U.S. patent application number 14/285047 was filed with the patent office on 2015-03-19 for serum-free mammalian cell culture medium, and uses thereof.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to David EPSTEIN, Richard FIKE, GLENN GODWIN, Stephen GORFIEN, Dale GRUBER, Don MCCLURE, Paul PRICE, JOYCE WOLANSKE.
Application Number | 20150079659 14/285047 |
Document ID | / |
Family ID | 27361964 |
Filed Date | 2015-03-19 |
United States Patent
Application |
20150079659 |
Kind Code |
A1 |
GORFIEN; Stephen ; et
al. |
March 19, 2015 |
SERUM-FREE MAMMALIAN CELL CULTURE MEDIUM, AND USES THEREOF
Abstract
The present invention provides a cell culture medium formulation
that supports the in vitro cultivation, particularly in suspension,
of mammalian cells, particularly epithelial cells and fibroblast
cells, and methods for cultivating mammalian cells in suspension in
vitro using these media. The media comprise a basal medium and a
polyanionic or polyanionic compound, preferably a polysulfonated or
polysulfated compound, and more preferably dextran sulfate. The
present invention also provides chemically defined, protein-free
eukaryotic cell culture media comprising an iron chelate and zinc,
which is capable of supporting the growth (and particularly the
high-density growth of mammalian cells) in suspension culture,
increasing the level of expression of recombinant protein in
cultured cells, and/or increasing virus production in cultured
cells.
Inventors: |
GORFIEN; Stephen;
(Williamsville, NY) ; FIKE; Richard; (Clarence,
NY) ; GODWIN; GLENN; (Holbrook, MA) ;
WOLANSKE; JOYCE; (Lancaster, NY) ; EPSTEIN;
David; (Philadelphia, PA) ; GRUBER; Dale;
(Leesburg, FL) ; MCCLURE; Don; (Indianapolis,
IN) ; PRICE; Paul; (Mount Pleasant, SC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
27361964 |
Appl. No.: |
14/285047 |
Filed: |
May 22, 2014 |
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Application
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12463371 |
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8785194 |
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14285047 |
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12463351 |
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8455246 |
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12463351 |
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09028514 |
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11151647 |
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Current U.S.
Class: |
435/201 ;
435/358; 435/384 |
Current CPC
Class: |
C12N 5/0603 20130101;
C12N 15/85 20130101; C12N 2500/22 20130101; C12Y 302/01023
20130101; C12N 5/005 20130101; C12N 2500/35 20130101; C12N 2500/74
20130101; C12N 2500/34 20130101; C12N 2501/91 20130101; C12N
2500/90 20130101; C12N 2500/38 20130101; C12N 2710/10051 20130101;
C12N 7/00 20130101; C12N 2501/90 20130101; C12N 5/0043 20130101;
C12N 9/2471 20130101; C07K 14/61 20130101; C12N 2500/14 20130101;
C12N 5/0037 20130101; C12N 2500/24 20130101; C12N 5/0682 20130101;
C12N 2500/16 20130101; C12N 9/2402 20130101; C12N 2500/76 20130101;
C12N 2500/32 20130101; C12N 2500/95 20130101 |
Class at
Publication: |
435/201 ;
435/384; 435/358 |
International
Class: |
C12N 5/071 20060101
C12N005/071; C12N 9/24 20060101 C12N009/24 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 2, 1997 |
US |
PCT/US97/15926 |
Claims
1-78. (canceled)
79. A method of producing a polypeptide comprising (a) obtaining a
mammalian cell that has been genetically engineered to produce a
polypeptide; and (b) cultivating said cell in a serum-free and
protein-free cell culture medium comprising an insulin substitute
and a transferrin substitute, under conditions favoring expression
of said polypeptide by said mammalian cell.
80. The method of claim 79, wherein said mammalian cell is an
epithelial cell.
81. The method of claim 79, wherein said mammalian cell is a human
cell.
82. The method of claim 81, wherein said human cell is a 293
embryonic kidney epithelial cell.
83. A polypeptide produced according to the method of claim 79.
84. A eukaryotic cell culture medium comprising a Fe.sup.2+ chelate
and a Zn.sup.2+ salt, wherein said medium is capable of supporting
the high-density growth of mammalian cells in suspension culture
and/or the expression of recombinant protein.
85. (canceled)
86. The eukaryotic cell culture medium according to claim 84,
wherein said mammalian cells are Chinese hamster ovary cells.
87. The eukaryotic cell culture medium according to claim 84,
wherein said medium is a 1.times. medium formulation.
88. The eukaryotic cell culture medium according to claim 84,
wherein said medium is a concentrated medium formulation.
89. The eukaryotic cell culture medium according to claim 88,
wherein said medium is a 10.times. medium formulation.
90. The eukaryotic cell culture medium according to claim 88,
wherein said medium formulation is greater than 10.times..
91-97. (canceled)
98. The eukaryotic cell culture medium according to claim 84,
further comprising one or more ingredients selected from the group
consisting of L-arginine, L-asparagine. H.sub.2O, L-aspartic acid,
L-glutamic acid, L-histidine, hydroxy-L-proline, L-isoleucine,
L-leucine, L-lysine.HCl, L-methionine, L-phenylalanine, L-proline,
L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine,
L-cystine.2HCl, Na.sub.2HPO.sub.4, pyridoxine. HCl, thiamine. HCl,
glutathione, cupric sulfate.7H.sub.2O, cadmium chloride.5H.sub.2O,
cobalt chloride.2H.sub.2O, stannous chloride. 2H.sub.2O, manganous
sulfate. H.sub.2O, nickel sulfate. 6H.sub.2O, sodium metavanadate,
ammonium molybdate. 4H.sub.2O, barium acetate, potassium bromide,
potassium iodide, chromium sulfate, sodium fluoride, silver
nitrate, rubidium chloride, zirconyl chloride, aluminum chloride,
germanium dioxide, titanium tetrachloride, sodium metasilicate,
magnesium chloride (anhydrous), D-calcium pantothenate, calcium
nitrate. 4H.sub.2O, potassium chloride, ascorbic acid magnesium
salt phosphate, pluronic F68 10% solution, Na.sub.2HPO.sub.4,
D-glucose, folic acid, riboflavin, biotin, choline chloride,
niacinamide, i-inositol, sodium pyruvate, vitamin B-12,
.beta.-mercaptoethanol, para-aminobenzoic acid,
.beta.-glycerophosphate, sodium selenite, ethanolamine.HCl,
spermine, putrescine.2HCl, monothioglycerol, and sodium
bicarbonate, wherein each of said ingredients is present in an
amount which supports the high-density growth of Chinese hamster
ovary cells in suspension culture and/or the expression of
recombinant protein.
99. A eukaryotic cell culture medium obtained by combining ferrous
sulfate.EDTA and ZnSO.sub.4.7H.sub.2O, together with a eukaryotic
medium, wherein said ferrous sulfate.EDTA and said
ZnSO.sub.4.7H.sub.2O are each present in an amount which supports
the high-density growth of Chinese hamster ovary cells in
suspension culture.
100. The eukaryotic cell culture medium according to claim 99,
further comprising a polyanionic or polycationic compound, wherein
said polyanionic or polycationic compound is present in an amount
sufficient to prevent cell clumping and/or increase the level of
recombinant protein expression.
101. The eukaryotic cell culture medium according to claim 100,
wherein said polyanionic compound is a polysulfonated compound or a
polysulfated compound.
102. The eukaryotic cell culture medium according to claim 101,
wherein said polysulfonated or polysulfated compound is selected
from the group consisting of dextran sulfate, heparin, heparan
sulfate, chondroitin sulfate, dermatan sulfate, pentosan sulfate
and a proteoglycan.
103. The eukaryotic cell culture medium according to claim 102,
wherein said polysulfonated or polysulfated compound is dextran
sulfate.
104. The eukaryotic cell culture medium according to claim 103,
wherein said dextran sulfate has an average molecular weight of
5,000 daltons.
105. The eukaryotic cell culture medium obtained according to claim
99 or 100, further comprising one or more ingredients selected from
the group consisting of L-arginine, L-asparagine.H.sub.2O,
L-aspartic acid, L-glutamic acid, L-histidine, hydroxy-L-proline,
L-isoleucine, L-leucine, L-lysine.HCl, L-methionine,
L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine, L-valine, L-cystine.2HCl, Na.sub.2HPO.sub.4,
pyridoxine.HCl, thiamine.HCl, glutathione, cupric
sulfate.5H.sub.2O, cadmium chloride.5H.sub.2O, cobalt
chloride.2H.sub.2O, stannous chloride.2H.sub.2O, manganous
sulfate.H.sub.2O, nickel sulfate.6H.sub.2O, sodium metavanadate,
ammonium molybdate.4H.sub.2O, barium acetate, potassium bromide,
potassium iodide, chromium sulfate, sodium fluoride, silver
nitrate, rubidium chloride, zirconyl chloride, aluminum. chloride,
germanium dioxide, titanium tetrachloride, sodium metasilicate,
magnesium chloride (anhydrous), D-calcium pantothenate, calcium
nitrate. 4H.sub.2O, potassium chloride, ascorbic acid magnesium
salt phosphate, pluronic F68 10% solution, Na.sub.2HP0.sub.4,
D-glucose, folic acid, riboflavin, biotin, choline chloride,
niacinamide, i-inositol, sodium pyruvate, vitamin B-12,
.beta.-mercaptoethanol, para-aminobenzoic acid,
.beta.-glycerophosphate, sodium selenite, ethanolamine.HCl,
monothioglycerol, and sodium bicarbonate, spermine,
putrescine.2HCl, wherein each of said ingredients is present in an
amount which supports the high-density growth of Chinese hamster
ovary cells in suspension culture and/or the expression of
recombinant protein.
106-128. (canceled)
129. A kit for the cultivation of a mammalian epithelial cell in
suspension in vitro, said kit comprising one or more containers,
wherein a first container contains a medium comprising a Fe.sup.2+
chelate and a Zn.sup.2+ salt, wherein said medium is capable of
supporting the high-density growth of mammalian cells in suspension
culture and/or the expression of recombinant protein.
130-139. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to cell culture
medium formulations. Specifically, the present invention provides
serum-free, low-protein or protein-free, defined cell culture
medium formulations that facilitate the in vitro cultivation of
mammalian cells in suspension. The culture media of the present
invention are particularly suitable for suspension culture of
epithelial cells, such as 293 human embryonic kidney cells, and
fibroblast cells, such as Chinese hamster ovary (CHO) cells.
BACKGROUND OF THE INVENTION
Cell Culture Media
[0002] The requirements of mammalian cell culture in vitro
comprise, in addition to basic nutritional substances, a complex
series of growth factors (Werner, R. G. et al., Mammalian Cell
Cultures Part I: Characterization, morphology and metabolism, in:
Arzneim.-Forsch./Drug Res. 43:1134-1139 (1993)). Usually, these are
added to the culture medium by supplying it with animal sera or
protein-fractions from animal sources. However, these chemically
non-defined mixtures exhibit variable lot to lot composition. Such
mixtures also represent a potential source of contaminants,
including viruses and mycoplasmas. For production on an industrial
scale, the high price of the supplements and difficulties in
downstream processing are additional considerations.
[0003] Cell culture media provide the nutrients necessary to
maintain and grow cells in a controlled, artificial and in vitro
environment. Characteristics and compositions of the cell culture
media vary depending on the particular cellular requirements.
Important parameters include osmolarity, pH, and nutrient
formulations.
[0004] Media formulations have been used to cultivate a number of
cell types including animal, plant and bacterial cells. Cells
cultivated in culture media catabolize available nutrients and
produce useful biological substances such as virus, monoclonal
antibodies, hormones, growth factors and the like. Such products
have therapeutic applications and, with the advent of recombinant
DNA technology, cells can be engineered to produce large quantities
of many of these products. Thus, the ability to cultivate cells in
vitro is not only important for the study of cell physiology, but
is also necessary for the production of useful substances which may
not otherwise be obtained by cost-effective means.
[0005] Cell culture media formulations have been well documented in
the literature and a number of media are commercially available. In
early cell culture work, media formulations were based upon the
chemical composition and physicochemical properties (e.g.,
osmolality, pH, etc.) of blood and were referred to as
"physiological solutions" (Ringer, S., J. Physiol. 3:380-393
(1880); Waymouth, C., In: Cells and Tissues in Culture, Vol. 1,
Academic Press, London, pp. 99-142 (1965); Waymouth, C., In Vitro
6:109-127 (1970)). However, cells in different tissues of the
mammalian body are exposed to different microenvironments with
respect to oxygen/carbon dioxide partial pressure and
concentrations of nutrients, vitamins, and trace elements;
accordingly, successful in vitro culture of different cell types
will often require the use of different media formulations. Typical
components of cell culture media include amino acids, organic and
inorganic salts, vitamins, trace metals, sugars, lipids and nucleic
acids, the types and amounts of which may vary depending upon the
particular requirements of a given cell or tissue type.
[0006] Typically, cell culture media formulations are supplemented
with a range of additives, including undefined components such as
fetal bovine serum (FBS) (10-20% v/v) or extracts from animal
embryos, organs or glands (0.5-10% v/v). While FBS is the most
commonly applied supplement in animal cell culture media, other
serum sources are also routinely used, including newborn calf,
horse and human. Organs or glands that have been used to prepare
extracts for the supplementation of culture media include
submaxillary gland (Cohen, S., J. Biol. Chem. 237:1555-1565
(1961)), pituitary (Peehl, D. M., and Ham, R. G., In Vitro
16:516-525 (1980); U.S. Pat. No. 4,673,649), hypothalamus (Maciag,
T., et al., Proc. Natl. Acad. Sci. USA 76:5674-5678 (1979);
Gilchrest, B. A., et al., J. Cell. Physiol. 120:377-383 (1984)),
ocular retina (Barretault, D., et al., Differentiation 18:29-42
(1981)) and brain (Maciag, T., et al., Science 211:1452-1454
(1981)). These types of chemically undefined supplements serve
several useful functions in cell culture media (Lambert, K. J. et
al., In: Animal Cell Biotechnology, Vol. 1, Spier, R. E. et al.,
Eds., Academic Press New York, pp. 85-122 (1985)). For example,
these supplements provide carriers or chelators for labile or
water-insoluble nutrients; bind and neutralize toxic moieties;
provide hormones and growth factors, protease inhibitors and
essential, often unidentified or undefined low molecular weight
nutrients; and protect cells from physical stress and damage. Thus,
serum or organ/gland extracts are commonly used as relatively
low-cost supplements to provide an optimal culture medium for the
cultivation of animal cells.
