioena. 42:1229-1237, 1993; Luan Y.T., Biotechnol. Letters 9:691:696, 1987).
  • serum has to be present in the feeding media which complicates subsequent purification procedures and increases production costs.
  • This invention relates to a serum-free eukaryotic cell culture medium supplement.
  • the supplement comprises carbon sources, vitamins, inorganic salts, amino acids and a protein digest.
  • the medium supplement of the present invention enables the maintenance of mammalian cell cultures at cell densities equal to or greater than that obtained with batch culture methods while increasing longevity and productivity.
  • Figure 1 shows a bar graph illustrating the monoclonal antibody production and cell density of one hybridoma cell line when fed with the medium supplement of the present invention versus the same culture without supplementation.
  • Figure 2 shows a graph of the final monoclonal antibody production of 16 different hybridomas using fed-batch method of cell culture with the addition of the medium supplement of the present invention versus that achieved through batch culture (i.e., without supplement feeding)
  • Figure 3 shows a graph of the maximum viable cell number of the 16 hybridomas in the fed-batch mode with supplementation using the medium supplement of the present invention versus the batch method of cell culture.
  • Figure 4 shows a graph of the longevity of the 16 hybridomas in the fed-batch mode plus the medium supplement of the present invention versus batch method.
  • This invention is based upon the discovery of a serum- free medium supplement which can be used to maintain a eukaryotic cell line viable in cell culture.
  • the supplement comprises carbon sources, vitamins, inorganic salts, amino acids and a protein digest.
  • supply is a buffered solution containing a concentrated amount of nutrients which when added to an in vitro eukaryotic cell line, maintains viability.
  • the supplement of the present invention can be added to an in vitro eukaryotic cell culture medium without the need to remove old or spent medium.
  • batch mode of cell culture describes a method of culturing cells where cells are seeded into a cell culture vessel containing an initial volume of nutrient medium (such as Dulbecco's Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS) or Protein-Free Hybridoma Medium (PFHM-II), wherein the initial volume of nutrient medium is not replenished with any medium.
  • DMEM Dulbecco's Modified Eagles Medium
  • FBS fetal bovine serum
  • PFHM-II Protein-Free Hybridoma Medium
  • fed-batch mode of cell culture describes a method of culturing cells where cells are seeded into a cell culture vessel containing an initial volume of nutrient medium and where supplements are added to the medium in a continuous or semi- continuous manner.
  • the medium of the present invention includes a carbon source.
  • Suitable carbon sources include L-glutamine and D-glucose.
  • a carbon source containing L-glutamine in a concentration of about 7.3 grams per liter . (g/L) and D-glucose in a concentration of 25 g/L are preferred.
  • the medium of the present invention comprises vitamins. Suitable vitamins include a biotin, choline chloride, a folic acid, an inositol, a niacinamide, benzoic acid, a pantothenic acid, a pyridoxine, a riboflavin, a thiamine and B vitamin or mixture thereof. A mixture containing the vitamins listed in Table 1 below is preferred. Table 1 VITAMIN MIXTURE
  • the medium of the present invention comprises inorganic salts.
  • Suitable inorganic salts include potassium chloride, potassium phosphate, sodium chloride and sodium phosphate or a mixture. A mixture containing the inorganic salts listed in Table 2 below is preferred.
  • the medium of the present invention further comprises amino acids.
  • Suitable amino acids include alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glycine, histidine, proline, isoleucine, lysine, methionine, serine, threonine, trytophan, tyrosine and valine or a mixture thereof.
  • a mixture containing the amino acids listed in Table 3 below is preferred. Table 3 AMINO ACID MIXTURE
  • the medium of the present invention comprises a protein digest.
  • Suitable protein digests include primatone CLT, casein or enzymatic hydrolysates.
  • the medium comprises primatone RL in the amount of 25 g/L of the water component of the medium.
  • Frozen hybridoma cells (16 distinct cell lines) were thawed quickly at 37°C and transferred into DMEM or PFHM (Gibco, N.Y.) with 10% fetal bovine serum and grown in t- flasks. The cells were maintained in the t-flasks at 37 ° C and 5- 10% CO2 until the cultures reached 40-70% maximum viable density.
  • the cells were then expanded into 1, 2, or 8-L spinner flasks with a starting viable cell density of at least 0.1-0.2 million/ml as determined by hemacytometer and trypan blue staining.
  • the 1-L and 3-L flasks were maintained at 37+ 1C and agitated at 50-80 rpm
  • D-Glucose 25.0 vitamin mixture (Table 1) 1.0914 inorganic salt mixture (Table 2) 2.3875 amino acid mixture (Table 3) 7.8829
  • Feeding was continued daily and D-glucose was maintained above 1 g/L (45% D-glucose solution) until the viable cell density had dropped below 0.3 million/ml.
  • the culture was harvested, the cells removed, and the product purified from the culture with the appropriate purification procedure.
  • a 0.5 to 1.0 ml sample of the culture was aseptically removed from the culture in a laminar flow hood and placed in a microfuge tube.
  • the cell suspension was diluted with Trypan blue/ PBS (0.4%) and mix thoroughly.
  • a cover slip was placed on a hemocytometer and a small amount of the cell mixture placed into the chambers. The chambers were allowed to be filled by capillary action.
  • the hemocytometer was viewed under a microscope under 10x10 power and cells in all 8 of the outer boxes were counted and recorded. Both viable (clear white) and non-viable (blue) cells were counted.
  • the wells were filled with 200 ⁇ l of 2% Bovine Serum Albumin (BSA) solution in PBS to block residual binding sites. After 30 minutes incubation at room temperature (RT), the blocking reagent was removed and 100 ⁇ l of the diluted culture supernatant (PBS containing 0.05% Tween 20 and 0.5% BSA diluant) or standard antibody preparation (0 to 20 ng/ml) was added to each well.
  • BSA Bovine Serum Albumin
  • Glucose, lactate, and ammonium concentrations in culture samples were measured by an IBI Biolyzer (International Biotechnologies, Inc., New Haven, CT) following the directions provided.

