Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/0018—Culture media for cell or tissue culture
  • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • 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
  • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
• • 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
  • C12N2500/34—Sugars
• • 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
  • C12N2500/70—Undefined extracts
  • C12N2500/74—Undefined extracts from fungi, e.g. yeasts

Definitions

• the present invention relates to the field of cell culture media. More particularly the invention relates to the field of mammalian cell culture media. Background of the Invention
• serum-free media Beyond a basal nutrient mixture of salts, sugars, amino acids, and vitamins, cells .in vitro have also been found to require for proliferation a supplement of poorly defined biological fluids or extracts. Because of availability and ease of storage, the most commonly used supplement is serum.
• the use of serum in cell culture media has several disadvantages. Serum is comparatively expensive. Since serum is not a defined component, different lots of serum may vary in the concentration of compounds present and thus result in unpredictable culture growth. Serum may also be contaminated with viruses or mycoplasms. The protein in serum may complicate the purification of cell products from the culture medium. In efforts to overcome the disadvantages of serum containing medium, researchers have attempted to provide serum-free media by substituting defined or better characterized components for serum.
• U.S. Patent 4,786,599 issued November 22, 1988 to Chessebeuf and Padieu discloses a serum-free animal tissue culture medium containing a mixture of six fatty acids and albumin or dextran.
• the medium is particularly adapted for the primary culture of rat liver epithelial cells and possibly in the presence of hormones and/or growth factors, for obtaining cell lines, in particular myeloma and hybridoma cell lines.
• Cytobiologica 2_6 123-132 report a serum-free medium for the culture of chick embryo cells containing dextran.
• Pietrzkowski and Korohoda (1988) Folia Histochemica et Cytobiologica _ ⁇ 143-154 report a serum-free medium containing dextran for the culture of chick embryo fibroblasts. In these two publications, the dextran was added to the medium to enhance cell attachment and spreading.
• Ohmori (1988) Journal of Immunological Methods 112: 227-233 reports a serum-free medium which is able to support primary antibody responses by cultured murine lymphocytes. This medium is based on a basal medium supplemented with ⁇ - cyclodextrin, insulin, transferrin, albumin, low density lipoprotein, putrescine and alanine.
• R-innmai- ⁇ of the Invention The present invention provides media for the culture of mammalian cells. The invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description. Detailed Description of the Invention The media of the invention are useful for the culture of mammalian cells.
• the media of the invention have been found to be useful in the culture of Chinese hamster ovary (CHO) cells, and HAK cells, a baby hamster kidney cell line.
• the media of the invention have been found not suitable for the culture of myeloma cell lines.
• Cells may be grown in batch and continuous culture with the serum-frae media of the invention.
• CHO cells grown in the media of the invention reach higher cell density and show increased recombinant product secretion when compared to CHO cells grown in a serum-containing medium.
• the cell culture media of the invention are prepared by adding components to a basal medium designed for mammalian cell culture.
• the media are prepared in accordance with standard procedures for preparing cell culture media.
• Suitable basal media include standard mammalian cell culture media such as Ham's medium, Waymouth MB 752/1 medium. Eagle's medium, Williams E medium, 199 medium and derived edia of the types MEM and MEM ⁇ and any combinations of these media.
• Other standard media used for the culture of mammalian cells are also suitable for use in the invention.
• a preferred basal medium is the basal medium of Example 1.
• the preferred basal medium supports cell growth and significantly reduces the size of cell clumps in the media during cell culture.
• a yeast hydrolysate such as Yeastolate is added to the basal medium in the amount of from about 0.1 to about 10.0 grams per liter, preferably in an amount of about 5 grams per liter.
• Albumin or dextran is added to the basal medium, in an amount of from about 0.1 to about 5.0 grams per liter.
• bovine serum albumin or dextran having a molecular weight of about 500,000 is added to the basal medium.
• Bovine serum albumin is preferably added in the amount of from about 0.1 to about 0.5 grams per liter.
• Dextran having a molecular weight of about 500,000 such as Dextran T500 is preferably added to the basal medium in the amount from about 0.1 to about 1.0 grams per liter.
• Insulin is added to the basal medium in the amount of from about 2.0 to about 20 milligrams per milliliter, preferably in the amount of about 10 milligrams per liter.
• Transferrin or transferrin substitute is added to the basal medium in the amount of from about 0 to about 100.0 micrograms per milliliter.
