Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
W092/05246 PCT/US91tO6837
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91~3
- MEDI~X FOR C~L~RE OF MAMMA~IAN CELLS
Field of the ~vention
The present invention relates to the field of cell
culture media. More particularly the invention relates to the
field of mammalian cell culture media.
Bac~round of the Invention
Beyond a basal nutrient mixture of salts, sugars,
amino acids, and vitamins, cells ln 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, however, 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.
; 20 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. Unfortunately, the
complexity of serum and the differing growth requirements of
different types of cells has made it difficult to provide such
media. For reviews on serum-free media for mammalian cell
culture see Rizzino et al. (1979) "Defined Media and the
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Determination of Nutritional and Hormonal Requirements of
Mammalian Cells in Culture" Nutrition Reviews 37: 369-378;
Barnes and Sato (1980) "Serum-free Cell Culture: a Unifying
Approach`', Cell 22: 649-655; Barnes and Sato (1980) "Methods
for Growth of Cultured Cells in Serum-Free Medium", Analyt.
Biochem. 102: 255-270; and Bodeker et al. (1985) "A Screening
Method To Develop Serum-Free Culture Media For Adherent Cell
Lines", Develop. Biol. Standard. 60: 93-100.
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.
Media for the serum-free culture of Chinese hamster
ovary cells (CHO) have been reported. Gasser et al (1985) In
Vitro Cellular & Developmental Biology 21: 588-592 discloses
a serum-free medium for the culture of CHO cells. The serum-
free medium is composed of a 1:1 mixture of Ham's F12 and
modified Eagle's minimum essential media supplemented with
transferrin, insulin, and selenium. Mendiaz et al. (1986) In
Vitro Cellular ~ Developmental Biology 22: 66-74 discloses a
serum-free medium for the culture of CH0 cells composed of a
basal medium supplemented with insulin, and ferric sulfate or
transferrin, selenium, trace elements, calcium chloride,
glutamine, linoleic acid, non-essential amino acids, and
insulin.
Pietrzkowski et al (1988) Folia Histochemica et
Cytobiologica 26: 123-132 report a serum-free medium for the
culture of chick embryo cells containing dextran.
Pietrz~owski and Xorohoda (1988) Folia Histochemica et
Cytobiologica 26: 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.
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W092/0~246 PCT/US91/06837
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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 densitylipoprotein, putrescine and alanine.
It is an object of the invention to provide serum-
free media for the culture of mammalian cells. It is also
object of the invention to provide serum-free media for the
culture of mammalian cells transformed to produce recom~inant
products that increase product yield. It is yet another
object of the invention to provide serum-free media for the
culture of CHO cells.
8ummary 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 n its
preferred embodiments in the following description.
Detailed Deqcription 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 HA~ 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-free media of the invention. C~O 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
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2091~43 -4~
media 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 l. 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.l to abou~ l0.0
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.l to about 5.0 grams per liter.
Preferably either 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.l 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.l to about l.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 l0 milligrams per liter.
Transferrin or transferrin substitute is added to
the basal medium in the amount of from about 0 to about l00.0
micrograms per milliliter. Transferrin may be substituted in
the medium with ferric fructose (from about l.0 to about l0.0
milligrams per liter), ferric citrate (from about l.0 to about
l00.0 milligrams per liter), or 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 l: linolenic 0.14 milligrams per liter of
medium. In preferred embodiments of the invention, keeping
this ratio of fatty acids, 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
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W092/05246 PCT/US91/06837
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5.0 milligra~s 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 t~e amount of from about 0 to about lO.0 milligrams
per liter.
In a preferred embodiment of the invention which is
described in further detail in Example 2, calcium chloride
(CaCl2)(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 lO0.0 milligrams per liter,
preferably in the amount of about 24 milligrams per liter.
The pH of the medium is preferably from abcut 6.8
to about 7.4. The osmolarity of the medium is preferab'y from
about 280 to 360 milliosmoles.
The basal medium may be stored as a powder at 4C
for onè year. The complete mediu~ (basal medium with added
supplements) in a liyuid form may be stored at 4 C for six
months.
Preferred embodiments of the invention are described
in the following Examples.