[0007] Unfortunately, the use of serum or organ/gland extracts in
tissue culture applications has several drawbacks (Lambert, K. J.
et al., In: Animal Cell Biotechnology, Vol 1, Spier, R. E. et al.,
Eds., Academic Press New York, pp. 85-122 (1985)). For example, the
chemical compositions of these supplements and sera vary between
lots, even from a single manufacturer. The supplements may also be
contaminated with infectious agents (e.g., mycoplasma and viruses)
which can seriously undermine the health of the cultured cells and
the quality of the final product. The use of undefined components
such as serum or animal extracts also prevents the true definition
and elucidation of the nutritional and hormonal requirements of the
cultured cells, thus eliminating the ability to study, in a
controlled way, the effect of specific growth factors or nutrients
on cell growth and differentiation in culture. Moreover, undefined
supplements prevent the researcher from studying aberrant growth
and differentiation and the disease-related changes in cultured
cells. Finally and most importantly to those employing cell culture
media in the industrial production of biological substances, serum
and organ/gland extract supplementation of culture media can
complicate and increase the costs of the purification of the
desired substances from the culture media due to nonspecific
co-purification of serum or extract proteins.
Defined Media
[0008] Improved levels of recombinant protein expression are
obtained from cells grown in serum-free medium, relative to the
level of expression seen in cells grown in medium supplemented with
serum (Battista, P. J. et al., Am. Biotech. Lab. 12:64-68 (1994)).
However, serum-free media may still contain one or more of a
variety of animal-derived components, including albumin, fetuin,
various hormones and other proteins. The presence of proteins or
peptides makes purification of recombinant protein difficult,
time-consuming, and expensive.
[0009] To overcome these drawbacks of the use of serum or
organ/gland extracts, a number of so-called "defined" media have
been developed. These media, which often are specifically
formulated to support the culture of a single cell type, contain no
undefined supplements and instead incorporate defined quantities of
purified growth factors, proteins, lipoproteins and other
substances usually provided by the serum or extract supplement.
Since the components (and concentrations thereof) in such culture
media are precisely known, these media are generally referred to as
"defined culture media." Often used interchangeably with "defined
culture media" is the term "serum-free media" or "SFM." A number of
SFM formulations are commercially available, such as those designed
to support the culture of endothelial cells, keratinocytes,
monocytes/macrophages, lymphocytes, hematopoietic stem cells,
fibroblasts, chondrocytes or hepatocytes which are available from
Life Technologies, Inc. (Rockville, Md.). The distinction between
SFM and defined media, however, is that SFM are media devoid of
serum and protein fractions (e.g., serum albumin), but not
necessarily of other undefined components such as organ/gland
extracts. Indeed, several SFM that have been reported or that are
available commercially contain such undefined components, including
several formulations supporting in vitro culture of keratinocytes
(Boyce, S. T., and Ham, R. G., J. Invest. Dermatol. 81:33 (1983);
Wille, J. J., et al., J. Cell. Physiol. 121:31 (1984); Pittelkow,
M. R., and Scott, R. E., Mayo Clin. Proc. 61:771 (1986); Pirisi,
L., et al., J. Virol. 61:1061 (1987); Shipley, G. D., and
Pittelkow, M. R., Arch. Dermatol. 123:1541 (1987); Shipley, G. D.,
et al., J. Cell. Physiol. 138:511-518 (1989); Daley, J. P., et al.,
FOCUS (GIBCO/LTI) 12:68 (1990); U.S. Pat. Nos. 4,673,649 and
4,940,666). SFM thus cannot be considered to be defined media in
the true definition of the term.
[0010] Defined media generally provide several distinct advantages
to the user. For example, the use of defined media facilitates the
investigation of the effects of a specific growth factor or other
medium component on cellular physiology, which may be masked when
the cells are cultivated in serum- or extract-containing media. In
addition, defined media typically contain much lower quantities of
protein (indeed, defined media are often termed "low protein
media") than those containing serum or extracts, rendering
purification of biological substances produced by cells cultured in
defined media far simpler and more cost-effective.
[0011] Some extremely simple defined media, which consist
essentially of vitamins, amino acids, organic and inorganic salts
and buffers have been used for cell culture. Such media (often
called "basal media"), however, are usually seriously deficient in
the nutritional content required by most animal cells. Accordingly,
most defined media incorporate into the basal media additional
components to make the media more nutritionally complex, but to
maintain the serum-free and low protein content of the media.
Examples of such components include serum albumin from bovine (BSA)
or human (HSA); certain growth factors derived from natural
(animal) or recombinant sources such as EGF or FGF; lipids such as
fatty acids, sterols and phospholipids; lipid derivatives and
complexes such as phosphoethanolamine, ethanolamine and
lipoproteins; protein and steroid hormones such as insulin,
hydrocortisone and progesterone; nucleotide precursors; and certain
trace elements (reviewed by Waymouth, C., in: Cell Culture Methods
for Molecular and Cell Biology, Vol. 1: Methods for Preparation of
Media, Supplements, and Substrata for Serum-Free Animal Cell
Culture, Barnes, D. W., et al., eds., New York: Alan R. Liss, Inc.,
pp. 23-68 (1984), and by Gospodarowicz, D., Id., at pp 69-86
(1984)).
[0012] The use of animal protein supplements in cell culture media,
however, also has certain drawbacks. For example, there is a risk
that the culture medium and/or products purified from it may be
immunogenic, particularly if the supplements are derived from an
animal different from the source of the cells to be cultured. If
biological substances to be used as therapeutics are purified from
such culture media, certain amounts of these immunogenic proteins
or peptides may be co-purified and may induce an immunological
reaction, up to and including anaphylaxis, in an animal receiving
such therapeutics.
[0013] To obviate this potential problem, supplements derived from
the same species as the cells to be cultured may be used. For
example, culture of human cells may be facilitated using HSA as a
supplement, while media for the culture of bovine cells would
instead use BSA. This approach, however, runs the risks of
introducing contaminants and adventitious pathogens into the
culture medium (such as Creutzfeld-Jakob Disease (CJD) from HSA
preparations, or Bovine Spongiform Encephalopathy ("Mad Cow
Disease") virus from BSA preparations), which can obviously
negatively impact the use of such media in the preparation of
animal and human therapeutics. In fact, for such safety reasons,
the biotechnology industry and government agencies are increasingly
regulating, discouraging and even forbidding the use of cell
culture media containing animal-derived proteins which may contain
such pathogens.
Non-Animal Peptide Supplements
[0014] To overcome the limitations of the use of animal proteins in
SFM, several attempts have been made to construct animal cell
culture media that are completely free of animal proteins. For
example, some culture media have incorporated extracts of yeast
cells into the basal medium (see, for example, U.K. Patent
Application No. GB 901673; Keay, L., Biotechnol. Bioengin.
17:745-764 (1975)) to provide sources of nitrogen and other
essential nutrients. In another approach, hydrolysates of wheat
gluten have been used, with or without addition of yeast extract,
to promote in vitro growth of animal cells (Japanese Patent
Application No. JP 2-49579). Still other media have been developed
in which serum is replaced by enzymatic digests of meat, or of
proteins such as .alpha.-lactalbumin or casein (e.g., peptone),
which have been traditionally used in bacterial culture
(Lasfargues, E. Y., et al., In Vitro 8(6):494-500 (1973); Keay, L.,
Biotechnol. Bioeng. 17:745-764 (1975); Keay, L., Biotechnol.
Bioeng. 19:399-411 (1977); Schlager, E.-J., J. Immunol. Meth.
194:191-199 (1996)). None of these approaches, however, provided a
culture medium optimal for the cultivation of a variety of animal
cells. Moreover, extracts from certain plants, including wheat,
barley, rye and oats have been shown to inhibit protein synthesis
in cell-free systems derived from animal cells (Coleman, W. H., and
Roberts, W. K., Biochim. Biophys. Acta 696:239-244 (1982)),
suggesting that the use of peptides derived from these plants in
cell culture media may actually inhibit, rather than stimulate, the
growth of animal cells in vitro. More recently, animal cell culture
SFM formulations comprising rice peptides have been described and
shown to be useful in cultivation of a variety of normal and
transformed animal cells (see co-pending, commonly owned U.S.
Application No. 60/028,197, filed Oct. 10, 1996, the disclosure of
which is incorporated herein by reference in its entirety).
Epithelial Cells
[0015] Overview
[0016] The epithelium lines the internal and external surfaces of
the organs and glands of higher organisms. Because of this
localization at the external interface between the environment and
the organism (e.g., the skin) or at the internal interface between
an organ and the interstitial space (e.g., the intestinal mucosal
lining), the epithelium has a major role in the maintenance of
homeostasis. The epithelium carries out this function, for example,
by regulating transport and permeability of nutrients and wastes
(Freshney, R. I., in: Culture of Epithelial Cells, Freshney, R. I.,
ed., New York: Wiley-Liss, pp. 1-23 (1992)).
[0017] The cells making up the epithelium are generically termed
epithelial cells. These cells may be present in multiple layers as
in the skin, or in a single layer as in the lung alveoli. As might
be expected, the structure, function and physiology of epithelial
cells are often tissue-specific. For example, the epidermal
epithelial cells of the skin are organized as stratified squamous
epithelium and are primarily involved in forming a protective
barrier for the organism, while the secretory epithelial cells of
many glands are often found in single layers of cuboidal cells that
have a major role in producing secretory proteins and
glycoproteins. Regardless of their location or function, however,
epithelial cells are usually regenerative. That is, under normal
conditions, or in response to injury or other activating stimulus,
epithelial cells are capable of dividing or growing. This
regenerative capacity has facilitated the in vitro manipulation of
epithelial cells, to the point where a variety of primary
epithelial cells and cell lines have been successfully cultivated
in vitro (Freshney, Id.).
[0018] 293 Cells
[0019] While the isolation and use of a variety of epithelial cells
and epithelial cell lines have been reported in the literature, the
human embryonic kidney cell line 293 ("293 cells"), which exhibits
epithelial morphology, has proven particularly useful for studies
of the expression of exogenous ligand receptors, production of
viruses and expression of allogeneic and xenogeneic recombinant
proteins. For example, U.S. Pat. No. 5,166,066 describes the
construction of a stable 293 cell line comprising functional GABA
receptors that include a benzodiazepine binding site, that have
proven useful in identification and screening of candidate
psychoactive drugs. 293 cells have also been used to produce
viruses such as natural and recombinant adenoviruses (Garnier, A.,
et al., Cytotechnol. 15:145-155 (1994); Bout, A., et al., Cancer
Gene Therapy 3(6):S24, abs. P-52 (1996); Wang, J.-W., et al.,
Cancer Gene Therapy 3(6):S24, abs. P-53 (1996)), which may be used
for vaccine production or construction of adenovirus vectors for
recombinant protein expression. Finally, 293 cells have proven
useful in large-scale production of a variety of recombinant human
proteins (Berg, D. T., et al., BioTechniques 14(6):972-978 (1993);
Peshwa, M. V., et al., Biotechnol. Bioeng. 41:179-187 (1993);
Garnier, A., et al., Cytotechnol. 15:145-155 (1994)).
Fibroblast Cells
[0020] Overview
[0021] Cells loosely called fibroblasts have been isolated from
many different tissues and are understood to be connective tissue
cells. It is clearly possible to cultivate cell lines, loosely
termed fibroblastic cells, from embryonic and adult tissues.
Fibroblasts cells characteristically have a "spindle" appearance.
Fibroblast-like cells have morphological characteristics typical of
fibroblast cells. Under a light microscope the cells appear pointed
and elongated ("spindle shaped") when they grow as a monolayer on
the surface of a culture vessel. Cell lines can be regarded as
fibroblast or fibroblast-like after confirmation with appropriate
markers, such as collagen, type I ((Freshney, R. I., in: Culture of
Epithelial Cells, Freshney, R. I., ed., New York: Wiley-Liss, pp.
1-23 (1987)).
[0022] CHO Cells
[0023] CHO cells have been classified as both epithelial and
fibroblast cells derived from the Chinese hamster ovary. A cell
line started from Chinese hamster ovary (CHO-K1) (Kao, F.-T. And
Puck, T. T., Proc. Natl. Acad. Sci. USA 60:1275-1281 (1968) has
been in culture for many years but its identity is still not
confirmed.
[0024] U.S. Pat. No. 5,316,938 discloses a medium for growing CHO
cells in suspension which is essentially free of protein, lipid,
and carbohydrate isolated from an animal source. This patent
teaches that zinc is an optional ingredient and that it is
preferable to supplement the medium with recombinant insulin.
[0025] U.S. Pat. No. 5,122,469 discloses a protein-free medium
which facilitates the expression of recombinant protein in CHO
cells. This patent teaches that it is preferable to supplement the
medium with both insulin and transferrin.
[0026] Zang, M. et al., Bio/Technology 13:389-392 (1995) discloses
a protein-free medium for growing CHO cells in suspension culture
for recombinant protein expression. See also U.S. Pat. Nos.
5,316,938 and 5,122,469.
[0027] U.S. Pat. No. 4,767,704 discloses a protein-free medium
which facilitates the long-term growth of antibody-producing
monolayer hybridoma cells.
[0028] Suspension Cells
[0029] As noted above, most primary mammalian epithelial cells,
mammalian fibroblast cells, epithelial cell lines, and fibroblast
cell lines are typically grown in monolayer culture. For some
applications, however, it would be advantageous to cultivate such
cells as suspension cultures. For example, suspension cultures grow
in a three-dimensional space. Monolayer cultures in similar-sized
vessels, however, can only grow two-dimensionally on the vessel
surface. Thus, suspension cultures typically result in higher cell
yields, and correspondingly higher yields of biologicals (e.g.,
viruses, recombinant polypeptides, etc.) compared to monolayer
cultures. In addition, suspension cultures are often easier to feed
and scale-up, via simple addition of fresh culture media (dilution
subculturing) to the culture vessel rather than trypsinization and
centrifugation as is often required with monolayer cultures.
[0030] Many anchorage-dependent cells, such as primary epithelial
cells, primary fibroblast cells, epithelial cell lines, and
fibroblast cell lines, however, are not easily adapted to
suspension culture. Since they are typically dependent upon
anchorage to a substrate for optimal growth, growth of these cells
in suspension may require their attachment to microcarriers such as
latex or collagen beads. Thus, cells grown in this fashion, while
capable of higher density culture than traditional monolayer
cultures, are still technically attached to a surface; subculturing
of these cells therefore requires similar steps as those described
above for monolayer cultures. Furthermore, when large batch or
fermenter cultures are established, a large volume of microcarriers
often settles to the bottom of the culture vessel, thereby
requiring a more complicated agitation mechanism to keep the
microcarriers (and thus, the cells) in suspension without causing
shear damage to the cells (Peshwa, M. V., et al., Biotechnol.