• Transferrin may be substituted in the medium with ferric fructose (from about 1.0 to about 10.0 milligrams per liter) , ferric citrate (from about 1.0 to about 100.0 milligrams per liter), or ferrous sulfate (from about 5.0 micromoles to about 200.0 micromoles per liter).
• ferric fructose from about 1.0 to about 10.0 milligrams per liter
• ferric citrate from about 1.0 to about 100.0 milligrams per liter
• ferrous sulfate from about 5.0 micromoles to about 200.0 micromoles per liter.
• a mixture of the fatty acids oleic, linoleic and linolenic are added to the basal medium in the ratio of oleic 0.6: linoleic 1: linole
• oleic acid is preferably added to the basal medium in the amount of from about 0.012 to about 0.12 milligrams per liter; linoleic acid is preferably added to the basal medium in the amount of from about 0.2 to about 5.0 milligrams per liter; linolenic acid is added to the medium in the amount of from about 0.028 to about 0.7 milligrams per liter. Cholesterol is added to the basal medium in the amount of from about 0 to about 10.0 milligrams per liter.
• CaCl 2 (anhydrous) is added to the basal medium in the amount of from about 0 to about 200 milligrams per liter, preferably in the amount of about 66.67 milligrams per liter.
• Magnesium sulfate (MgSO (anhydrous) is added to the basal medium in the amount of from about 0 to about 100.0 milligrams per liter, preferably in the amount of about 24 milligrams per liter.
• the pH of the medium is preferably from about 6.8 to about 7.4.
• the osmolarity of the medium is preferably from about 280 to 360 milliosmoles.
• the basal medium may be stored as a powder at 4 * C for one year.
• the complete medium (basal medium with added supplements) in a liquid form may be stored at 4 ⁇ C for six months.
• the components in the basal media are mixed and ball-mill ground to formulate a homogeneous powder.
• the powdered media is then dispensed into 100L packets and stored at 4'C.
• L-Alanine 41 300000 L-Arginine HCl 112.546700 L-Arginine FB 16.666000 L-Asparagine H20 28.336700 L-Aspartic Acid 24.433300 L-Cystine 2HC1 19.116600 L-Cysteine HC1.H20 45.040000 L-Cysteine FB 13.333300 L-Glutamic Acid 46.566700 L-Glutamine 292.000000 Glycine 35.833300
• Vitamin B12 0.973300
• Methyl Linoleate 0.010000 Vitamin A Acetate 0.033000
• Medium MRl-3 contains the basal medium of Example 1 supplemented with 5,000 mg/1 TC Yeastolate (Difco, Detroit, Michigan) , 500 mg/1 bovine serum albumin (BSA) (Armour, Kankakee, Illindis) 10 mg/1 bovine insulin (Waitaki, Toronto, Canada), 10 mg/1 bovine transferrin (Sigma Chemical Co., St.
• the medium is prepared as follows:
• Medium MR1-6 is contains the basal medium of Example 1 supplemented with 5,000 mg/1 TC Yeastolate (Difco, Detroit, Michigan) , 500 mg/1 bovine serum albumin (Armour, Kankakee, Illinois) , 10 mg/1 bovine insulin (Waitaki, Toronto, Canada) , 10 mg/1 bovine transferrin (Sigma Chemical Co., St. Louis, Missouri), 0.12 mg/1 oleic acid (Ameresco, Cleveland, Ohio), 0.20 mg/1 linoleic acid (Ameresco), 0.028 mg/1 linolenic acid (Ameresco) , and 2 mg/1 cholesterol (Ameresco) .
• the medium is prepared in the same way as medium MRl-3 in Example 2 except that steps 3 and 4 are omitted. In this medium no additional MgS0 4 or CaCl 2 is added.
• Example 4 Preparation of Medium MRl-7.
• Medium MRl-7 contains the basal medium of Example 1 supplemented with 5,000 mg/1 TC Yeastolate (Difco, Detroit, Michigan), 1,000 mg/1 Dextran T-500 (Pharmacia, Piscataway, New Jersey), 10 mg/1 bovine insulin (Waitaki, Toronto, Canada) , 10 mg/1 bovine transferrin (Sigma Chemical Co, St.
• Mediun MRl-7 is prepared in the same way as medium MRl-3 in Example 2 except that steps 3 and 4 are omitted and Dextran T-500 replaces bovine serum albumin in step 6. At step 6, 100 grams of Dextran T-500 are added and mixed until dissolved. • By.» ⁇ t ⁇ * ⁇ «» 5 Cell Culture
• CHO cells transformed to produce soluble T4 lymphocytic cell receptor (cell line 37-80N) were cultured in four different media: serum containing medium Alpha (-) MEM/5% Fetal bovine serum (FBS) , and the media described in Examples 2, 3, and 4. 5 x 10 5 cells per milliliter were cultured for 7 days after seeding in 250 ml SP flasks with 150 ml of medium. Total cell number was determined by Coulter counter, and viability was determined by trypan blue dye exclusion using a hemocytometer. Concentration of ST4 was determined by an ELISA-based assay. At day two after seeding, the serum-free media showed greater number of cells than the serum containing medium.

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• 1991
  • 1991-09-20 CA CA 2091443 patent/CA2091443A1/fr not_active Abandoned
  • 1991-09-20 WO PCT/US1991/006837 patent/WO1992005246A1/fr not_active Application Discontinuation
  • 1991-09-20 JP JP3516989A patent/JPH06500918A/ja active Pending
  • 1991-09-20 AU AU87348/91A patent/AU662491B2/en not_active Ceased
  • 1991-09-20 EP EP19910918096 patent/EP0550638A4/en not_active Ceased
  • 1991-09-23 ZA ZA917561A patent/ZA917561B/xx unknown
  • 1991-09-23 NZ NZ23990091A patent/NZ239900A/xx unknown
  • 1991-09-24 IE IE334591A patent/IE913345A1/en unknown
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