Exa~ple 1 Preparation of B~sAl XeCium
The components in the basal media are mixed and
ball-mill ground to formulate a homogeneous powder. The
powdered media is then dispensed in~o lOOL packets and stored
at 4-C.
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BASAL MEDIUM COMPONENTS: MRl SERUM-FREE MEDIA
COMPONENTS milligrams/liter
INORGANIC SALTS/TRACE ELEMENTS
NaCl 7066.333000
KCL 341.200000
NaH2PO4.H20 93.333000
Na2HPO4 47.347000
MgC12 6H20 4.050000
MgSO4 (anhydrous) 6.510000
CuS04.5H20 0.000866
Fe(NO3)3.9H20 0.000033
FeSO4.7H20 0.278000
ZnS04.7H20 0.287700
MnC12.4H20 0.000033
Na2SeO3 (anhyd) 0.172900
AMINO ACIDS
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 HCl.H20 45.040000
L-Cysteine FB 13.333300
L-Glutamic Acid 46.566700
L-Glutamine 292.000000
Glycine 35.833300
L-Histidine HCl.H20 20.986700
L-Histidine FB 5.000000
L-Isoleucine 35.480000
L-Leucine 46.833300
L-Lysine HCl 65.486600
L-Methionine 11.493300
L-Phenylalanine 20.653300
L-Proline 34.833300
L-Serine 15.166700
L-Threonine 33.3000D0
L-Tryptophan 7.346700
L-Tyrosine 2Na2H20 36.776700
L-Valine 35.90000C
VITAMINS/MISC. COMPONENTS
Dextrose 4500.000000
Putrescine 2HC1 0.053700
Sodium Pyruvate 81.666700
Ascorbic Acid 17.333300
Biotin 0.202400
D-Calcium Pantothenate 0.160000
Sodium Pantothenate 0.337330
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W092/05246 ~7~ r - I ~ D ~ 1 4 4 3
~ Choline Chloride 5 486700
Folic Acid 1 100000
i-Inositol 7.333300
Nicotinamide o 679000
Na2 alpha Tocopherol PO4 0 003300
Glutathione (Reduced) 0. 016700
Menadione Na Bisulfite 0 003300
Pyridoxine HCl 0 020700
Pyridoxal HCl 0.666700
Riboflavin 0 079300
Thiamine HCl 0 780000
Vitamin B12 0.973300
Calciferol 0 033300
Methyl Linoleate 0 010000
Vitamin A Acetate 0.033000
Linoleic ~cid 0 028000
Lipoic Acid 0 136700
Preparation of Basal Medium - for a final volume of 100L
Ninety liters of deionized-distilled water is measured into
an appropriate mixing vessel. One 100L packet of ball-mill
ground powdered media (see above) is added. The pH of the
medium is adjusted to 7.2 using lN HCl. The volume of the
medium is brought to 100L by the addition of water. The
medium may then be sterilized by membrane filtration using a
0.2 micron cellulose acetate filter.
Example 2 Preparation of Medium MR1-3__
Medium MR1-3 contains the basal medium of Example
supplemented with 5,000 mg/l TC Yeastolate (Difco, Detroit,
Michigan), 500 mg/l bovine serum albumin (BSA) (Armour,
Kankakee, Illinais) 10 mg/l bovine insulin (Waitaki, Toronto,
Canada), 10 mg/l bovine transferrin (Sigma Chemical Co., St.
Louis, Missouri), 0.12 mg/l oleic acid (Ameresco, Cleveland,
Ohio), 0.20 mg/l linoleic acid (Ameresco), 0.028 mg/l
linolenic acid (Ameresco), 2 mg/l cholesterol (Ameresco),
66.67 mg/l anhydrous calcium chloride, and 24 mg/l anhydrous
magnesium sulfate. The medium is prepared as follows:
~or a final volume of 100L
1. Measure 90 liters of deionized-distilled water into an
appropriate mixing vessel.
2. Add one l~OL pac~et of ball-mill ground powdered media
(from Example 1).
3. Add 2.4 grams of MgSO4 (anhydrous) and mix until
dissolved .
4. Add 6.7 grams of CaCl2 (anhydrous) and mix until
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dissolved.