Bioeng. 41:179-187 (1993)).
[0031] Although many transformed cells are capable of being grown
in suspension (Freshney, R. I., Culture of Animal Cells: A Manual
of Basic Technique, New York: Alan R. Liss, Inc., pp. 123-125
(1983)), successful suspension cultures often require relatively
high-protein media or supplementation of the media with serum or
serum components (such as the attachment factors fibronectin and/or
vitronectin), or sophisticated perfusion culture control systems
(Kyung, Y.-S., et al., Cytotechnol. 14:183-190 (1994)), which may
be disadvantageous for the reasons discussed above. In addition,
many epithelial cells when grown in suspension form aggregates or
"clumps" which may interfere with successful subculturing and
reduce growth rate and production of biologicals by the cultures.
When clumping occurs, the overall cellular surface area exposed to
medium is decreased and the cells are deprived of nutrition. As a
result, growth slows, diminished cell densities are obtained, and
protein expression is compromised.
[0032] Thus, there remains a need for a chemically defined,
protein-free medium which facilitates the growth of mammalian cells
to high density and/or increases the level of expression of
recombinant protein, reduces cell clumping, and which does not
require supplementation with animal proteins, such as transferrin
and insulin.
[0033] There also remains a need remains for defined culture media,
that are serum-free, and low-protein or protein-free, for the
suspension cultivation of mammalian cells that are normally
anchorage-dependent, including epithelial cells and fibroblast
cells, such as 293 cells and CHO cells. Such culture media will
facilitate studies of the effects of growth factors and other
stimuli on cellular physiology, will allow easier and more
cost-effective production and purification of biological substances
(e.g., viruses, recombinant proteins, etc.) produced by cultured
mammalian cells in the biotechnology industry, and will provide
more consistent results in methods employing the cultivation of
mammalian cells.
SUMMARY OF THE INVENTION
[0034] The present invention provides a method of cultivating a
mammalian cell in suspension in vitro, the method comprising (a)
obtaining a mammalian cell to be cultivated in suspension; and (b)
contacting the cell with a serum-free cell culture medium
comprising at least one polyanionic or polycationic compound,
wherein the medium supports the cultivation of said cell in
suspension. The present invention also relates to media for
suspension culture and to compositions comprising mammalian cells
in such suspension culture.
[0035] The present invention also relates to a method of replacing
protein (particularly animal derived protein) in mammalian cell
culture media. In particular, the invention relates to replacing
transferrin and/or insulin, to media containing such replacements,
and to compositions comprising mammalian cells in such media.
[0036] The present invention relates in particular to a medium
referred to herein as the "suspension medium" and to a medium
referred to herein as the "replacement medium."
The Suspension Medium
[0037] The present invention is directed to a serum-free cell
culture medium comprising one or more polyanionic or polycationic
compounds, wherein the medium is capable of supporting the
suspension cultivation of a mammalian epithelial of fibroblast
cells in vitro. In the suspension medium, the polyanionic compound
is preferably a polysulfonated or polysulfated compound, more
preferably heparin, dextran sulfate, heparan sulfate, dermatan
sulfate, chondroitin sulfate, pentosan polysulfate, a proteoglycan
or the like, and most preferably dextran sulfate, which preferably
has a molecular weight of about 5,000 daltons.
[0038] In particular, the invention is directed to such culture
media that further comprise one or more ingredients selected from
the group of ingredients consisting of one or more amino acids, one
or more vitamins, one or more inorganic salts, one or more sugars,
one or more buffering salts, one or more lipids, one or more
insulins (or insulin substitutes) and one or more transferrins (or
transferrin substitutes). The preferred sugar used in the media of
the invention is D-glucose, while the preferred buffer salt is
N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES).
The invention is also directed to such culture media which may
optionally comprise one or more supplements selected from the group
of supplements consisting of one or more cytokines, heparin, one or
more animal peptides, one or more yeast peptides and one or more
plant peptides (which are preferably one or more rice peptides or
one or more soy peptides). The amino acid ingredient of the present
media preferably comprises one or more amino acids selected from
the group consisting of L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,
L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine and L-valine. The vitamin ingredient of the present
media preferably comprises one or more vitamins selected from the
group consisting of biotin, choline chloride,
D-Ca.sup.++-pantothenate, folic acid, i-inositol, niacinamide,
pyridoxine, riboflavin, thiamine and vitamin B.sub.12. The
inorganic salt ingredient of the present media preferably comprises
one or more inorganic salts selected from the group consisting of
one or more calcium salts, Fe(NO.sub.3).sub.3, KCl, one or more
magnesium salts, one or more manganese salts, NaCl, NaHCO.sub.3,
Na.sub.2HPO.sub.4, one or more selenium salts, one or more vanadium
salts and one or more zinc salts.
[0039] The invention is also directed to a cell culture medium
comprising the ingredients ethanolamine, D-glucose,
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES),
insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68,
putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine,
L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,
L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline
chloride, D-Ca.sup.++-pantothenate, folic acid, i-inositol,
niacinamide, pyridoxine, riboflavin, thiamine, vitamin B.sub.12,
one or more calcium salts, Fe(NO.sub.3).sub.3, KCl, one or more
magnesium salts, one or more manganese salts, NaCl, NaHCO.sub.3,
Na.sub.2HPO.sub.4, one or more selenium salts, one or more vanadium
salts and one or more zinc salts, wherein each ingredient is
present in an amount which supports the suspension cultivation of a
mammalian epithelial cell in vitro. The invention is also directed
to such media which further comprise dextran sulfate, and which
optionally comprise one or more supplements selected from the group
consisting of one or more cytokines, heparin, one or more animal
peptides, one or more yeast peptides and one or more plant peptides
(most preferably one or more rice peptides or one or more soy
peptides).
[0040] The invention is also directed to a mammalian cell culture
medium obtained by combining a basal medium with dextran sulfate
(which preferably has a molecular weight of about 5,000 daltons),
wherein the medium is capable of supporting the suspension
cultivation of a mammalian epithelial or fibroblast cell in vitro.
In one preferred such medium, the basal medium is obtained by
combining one or more ingredients selected from the group
consisting of ethanolamine, D-glucose,
N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES),
insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68,
putrescine, sodium pyruvate, transferrin, L-alanine, L-arginine,
L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,
L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine,
L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,
L-threonine, L-tryptophan, L-tyrosine, L-valine, biotin, choline
chloride, D-Ca.sup.++-pantothenate, folic acid, i-inositol,
niacinamide, pyridoxine, riboflavin, thiamine, vitamin B.sub.12,
one or more calcium salts, Fe(NO.sub.3).sub.3, KCl, one or more
magnesium salts, one or more manganese salts, NaCl, NaHCO.sub.3,
Na.sub.2HPO.sub.4, one or more selenium salts, one or more vanadium
salts and one or more zinc salts, wherein each ingredient is added
in an amount which supports the suspension cultivation of a
mammalian epithelial or fibroblast cell in vitro. The invention is
also directed to a medium obtained by combining the media obtained
as described above and one or more supplements selected from the
group consisting of one or more cytokines, heparin, one or more
animal peptides, one or more yeast peptides and one or more plant
peptides (preferably one or more rice peptides or one or more soy
peptides).
[0041] The media provided by the present invention may be
protein-free, and may be a 1.times. formulation or concentrated as
a 10.times. or higher formulation. The basal medium of the present
invention comprises a number of ingredients, including amino acids,
vitamins, organic and inorganic salts, sugars and other components,
each ingredient being present in an amount which supports the
cultivation of a mammalian epithelial cell in vitro. The medium may
be used to culture a variety of mammalian cells, including primary
epithelial cells (e.g., keratinocytes, cervical epithelial cells,
bronchial epithelial cells, tracheal epithelial cells, kidney
epithelial cells and retinal epithelial cells) and established cell
lines (e.g., 293 embryonic kidney cells, HeLa cervical epithelial
cells and PER-C6 retinal cells, MDBK (NBL-1) cells, CRFK cells,
MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells,
HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells,
LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24
cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,
LLC-MK.sub.2 cells, Clone M-3 cells, I-10 cells, RAG cells, TCMK-1
cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1 cells,
GH.sub.3 cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1 cells,
XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives
thereof), fibroblast cells from any tissue or organ (including but
not limited to heart, liver, kidney, colon, intesting, esophagus,
stomach, neural tissue (brain, spinal cord), lung, vascular tissue
(artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,
tonsil, bone marrow, and blood), spleen, and fibroblast and
fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33
cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells,
Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit
529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,
Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38
cells, WI-26 cells, MiCl.sub.1 cells, CHO cells, CV-1 cells, COS-1
cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells,
BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB
cells, BLO-11 cells, NOR-10 cells, C.sub.3H/IOTI/2 cells,
HSDM.sub.1C.sub.3 cells, KLN205 cells, McCoy cells, Mouse L cells,
Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells,
L-MTK.sup.- (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1
cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C.sub.II
cells, and Jensen cells, or derivatives thereof).
[0042] Cells supported by the medium of the present invention may
be derived from any animal, preferably a mammal, and most
preferably a human. The cells cultivated in the present media may
be normal cells or abnormal cells (i.e., transformed cells,
established cells, or cells derived from diseased tissue samples).
The media of the invention may also be prepared in different forms,
such as dry powder media ("DPM"), as liquid media or as media
concentrates.
[0043] The present invention also provides methods of cultivating
mammalian epithelial or fibroblast cells using the culture medium
formulations disclosed herein, comprising (a) contacting the cells
with the cell culture media of the invention; and (b) cultivating
the cells under conditions suitable to support cultivation of the
cells. Preferably, cells cultivated according to these methods
(which may include any of the cells described above) are cultivated
in suspension.
[0044] The invention also provides kits for use in the cultivation
of a mammalian epithelial cell. Kits according to the present
invention comprise one or more containers, wherein a first
container contains the culture medium of the invention. Additional
kits of the invention comprise one or more containers wherein a
first container contains a basal culture medium as described above
and a second container contains one or more polyanionic or
polycationic compounds, preferably a polysulfonated or polysulfated
compound, more preferably heparin, dextran sulfate, heparan
sulfate, dermatan sulfate, chondroitin sulfate, pentosan
polysulfate, a proteoglycan or the like, and most preferably
dextran sulfate which preferably has a molecular weight of about
5,000 daltons. These kits may further comprise one or more
additional containers containing one or more supplements selected
from the group consisting of one or more cytokines, heparin, one or
more animal peptides, one or more yeast peptides and one or more
plant peptides (which are preferably one or more rice peptides or
one or more soy peptides).
[0045] The invention further provides compositions comprising the
culture media of the present invention, which optionally may
further comprise one or more mammalian epithelial or fibroblast
cells, such as those described above, particularly one or more 293
embryonic kidney cells, PER-C6 retinal cells, and CHO cells.
[0046] The present invention further relates to methods of
cultivating mammalian cells (particularly those described above and
most particularly 293 embryonic kidney epithelial cells, PER-C6,
and CHO cells) in suspension comprising (a) obtaining a mammalian
cell to be cultivated in suspension; and (b) contacting the cell
with the culture media of the invention under conditions sufficient
to support the cultivation of the cell in suspension.
[0047] The present invention further relates to methods of
producing a virus, and to viruses produced by these methods, the
methods comprising (a) obtaining a mammalian cell, preferably a
mammalian cell described above and most preferably a 293 embryonic
kidney epithelial cell, PER-C6, and CHO cells, to be infected with
a virus; (b) contacting the cell with a virus under conditions
suitable to promote the infection of the cell by the virus; and (c)
cultivating the cell in the culture medium of the invention under
conditions suitable to promote the production of the virus by the
cell. Viruses which may be produced according to these methods
include adenoviruses, adeno-associated viruses and
retroviruses.
[0048] The present invention further relates to methods of
producing a polypeptide, and to polypeptides produced by these
methods, the methods comprising (a) obtaining a mammalian cell,
preferably a mammalian cell described above and most preferably a
293 embryonic kidney epithelial cell, PER-C6, and CHO cell, that
has been genetically engineered to produce a polypeptide; and (b)
cultivating the mammalian cell in the culture medium of the
invention under conditions favoring the expression of the desired
polypeptide by the mammalian cell.
The Replacement Medium
[0049] The present invention also provides the replacement medium,
a chemically defined, protein-free eukaryotic (e.g., mammalian)
cell culture medium comprising a Fe.sup.2+ and/or Fe.sup.3+ chelate
and/or a Zn.sup.2+ salt, and optionally at least one polyanionic or
polycationic compound as defined herein, which is capable of
supporting the growth (in particular, the high-density growth of
any of mentioned mammalian cells, and particularly those described
above, and preferably, CHO cells, PER-C6 cells, and 293 cells) in
suspension culture, increasing the level of expression of
recombinant protein in cultured cells, and/or increasing virus
production in cultured cells. Further, the present invention
provides a eukaryotic cell culture medium, obtained by combining an
iron chelate and zinc, which is capable of supporting the density
growth (in particular, the high-density growth of any of mentioned
mammalian cells, and particularly those described above, and
preferably, CHO cells, PER-C6 cells, and 293 cells) in suspension
culture, increasing the level of expression of recombinant protein
in cultured cells, and/or increasing virus production in cultured
cells. Further, the present invention provides a method of
cultivating mammalian cells, and particularly CHO cells, in
suspension culture such that said cells express a recombinant
protein comprising the steps of contacting said cells with the
eukaryotic cell culture medium, wherein the iron chelate and zinc
are present in an amount which supports the growth of mammalian
cells in culture, and optionally together with a polyanionic or
polycationic compound in an amount effective to reduce clumping of
the cells compared to when the compound is not added, and
cultivating the cells under conditions suitable to support both the
growth (in particular, the high-density growth of any of mentioned
mammalian cells, and particularly those described above, and
preferably, CHO cells, PER-C6 cells, and 293 cells) in suspension
culture, increasing the level of expression of recombinant protein
in cultured cells, and/or increasing virus production in cultured
cells.