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  • 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

This invention relates to a serum-free eukaryotic cell culture medium supplement. The supplement comprises carbon sources, vitamins, inorganic salts, amino acids and a protein digest. The medium supplement of the present invention enables the maintenance of mammalian cell cultures at cell densities equal to or greater than that obtained with batch culture methods while increasing longevity and productivity.

Description

SERUM-FREE MEDIUM SUPPLEMENT
Background of Invention
Since the development of the in vitro cultivation of mammalian cells the demand for large scale production of these cells has increased due to diagnostic and therapeutic potential of many of the products they produce. These useful agents include monoclonal antibodies, human growth hormone, lymphokines, erythropoietin, blood clotting factors and tissue plasminogen activators.
For many of these cellular agents mammalian cell culture provides the only viable production source. Mammalian cells have the capability to synthesize such agents with the proper configuration, correct disulfide bonding, and arrays of sugar side chains, all of which result in the desired activity of the naturally occurring agent. Therefore, many agents derived from mammalian cells are more likely to be efficacious and are less likely to be immunogenic in target mammals if expressed by bacterial or yeast fermentation.
To improve productivity, many medium formulations for feeding of mammalian cultures have been suggested (Fike et ah, BioPharm. Oct.: 49-54, 1993). Some suggested medium formulations use unconcentrated nutrients which significantly increase the final culture volume and thus complicate the process of recovery Reuveny et al. Develop. Biol Standard 60: 185-197, 1985).
Feeding with concentrated nutrients (i.e. supplements) is the preferred in vitro cultivation strategy because the product can be recovered more economically from a smaller volume of liquid, i.e., more concentrated. Methods of supplementation involve either boosting the concentration of nutrients in a basal formulation or feeding the culture with supplements (Jo E.C. et al, UK Patent Application #2251249A, 1992; Jo E.C. et al, Biotechnol & Bioena. 42:1229-1237, 1993; Luan Y.T., Biotechnol. Letters 9:691:696, 1987). There have been only a few reports on such feeding strategy. However, serum has to be present in the feeding media which complicates subsequent purification procedures and increases production costs. A need exists to develop a serum-free supplement for use in mammalian cell cultures.
Summary of the Invention This invention relates to a serum-free eukaryotic cell culture medium supplement. The supplement comprises carbon sources, vitamins, inorganic salts, amino acids and a protein digest.
The medium supplement of the present invention enables the maintenance of mammalian cell cultures at cell densities equal to or greater than that obtained with batch culture methods while increasing longevity and productivity.
Brief Description of the Drawings
Figure 1 shows a bar graph illustrating the monoclonal antibody production and cell density of one hybridoma cell line when fed with the medium supplement of the present invention versus the same culture without supplementation.
Figure 2 shows a graph of the final monoclonal antibody production of 16 different hybridomas using fed-batch method of cell culture with the addition of the medium supplement of the present invention versus that achieved through batch culture (i.e., without supplement feeding)
Figure 3 shows a graph of the maximum viable cell number of the 16 hybridomas in the fed-batch mode with supplementation using the medium supplement of the present invention versus the batch method of cell culture.
Figure 4 shows a graph of the longevity of the 16 hybridomas in the fed-batch mode plus the medium supplement of the present invention versus batch method. Detailed Description of the Invention