5. Add 500 grams of TC Yeastolate, mix until dissolved.
6. Add 50 grams of BSA, mix until dissolved.
7. Add 220 grams of NaHCO3, mix until dissolved.
8. Add l gram of insulin, l gram of transferrin (or l00 ml
of ferric fructose) and mix until dissolved.
9. Dissolve 12 mg of Oleic acid, 20 mg of Linoleic acid, 2.8
mg of Linolenic acid, and 200 mg of cholesterol in l00 mls
of absolute ethanol, and add this fatty acid mix to the
mixing vessel.
l0. Adjust the pH to 7.2 using lN HCl.
ll. Bring the volume to l00 liters and mix thoroughly.
12. Filter sterilize using a 0.2 micron cellulose acetate
filter.
13. Check osmolarity and record.
14. Store at 4~C for up to six months.
Example 3 PreParation of Medium MRl-6
Medium MR1-6 is contains the basal medium of Example
supplemented with 5,000 mg/l TC Yeastolate (Difco, Detroit,
MIchigan), 500 mg/l bovine serum albumin (Armour, Kankakee,
Illinois), l0 mg/l bovine insulin (Waitaki, Toronto, Canada),
lO mg/l bovine transferrin (Sigma Chemical Co., St. Louis,
Missouri), 0.12 ~g/l oleic acid (Ameresco, Cleveland, Ohio),
0.20 mg/l linoleic acid (Ameresco), 0.028 mg/l linolenic acid
(Ameresco), and 2 mg/l cholesterol (Ameresco). The medium is
prepared in the same way as medium MR1-3 in Example 2 except
that steps 3 and 4 are omitted. In this medium no additional
MgSO4 or CaClz is added.
Bxa~ple_4 Preparation of Medium MRl-7.
Medium MR1-7 contains the basal medium of Example
supplemented with 5,000 mg/l TC Yeastolate (Difco, Detroit,
Michigan), l,000 mg/l Dextran T-500 (Pharmacia, Piscataway,
New Jersey), l0 mg/l bovine insulin (Waitaki, Toronto,
Canada), l0 mg/l bovine transferrin (Sigma Chemical Co, St.
Louis, Missouri), 0.12 mg/l oleic acid (Ameresco, Cleveland,
Ohio)j 0.20 mg/l linoleic acid (Ameresco), 0.028 mg/l
linolenic acid (Ameresco), and 2 mg/l cholesterol (Ameresco).
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, l00
grams of Dextran T-500 are added and mixed until dissolved.
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W092/05246 PCT/US91/06837 l
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~xample S Cell Culture
1'
C~O cells transformed to produce soluble T4, a soluble
form of the T-4 lymphocytic cell receptor (cell llne 37-80N),
were cultured in four different media: serum containing medium
Alpha (-) MEM/5% Fetal bovine sexum (FBS), and the media
described in Examples 2, 3, and 4. 5 x 105 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. In serum-
containing medium, there were approximately 1. 3 X 106 cells,
whereas in the serum-free media there were approximately 1.6
x 106 cells. At days 3 through 7 significantly more cells
were present in the serum-free media t~an the serum containing
medium. At day 3, there were approximately 2.4 x lo6 cells
in the serum-containing medium and approximately 3.3 x 106
cells in the serum-free media. At day 4, the total number of
cells in the serum-containing medium had dropped slightly to
about 2.25 x 106 cells. In contrast, the number of cells in
the serum-free media had increased to approximately 3.6 x 106
cells in MR1-7, 4.1 x 106 cells in MR1-3, and 4.3 x 106 cells
in MR1-6. By day 7, the total number of cells in medium MRl-
7 had increased to approximately 4.0 x 106 cell, and the
number of cels in the other med:~.a remained at levels
comparable to the levels at day 4.
By three days post seeding, cells grown in the serum-
free media produced significantly more sT4 than did cellsgrown in the serum containing medium. The difference in
amount of sT4 product became more pronounced at days 4-7. At
day 7, cells cultured in the serum free media produced from
about 75 to 37 micro~ra~s of sT4 per milliliter o~ medium,
whereas cells cultured in the serum containing medium produced
about 35 micrograms of sT4 per milliliter of medium.
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