[0050] The media provided by the present invention may be
protein-free, and may be a 1.times. formulation or concentrated as
a 10.times. or higher formulation. The basal medium of the present
invention comprises a number of ingredients, including amino acids,
vitamins, organic and inorganic salts, sugars and other components,
each ingredient being present in an amount which supports the
cultivation of a mammalian epithelial cell in vitro. The medium may
be used to culture a variety of mammalian cells, including primary
epithelial cells (e.g., keratinocytes, cervical epithelial cells,
bronchial epithelial cells, tracheal epithelial cells, kidney
epithelial cells and retinal epithelial cells) and established cell
lines (e.g., 293 embryonic kidney cells, HeLa cervical epithelial
cells and PER-C6 retinal cells, MDBK (NBL-1) cells, CRFK cells,
MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells,
HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells,
LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24
cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,
LLC-MK.sub.2 cells, Clone M-3 cells, I-10 cells, RAG cells, TCMK-1
cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1 cells,
GH.sub.3 cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1 cells,
XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives
thereof), fibroblast cells from any tissue or organ (including but
not limited to heart, liver, kidney, colon, intesting, esophagus,
stomach, neural tissue (brain, spinal cord), lung, vascular tissue
(artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,
tonsil, bone marrow, and blood), spleen, and fibroblast and
fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33
cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells,
Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit
529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,
Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38
cells, WI-26 cells, MiCl.sub.1 cells, CHO cells, CV-1 cells, COS-1
cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells,
BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB
cells, BLO-11 cells, NOR-10 cells, C.sub.3H/IOTI/2 cells,
HSDM.sub.1C.sub.3 cells, KLN205 cells, McCoy cells, Mouse L cells,
Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells,
L-MTK.sup.- (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1
cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C.sub.II
cells, and Jensen cells, or derivatives thereof).
[0051] Cells supported by the medium of the present invention may
be derived from any animal, preferably a mammal, and most
preferably a human. The cells cultivated in the present media may
be normal cells or abnormal cells (i.e., transformed cells,
established cells, or cells derived from diseased tissue samples).
The media of the invention may also be prepared in different forms,
such as dry powder media ("DPM"), as liquid media or as media
concentrates.
[0052] The present invention also provides methods of cultivating
mammalian epithelial or fibroblast cells using the culture medium
formulations disclosed herein, comprising (a) contacting the cells
with the cell culture media of the invention; and (b) cultivating
the cells under conditions suitable to support cultivation of the
cells. Preferably, cells cultivated according to these methods
(which may include any of the cells described above) are cultivated
in suspension.
[0053] The medium of the present invention is a chemically defined
formulation which contains no protein or hydrolysates of either
plant or animal origin. Although the invention is not bound by any
particular theory, it is believed that the ability of the medium of
the present invention to facilitate the growth of mammalian cells
is due to the replacement of insulin by zinc and/or the replacement
of transferrin with an iron chelate. Moreover, when supplemented
with dextran sulfate, the medium facilitates growth (in particular,
the high-density growth of any of mentioned mammalian cells, and
particularly those described above, and preferably, CHO cells,
PER-C6 cells, and 293 cells) in suspension culture, increases the
level of expression of recombinant protein in cultured cells,
and/or increases virus production in cultured cells without
clumping.
[0054] The medium of the present invention can be used to grow
mammalian cells (in particular, to high-density) of any of
mentioned mammalian cells, and particularly those described above,
and preferably, CHO cells, PER-C6 cells, and 293 cells) to high
density, to facilitate the expression of recombinant protein in
such cells, and/or to increase virus production in cultured cells
without clumping. The medium is advantageous because it is
chemically defined, it is protein free, and it does not require
supplementation with transferrin, insulin, or other proteins to
facilitate cell growth and/or expression of recombinant protein. In
addition, the protein-free nature of the medium of the present
invention greatly simplifies the purification of recombinant
protein.
[0055] The invention also provides kits for use in the cultivation
of a mammalian epithelial cell. Kits according to the present
invention comprise one or more containers, wherein a first
container contains the culture medium of the invention. Additional
kits of the invention comprise one or more containers wherein a
first container contains a basal culture medium as described above
and a second container contains one or more polyanionic or
polycationic compounds, preferably a polysulfonated or polysulfated
compound, more preferably heparin, dextran sulfate, heparan
sulfate, dermatan sulfate, chondroitin sulfate, pentosan
polysulfate, a proteoglycan or the like, and most preferably
dextran sulfate which preferably has a molecular weight of about
5,000 daltons.
[0056] Other preferred embodiments of the present invention will be
apparent to one of ordinary skill in light of the following
drawings and description of the invention, and of the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1 depicts a line graph demonstrating cell growth
(.box-solid.) and percent viable cells ( ), over a five-day time
course, of 293 cells cultured in suspension in the suspension
culture media of the invention.
[0058] FIG. 2 depicts a line graph demonstrating viable cell
density, over a seven-day time course, of 293 cells cultured in
suspension in the suspension culture media without transferrin ( ),
in the present culture media with human transferrin (.box-solid.),
or in the present culture media in which transferrin was replaced
with 60 .mu.M ferric chloride/sodium citrate chelate
(.tangle-solidup.) or with 40 .mu.M ferrous sulfate/EDTA chelate
(.gradient.).
[0059] FIG. 3 depicts a bar graph demonstrating the production of
adenovirus-5 in 293 cells cultured in the suspension culture media
of the invention over a five-day time course. TCID50=tissue culture
infectious dose-50%.
[0060] FIG. 4 depicts a line graph demonstrating
.beta.-galactosidase production, as a function of viable cell
number over a six-day time course, by 293 cells cultured in the
suspension culture media of the invention.
[0061] FIG. 5A depicts a bar graph showing the effect of
low-protein/serum-free, essentially protein-free, and
protein-free/chemically defined media on the growth of r.beta.-gal
CHO cells. FIG. 5B depicts a bar graph showing the effect of low
protein/serum-free, essentially protein-free, and
protein-free/chemically defined media on the expression of
r.beta.-galactosidase in r.beta.-gal CHO cells.
[0062] FIG. 6A depicts a bar graph showing the effect of low
protein/serum-free, essentially protein-free, and protein
free/chemically defined media on the growth of r.beta.-gal CHO
cells. In this figure, "g" refers to the growth phase and "p"
refers to the production phase. FIG. 6B depicts a bar graph showing
the effect of low protein/serum-free, essentially protein-free, and
protein-free/chemically defined media on the expression of
r.beta.-galactosidase in r.beta.-gal CHO cells. In this figure, "g"
refers to the growth phase and "p" refers to the production
phase.
[0063] FIG. 7A depicts a bar graph showing the effect of
methotrexate on the growth of r.beta.-gal CHO cells. FIG. 7B
depicts a bar graph showing the effect of methotrexate on the
expression of r.beta.-galactosidase in r.beta.-gal CHO cells.
[0064] FIG. 8A depicts a graph showing the effect of essentially
protein-free ( ) and protein-free/chemically defined (.box-solid.)
media on rbGH CHO cells. FIG. 8B depicts a graph showing the effect
of essentially protein-free ( ) and protein-free/chemically defined
(.box-solid.) media on rbGH expression in rbGH CHO cells.
[0065] FIG. 9A depicts a bar graph showing the effect of
low-protein, insulin- and transferrin-containing medium and a
protein-free/chemically defined medium on the growth of r.beta.-gal
CHO cells. FIG. 9B depicts a bar graph showing the effect of
low-protein, insulin- and transferrin-containing medium and a
protein-free/chemically defined medium on r.beta.-galactosidase
expression in r.beta.-gal CHO cells.
[0066] FIG. 10 depicts a bar graph showing the effect of CD CHO,
CHO III, and FMX-8 media on the growth of r.beta.-gal CHO
cells.
[0067] FIG. 11 depicts a bar graph showing the effect of CD CHO,
CHO III, and FMX-8 media on r.beta.-gal expression in r.beta.-gal
CHO cells.
[0068] FIG. 12A shows the level of growth of r.beta.-gal CHO cells
cultured in CD CHO medium in a shake flask (.box-solid.) and in a
bioreactor ( ). FIG. 12B shows the level of r.beta.-gal expression
in cells cultured in CD CHO medium in a shake flask and in a
bioreactor.
[0069] FIG. 13 depicts a bar graph showing the effect of dextran
sulfate on the growth of r.beta.-gal CHO cells. In the figure, "A"
is dextran sulfate (m.w. 5,000) and "C" is dextran sulfate (m.w.
500,000).
[0070] FIG. 14 depicts a bar graph showing the effect of dextran
sulfate on r.beta.-gal expression in r.beta.-gal CHO cells. In the
figure, "A" is dextran sulfate (m.w. 5,000) and "C" is dextran
sulfate (m.w. 500,000).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0071] In the description that follows, a number of terms
conventionally used in the field of cell culture media are utilized
extensively. In order to provide a clear and consistent
understanding of the specification and claims, and the scope to be
given such terms, the following definitions are provided.
[0072] The term "batch culture" refers to a culture allowed to
progress from inoculation to conclusion without refeeding the
cultured cells with fresh medium.
[0073] The term "cytokine" refers to a compound that induces a
physiological response in a cell, such as growth, differentiation,
senescence, apoptosis, cytotoxicity or antibody secretion. Included
in this definition of "cytokine" are growth factors, interleukins,
colony-stimulating factors, interferons, lymphokines and the
like.
[0074] By "cell culture" or "culture" is meant the maintenance of
cells in an artificial, in vitro environment. It is to be
understood, however, that the term "cell culture" is a generic term
and may be used to encompass the cultivation not only of individual
cells, but also of tissues, organs, organ systems or whole
organisms, for which the terms "tissue culture," "organ culture,"
"organ system culture" or "organotypic culture" may occasionally be
used interchangeably with the term "cell culture." The media of the
present invention can be used to culture any adherent mammalian
cell (i.e., a cell which adheres to the culture vessel) and any
mammalian cell which grows in suspension culture.
[0075] By "cultivation" is meant the maintenance of cells in vitro
under conditions favoring growth, differentiation or continued
viability, in an active or quiescent state, of the cells. In this
sense, "cultivation" may be used interchangeably with "cell
culture" or any of its synonyms described above.
[0076] By "culture vessel" is meant a glass, plastic, or metal
container that can provide an aseptic environment for culturing
cells.
[0077] The phrases "cell culture medium," "culture medium" (plural
"media" in each case) and "medium formulation" refer to a nutritive
solution for cultivating cells and may be used interchangeably.
[0078] The term "contacting" refers to the placing of cells to be
cultivated in vitro into a culture vessel with the medium in which
the cells are to be cultivated. The term "contacting" encompasses
mixing cells with medium, pipetting medium onto cells in a culture
vessel, and submerging cells in culture medium.
[0079] The term "combining" refers to the mixing or admixing of
ingredients in a cell culture medium formulation.
[0080] A "chemically defined" medium is one for which every
ingredient is known. A chemically defined medium is distinguished
from serum, embryonic extracts, and hydrolysates, each of which
contain unknown components. The medium of the present invention is
chemically defined and is free of proteins and peptides.
[0081] The term "high density" refers to a cellular density of
about 1.times.10.sup.6 to about 2.times.10.sup.7 cells/ml. In a
preferred embodiment, the term refers to a cellular density of
about 1.times.10.sup.6 to about 5.times.10.sup.6 cells/ml in batch
culture.
[0082] The term "ingredient" refers to any compound, whether of
chemical or biological origin, that can be used in cell culture
media to maintain or promote the growth of proliferation of cells.
The terms "component," "nutrient" and ingredient" can be used
interchangeably and are all meant to refer to such compounds.
Typical ingredients that are used in cell culture media include
amino acids, salts, metals, sugars, lipids, nucleic acids,
hormones, vitamins, fatty acids, proteins and the like. Other
ingredients that promote or maintain cultivation of cells ex vivo
can be selected by those of skill in the art, in accordance with
the particular need.
[0083] Each ingredient used in cell culture media has unique
physical and chemical characteristics. By "compatible ingredients"
is meant those media nutrients which can be maintained in solution
and form a "stable" combination. A solution containing "compatible
ingredients" is said to be "stable" when the ingredients do not
degrade or decompose substantially into toxic compounds, or do not
degrade or decompose substantially into compounds that can not be
utilized or catabolized by the cell culture. Ingredients are also
considered "stable" if degradation can not be detected or when
degradation occurs at a slower rate when compared to decomposition
of the same ingredient in a 1.times. cell culture media
formulation. Glutamine, for example, in 1.times. media
formulations, is known to degrade into pyrrolidone carboxylic acid
and ammonia. Glutamine in combination with divalent cations are
considered "compatible ingredients" since little or no
decomposition can be detected over time. See U.S. Pat. No.
5,474,931.
[0084] Compatibility of media ingredients, in addition to stability
measurements, are also determined by the "solubility" of the
ingredients in solution. The term "solubility" or "soluble" refers
to the ability of a ingredient to form a solution with other
ingredients. Ingredients are thus compatible if they can be
maintained in solution without forming a measurable or detectable
precipitate. Thus, the term "compatible ingredients" as used herein
refers to the combination of particular culture media ingredients
which, when mixed in solution either as concentrated or 1.times.
formulations, are "stable" and "soluble."
[0085] A "protein-free" medium is one which contains no proteins or
peptides. A protein-free medium is distinguished from low-protein
and essentially protein-free media, both of which contain proteins
and/or peptides.
[0086] A cell culture medium is composed of a number of ingredients
and these ingredients vary from one culture medium to another. A
"1.times. formulation" is meant to refer to any aqueous solution
that contains some or all ingredients found in a cell culture
medium at working concentrations. The "1.times. formulation" can
refer to, for example, the cell culture medium or to any subgroup
of ingredients for that medium. The concentration of an ingredient
in a 1.times. solution is about the same as the concentration of
that ingredient found in a cell culture formulation used for
maintaining or cultivating cells in vitro. A cell culture medium
used for the in vitro cultivation of cells is a 1.times.
formulation by definition. When a number of ingredients are
present, each ingredient in a 1.times. formulation has a
concentration about equal to the concentration of those ingredients
in a cell culture medium. For example, RPMI-1640 culture medium
contains, among other ingredients, 0.2 g/L L-arginine, 0.05 g/L
L-asparagine, and 0.02 g/L L-aspartic acid. A "1.times.
formulation" of these amino acids contains about the same
concentrations of these ingredients in solution. Thus, when
referring to a "1.times. formulation," it is intended that each
ingredient in solution has the same or about the same concentration
as that found in the cell culture medium being described. The
concentrations of ingredients in a 1.times. formulation of cell
culture medium are well known to those of ordinary skill in the
art. See Methods For Preparation of Media, Supplements and
Substrate For Serum-Free Animal Cell Culture, New York: Allen R.
Liss (1984), which is incorporated by reference herein in its
entirety. The osmolarity and/or pH, however, may differ in a
1.times. formulation compared to the culture medium, particularly
when fewer ingredients are contained in the 1.times.
formulation.