This invention is based upon the discovery of a serum- free medium supplement which can be used to maintain a eukaryotic cell line viable in cell culture. The supplement comprises carbon sources, vitamins, inorganic salts, amino acids and a protein digest.
By the use of the term "supplement" what is intended is a buffered solution containing a concentrated amount of nutrients which when added to an in vitro eukaryotic cell line, maintains viability. The supplement of the present invention can be added to an in vitro eukaryotic cell culture medium without the need to remove old or spent medium.
The term "batch mode of cell culture" as used herein describes a method of culturing cells where cells are seeded into a cell culture vessel containing an initial volume of nutrient medium (such as Dulbecco's Modified Eagles Medium (DMEM) with 10% fetal bovine serum (FBS) or Protein-Free Hybridoma Medium (PFHM-II), wherein the initial volume of nutrient medium is not replenished with any medium. The term "fed-batch mode of cell culture" as used herein describes a method of culturing cells where cells are seeded into a cell culture vessel containing an initial volume of nutrient medium and where supplements are added to the medium in a continuous or semi- continuous manner. The medium of the present invention includes a carbon source. Suitable carbon sources include L-glutamine and D-glucose. A carbon source containing L-glutamine in a concentration of about 7.3 grams per liter. (g/L) and D-glucose in a concentration of 25 g/L are preferred. The medium of the present invention, in addition, comprises vitamins. Suitable vitamins include a biotin, choline chloride, a folic acid, an inositol, a niacinamide, benzoic acid, a pantothenic acid, a pyridoxine, a riboflavin, a thiamine and B vitamin or mixture thereof. A mixture containing the vitamins listed in Table 1 below is preferred. Table 1 VITAMIN MIXTURE
Vitamins g/
D-Biotin 0.005
Choline Chloride 0.075
Folic Acid 0.025 myo-Inositol 0.875
Niacinamide 0.025 p-Amino Benzoic Acid 0.025
D-Pantothenic Acid 0.00625
(hemicalcium)
Pyridoxine HC1 0.025
Riboflavin 0.005
Thiamine HC1 0.025
Vitamin B12 0.000125
Furthermore, the medium of the present invention comprises inorganic salts. Suitable inorganic salts include potassium chloride, potassium phosphate, sodium chloride and sodium phosphate or a mixture. A mixture containing the inorganic salts listed in Table 2 below is preferred.
Table 2 INORGANIC SALT MIXTURE
Inorganic Salts g/L Potassium Chloride 0.05
Potassium Phosphate Monobasic 0.05
(anhydrous)
Sodium Chloride 2.0
Sodium Phosphate Dibasic 0.2
(anhydrous)
The medium of the present invention, further comprises amino acids. Suitable amino acids include alanine, arginine, asparagine, aspartic acid, cystine, glutamic acid, glycine, histidine, proline, isoleucine, lysine, methionine, serine, threonine, trytophan, tyrosine and valine or a mixture thereof. A mixture containing the amino acids listed in Table 3 below is preferred. Table 3 AMINO ACID MIXTURE
Amino Acids g L
L-Arginine (free base) 2.5
L-Asparagine (anhydrous) 0.625
L-Aspartic Acid 0.25
L-Cystine 0.625
L-Glutamic Acid 0.25
Glycine 0.125
L-Histidine (free base) 0.1875
Hydroxy- L- Proline 0.25
L-Isoleucine 0.625
L-Lysine-HCl 0.5
L-Methionine 0.1875
L-Phenylalanine 0.1875
L-Proline 0.25
L-Serine 0.375
L-Threonine 0.25
L-Trytophan 0.0625
L-Tyrosine-2Na2H2θ 0.3604
L-Valine 0.2725
In addition, the medium of the present invention comprises a protein digest. Suitable protein digests include primatone CLT, casein or enzymatic hydrolysates. In a preferred embodiment the medium comprises primatone RL in the amount of 25 g/L of the water component of the medium.
Exemp lification
Material and Method:
MAINTENANCE. EXPANSION AND FEEDING OF CULTURES
Frozen hybridoma cells (16 distinct cell lines) were thawed quickly at 37°C and transferred into DMEM or PFHM (Gibco, N.Y.) with 10% fetal bovine serum and grown in t- flasks. The cells were maintained in the t-flasks at 37°C and 5- 10% CO2 until the cultures reached 40-70% maximum viable density.
