CELL GROWTH MEDIUM SUPPLEMENT

SUPPLEMENT POUR MILIEU DE CULTURE CELLULAIRE

English Abstract

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ABSTRACT OF THE DISCLOSURE
Cell growth medium supplement is provided as a
replacement for a large proportion of all animal
serum previously used in connection with in vitro
cell culture. The cell growth medium supplement of
this invention contains a minor amount of animal
serum and a major amount of components necessary to
act as a substantially universal supplement for
growing a large variety of cells. The components
include estrogens, thyroid hormones, growth factors,
corticoids, transport factors, nutrients, androgens,
peptide hormones, inorganic salts, amino acids,
vitamins, sugar and minor amounts of other known
tissue culture additives.

Claims

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The embodiments of the invention, in which an
esclusive property or privilege is claimed, are
defined as follows:-
1. A cell growth medium supplement for growing
cells in vitro,
said supplement consisting essentially of a
minor amount of animal serum and a major amount of a
mixture of, peptide and thyroid hormones, estrogens,
androgens, corticoids, nutrients, growth factors,
transport factors, inorganic salts, amino acids,
vitamins, sugar and minor other components.
2. A cell growth medium supplement in
accordance with claim 1 wherein said animal serum,
hormones, estrogens, androgens, corticoid, growth
factors, nutrients and transport factors consist
essentially of mammalian serum, insulin,
transferrin, EGF, ECGS, selenous acid,
o-phosphoryl-ethanolamine, triiodothyroxine,
hydrocortisone, estradiol 17, progesterone and
testosterone.
3. A cell growth medium supplement in
accordance with claim 2 wherein said serum is bovine
serum.

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4. A cell growth medium supplement in
accordance with claim 3 wherein said components are
used in amounts of said supplement as follows:
<IMG>

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5. A cell growth medium supplement consisting
essentially of the following: a minor amount of
bovine serum, and a major amount of a mixture of,
peptide and thyroid hormones, estrogens, androgens,
corticoids, nutrients, growth factors, transport
factors, inorganic salts, amino acids, vitamins,
sugar and minor other components,
said supplement being useful to aid growth of a
wide variety of different types of cells including
transformed and non-transformed primaries of both
fibroblastoid and epithelioid origin, and
transformed and non-transformed continuous cell
lines.
6. A cell growth medium supplement in
accordance with claim 5 wherein said bovine serum,
hormones, estrogens, androgens, corticoid, growth
factor, nutrients and transport factor consist
essentially of bovine serum, insulin, transferrin,
EGF, ECGS, selenous acid, o-phosphoryl-ethanolamine,
triiodothyroxine, hydrocortisone, estradiol 17,
progesterone and testosterone.
7. A cell growth medium supplement in
accordance with claim 6 whrein said serum is newborn
calf serum.

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8. A cell growth medium supplement consisting
essentially of
<IMG>

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Claim 8 continued:
<IMG>

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9. A cell growth medium supplement in
accordance with claim 8 wherein said animal serum is
newborn calf serum in amount of 5 to 25% by volume.
10. A cell growth medium supplement in
accordance with claim 9 wherein said newborn calf
serum is used in an amount of 25%.
11. A combination comprising,
from 99.9 to 60% by volume of
(a) a cell culture medium having all growing
components necessary for animal cell growth when
combined with animal serum, and
(b) from 0.1 to 40% by volume of a cell growth
medium supplement consisting essentially of a minor
amount of animal serum and a major amount of a
mixture of, peptide and thyroid hormones, estrogens,
androgens, corticoids, nutrients, growth factors,
transport factors, inorganic salts, amino acids,
vitamins, sugar and minor other components.