[0087] A "10.times. formulation" is meant to refer to a solution
wherein each ingredient in that solution is about 10 times more
concentrated than the same ingredient in the cell culture medium.
For example, a 10.times. formulation of RPMI-1640 culture medium
may contain, among other ingredients, 2.0 g/L L-arginine, 0.5 g/L
L-asparagine, and 0.2 g/L L-aspartic acid (compare 1.times.
formulation, above). A "10.times. formulation" may contain a number
of additional ingredients at a concentration about 10 times that
found in the 1.times. culture medium. As will be readily apparent,
"25.times. formulation," "50.times. formulation," "100.times.
formulation," "500.times. formulation," and "1000.times.
formulation" designate solutions that contain ingredients at about
25-, 50-, 100-, 500-, or 1000-fold concentrations, respectively, as
compared to a 1.times. cell culture medium. Again, the osmolarity
and pH of the media formulation and concentrated solution may
vary.
[0088] Tissues, organs and organ systems derived from animals or
constructed in vitro or in vivo using methods routine in the art
may similarly be cultivated in the culture media of the present
invention. Animals from which cells can originate include human,
monkey, ape, mouse, rat, hamster, rabbit, guinea pig, cow, swine,
dog, horse, cat, goat, and sheep.
[0089] The media of the present invention can be used to grow
mammalian cells in suspension culture in bioreactors, roller
bottles, and microcarrier systems.
Formulation of the Suspension Culture Media
[0090] The suspension media of the present invention is generally
directed to a serum-free cell culture medium comprising one or more
polyanionic or polycationic compounds, wherein the medium is
capable of supporting the suspension cultivation of mammalian
epithelial cells (epithelial or fibroblast) in vitro. In the
present media, the polyanionic compound is preferably a
polysulfonated or polysulfated compound, more preferably heparin,
dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin
sulfate, pentosan polysulfate, a proteoglycan or the like, and most
preferably dextran sulfate, which preferably has a molecular weight
of about 5,000 daltons. The invention also relates generally to
serum-free culture media for use in suspension cultivation of a
mammalian cell, comprising one or more of the above-described
polyanionic or polycationic compounds, particularly dextran
sulfate. In addition, the invention relates to serum-free culture
media for use in producing a virus, the media comprising one or
more of the above-described polyanionic or polycationic compounds,
particularly dextran sulfate, wherein a virus-infected mammalian
cell cultivated in suspension in the media produces a higher virus
titer than a mammalian cell not cultivated in suspension in the
media.
[0091] Basal and Complete Media
[0092] Any basal medium may be used in accordance with the present
invention. Ingredients which the basal media of the present
invention may include are amino acids, vitamins, inorganic salts,
sugars, buffering salts, lipids, insulin (or insulin substitute)
and transferrin (or transferrin substitute). More specifically, the
basal media may contain ethanolamine, D-glucose,
N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic acid] (HEPES),
insulin, linoleic acid, lipoic acid, phenol red, PLURONIC F68,
putrescine, sodium pyruvate. This culture medium contains no serum
and therefore is considered an SFM; it is also a low-protein medium
since the only protein components are insulin and transferrin.
Transferrin may be in the iron-free form (i.e., apotransferrin) or
in the iron-complexed form (i.e., ferrotransferrin or
holotransferrin), and insulin, if present, may be human- or
animal-derived and may be natural or recombinant. The medium may,
of course, be made completely protein-free by not including
transferrin and insulin in the formulation. Transferrin may be
replaced by ferric citrate chelates at a concentration of about
10-100 .mu.M (preferably FeCl.sub.3-sodium citrate chelate at about
60 .mu.M) or ferrous sulfate chelates at a concentration of about
10-100 .mu.M (preferably FeSO.sub.4EDTA chelate at about 40 .mu.M).
Insulin may be replaced by one or more zinc-containing compounds
such alone or more zinc salts. Zinc-containing compounds which may
be used include but are not limited to ZnCl, Zn(NO.sub.3).sub.2,
ZnBr, and ZnSO.sub.4, any of which may be present in their
anhydrous or hydrated (i.e., ".H.sub.2O") forms. Preferably, the
zinc-containing compound used is ZnSO.sub.4.7H.sub.2O. In the
protein-free medium of the present invention, the concentration of
zinc can be optimized using only routine experimentation.
Typically, the concentration of zinc in the 1.times. medium of the
present invention may be about 0.07 to 0.73 mg/L, and is preferably
about 0354 mg/L. Each of these ingredients may be obtained
commercially, for example from Sigma (Saint Louis, Mo.).
[0093] Amino acid ingredients which may be included in the media of
the present invention include L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,
L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,
L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine and L-valine. These amino acids may be obtained
commercially, for example from Sigma (Saint Louis, Mo.).
[0094] Vitamin ingredients which may be included in the media of
the present invention include biotin, choline chloride,
D-Ca.sup.++-pantothenate, folic acid, i-inositol, niacinamide,
pyridoxine, riboflavin, thiamine and vitamin B.sub.12. These
vitamins may be obtained commercially, for example from Sigma
(Saint Louis, Mo.).
[0095] Inorganic salt ingredients which may be used in the media of
the present invention include one or more calcium salts (e.g.,
CaCl.sub.2), Fe(NO.sub.3).sub.3, KCl, one or more magnesium salts
(e.g., MgCl.sub.2 and/or MgSO.sub.4), one or more manganese salts
(e.g., MnCl.sub.2), NaCl, NaHCO.sub.3, Na.sub.2HPO.sub.4, and ions
of the trace elements selenium, vanadium and zinc. These trace
elements may be provided in a variety of forms, preferably in the
form of salts such as Na.sub.2SeO.sub.3, NH.sub.4VO.sub.3 and
ZnSO.sub.4. These inorganic salts and trace elements may be
obtained commercially, for example from Sigma (Saint Louis,
Mo.).
[0096] To this basal medium, one or more polyanionic or
polycationic compounds are added to formulate the complete culture
media of the present invention; these compounds prevent the cells
from clumping and promote growth of the cells in suspension. Thus,
the complete media of the invention are capable of supporting the
suspension cultivation of a mammalian cells in vitro. In the
present media, the polyanionic compound is preferably a
polysulfonated or polysulfated compound, more preferably heparin,
dextran sulfate, heparan sulfate, dermatan sulfate, chondroitin
sulfate, pentosan polysulfate, a proteoglycan or the like, which
may be obtained from a number of commercial sources (such as Sigma;
St. Louis, Mo.; Life Technologies, Inc.; Rockville, Md.).
Particularly preferred for use in the present culture media is
dextran sulfate. Dextran sulfate may be added to freshly formulated
basal medium, or it may be formulated as described in detail in
Example 1 in a solution of basal medium (prepared as described
above). This solution of dextran sulfate may also be prepared as a
1.times.-1000.times. formulation, most preferably as a 1.times.,
10.times., 100.times., 500.times. or 1000.times. formulation, which
is then diluted appropriately into culture medium to provide a
1.times. final formulation in the complete media of the present
invention as described in detail in Example 1.
[0097] Dextran sulfate may be obtained commercially, for example
from Sigma (Saint Louis, Mo.), and is preferably of an average
molecular weight of about 5,000 to about 500,000 daltons, about
5,000 to about 250,000 daltons, about 5,000 to about 100,000
daltons, about 5,000 to about 50,000 daltons, about 5,000 to about
25,000 daltons, or about 5,000 to about 10,000 daltons. Most
preferably the dextran sulfate used in the present culture media is
of a molecular weight of about 5,000 daltons.
[0098] Polyanionic and polycationic compounds can be used in
accordance with the invention for any cell, particularly epithelial
cells or cell lines and fibroblast cells or cell lines, to prevent
aggregation of clumping of cells cultivated in suspension. Such
cells include epithelial and fibroblast cells and cell lines. Such
epithelial and fibroblast cells are particularly those cells and
cell lines described above, and preferably, CHO cells, PER-C6
cells, and 293 cells).
[0099] To formulate the medium of the present invention, a
polyanionic or polycationic compound (and particularly, dextran
sulfate) is added to the above-described basal medium in an amount
effective to prevent clumping, or in an amount to effectively
provide a suspension culture. e.g., at a concentration of about
0-500 mg/liter, about 1-250 mg/liter, about 5-200 mg/liter, about
10-150 mg/liter or about 50-125 mg/liter, and most preferably at a
concentration of about 100 mg/liter. Similar concentrations may be
used for other polyanionic and polycationic compounds, such as
those described above, for formulating the complete media of the
invention.
[0100] The specific combinations of the above ingredients, their
concentration ranges and preferred concentrations, in one example
of the culture media of the invention are shown in Table 1.
Although this specific example uses dextran sulfate, it is to be
understood that any of the above-described polyanionic or
polycationic compounds may be used in the present media.
[0101] The above ingredients listed in Table 1, when admixed
together in solution, form a complete culture medium of the present
invention. These complete media are suitable for use in the culture
of a variety of mammalian cells, as described in more detail below.
In particular, the complete media of the invention may be used to
support cultivation of mammalian cells in suspension, particularly
those that ordinarily are cultivated in monolayers, as described
below. However, the present media are also suitable for cultivating
mammalian cells under standard monolayer conditions.
TABLE-US-00001 TABLE 1 TYPICAL ANIMAL EPITHELIAL CELL CULTURE
MEDIUM COMPONENT CONCENTRATIONS. Component A Preferred Most
Preferred Ranges Embodiment Embodiment (mg/L) (mg/L) (mg/L)
Component about: about: about: Amino Acids L-Alanine 0-100 0 0.00
L-Arginine 200-600 360 355.6 L-Asparagine 5-150 26 26.40 L-Aspartic
Acid 10-350 75 75.00 L-Cysteine 15-150 58 57.6 L-Glutamic Acid
5-150 30 29.40 L-Glutamine 300-1200 600 585.00 Glycine 0-100 0 0.00
L-Histidine 25-200 42 42.2 L-Isoleucine 50-400 190 190.00 L-Leucine
100-500 280 280.00 L-Lysine 100-500 200 204.0 L-Methionine 25-250
115 115.00 L-Phenylalanine 15-200 70 70.00 L-Proline 0-100 0 0.00
L-Serine 5-500 250 250.00 L-Threonine 15-400 60 60.00 L-Tryptophan
5-100 20 20.00 L-Tyrosine 15-150 70 69.2 L-Valine 50-500 200 190.00
Other Components Ethanolamine 0.5-5 3 3.2 D-Glucose 2500-9000 4500
4500.00 HEPES 1000-7000 3000 2980.00 Insulin 5-25 10 10.00 Linoleic
Acid 0.01-5.0 0.1 0.06 Lipoic Acid 0.2-15 2 2.00 Phenol Red 0.5-30
1 1.00 PLURONIC F68 0-750 300 300.00 Putrescine 0.01-1 0.1 0.087
Sodium Pyruvate 10-500 110 110.00 Transferrin 3-100 5 5.00 Vitamins
Biotin 0.001-1 0.1 0.097 Choline Chloride 1-100 14 14.00
D-Ca.sup.++- 0.2-10 1 1.19 Pantothenate Folic Acid 1-100 5 5.00
i-Inositol 2-200 18 18.00 Niacinamide 0.1-10 1 1.22 Pyridoxine
0.1-10 0.9 0.85 Riboflavin 0.02-5 0.2 0.22 Thiamine 0.1-10 1 1.00
Vitamin B12 0.05-10 1 1.03 Dextran Sulfate, 50-250 100 100 MW 5000
Inorganic Salts calcium salt 0-100 10 11.10 (e.g., CaCl.sub.2)
Fe(NO.sub.3).sub.3 0.25-1.5 0.8 0.810 KCl 10-500 275 276.30
MgCl.sub.2 25-150 75 76.20 MgSO.sub.4 5-150 25 24.10 manganese salt
0.00001-0.0005 0.0001 0.0001 (e.g., MnCl.sub.2) NaCl 3000-9000 4400
4410.00 NaHCO.sub.3 100-4000 2400 2400.00 Na.sub.2HPO.sub.4 10-750
125 125.00 selenium salt 0.0000005-0.00002 0.000005 0.0000067
(e.g., Na.sub.2SeO.sub.3) vanadium salt 0.00005-0.002 0.0006 0.0006
(e.g., NH.sub.4VO.sub.3) zinc salt (e.g., 0.001-0.15 0.1 0.0874
ZnSO.sub.4)* *Concentrations of Fe(NO.sub.3).sub.3 and zinc salt(s)
may be higher in protein-free complete media (see above).
[0102] For some applications, it may be preferable to further
enrich the nutritional content of the complete media to support
faster growth and enhanced production of biologicals by the
cultured cells, and to provide a more suitable environment for the
culture of fastidious mammalian cells. To accomplish such
enrichment, one or more supplements may optionally be added to the
basal media or the complete media of the invention. Supplements
which may advantageously be added to the present media include one
or more cytokines (e.g., growth factors such as EGF, aFGF, bFGF,
IGF-1, IGF-2, HB-EGF, KGF, HGF and the like), heparin (to stabilize
heparin-binding growth factors such as the FGFs, HB-EGF, KGF and
HGF) and one or more peptides derived from animals (e.g., HSA or
BSA), yeasts (e.g., yeast extract, yeastolate or yeast extract
ultrafiltrate) or plants (e.g., rice or soy peptides). Cytokines,
which may be natural or recombinant, are available commercially,
for example from Life Technologies, Inc. (Rockville, Md.) or
R&D Systems, Inc. (Rochester, Minn.) and may be added to the
basal media at concentrations recommended by the manufacturer for
the particular cell type to be cultured (typically a final
concentration of about 0.00001-10 mg/liter). Heparin is available
commercially, for example from Sigma (St. Louis, Mo.), and is
preferably porcine mucosa heparin used at a final concentration in
the media of about 1-500 U.S.P. units/liter. Animal, yeast and
plant peptides may be obtained commercially (e.g., from Sigma for
animal peptides; from Difco, Norwell, Mass., for yeast peptides;
and from Quest International, Norwich, N.Y., for plant peptides),
or may be derived and formulated into the present culture media as
described in detail in co-pending, commonly owned U.S. Application
No. 60/028,197, filed Oct. 10, 1996, the disclosure of which is
incorporated herein by reference in its entirety.