The cells were then expanded into 1, 2, or 8-L spinner flasks with a starting viable cell density of at least 0.1-0.2 million/ml as determined by hemacytometer and trypan blue staining. The 1-L and 3-L flasks were maintained at 37+ 1C and agitated at 50-80 rpm
(stir bar) with an overlay of mixed gas (5% CO2, 20-40% O2, balance
N2) at 10-20 ml/min/L. The 8-10-L flasks were agitated at 50 rpm with overhead drive and overlay with the same gas mixture at 10 ml/min/L. The cultures were monitored daily as to viable cell number, cell viability, glucose concentration, ammonia concentration, and lactate concentration.
When the viable cell density reached 50%± 20% of peak density, 20 ml/L of the serum-free medium supplement, components of which are shown in Table 4 below, were added to the culture.
Table 4: SERUM-FREE MEDIUM SUPPLEMENT
Component g/L
L-Glutamine 7.3
D-Glucose 25.0 vitamin mixture (Table 1) 1.0914 inorganic salt mixture (Table 2) 2.3875 amino acid mixture (Table 3) 7.8829
Primatone RL* 25.0
Feeding was continued daily and D-glucose was maintained above 1 g/L (45% D-glucose solution) until the viable cell density had dropped below 0.3 million/ml. The culture was harvested, the cells removed, and the product purified from the culture with the appropriate purification procedure.
Viable Cell Count
A 0.5 to 1.0 ml sample of the culture was aseptically removed from the culture in a laminar flow hood and placed in a microfuge tube. The cell suspension was diluted with Trypan blue/ PBS (0.4%) and mix thoroughly. A cover slip was placed on a hemocytometer and a small amount of the cell mixture placed into the chambers. The chambers were allowed to be filled by capillary action. The hemocytometer was viewed under a microscope under 10x10 power and cells in all 8 of the outer boxes were counted and recorded. Both viable (clear white) and non-viable (blue) cells were counted.
viable cells #/ml = # of viable cells counted x 1 box x Dilution Factor
8 boxes 10-4ml
(%) viability •**- # viable cells x 100
# viable cells + # non-viable cells
ELISA assay
Wells in polystyrene plates (cat #25801-96 high-binding flat bottom, Corning, Corning, N.Y.) were coated with 100 μl of goat anti-mouse IgG (H'+L) (Cat # 115-005-003, Jackson ImmunoResearch, West Grove, Pennsylvania), 1:200 dilution into 0.1M sodium carbonate pH 9.0, overnight at 4°C. After the overnight incubation, the liquid was removed and the plate was washed four times with phosphate buffered saline (P3S) pH 7.4 containing 0.05% Tween 20. The wells were filled with 200 μl of 2% Bovine Serum Albumin (BSA) pH 7.4 containing 0.05% Tween 20. The wells were filled with 200 μl of 2% Bovine Serum Albumin (BSA) solution in PBS to block residual binding sites. After 30 minutes incubation at room temperature (RT), the blocking reagent was removed and 100 μl of the diluted culture supernatant (PBS containing 0.05% Tween 20 and 0.5% BSA diluant) or standard antibody preparation (0 to 20 ng/ml) was added to each well. After 30 minutes incubation at RT, the plate was washed four times with the washing solution and 100 μl of peroxidase conjugated goat anti-mouse IgG (cat #115-035-062, Jackson ImmunoResearch) diluted 1 :2000 in PBS containing 0.05% Tween 20 and 0.5% BSA was added to each well. After 30 minutes incubation at RT, the plate was washed four times with the washing solution and 100 μl of OPD substrate (Cat #CIN4905/CIN4805, Medix Biotech, Inc. Foster City, CA) was added to each well. After 5 minutes incubation at RT, 100 μl 0.2 N Sulferic Acid was added to each well. Absorbance at 492 nm was measured in an ELISA reader and concentrations determined from the linear standard curve.
Glucose, lactate. and ammonium analysis
Glucose, lactate, and ammonium concentrations in culture samples were measured by an IBI Biolyzer (International Biotechnologies, Inc., New Haven, CT) following the directions provided.