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12. A combination in accordance with claim 11
wherein said animal serum, hormones, estrogens,
androgens, cortocoid, growth factors, nutrients and
transport factor consist essentially of mammalian
serum, insulin, transferrin, EGF, ECGS, selenous
acid, o-phosphoryl-ethanolamine, triiodothyroxine,
hydrocortisone, estradiol 17, progesterone and
testosterone.
13. A combination in accordance with claim 11
wherein said serum is newborn calf serum.
14. A combination in accordance with claim 11
wherein said supplement has the following formula:
<IMG>

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Claim 14 continued:
<IMG>

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Claim 14 continued:
<IMG>
distilled water to volume of 1 liter.
15. A method of growing cells in vitro,
said method comprising growing cells in the
presence of a cell culture medium having mixed
therewith a cell growth medium supplement in an
amount of from 0.1 to 40% by volume of the mixture,
said supplement consisting essentially of a
minor amount of animal serum and a major amount of a
mixture of, peptide and thyroid hormones, estrogens,
androgens, corticoids, nutrients, growth factors,
transport factors, inorganic salts, amino acids,
vitamins, sugar and minor other components to permit
rapid and prolific growth of cells.

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16. A method in accordance with the method of
claim 15 wherein said supplement animal serum,
hormones, estrogens, androgens, corticoids, growth
factors, nutrients and transport factors consist
essentially of mammalian serum, insulin,
transferrin, EGF, ECGS, selenous acid,
o-phosphoryl-ethanolamine, triiodothyroxine,
hydrocortisone, estradiol 17, progesterone and
testosterone.
17. A method in accordance with the method of
claim 16 wherein said animal serum is newborn calf
serum.
18. A method in accordance with the method of
claim 16 wherein said supplement is present in an
amount of 5 to 25% by volume of the mixture.
19. A method in accordance with the method of
claim 16 wherein said cell growth medium supplement
consists essentially of
<IMG>

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Claim 19 continued:
<IMG>

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Claim 19 continued:
<IMG>

Description

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CELL GROWTH MEDI~M SUPPLEMENT
BACKGROUND OF THE Ii~VENTION
Animal serum and particularly fetal calf serum
is known as a supplement to conventional tissue
culture media. Thus, it is known to add up to 20
by volume, and often 10%, bovine serum to tissue
culture of animal cells in such standard tissue
growth media as Ham's F12, Dulbecco's Modified
Eagle's Media and others. The serum appears to
provide some essential materials for tissue culture
which are not provided easily, if at all, by known
substitutes. The use of animal serum as a
supplement to growth media often aids cells in
expressing a differentiated function, growing to
desired concentrations and maintaining growth and
viability for long time periods.
However, the use of animal serum can present
certain problems in tissue culture. Chief among the
problems is the variability in the composition of
serum. Thus, growth promotability of individual
lots of serum can vary greatly. Suppliers of animal
serum attempt to minimize this variation by pooling
serum from many animals and maintaining donor herds
~'` .
.

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of animals of known quantity and/or screening or
assaying the levels of hormones, growth factor and
other serum components. However, such attempts can
be costly and are not always successful in reducing
the problems of variability. Tnese problems can
restrict use by users and can lead to variations in
investigational results. Often users must do
extensive testing of serum prior to use which can be
costly in time as well as money. The availability
of bovine serum in quantity can be a problem since
it varies depending upon the ups and downs of the
beef industry. Thus, fetal calf serum as well as
other bovine serum has an availability as a
by-product and is linked to herd sizes which vary
cyclically.
It has been recognized that the use of animal
serum as a supplement to normal growth media has
drawbacks. Others have attempted to reduce the
amount of serum used in tissue culture or eliminate
serum use entirely. Serum-free growth media have
been designed which are specific to particular types
of cells. Similarly, reduction in serum normally
added has been used along with addition of growth
factors in other supplements designed for specific
cell lines. Thus, where serum amounts have been cut
down or eliminated in normal tissue growth in the
past, this has been in connection with particular

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types of cells. The prior art has not provided a
substantially universal supplement for tissue
culture which has been accepted by the industry for
use with a wide variety of different types of cells
including transformed and non-transformed primaries
of both fibroblastoid and epithelioid origin, and
transformed and non-transformed continuous cell
lines.
SUMMA}~ OF THE INVENTION
It is an object of this invention to provide a
cell growth medium supplement containing animal
serum and a combination of substitutes for animal
serum components which supplement is useful in
promoting cell growth in culture.
Still another object of this invention is to
provide a cell growth medium supplement in
accordance with the preceding object which can be
produced with substantially consistent properties in
desired volumes for use in connection with cell
growth in a variety of media which cells can be
selected from a large variety of cell types.
Still another object of this invention is to
provide cell growth medium supplements in accordance
with the preceding objects in combination with
conventional tissue culture media for use in growing
cells.
Still another object of this invention is to