[0103] The basal and complete medium ingredients and optional
supplements can be dissolved in a liquid carrier or maintained in
dry form. If dissolved in a liquid carrier at the preferred
concentrations shown in Table 1 (i.e., a "1.times. formulation"),
the pH of the medium should be adjusted to about 7.0-7.6,
preferably about 7.1-7.5, and most preferably about 7.2-7.4. The
osmolarity of the medium should also be adjusted to about 260 to
about 300 mOsm, preferably about 265 to about 280 mOsm, and most
preferably about 265 to about 275 mOsm. The type of liquid carrier
and the method used to dissolve the ingredients into solution vary
and can be determined by one of ordinary skill in the art with no
more than routine experimentation. Typically, the medium
ingredients can be added in any order.
[0104] Preferably, the solutions comprising individual ingredients
are more concentrated than the concentration of the same
ingredients in a 1.times. media formulation. The ingredients can be
10-fold more concentrated (10.times. formulation), 25-fold more
concentrated (25.times. formulation), 50-fold more concentrated
(50.times. concentration), or 100-fold more concentrated
(100.times. formulation). More highly concentrated formulations can
be made, provided that the ingredients remain soluble and stable.
See U.S. Pat. No. 5,474,931, which is directed to methods of
solubilizing culture media components at high concentrations.
[0105] If the individual medium ingredients are prepared as
separate concentrated solutions, an appropriate (sufficient) amount
of each concentrate is combined with a diluent to produce a
1.times. medium formulation. Typically, the diluent used is water
but other solutions including aqueous buffers, aqueous saline
solution, or other aqueous solutions may be used according to the
invention.
[0106] The culture media of the present invention are typically
sterilized to prevent unwanted contamination. Sterilization may be
accomplished, for example, by filtration through a low
protein-binding membrane filter of about 0.22 .mu.m or 0.45 .mu.m
pore size (available commercially, for example, from Millipore,
Bedford, Mass.) after admixing the concentrated ingredients to
produce a sterile culture medium. Alternatively, concentrated
subgroups of ingredients may be filter-sterilized and stored as
sterile solutions. These sterile concentrates can then be mixed
under aseptic conditions with a sterile diluent to produce a
concentrated 1.times. sterile medium formulation. Autoclaving or
other elevated temperature-based methods of sterilization are not
favored, since many of the components of the present culture media
are heat labile and will be irreversibly degraded by temperatures
such as those achieved during most heat sterilization methods.
[0107] The optimal concentration ranges for the basal medium
ingredients are listed in Table 1. These ingredients can be
combined to form the basal mammalian cell culture medium which is
then supplemented as described above with polyanionic or
polycationic compounds (e.g., dextran sulfate), and optionally with
one or more supplements such as one or more cytokines, heparin,
and/or one or more animal, yeast or plant peptides, to formulate
the complete media of the present invention. As will be readily
apparent to one of ordinary skill in the art, the concentration of
a given ingredient can be increased or decreased beyond the range
disclosed and the effect of the increased or decreased
concentration can be determined using only routine experimentation.
In a preferred embodiment, the concentrations of the ingredients of
the medium of the present invention are the concentrations listed
in the far right column of Table 1, supplemented with polyanionic
or polycationic compounds (e.g., dextran sulfate), and optionally
with one or more supplements such as one or more cytokines,
heparin, and one or more animal, yeast or plant peptides, as
described above.
[0108] As will be readily apparent to one of ordinary skill in the
art, each of the components of the culture medium may react with
one or more other components in the solution. Thus, the present
invention encompasses the formulations disclosed in Table 1,
supplemented as described above, as well as any reaction mixture
(i.e., a culture medium or other reaction mixture) which forms
after, or which is obtained by, combining these ingredients.
[0109] The optimization of the present media formulations was
carried out using approaches described by Ham (Ham, R. G., Methods
for Preparation of Media, Supplements and Substrata for Serum-Free
Animal Culture, Alan R. Liss, Inc., New York, pp. 3-21 (1984)) and
Waymouth (Waymouth, C., Methods for Preparation of Media,
Supplements and Substrata for Serum-Free Animal Culture, Alan R.
Liss, Inc., New York, pp. 23-68 (1984)). The optimal final
concentrations for medium ingredients are typically identified
either by empirical studies, in single component titration studies,
or by interpretation of historical and current scientific
literature. In single component titration studies, using animal
cells, the concentration of a single medium component is varied
while all other constituents and variables are kept constant and
the effect of the single component on viability, growth or
continued health of the animal cells is measured.
Formulation of the Replacement Culture Media
[0110] In the replacement media of the invention, any basal media
may be used. Such basal media may contain one or more amino acids,
one or more vitamins, one or more inorganic salts, one or more
buffer salts, and one or more lipids. In accordance with the
invention, transferrin is replaced with iron or an iron-containing
compound and/or insulin is replaced with zinc or a zinc containing
compound. Preferably, iron chelate compounds are used in accordance
with the invention
[0111] Fe.sup.2+ and/or Fe.sup.3+ chelate compounds which may be
used include but are not limited to compounds containing an
Fe.sup.2+ and/or Fe.sup.3+ salt and a chelator such as
ethylenediaminetetraacetic acid (EDTA), ethylene
glycol-bis(.beta.-aminoethyl ether)-N,N,N',N'-tetraacetic acid
(EGTA), deferoxamine mesylate, dimercaptopropanol,
diethylenetriaminepentaacetic acid (DPTA), and
trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA). For
example, the iron chelate compound may be a ferric citrate chelate
or a ferrous sulfate chelate. Preferably, the iron chelate compound
used is ferrous sulphate-7H.sub.2O.EDTA (FeSO.sub.4.7H.sub.2O.EDTA,
e.g., Sigma F0518, Sigma, St. Louis, Mo.). In the medium of the
present invention, the concentration of Fe.sup.2+ and/or Fe.sup.3+
can be optimized using only routine experimentation. Typically, the
concentration of Fe.sup.2+ and/or Fe.sup.3+ in the 1.times. medium
of the present invention can be about 0.00028 to 0.011 g/L.
Preferably, the concentration of iron is about 0.0011 g/L.
[0112] Zn.sup.2+-containing compounds which may be used include but
are not limited to ZnCl, Zn(NO.sub.3).sub.2, ZnBr, and
ZnSO.sub.4.7H.sub.2O. Preferably, the Zn.sup.2+ compound used is
zinc sulfate.7H.sub.2O (ZnSO.sub.4.7H.sub.2O). In the medium of the
present invention, the concentration of Zn.sup.2+ can be optimized
using only routine experimentation. Typically, the concentration of
Zn.sup.2+ in the 1.times. medium of the present invention can be
about 0.00007 to 0.00073 g/L. Preferably, the concentration of
Zn.sup.2+ is about 0.000354 g/L.
[0113] The term "anticlumping agent" refers to a compound which
reduces the degree to which of cells in culture clump together.
Preferably, the anticlumping agent is a polyanionic or polycationic
compound. The polyanionic compound is preferably a polysulfonated
or polysulfated compound, preferably dextran sulfate, pentosan
polysulfate, heparin, heparan sulfate, dermatan sulfate,
chondroitin sulfate, a proteoglycan or the like. More preferably,
the anticlumping agent is dextran sulfate or pentosan polysulfate.
Most preferably, the anticlumping agent is dextran sulfate, which
preferably has a molecular weight of about 5,000 daltons.
[0114] Anticlumping agents may be used to decrease clumping of
cells grown in suspension culture, increase the level of
recombinant protein expression, and/or virus production. The
present invention provides a eukaryotic cell culture medium
containing polyanionic or polycationic compounds (preferably,
dextran sulfate) in an amount sufficient to prevent cell clumping
and/or increase the level of recombinant protein expression.
[0115] The inclusion of polyanionic or polycationic compounds
(preferably, dextran sulfate) in the present media inhibits cell
aggregation; thus, unlike traditional serum-free media in which
suspension cells tend to aggregate or form clumps, the present
media promote the cultivation of single cells in suspension. The
ability to cultivate cells under these suspension culture
conditions provides for rapid subculturing and high-density
culture, which are advantageous for applications in which mammalian
cells are used to produce a variety of products such as in the
biotechnology industry, as described below. Furthermore, since the
present media are serum-free and low-protein or protein-free, the
media may be used for rapid production and isolation of biologicals
(e.g., viruses, recombinant polypeptides, etc.), and in assays
measuring the binding and/or activity of a variety of ligands such
as proteins, hormones, synthetic organic or inorganic drugs, etc.,
on mammalian cells in vitro.
[0116] Dextran sulfate and pentosan polysulfate may be obtained
commercially, for example from Sigma (St. Louis, Mo.). Dextran
sulfate is preferably of an average molecular weight of about 5,000
to about 500,000 daltons, about 5,000 to about 250,000 daltons,
about 5,000 to about 100,000 daltons, about 5,000 to about 50,000
daltons, about 5,000 to about 25,000 daltons, or about 5,000 to
about 10,000 daltons. Most preferably the dextran sulfate used in
the present culture media is of a molecular weight of about 5,000
daltons.
[0117] The pH of the 1.times. medium of the present invention
should preferably be between about 6.9 to about 7.3. The osmolarity
of the 1.times. medium of the present invention should preferably
be between about 270 to about 350 mOsm. If desired, the osmolarity
can be increased by adding a suitable salt such as NaCl. If the
preferred concentrations of ingredients are used (Table 2), the
osmolarity should not have to be adjusted.
[0118] Medium ingredients can be dissolved in a liquid carrier or
maintained in dry form. The type of liquid carrier and the method
used to dissolve the ingredients into solution vary and can be
determined by one of ordinary skill in the art with no more than
routine experimentation.
[0119] Preferably, the solutions comprising ingredients are more
concentrated than the concentration of the same ingredients in a
1.times. media formulation. The ingredients can be 10 fold more
concentrated (10.times. formulation), 25 fold more concentrated
(25.times. formulation), 50 fold more concentrated (50.times.
concentration), or 100 fold more concentrated (100.times.
formulation). More highly concentrated formulations can be made,
provided that the ingredients remain soluble and stable. See U.S.
Pat. No. 5,474,931.
[0120] If the media ingredients are prepared as separate
concentrated solutions, an appropriate (sufficient) amount of each
concentrate is combined with a diluent to produce a 1.times. medium
formulation. Typically, the diluent used is water, but other
solutions including aqueous buffers, aqueous saline solution, or
other aqueous solutions may be used according to the invention.
[0121] The culture medium of the present invention is typically
sterilized to prevent unwanted contamination of the cell culture
media. Sterilization may be accomplished, for example, by
filtration after admixing the concentrated ingredients to produce a
sterile culture medium. Alternatively, concentrated subgroups of
ingredients may be filter-sterilized and stored as sterile
solutions. These sterile concentrates can then be mixed with a
sterile diluent to produce a concentrated 1.times. sterile medium
formulation.
[0122] The medium of the present invention facilitates the growth
of mammalian cells, and particularly epithelial cells and cell
lines, and fibroblast cells and cell lines, as described above, and
particularly to high density, increases the level of expression of
recombinant protein in cultured cells, and/or increases virus
production in cultured cells. Cellular metabolic energy is expended
on both cell growth and recombinant protein expression. Depending
on the culture conditions and the particular cell line, either cell
growth or recombinant protein expression can be facilitated at the
expense of the other activity.
[0123] To shift the distribution of metabolic energy from
supporting cell growth to supporting protein expression, cells can
be treated with sodium butyrate (De Gros, G. S. et al., Lymphokine
Res. 4:221-227 (1985)). About 10 .mu.M to about 10-mM sodium
butyrate can be used. Preferably, about 100 .mu.M to 1.0 mM is
used. Cells can be grown to the desired density prior to the
addition of sodium butyrate. After sodium butyrate has been added,
cell growth slows and recombinant protein expression increases.
Although treatment with sodium butyrate decreases the rate of cell
growth, this decrease in growth rate is outweighed by the increase
in recombinant protein production. Moreover, because the medium of
the present invention is protein-free, purification of recombinant
protein can be performed more quickly, more easily and less
expensively than purification can be done from cells that were
grown in media containing serum or protein. See also U.S. Pat. No.
5,393,558.
[0124] Dihydrofolate reductase (DHFR) catalyzes the conversion of
folate to tetrahydrofolate, which is required for purine, amino
acid, and nucleoside biosynthesis. The folic acid analogue
methotrexate binds and inhibits DHFR, causing cell death. DHFR
deficient cells (DHFR) which have been transfected with a gene of
interest and a methotrexate resistance gene can be treated with
methotrexate to amplify recombinant cells (Bebbington, C. R. et
al., The Use of Vectors Based on Gene Amplification for the
Expression of Cloned Genes n Mammalian Cells, In: DNA Cloning, Vol.
III, Glover, D., ed., Academic Press (1987), pp. 163-188).
Surviving populations of cells exposed to sequentially increasing
concentrations of methotrexate contain increased levels of DHFR
that result from gene amplification.
[0125] About 50 nM to about 2 .mu.M methotrexate can be used.
Preferably, about 100 nM to about 500 nM methotrexate is used.
Although treatment with methotrexate may decrease overall cell
density, this decrease in cell density is outweighed by the
increase in recombinant protein production. Again, because the
medium of the present invention is protein-free, purification of
recombinant protein can be performed more quickly, more easily and
less expensively than purification can be done from cells that were
grown in media containing serum or protein.
[0126] The concentration ranges within which ingredients of the
1.times. medium are believed to support growth (and particularly
the high-density growth), increase the level of expression of
recombinant protein in cultured cells, and/or increases virus
production in cultured, are listed in the second column of Table 2.
These ingredients can be combined to form the 1.times. eukaryotic
cell culture medium of the present invention. As will be apparent
to one of ordinary skill in the art, the concentration of a given
ingredient can be increased or decreased beyond the range disclosed
and the effect of the increased or decreased concentration can be
determined using only routine experimentation. The concentration of
each ingredient in a preferred embodiment of the medium of the
present invention is listed in the third column of Table 2. The
concentration of each ingredient in a particularly preferred
embodiment is shown in the fourth column of Table 2.
[0127] The 1.times. medium of the present invention can be made
using a medium concentrate technology. See U.S. Pat. No. 5,474,931.
Ingredients can be stored in solution. Preferably, ingredients are
grouped in concentrated solutions and stored. For example, Table 2
shows suitable groups of ingredients. Stock solutions of the
grouped ingredients can be made as concentrated stocks. For
example, it is possible to make 10.times. to 100.times. chemical
stock solutions, which can be stored as liquids or frozen in the
appropriate aliquot sizes for later use.
[0128] Stock solutions offer a number of advantages. For example, a
higher final concentration of a given ingredient can be used in the
1.times. medium. In addition, some ingredients are more stable when
stored in a concentrated stock solution. Moreover, less storage
volume is required for a concentrated stock solution than is
required for a 1.times. medium. See U.S. Pat. No. 5,474,931.