Claims

1. A serum-free eukaryotic cell culture medium supplement, comprising: a. carbon sources; b. vitamins; c. inorganic salts; d. amino acids; and e. a protein digest.
2. The serum-free eukaryotic cell culture medium supplement in claim 1 wherein the carbon sources contain a glutamine and a glucose.
3. The serum-free eukaryotic cell culture medium supplement in claim 1 wherein the vitamins are a mixture containing the vitamins listed in Table 1.
4. The serum-free eukaryotic cell culture medium supplement in claim 1 wherein the inorganic salts are a mixture containing the inorganic salts listed in Table 2.
5. The serum-free eukaryotic cell culture medium supplement in claim 1 wherein the amino acids are a mixture containing the amino acids listed in Table 3.
6. The serum-free eukaryotic cell culture medium supplement in claim 1 wherein the protein digest is Primatone RL.
7. A method of culturing a eukaryotic cell line, comprising adding a medium supplement comprising: a. carbon sources; b. vitamins; c. inorganic salts; d. amino acids; and e. a protein digest, wherein the supplement maintains eukaryotic cell viability when added to the culture medium.
8. The serum-free eukaryotic cell culture medium supplement in claim 7 wherein the carbon sources are a mixture containing a glutamine and a glucose.
9. The serum- free eukaryotic cell culture medium supplement in claim 7 wherein the vitamins are a mixture containing the vitamins listed in Table 1.
10. The serum-free eukaryotic cell culture medium supplement in claim 7 wherein the inorganic salts are a mixture containing the inorganic salts listed in Table 2.
11. The serum-free eukaryotic cell culture medium supplement in claim 7 wherein the amino acids are a mixture containing the amino acids listed in Table 3.
12. The serum-free eukaryotic cell culture medium supplement in claim 7 wherein the protein digest is Primatone RL.
PCT/US1995/004885 1994-04-21 1995-04-21 Serum-free medium supplement Ceased WO1995029231A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU23598/95A AU702402B2 (en) 1994-04-21 1995-04-21 Serum-free medium supplement
JP52777095A JPH09512171A (en) 1994-04-21 1995-04-21 Serum-free medium additive
EP95917608A EP0763102A4 (en) 1994-04-21 1995-04-21 Serum-free medium supplement

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US23093394A 1994-04-21 1994-04-21
US08/230,933 1994-04-21