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provide advantageous methods of making and using the
cell growth medium supplement of this invention.
According to the invention, a cell growth
medlum supplement comprises a minor amount of animal
serum, preferably newborn calf serum, along wlth a
major amount of selected thyroid and peptide
hormones, corticoids, androgens, estrogens, growth
factors, nutrients and transport factors, inorganic
salts, amino acids, vitamins, sugar and minor other
components in an aqueous base to permit rapid and
prolific growth of a large variety of cell types in
primary and continuous cell culture lines,
transformed and non-transformed cell lines from
tissues including fibroblastoid and epithelioid
cells.
In a preferred embodiment, the serum used is
newborn calf serum, the peptide hormone is insulin,
the transport factor is transferrin, the growth
factors are epidermal growth factor and endothelial
cell growth supplement while nutrients include
; selenous acid and 0-phosphoryl-ethanolamine and the
thyrola hormone is triiodothyroxine, the corticoid
is hydrocortisone, estrogen is estradiol 17,
progesterone and the androgen is testosterone.
Preferably the supplement of this invention is
formulated, thoroughly mixed and a filtering step is
used to sterilize the mixture. Filtering is
.
.
-
:. :
.: -
:

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preferably carried out as a last step in formation
of the supplement.
It is a feature of this invention that the
supplement can reduce or eliminate problems
associated with variability of serum composition
from lot to lot. The small amount of serum used can
vary without substantial effect since it is only a
small component of the tissue culture, frequently,
one quarter or less, than that amount normally
used. Surprisingly, even though only a small amount
of serum is used, it provides necessary components
for a universal tissue growth medium as for example,
cell attachment factors ana carrier proteins for
water insoluble substances. The small serum base
aadition contributes other factors for cell
multiplication. S~nce the serum amount is small,
there are decreased levels of toxic substances
normally carried in serum. Since the protein level
in the ultimate cell culture is reduced over the use
of serum alone, separation of protein from the final
product is made easier. The concentration of cell
product grown in culture media can be increased when
the supplement of this invention is used to enhance
growth. The reduction of total protein content of
the cell growth medium also can result in increased
efficiency and ease of recovery and purification of
the desired cell products. This can be extremely

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important in the pharmaceutiCal and vaccine
industry. The reproducibility of a constant product
is enhanced in the supplement of this invention.
This can eliminate variability in growth promotion
customarily observed in different lots of whole
anlmal serum and eliminate tne necessity to test
multiple lots of serum to find a desirable lot.
Cost and time reduction in the overall operation of
the tissue culture facility is obtainable.
DE5CRIPTION OF PREFFERED EMBODIMENTS
The cell growth medium supplement of this
invention is used in place of animal serum in cell
and tissue culture. Customarily, animal serum has
been used in cell culture in amounts of from 5 to
20~ by volume of normal tissue culture media;
however, it is preferred to use the cell growth
medium supplement of this invention in amounts of
from 1 to 25% by volume of conventional tissue
culture media. In limlted cell lines higher or
lower proportions of the supplement to conventional
medium c~n be used as for example from 0.1~ to 40~
supplement to from 99.9 to 60~ conventional medium.
Such normal tissue culture media suitable for use
with the supplement of this invention which were
prevlously used with serum alone include, but are
not limited to, the following:

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McCoy's 5a Medium
McCoy, T.A., Maxwell, M. and Kruse, P.F., Proc.
Soc. Exper. Biol. and Med., 100, 115-118
(1959)-
Minimum Essential Medium - MEM
Eagle, H., Science 130, 432 (1959)
Modified Eagle Medium
Daniel, M.D. and Melendez, L.V., Proc. of the
Soc for Ex~. Biol. and Med. 127, No. 3 (March
Dulbecco's Modified Eagle Medium
Dulbecco, R. and Freeman, G. Virology 8, 396
(1959)
Smith, J.D., Freeman, G., Vogt, M. and
Dulbecco, R., virology 12, 185-196 (1960)
Tlssue Culture Standards Committee, In Vitro 6,
No. 2, 93
L-15 (Leibovitz) Medium
Leibovitz, A., Am. J. HYq. 78, 173-180 (1963)
Nutrient Mlxtures - F-10 and F-12 Media
Ham, R.G., ~xp. Cell Res. 29, 515-526 (1963)
Ham, R.G., Proc. Nat. Acad. Sci. 53, 288-293
(1965)
Waymouth's Medium
Waymouth, C., J. Nat. Cancer Inst. 22,
1003-1017 (1959)
Examples of aqueous formulations of such medium
are as follows:
HAM'S NUTRIENT MEDIUM F-l~ (Available
from Gibco Laboratories)

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COMPONENT mq/L
Inorganic Salts:
CaC12 . 2H2O 44.00
CuSO4 . 5H208 0.00249
FeSO4 : 7H2O 0.834
KCl 223.60
MgC12 6H2 122.00
NaCl 7599.00
NaHCO3 1176.00
Na2HPO4 . 7H~O 268.00
ZnSO4 . 7H~O 0.863
Other Components:
D-Glucose 1802.00
Hypoxanthine 4.10
L5 LinoleiC acid 0.084
Lipoic acid 0.21
Phenol red 1.20
Putrescine 2HC1 0.161
Sodium pyruvate 110.00
20 Thymidine o 73
Amino Acids:
L-Alanine 8.90
L-Arginine HCl 211.00
L-Asparagine . H2Ob 15.01
25 L-Aspartic acid 13.30
L-Cysteine HCl . H2O 35.12
L-Glutamic acid 14.70
L-Glutamine 146.00
Glyclne 7.50
30 L-Histidine HCl . H2O 20.96
! L-Isoleucine 3.94
L-Leucine 13.10
L-Lysine HCl 36.50
L-~etnlonine 4.48
35 L-Phenylalanine 4.96
L-Proline 34.50
L-Serine 10.50

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L-Threonine 11.90
L-Try~tophan 2.04
L-Tyrosine 5.40
L-Valine 11.70
Vitamins: -
Biotin
D-Ca pantothenateb 0.4800
Choline chloride 13.9600
Folic acid 1.300
i-Inositol 18.000
Niacinamide 0.0370
Pyridoxine HCl 0.0620
Riboflavin 0.0380
Thiamine HCl 0.3400
vitamin B12 1.3600
DULBECCO'S MODIFIED EAGLE MEDIUM (in H2O)
COMPON~NT mq/L
Inorganic Salts:
CaC12 (anhyd.) 200.00
Fe~NO3)3 9~20 0.10
KCl 400
MgSO4 7H2~ 200.00
NaCl 6400.00
NaHCO3 3700.00
Na2HPO4 H2~ 125.00a
Other Components:
D-Glucose 1000.00
Phenol red 15.00
Sodium pyruvate 110.00
Amino Acids:
L-Arginine . HCl 84.00
L-Cystine 48.00
L-Glutamine 584.00
~lycine 30.00

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--10--
L-Histidine HCl . H2O 4~.00
L-Isoleucine 105.00
L-Leucine 105.00
L-Lysine HCl 146.00
L-Methionine 30.00
L-Phenylalanine 66.00
L-Serine 42.00
L-Threonine 95.00
L-Tryptophane 16.00
L-Tyrosine 72.00
L-Valine 94.00
Vitamins:
D-Ca pantothenate 4.00
Choline chlorlde 4.00
Folic acid 4.00
Myo-Inositol 7.20
Nicotlnamide 4.00
Pyridoxal HCl 4 00
Ri~oflavin 0.40
Thiamine HCl 4.00
vitamln B12 --
aValues shown are in conformance with the Tissue
Culture Standards Committee, In Vitro, 9, No. 6
(1970).
Other standard tissue culture media can be used
along with the supplement of this invention to grow
cells. The variations in such media are numerous.
The limitation is only one of using the supplement
with media known to promote growth in the presence
of animal serum.
Newborn calf serum is preferred as the serum
portion of the supplement of this invention ,
because it is more universal than other animal