[0129] In a preferred embodiment, 27.63.times. concentrated stock
solutions of the groups of ingredients in Table 2 are prepared as
followed. To prepare a 26.63.times. concentrated stock solution of
the Acid Soluble I group of ingredients, each of the ingredients in
the Acid Soluble I group of ingredients in Table 2 is added to
approximately 19.33 mL of distilled water. The ingredients in the
Acid Soluble I group can be added in any order. The pH of the
solution is reduced to 0.80 with 5N HCl (approximately 7.4 mL) and
the solution is mixed until all of the ingredients are dissolved.
The final volume of the solution is 36.192 mL.
[0130] To prepare a 27.63.times. concentrated stock solution of the
Acid Soluble II group of ingredients, sodium phosphate is first
added to approximately 39.33 mL of distilled water and the solution
is mixed until the sodium phosphate is completely dissolved. The pH
is adjusted to 1.00 using 5N HCl (approximately 3.5 mL). The rest
of the ingredients in the Acid Soluble II group in Table 2 are
added (the order of addition of the rest of these ingredients is
not critical). A concentrated stock solution of the trace elements
can be used. The Acid Soluble II solution is mixed until all of the
ingredients are dissolved. The pH final should be 1.00. If
necessary, the pH should be adjusted to 1.00. The final volume of
the solution is 36.192 mL.
[0131] To prepare a 27.63.times. concentrated stock solution of the
Salts I solution, MgCl.sub.2 and ascorbic acid Mg salt phosphate
are added to approximately 35.83 mL of distilled water. The
solution is mixed until the ingredients are dissolved. The pH is
lowered to 5.5 using 5N HCl (approximately 6.0 mL).
D-Ca-pantothenate, calcium nitrate, and KCl are added and mixed.
After mixing, the pH should be 5.50. If necessary, the pH should be
adjusted to 5.50. The final volume of the solution is 36.192
mL.
[0132] To prepare a 27.63.times. concentrated stock solution of the
Salts II solution, pluronic F-68 is added to approximately 28.23 mL
of distilled water, followed by sodium phosphate. After mixing, the
pH is reduced to 7.00 using 5N HCl (approximately 0.06 mL). Glucose
is added, followed by the rest of the ingredients in the Salts II
group of ingredients in Table 2, except for folic acid and
riboflavin (the order of addition of the rest of these ingredients,
except for folic acid and riboflavin, is not critical). Preferably,
a 10,000.times. stock solution of sodium selenite in distilled
water is used. The density of .beta.-mercaptoethanol is preferably
1.114 g/mL and the density of monothioglycerol is preferably 1.250
g/mL.
[0133] In a separate container of 2 mL of distilled water protected
from light, folic acid and riboflavin are added. The pH is adjusted
to 11.5 using 5N HCl (approximately 7 .mu.L) and the solution is
mixed until the folic acid and riboflavin are dissolved. This
solution of folic acid and riboflavin is then added to the Salts II
ingredient solution. The final volume of the Salts II solution is
36.193 mL and the final pH should be 7.00. If necessary, the pH can
be adjusted to 7.00.
[0134] For the Salts II solution, the pluronic F-68 can be in
liquid form or in powder form. For the Acid Soluble I, Acid Soluble
II, Salts I or Salts H solutions, unless other wise noted above,
components can be added to solution singly or in combination.
[0135] To prepare the 1.times. medium of the present invention, the
following procedure is preferably used. To 840.000 mL of distilled
water (pH 5.61) is added 36.192 mL of a 27.63.times. concentrate of
the Acid Soluble I group of ingredients (pH 1.77), followed by
36.192 mL of a 27.63.times. concentrate of the Acid Soluble II
group of ingredients (pH 1.71), followed by about 9.800 mL of 5N
NaOH (pH 6.8), followed by 36.192 mL of a 26.63.times. concentrate
of the Salts I group of ingredients (pH 6.8), followed by 36.192 mL
of a 26.63.times. concentrate of the Salts II group of ingredients
(pH 6.85), followed by 1.810 mL of ferrous sulfate chelate (pH
6.85), followed by 2.22 g of NaHCO.sub.3 (pH 7.16), followed by
about 0.400 mL of 5N HCl (pH 7.00), followed by 0.427 g of NaCl.
The final pH of the solution should be 7.00 and the final volume
should be 1000 mL. The osmolarity range of the solution should be
between about 320 to 330 mOsm. 40 mL of a 200 mM glutamine solution
(100.times.) is added to the 1.times. medium at the time of
use.
[0136] The iron chelate compound is preferably added to the
1.times. medium prior to filter sterilization.
[0137] Dextran sulfate can be added to the 1.times. medium to a
final concentration of about 1 .mu.g/ml to about 1 mg/ml.
Preferably, the final concentration of dextran sulfate is about 10
to about 25 Dextran sulfate can be added before filter
sterilization of the 1.times. medium. Alternatively, presterilized
dextran sulfate can be added to sterile 1.times. medium. If dextran
sulfate is to be included in a concentrated stock solution, it can
be included in the Salts II group of ingredients (see Table 2). The
concentration of other anticlumping agents can be determined using
only routine experimentation.
[0138] As will be apparent to one of ordinary skill in the art, the
ingredients may react in solution. Thus, the present invention
encompasses the formulations disclosed in Table 2 as well as any
reaction mixture which forms after the ingredients in Table 2 are
combined.
TABLE-US-00002 TABLE 2 The 1X Replacement Medium Formulation
PREFERRED PARTICULARLY CONCENTRATION EMBODIMENT PREFERRED RANGE
(G/L) EMBODIMENT INGREDIENT (G/L) ABOUT (G/L) Acid Soluble I
L-arginine 0.1000-0.7200 0.4 0.36192 L-asparagine.cndot.H.sub.2O
0.1000-1.8000 0.9 0.90480 L-aspartic acid 0.0100-0.3600 0.2 0.18096
L-glutamic acid 0.1000-0.6000 0.3 0.27144 L-histidine 0.0600-0.3600
0.2 0.18096 hydroxy-L-proline 0.0040-0.3600 0.2 0.18096
L-isoleucine 0.1000-0.7200 0.4 0.36192 L-leucine 0.1000-1.1000 0.5
0.54288 L-lysine.cndot.HCl 0.2000-1.1000 0.5 0.54288 L-methionine
0.0500-0.2400 0.1 0.12667 L-phenylalanine 0.0900-0.4200 0.2 0.21715
L-proline 0.0500-1.1000 0.5 0.54288 L-serine 0.1000-1.1000 0.5
0.54288 L-threonine 0.1000-0.7200 0.4 0.36192 L-tryptophan
0.0200-0.4200 0.2 0.20810 L-tyrosine 0.1000-0.3600 0.2 0.18096
L-valine 0.1000-0.7200 0.4 0.36192 L-cystine.cndot.2HCl
0.0200-0.2200 0.1 0.10496 Acid Soluble II Na.sub.2HPO.sub.4
(anhydrous) 0.2000-2.5000 0.6 0.63336 pyridoxine.cndot.HCl
0.0010-0.0072 0.004 0.00362 thiamine.cndot.HCl 0.0010-0.0072 0.004
0.00362 glutathione 0.0006-0.0036 0.002 0.00181 zinc
sulfate.cndot.7H.sub.2O 0.0003-0.0032 0.002 0.00156 cupric
sulfate.cndot.5H.sub.2O 0.000001-0.000009 0.000005 0.000004524
cadmium chloride.cndot.5H.sub.2O 0.000004-0.000040 0.00002
0.000020629 cobalt chloride.cndot.6H.sub.2O 0.0000006-0.0000086
0.000004 0.000004343 stannous chloride.cndot.2H.sub.2O
0.00000001-0.00000020 0.0000001 0.000000101 manganous
sulfate.cndot.H.sub.2O 0.00000001-0.00000030 0.0000002 0.000000152
nickel sulfate.cndot.6H.sub.2O 0.00000005-00000024 0.0000001
0.000000118 sodium metavanadate 0.0000003-0000012 0.0000006
0.000000561 ammonium molybdate.cndot.4H.sub.2O 0.00000300-0.0000110
0.000005 0.000005429 barium acetate 0.00000065-0.00000240 0.000001
0.000001176 potassium bromide 0.00000003-0.00000011 0.00000005
0.000000054 potassium iodide 0.000000045-0.00000016 0.00000008
0.000000081 chromium sulfate 0.000000165-0.00000060 0.0000003
0.000000299 sodium fluoride 0.00000105-0.00000360 0.000002
0.000001810 silver nitrate 0.000000045-0.00000016 0.00000008
0.000000081 rubidium chloride 0.00000035-0.0000013 0.0000006
0.000000633 zirconyl chloride 0.0000008-0.0000029 0.000001
0.000001448 aluminum chloride 0.0000003-0.0000011 0.0000005
0.000000543 germanium dioxide 0.000000135-0.00000049 0.0000002
0.000000244 titanium tetrachloride 0.00000025-0.0000009 0.0000005
0.000000452 sodium metasilicate 0.00005-0.00095 0.0005 0.000452400
Salts I MgCL.sub.2 (anhydrous) 0.0100-0.1400 0.07 0.06985 D-Calcium
pantothenate 0.0020-0.0060 0.004 0.00362 Calcium
nitrate.cndot.4H.sub.2O 0.01800-0.3600 0.09 0.09048 KCl
0.3340-1.4500 0.7 0.72384 Ascorbic acid, 0.00199-0.040 0.02 0.01991
Mg salt phosphate Salts II Pluronic F68, 5.0 mL-40.0 mL/L 18 mL/L
18.096 mL/L 10% Solution (0.5-4.0 g/L) (2 g/L) (1.8096 g/L)
Na.sub.2HPO.sub.4 (anhydrous) 0.018-0.360 0.09 0.09048 D-glucose
1.000-12.60 6 6.33360 folic acid 0.002-0.0072 0.004 0.00362
riboflavin 0.0002-0.00072 0.0004 0.000362 biotin 0.000575-0.00360
0.002 0.00181 choline chloride 0.0280-0.1810 0.09 0.09048
niacinamide 0.0003-0.00724 0.004 0.00362 i-inositol 0.0260-0.127
0.06 0.06334 sodium pyruvate 0.070-0.400 0.2 0.19906 vitamin B-12
0.0005-0.0018 0.0009 0.00090 .beta.-mercaptoethanol 0.00014-0.00282
0.001 0.00141 para-aminobenzoic acid 0.0010-0.00362 0.002 0.00181
.beta.-glycerophosphate 0.090-1.800 0.9 0.90480 sodium selenite
0.00000157-0.000032 0.00002 0.0000157 ethanolamine.cndot.HCl
0.0075-0.0280 0.01 0.01357 spermine 0.0009-0.0181 0.009 0.00905
putrescine.cndot.2HCl 0.00012-0.00110 0.0005 0.000543
monothioglycerol 0.0100-0.0362 0.02 0.01810 Dried powder medium
NaHCO.sub.3 1-4 2 2.22
[0139] The iron chelate compound is preferably added to the
1.times. medium prior to filter sterilization.
[0140] If glutamine is to be used, it can be added to the 1.times.
medium prior to filter sterilization. For example, 40 ml of 200 mM
L-glutamine can be added per liter of 1.times. medium (the final
concentration of glutamine is 8 mM). If glutamine is not added,
then preferably the concentrations of each of the above ingredients
should be diluted 1.04-fold/L with diluent, although dilution is
not necessary. Alternatively, a sterile 200 mM stock solution of
L-glutamine can be added after the 1.times. medium has been filter
sterilized.
[0141] The 1.times. medium does not need to be supplemented with
glutamine if the glutamine synthase expression system (Celltech,
Slough, UK) is used. Using this system, cells are engineered to
express glutamine synthetase, which catalyzes the synthesis of
glutamine from glutamate and ammonia. See U.S. Pat. No.
5,122,464.
[0142] For the 1.times. medium to be effective for culturing NS/O
myeloma cells, a lipid mixture supplement may need to be added to
the 1.times. medium. The lipid supplement formulation of Table 3
can be added to the 1.times. medium prior to filter sterilization.
Preferably, a 200-400.times. concentrated stock solution of the
lipid mixture supplement is used. In a preferred embodiment, the
lipid mixture supplement is prepared as a 379.times. concentrated
stock solution of which 2.64 ml/L is added to the 1.times. medium.
To make the lipid supplement, ethanol is added to Pluronic F68 with
constant stirring at 60.degree. C. The mixture is cooled to
37-40.degree. C. and the sterol is added. The mixture is overlaid
with argon gas and is mixed until the sterol dissolves. After the
sterol has dissolved, the rest of the ingredients in Table 3 are
added. This lipid mixture supplement can be stored for future use.
The sterol can be cholesterol or a plant sterol, such as sitosterol
or stigmasterol or other plant sterol known to those of ordinary
skill in the art.
TABLE-US-00003 TABLE 3 Lipid supplement (final concentrations in 1X
Replacement medium). PARTICULARLY CONCENTRATION PREFERRED PREFERRED
RANGE EMBODIMENT (G/L) EMBODIMENT INGREDIENT (G/L) ABOUT (G/L)
Pluronic F68 0.1-4.0 1.7 1.74 ethanol 0.09-2.0 0.9 0.87 mL
cholesterol 0.0007-0.07 0.007 0.00696 lipoic acid 0.00003-0.003
0.0003 0.000296 linoleic acid 0.00001-0.001 0.0001 0.000122
.alpha.-tocopherol 0.00002-0.002 0.0002 0.000231 palmitic acid
0.0002-0.02 0.002 0.00174 oleic acid 0.0002-0.02 0.002 0.00174
dilinoleoyl 0.0002-0.02 0.002 0.00174 phosphatidylcholine stearic
acid 0.0002-0.02 0.002 0.00174 linolenic acid 0.0002-0.02 0.002
0.00174 palmitoleic acid 0.0002-0.02 0.002 0.00174 myristic acid
0.0002-0.02 0.002 0.00174
Kits
[0143] The invention also provides kits for use in the cultivation
of a mammalian cell, and in particular a mammalian epithelial cell.
Kits according to the present invention comprise one or more
containers containing one or more of the media formulations of the
invention. For example, a kit of the invention may comprise one or
more containers wherein a first container contains a complete
serum-free, low-protein or protein-free culture medium prepared as
described above.