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JP (1) JPH09512171A (en)
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CA (1) CA2186608A1 (en)
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996040866A1 (en) * 1995-06-07 1996-12-19 Novartis Ag Serum-free media for primitive hematopoietic cells and methods of use thereof
WO2000002999A3 (en) * 1998-07-10 2000-04-20 Encelle Inc Medium and matrix for long-term proliferation of cells
US6352707B1 (en) 1992-02-24 2002-03-05 Anton-Lewis Usala Transplant encapsulation in a hydrogel matrix to obscure immune recognition

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* Cited by examiner, † Cited by third party
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US6506576B2 (en) 2001-03-14 2003-01-14 Board Of Trustees Of The University Of Arkansas Serum-and steroid-free culture media for cerebellar granule neurons
WO2002090479A1 (en) * 2001-05-04 2002-11-14 The Procter & Gamble Company Perfumed particles and articles containing the same
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US7299805B2 (en) * 2002-06-07 2007-11-27 Marctec, Llc Scaffold and method for implanting cells
JP2008541746A (en) * 2005-06-03 2008-11-27 ビオヴィトルム・アクチボラゲット（プブリクト） New process
US20130281355A1 (en) 2012-04-24 2013-10-24 Genentech, Inc. Cell culture compositions and methods for polypeptide production
WO2026006162A2 (en) 2024-06-24 2026-01-02 Genentech, Inc. B vitamin modulation

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4282326A (en) * 1978-10-12 1981-08-04 Jeanne Moldenhauer Cell culture medium supplement
US5122469A (en) * 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4657866A (en) * 1982-12-21 1987-04-14 Sudhir Kumar Serum-free, synthetic, completely chemically defined tissue culture media
CS262822B1 (en) * 1986-10-03 1989-04-14 Kovar Jan Synthetic medium for the cultivation of myelomic cells
GB9022545D0 (en) * 1990-10-17 1990-11-28 Wellcome Found Culture medium
GB2251249B (en) * 1990-12-28 1995-06-21 Mogam Biotech Res Inst High-density medium for animal cell culture
DE4219250A1 (en) * 1992-06-12 1993-12-16 Behringwerke Ag Serum-free cell culture medium

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4282326A (en) * 1978-10-12 1981-08-04 Jeanne Moldenhauer Cell culture medium supplement
US5122469A (en) * 1990-10-03 1992-06-16 Genentech, Inc. Method for culturing Chinese hamster ovary cells to improve production of recombinant proteins

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0763102A4 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6231881B1 (en) 1992-02-24 2001-05-15 Anton-Lewis Usala Medium and matrix for long-term proliferation of cells
US6315994B2 (en) 1992-02-24 2001-11-13 Anton-Lewis Usala Medium and matrix for long-term proliferation of cells
US6352707B1 (en) 1992-02-24 2002-03-05 Anton-Lewis Usala Transplant encapsulation in a hydrogel matrix to obscure immune recognition
US6713079B2 (en) 1992-02-24 2004-03-30 Encelle, Inc. Methods for increasing vascularization and promoting wound healing
US6730315B2 (en) 1992-02-24 2004-05-04 Encelle, Inc. Medium and matrix for long-term proliferation of cells
WO1996040866A1 (en) * 1995-06-07 1996-12-19 Novartis Ag Serum-free media for primitive hematopoietic cells and methods of use thereof
WO2000002999A3 (en) * 1998-07-10 2000-04-20 Encelle Inc Medium and matrix for long-term proliferation of cells
US6261587B1 (en) 1998-07-10 2001-07-17 Anton-Lewis Usala Methods for increasing vascularization and promoting wound healing

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EP0763102A1 (en) 1997-03-19
US5512477A (en) 1996-04-30
US5691202A (en) 1997-11-25
EP0763102A4 (en) 1998-12-16
CA2186608A1 (en) 1995-11-02
AU702402B2 (en) 1999-02-18
AU2359895A (en) 1995-11-16
JPH09512171A (en) 1997-12-09

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