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serums. "Newborn" calf serum is taken from 10-day
old or younger calves. In some cases, the newborn
calf serum can be replaced with o~her bovine serum
or other animal serum such as horse serum, bovine
serum, chicken serum, rabbit serum, porcine serum,
ovine serum, or goat serum or a combination
thereof. When growing cell lines that normally grow
in other serums than newborn calf, that other serum
can be used as the serum portion of the cell growth
supplement of this invention.
In all cases, it is preferred to obtain a
combination of ingredients in the supplement which
has great versatility and is useful on a large
variety of cell lines. Thus, the hormones and other
ingredients are selected such that although some may
not be required for use in all cell lines, none
lnterfere with action of the other in any cell
line.
The peptide hormone components of the present
invention can include insulin. The transport factor
is transferrin which is also a peptide hormone. The
thyroid hormones are triiodothyroxine and
thyroxine.
The growth factors used are preferably
epidermal growth factor, as sold by Collaborative
Research, Inc. of Waltham, Massachusetts under its
designation CR-EGF. Such EGF is as described in the

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literature reference Savage, C.R. and Cohen, S. J.
Biol Chem , 247, 7609-7611 (1972). Endothelial
-
cell growth supplement useful in this invention
containing endothelial cell growth factor is
available from Collaborative Research, Inc. of
Waltham, Massachusetts under its designation
CR-ECGS. This growth factor is further described in
literature reference Maciag, T., Cerundolo, J.,
Ilsley, S., Kelley, P.R., and Forand, R., Proc. Nat.
10Acad. Sci. USA _ , 5674-5678 (1979).
The androgen used can be testosterone,
dihydrotestosterone, nandrolone and oxymetholone,
the estrogen used can be progesterone, 17
(beta)estradiol, estrone.
15Tne corticoid is preferably hydrocortisone but
other materials such as cortisone, dexamethasone,
prednisolone and corticosterone can be used.
- Nutrients preferably include include selenous acid,
0-phosphoryl-ethanolamine and other materials which
are involved in cellular enzymatic functions.
Inorganic salts useful in this invention are
those commonly used in tissue culture media as for
example, calcium, copper, iron, manganese, ammonia,
potassium, magnesium, sodium and zinc salts of
chlorine, sulphate, phosphate, and the like, some of
which may be hydrated.
Conventional amino acia additives to tissue

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culture media such as L-alanine, L~arginine HCl,
L-asparagine, L-aSpartiC acid, L-cysteine HCl.H2O,
L-isoleucine, L-lysine H~l, L-methionine,
L-pnenylalanine~ L-proline, L-serine, L-threonine,
L-tryptophan, L-tyrosine, L-valine, L-glutamic acid,
L-glycine, L-glutamine, L-histidine and L-leucine
are also useful.
Essential vitamins include d-Biotin, D-Ca
Pantothenate, Choline Chloride, Folic Acid,
Myo-inositol, Nicotinamide, Pyridoxine HCl,
Riboflavin, Thiamine HCl and Vitamin B12. Other
miscellaneous components include sugars such as
glucose, hypoxanthine, linoleic acid, phenol red,
olltrescine 2 HCl, sodium pyruvate, thymidine,
~scorbic acid, calciferol, niacin, para-aminobenzoic
acid, pyridoxal HCl, vitamin A. Preferably
delonized distilled water with a preferred
resistivity greater than 8 mega-ohms is used.
However, any water as customarily used in tissue
culture can be employea.
It should be understood that the components can
be used in their water soluble form as for example
salt forms of the hormones, amino acids, vitamins
and the like.
The supplement of this invention is preferably
used in place of serum as ordinarily used in tissue
culture. Thus, preferably the supplement can be

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used in amounts of from 1 to 25~ by volume of the
total solution of a conventional tissue culture
media as for example in Ham's F12 or Eagle's media
as for example in a proportion of 1:99 to 1:4.
Thus, the level of calf serum when the animal serum
is calf serum, is preferably at a level of from .25%
to 6.25~ by volume of the overall tissue culture
media. Thus, this level of serum in this invention
is low enough to avoid any substantial addition of
toxic factors, reduces variation in serum
consistency from having an effect and provides
required materials for cell growth other than from
an entirely protein based material.
In the preferrea formulation of this invention,
a cell growth medium supplement is formulated as
follows and considered the preferred universal serum
substitute of this invention:
FORM~LATION 1
Component Concentration
20 Newborn Calf Serum 25~ by volume of the
supplement
mg/Liter
Insulin 25
Transferrin 50
25 EGF
ECGS 77 5
Selenous Acid 0.1612
O-phosphoryl-ethanolamine 56.44

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L-3,5,3'triiodothyroxine 1.683 x 10-4
Hydrocortisone4.33 x 10-3
Estradiol 173.41 x 10-3
Progesterone3.93 x 10-3
Testosterone3.61 x 10-3
Inoryanic Saltsmg~Liter
CaC12 23.24
CuS~4~5~20 0.00174
FeS~4~7~20 0.58
KCl 156.56
M9C12'6~2 85.40
NaCl 5,319.30
Na2HPO4 99.40
znSO4-7H2O 0.60
Amino Acids
L-alanine 6.24
L-arginine HCl 153.73
L-asparagine 10.50
L-aspartic acid 9.32
L-cysteine HCl H2O24.58
L-glutamic acid 10.30
Glycine 5.26
L-Histidine HCL H2O14.67
L-isoleucine 2.76
L-Lysine HCl 25.56
L-methionine 3.13
L-phenyloilanine 3.47
L-proline 24.17
L-serine 7.36
L-threonine 8.34
: L-tryptophon 1.43
L-tyrosine 3.81
L-valine 8.20
Vitamins
d-biotin 0.0051
D-Ca Pantothenate 0.18
Choline Chloride 9.77

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Folic ~c id O . 93
Myo-inositol 12.61
Nicotinamide 0.026
Pyridoxine HCl 0.043
Riboflavin 0.026
Thiamine HCl 0.24
vitamin B12
Miscellaneous Components
G ucose 1,261.12
Hypoxanthine 2.86
Linoleic acid 0.059
Phenol red 0.87
Putrescine 2HCl 0.11
Sodium pyruvate 77.07
Thymidine 0.51
Sodium Bicarbonate 1176.
Deionized distilled water with
resistivity greater than 8 mega-ohms to volume of
1 liter.
The preferred formulation can be made in large
batch or small amounts as for example 1 liter wlth
the amount given being maintained in the proportion
set forth in preceding Formulation 1.
While this specific formulation has been found
to be preferred, variations are possible. As
previously described, other animal serums can be
used in place of newborn calf serum. The specific
formulation above can be used wherever bovine serum
was previously used as a supplement to conventional
tissue culture media. Thus, from 0.1 to 40% by
volume of the above formulation can be added to
normal tissue culture media to a large variety of

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cell lines that would otherwise have grown with an
equal volume addition of animal serum. The growing
conditions used for the cells are substantially the
same as those used when animal serum alone is used
as the supplement.
Conventlonal tissue culture conditions are used
when the supplements of this invention are admixed
wlth tlssue culture media an~ used ~o grow cells in
tissue culture. For example, conventional growing
temperatures of from 30C to 38C can be used. Time
periods of cell growth vary considerably usually
from 6 hours or more. The types of cell culture can
be any of the normal types used including suspension
culture, substrate at~ached cell growth,
microcarrier bead attached cell growth, growth in
soft agar, cell growth with collagen and other
biomatrix supports. Roller, shaker or stationary
suspension culture techniques can be used. The
media supplemented with the supplement of this
invention preferably is maintained at a pH of from
about 6.5 to about 7.8 for maximized cell growth.
Normally buffers in ordinary media such as F12 or
modified Eagle media buffers animal cell tissue
growth media to a pH in the physiological range.
The gaseous atmosphere above the growing mixtures of
this invention can be as conventionally used for the
particular cell line being grown. Thus carbon

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dioxide atmospheres of from 0 to 15~ can be used as
can mixed gas atmospheres of oxygen, nltrogen,
carbon dioxide and air.
Preferred variations from the amounts indicated
in the above preferred supplement formulation can be
made and are listed below:
comPonent Range
~ewborn Calf Serum 5-25% by volume
ma/Liter
Insulin 5-10
Transferrin 20-100
EGF 2-100
ECGS 2-100
Selenous Acid 0.013-0.13
~-phosphoryl-ethanolamine 1.4-140
L-3,5,3'triiodothyroxine 6.73 x 10-7 - 6.73 x
10-4
Hydrocortisone 3.46 x 10-4 - 3.46 x
10-2
Estradiol 17 2.72 x 10-4 - 2.72 x
10-2
Progesterone 3.15 x 10-4 - 3.15 x
10-2
Testosterone 2.88 x 10-4 - 2.88 x
10-2
The remaining components which are conventional
additives to tissue culture media can vary in amount
to a greater degree with greater substitution
possible than for the above listed components.
These remaining components are those additionally
listed in Formulation 1 under Inorganic Salts, Amino
Acids, Vitamins and Miscellaneous Components.

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The amounts cf each of the components can vary
outside the preferred ranges with certain cell
lines.
In formulating the preferred cell growth medium
supplement in accordance with this invention, the
materials are a~mixed in any conventional manner.
However, it is preferred to filter the material
after admixture ln order to provide for final
sterilization.
The following non-limiting examples illustrate the
invention, reference being made to the appended drawings
in which the single figure shows the test results of
example 2.
xample 1
This example illustrates a preferred method of
fonming the preferred Formulation 1.
The cell growth medium supplement of this
invention with the amounts of components noted in
Formulation 1 is formulated as follows:
1. Frozen newborn calf serum is thawed and
warmed to 37C to insure solubilization of cold
insoluble globulin.
2. Inorganic salts, amino acids, vitamins and
miscellaneous components are dissolved in the
deionized distilled water at a temperature in the
range of 31-35C to form a solution.
3. An aliquot ( ~25%) of the solution in step
2 is removed for solubilization of remaining
components. To this aliquot, newborn calf serum is
added to a final concentration of 2% by volume.
4. Stock solutions of the following components
:

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are prepared using distilled deionized water:
Bovine Insulin (25 gm/L); Transferrin (25 gm/L);
EGF, Receptor Grade (10 mg/L); ECGS (1.5 gm/L);
selenous acid (16.12 mg/L); 0-phosphorylethanolamine
(56.44 gm/L) in sodium hydroxide; (triiodothyroxine
168.25 ug/L) and in absolute ethanol:
hydrocortisone (4.12 mg/L); Estradiol 17 (3.41
mg/L); progesterone (3.93 mg/L); testosterone (3.61
mg/L).
5. The proper volume of each stock solution is
added slowly with constant stirring to the solution
of Step 2 in the following order: insulin,
~ransferrin, EGF, ECGS, Selenous acid,
0-phosphoryl-ethanolamine~ triiodothyroxine are
diluted first in a small volume of the solution of
Step 2 before addition to the final formulation. In
a similar manner, hydrocortisone, estradiol,
progesterone, and testosterone are diluted in the
solution of Step 2 before addition to final
formulation.
6. The 37C heated newborn calf serum is added
slowly with stirring.
7. The final pH of the formulation is in the
range of pH 7.2-8.0 and has an osmolarity of from
330-350 milliosmoleS.
8. The final solution is double filtered
through a 0.2 micron filter to provide a sterilized

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final product having the composition of Formulation
1 above.
In specific examples of using the preferred
- universal serum supplement of the first formulation
above, several cell lines have been grown as follows:
Example 2
The growth medium supplement of Formulation 1
is used in connection with the growth of B~K-21
cells American Type Culture Collection, Xockville,
~D A~'CC NoO CCL-10 (baby hamster kldney cell).
Dulbecco's Modified Eagle's medium (DMEM) in a
volume of 5 mls each in a first set of 60 mm petri
plates is supplemented with 10~ calf serum. The
; cells are grown for 6 days at 37C in a humidified
5% CO2-95% air environment.
In a second set of 60 mm petri plates, BHK-21
cells are grown under the same conditions in DMEM
supplemented with 10% of the growth supplement of
Formulation 1 of this invention.
The first and second sets are made up of
several petri dishes in each of Lot A, B and C of
the supplement and Lot A, B and C of the calf serum
modified medium. At 3 days and about 5 days, the
Formulation 1 supplement-media and serum-media are
replaced with fresh stock of each in each dish.
The sultability of the substitution of the
'

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supplement of this invention for ~HK-21 cell growth
is illustrated in the graph of FIG. lo Tnus, BHK-21
generation time in cell growth medium supplement of
thls invention is found to be 13.1 nours as compared
to 16.5 hours in medium supplemented with 10% calf
serum. Cell yeneratlon time or cell aoubling time
is used to measure the growth rate or doubling rate
of the cell population. Cell generation time is
thus a quantitative measure of growth promotability
of the tissue culture medium supplement. Cell
density at day six is 2.23 X 10 5 cells per square
centimeter in 10% cell growth supplement of
Formulation 1 in DMEM, and 1.59 X 10 5 cells per
square centimeter in 10% calf serum in DMEM.
Thus, Formulation 1 has been shown to be a
substantially universal cell growth supplement for
addition to a tissue culture media.
Other cells which have been grown with positive
results using the formulation of Formulation 1 as a
supplement of this invention include the following:
A431 - ~uman Epidermoid Adenocarcinoma
~AL~ 3T3 - Mouse Embryo Fibroblast
C6 - Rat Glioma
CV-l - African Green Monkey Kidney
Endothelial (non transformed) - Mouse Aortic
Endothelial
F9 - Mouse Embryonic Carcinoma
FS-1230 - Human Diploid Fibroblasts
HeLa - Human Cervical Carcinoma
Hybridoma Sp-2 - Mouse Myeloma Parent -

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continued antibody production
Hybridoma - NS-l Parent Mouse Myeloma
KB - Human Oral Epidermoid Carcinoma
L6 - Rat Skeletal Muscle Myoblast
MCF-7 - Human Breast Carcinoma
Mouse Sarcoma
Myoblastoid (non-transformed) - Murine
SV403T3 - SV-40 Virus Transformed BALB C 3T3
TR-M Rat Testicular Tumor
TR-ST Rat Testicular Tumor
TM-4 Mouse Sertoli
TK-l Rat Testicular Cell (non-transformed)
VERO - African Green Monkey Kidney
WI-38 - Human Diploid Fibroblast
M~C-5 - Human Diploid Fibroblast
~MTW9PL - Rat Mammary Tumor - 6 Independent
Clones
Primary Cultures or Human Fibroblast
Primary Cultures From Human Tumor Biopsy
SpecimenS
While some cell lines such as rat embryo
fibroblast cells do not grow ordinarily in the cell
growth medium of this invention, it is still
considered a universal supplement since it does5 promote growth in a wide variety of cell cultures.
"Growing" as used in this application refers to
proliferation of cell multiplication to higher
densities in media when grown on solid supports or
freely in suspension. When the supplement of this
invention is used with conventional media of the
type described above, in the proportions noted
broadly, 0.1 to 40% cell growth medium supplement of
this invention to 99.9% to 60% medium and preferably

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1 to 25% by volume supplement of this invention to
99 to 75~ by volume conventional medium, cell growth
of a wide variety of cell lines is promoted. The
supplement of this invention can be stored for long
periods of time without significant loss of
activity. For example, the supplement can be frozen
at -20C. The supplement has been found to be
stable at temperatures of 4C for at least 4-week
periods. Even after admixture with conven~ional
media, the supplement maintains its cell growth
promoting activities for long periods of time.
While the supplement has been described as a
separate solution to be added to conventional
medium, it should be understood that the supplement
formulation itself can be formed directly in
conventional media by addition of portions thereto.
In all cases, it is preferred to add the serum used
as a final step after first solubilizing at least a
portion of the serum in an aliquot of a mixture of
inorganic salts, amino acids, vitamins and
miscellaneous components.