[0144] Additional kits of the invention may comprise one or more
containers wherein a first container contains a basal culture
medium prepared as described above and a second container contains
a polyanionic or polycationic compound, preferably a polysulfonated
or polysulfated compound, more preferably heparin, dextran sulfate,
heparan sulfate, dermatan sulfate, chondroitin sulfate, pentosan
polysulfate, a proteoglycan or the like, and most preferably
dextran sulfate. The complete media, basal media and/or dextran
sulfate contained in the containers of these kits may be present as
1.times. ready-to-use formulations, or as more concentrated
solutions (for example 2.times., 5.times., 10.times., 20.times.,
25.times., 50.times., 100.times., 500.times., 1000.times. or
higher). Additional kits of the invention may further comprise one
or more additional containers containing one or more supplements
selected from the group consisting of one or more cytokines,
heparin, one or more animal peptides, one or more yeast peptides
and one or more plant peptides. Preferred cytokines, heparin,
animal peptides, yeast peptides and plant peptides for inclusion in
the containers of the kits of the invention are as described above.
The kits of the invention may be used to produce one or more of the
culture media of the invention for use in a variety of applications
as described below.
Compositions
[0145] The invention further provides compositions comprising the
media of the present invention. Compositions according to this
aspect of the invention may consist of one or more of the present
media and optionally one or more additional components, such as one
or more cells, one or more tissues, one or more organs, one or more
organisms, one or more viruses, one or more proteins or peptides
(such as one or more enzymes), one or more hormones, one or more
nucleic acids, one or more enzyme substrates, one or more
cytokines, one or more extracellular matrix components (including
attachment factors), one or more antibodies, one or more detectable
labeling reagents (such as fluorophores, phosphors or radiolabels),
and the like.
[0146] Particularly preferred compositions of the invention
comprise one or more of the present culture media and one or more
mammalian cells. Mammalian cells and cell lines, as described
above, can be used in the compositions of the present invention.
Mammalian cells particularly suitable for use in formulating the
present cell culture compositions comprising the suspension medium
of the present invention include epithelial cells of human origin,
which may be primary cells derived from a tissue sample such as
keratinocytes, cervical epithelial cells, bronchial epithelial
cells, tracheal epithelial cells, kidney epithelial cells or
retinal epithelial cells, or transformed cells or established cell
lines (e.g., 293 human embryonic kidney cells, HeLa cervical
epithelial cells or derivatives thereof (e.g., HeLaS3), PER-C6
human retinal cells and HCAT human keratinocytes), or derivatives
thereof. Most preferable for use in the present compositions of the
suspension medium of the present invention are 293 human embryonic
kidney cells. The cells used in such preferred compositions of the
invention may be normal cells, or may optionally be diseased or
genetically altered. Other mammalian cells, such as CHO cells, COS
cells, VERO cells, BHK cells (including BHK-21 cells) and
derivatives thereof, are also suitable for use in formulating the
present cell culture compositions.
[0147] Mammalian cells and cell lines, as described above, can be
used in the present compositions. Most preferable for use in the
present compositions of the replacement medium of the present
invention are CHO cells. The cells used in such preferred
compositions of the invention may be normal cells, or may
optionally be diseased or genetically altered. Other mammalian
cells, such as 293 cells, COS cells, VERO cells, BHK cells
(including BHK-21 cells) and derivatives thereof, are also suitable
for use in formulating the present cell culture compositions.
[0148] Epithelial tissues, organs and organ systems derived from
animals or constructed in vitro or in vivo using methods routine in
the art may similarly be used to formulate the suspension and
replacement compositions of the present invention.
[0149] The compositions of the invention may be used in a variety
of medical (including diagnostic and therapeutic), industrial,
forensic and research applications requiring ready-to-use cultures,
particularly suspension cultures, of mammalian cells in serum-free,
low-protein or protein-free media. Non-limiting examples of uses of
these compositions include providing stock cell cultures; short- or
long-term storage of cells, tissues, organs, organisms and the
like; providing vaccination reagents; etc.
Use of Culture Media
[0150] The present cell culture media may be used to facilitate
cultivation of a variety of mammalian cells in suspension or in
monolayer cultures. In particular, these media may be used to
cultivate mammalian epithelial cells or cell lines, as described
above, particularly human epithelial cells and cell lines and
fibroblast cells and cell lines. The present media advantageously
facilitate the suspension cultivation of cells which are typically
anchorage-dependent or grown in monolayer cultures, without the use
of microcarriers such as latex or collagen beads (although cells
may be cultivated on such microcarriers or beads in the present
media). Methods for isolation, and suspension and monolayer
cultivation, of a variety of animal cells including mammalian
epithelial cells are known in the art (see, e.g., Freshney, R. I.,
Culture of Animal Cells: A Manual of Basic Technique, New York:
Alan R. Liss, Inc. (1983)) and are described in further detail
below and in the Examples. While the present media are particularly
useful for culturing mammalian cells in suspension, it is to be
understood that the media may be used in any standard cell culture
protocol whether the cells are grown in suspension, in monolayers,
in perfusion cultures (e.g., in hollow fiber microtube perfusion
systems), on semi-permeable supports (e.g., filter membranes), in
complex multicellular arrays or in any other method by which
mammalian cells may be cultivated in vitro.
[0151] In a preferred embodiment, the replacement medium of the
present invention is used to grow CHO cells in suspension culture.
In another preferred embodiment, the replacement medium of the
present invention is used to grow hybridoma cells in suspension
culture. In yet another preferred embodiment, the replacement
medium of the present invention can be used to culture NS/O myeloma
cells in suspension culture. If NS/O myeloma cells are cultured,
the replacement 1.times. medium of the present invention can be
supplemented with a lipid mixture supplement (see Table 3).
[0152] The inclusion of a polyanionic or polycationic compound,
such as dextran sulfate, in the present media inhibits the
aggregation of 293 cells, CHO cells, as well as the aggregation of
other mammalian cells; thus, unlike traditional serum-free media in
which suspension cells tend to aggregate or form clumps, the
present media promote the cultivation of single cells in
suspension. The ability to cultivate cells under these suspension
culture conditions provides for rapid subculturing and high-density
culture, which are advantageous for applications in which mammalian
cells are used to produce a variety of products such as in the
biotechnology industry, as described below. Furthermore, since the
present media are serum-free and low-protein or protein-free, the
media may be used for rapid production and isolation of biologicals
(e.g., viruses, recombinant polypeptides, etc.), and in assays
measuring the binding and/or activity of a variety of ligands such
as proteins, hormones, synthetic organic or inorganic drugs, etc.,
on mammalian cells in vitro.
[0153] Cells which can be grown in the media of the present
invention are those of animal origin, including but not limited to
cells obtained from mammals. Mammalian cells particularly suitable
for cultivation in the present media include epithelial cells and
cell lines, which may be primary cells derived from a tissue
sample.
[0154] The media of the present invention may be used to culture a
variety of mammalian cells, including primary epithelial cells
(e.g., keratinocytes, cervical epithelial cells, bronchial
epithelial cells, tracheal epithelial cells, kidney epithelial
cells and retinal epithelial cells) and established cell lines
(e.g., 293 embryonic kidney cells, HeLa cervical epithelial cells
and PER-C6 retinal cells, MDBK (NBL-1) cells, CRFK cells, MDCK
cells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa
229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS
174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24
cells, WI-28 VA13, 2RA cells, WISH cells, BS-C-I cells,
LLC-MK.sub.2 cells, Clone M-3 cells, I-10 cells, RAG cells, TCMK-1
cells, Y-1 cells, LLC-PK.sub.1 cells, PK(15) cells, GH.sub.1 cells,
GH.sub.3 cells, L2 cells, LLC-RC 256 cells, MH.sub.1C.sub.1 cells,
XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives
thereof), fibroblast cells from any tissue or organ (including but
not limited to heart, liver, kidney, colon, intesting, esophagus,
stomach, neural tissue (brain, spinal cord), lung, vascular tissue
(artery, vein, capillary), lymphoid tissue (lymph gland, adenoid,
tonsil, bone marrow, and blood), spleen, and fibroblast and
fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33
cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells,
Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit
529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,
Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38
cells, WI-26 cells, MiCl.sub.1 cells, CHO cells, CV-1 cells, COS-1
cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells,
BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB
cells, BLO-11 cells, NOR-10 cells, C.sub.3H/IOTI/2 cells,
HSDM.sub.1C.sub.3 cells, KLN205 cells, McCoy cells, Mouse L cells,
Strain 2071 (Mouse L) cells, L-M strain (Mouse L) cells,
L-MTK.sup.- (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1
cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C.sub.II
cells, and Jensen cells, or derivatives thereof).
[0155] Cells may be normal cells, or may optionally be abnormal
(e.g., diseased or genetically altered). Other mammalian cells,
such as leukemic cell lines such as K562 cells, MOLT-4 cells, M1
cells and the like, and derivatives thereof, are also suitable for
cultivation in the present media.
[0156] 293 human embryonic kidney cells and HeLaS3 cells are
particularly preferred for growth in the suspension medium of the
present invention. Chinese hamster ovary (CHO) cells, NS/O cells,
and hybridoma cells are particularly preferred for growth in the
replacement medium of the present invention. Especially preferred
are CHO cells.
[0157] Cell lines and hybridoma lines are well known to those of
ordinary skill in the art. See, for example, the ATCC Catalogue of
Cell Lines and Hybridomas, 7th Edition, 1992 (American Type Culture
Collection, Rockville, Md., USA), and the ATCC Catalogue of Cell
Lines and Hybridomas, 8th Edition, 1996 (American Type Culture
Collection, Rockville, Md., USA).
[0158] Epithelial tissues, organs and organ systems derived from
animals or constructed in vitro or in vivo using methods routine in
the art may similarly be cultivated in the culture media of the
present invention.
[0159] Isolation of Cells
[0160] Animal cells for culturing in the media of the present
invention may be obtained commercially, for example from ATCC
(Rockville, Md.), Quantum Biotechnologies (Montreal, Canada) or
Invitrogen (San Diego, Calif.). Alternatively, cells may be
isolated directly from samples of animal tissue obtained via
biopsy, autopsy, donation or other surgical or medical
procedure.
[0161] Tissue should be handled using standard sterile technique
and a laminar flow safety cabinet. In the use and processing of all
human tissue, the recommendations of the U.S. Department of Health
and Human Services/Centers for Disease Control and Prevention
should be followed (Biosafety in Microbiological and Biomedical
Laboratories, Richmond, J. Y. et al., Eds., U.S. Government
Printing Office, Washington, D.C. 3rd Edition (1993)). The tissue
should be cut into small pieces (e.g., 0.5.times.0.5 cm) using
sterile surgical instruments. The small pieces should be washed
twice with sterile saline solution supplemented with antibiotics as
above, and then may be optionally treated with an enzymatic
solution (e.g., collagenase or trypsin solutions, each available
commercially, for example, from Life Technologies, Inc., Rockville,
Md.) to promote dissociation of cells from the tissue matrix.
[0162] The mixture of dissociated cells and matrix molecules are
washed twice with a suitable physiological saline or tissue culture
medium (e.g., Dulbecco's Phosphate Buffered Saline without calcium
and magnesium). Between washes, the cells are centrifuged (e.g., at
200.times.g) and then resuspended in serum-free tissue culture
medium. Aliquots are counted using an electronic cell counter (such
as a Coulter Counter). Alternatively, the cells can be counted
manually using a hemacytometer.
[0163] Cultivation of Cells
[0164] The isolated cells and cell lines can be cultivated
according to the experimental conditions determined by the
investigator. The examples below demonstrate at least one
functional set of culture conditions useful for cultivation of
certain mammalian cells, particularly under suspension conditions.
It is to be understood, however, that the optimal plating and
culture conditions for a given animal cell type can be determined
by one of ordinary skill in the art using only routine
experimentation. For routine monolayer culture conditions, using
the media of the present invention, cells can be plated onto the
surface of culture vessels without attachment factors.
Alternatively, the vessels can be precoated with natural,
recombinant or synthetic attachment factors or peptide fragments
(e.g., collagen, fibronectin, vitronectin, laminin and the like, or
natural or synthetic fragments thereof), which are available
commercially for example from Life Technologies, Inc. (Rockville,
Md.), R&D Systems, Inc. (Rochester, Minn.), Genzyme (Cambridge,
Mass.) and Sigma (St. Louis, Mo.). Isolated cells can also be
seeded into or onto a natural or synthetic three-dimensional
support matrix such as a preformed collagen gel or a synthetic
biopolymeric material. For suspension cultivation, cells are
typically suspended in the present culture media and introduced
into a culture vessel that facilitates cultivation of the cells in
suspension, such as a spinner flask, perfusion apparatus, or
bioreactor (see Freshney, R. I., Culture of Animal Cells: A Manual
of Basic Technique, New York: Alan R. Liss, Inc., pp. 123-125
(1983)). Ideally, agitation of the media and the suspended cells
will be minimized to avoid denaturation of media components and
shearing of the cells during cultivation.
[0165] The cell seeding densities for each experimental condition
can be optimized for the specific culture conditions being used.
For routine monolayer culture in plastic culture vessels, an
initial seeding density of 1-5.times.10.sup.5 cells/cm.sup.2 is
preferable, while for suspension cultivation a higher seeding
density (e.g., 5-20.times.10.sup.5 cells/cm.sup.2) may be used.
[0166] Mammalian cells are typically cultivated in a cell incubator
at about 37.degree. C. The incubator atmosphere should be
humidified and should contain about 3-10% carbon dioxide in air,
more preferably about 8-10% carbon dioxide in air and most
preferably about 8% carbon dioxide in air, although cultivation of
certain cell lines may require as much as 20% carbon dioxide in air
for optimal results. Culture medium pH should be in the range of
about 7.1-7.6, preferably about 7.1-7.4, and most preferably about
7.1-7.3.
[0167] Cells in closed or batch culture should undergo complete
medium exchange (i.e., replacing spent media with fresh media) when
the cells reach a density of about 1.5-2.0.times.10.sup.6 cells/ml.
Cells in perfusion culture (e.g., in bioreactors or fermenters)
will receive fresh media on a continuously recirculating basis.
[0168] Virus Production
[0169] In addition to cultivation of mammalian cells in suspension
or in monolayer cultures, the present media may be used in methods
for producing viruses from mammalian cells. Such methods according
to this aspect of the invention comprise (a) obtaining a mammalian
cell to be infected with a virus; (b) contacting the cell with a
virus under conditions suitable to promote the infection of the
cell by the virus; and (c) cultivating the cell in the culture
media of the invention under conditions suitable to promote the
production of virus by the cell. According to the invention, the
cell may be contacted with the virus either prior to, during or
following cultivation of the cell in the culture media of the
invention; optimal methods for infecting a mammalian cell with a
virus are well-known in the art and will be familiar to one of
ordinary skill. Virus-infected mammalian cells cultivated in
suspension in the media of the invention may be expected to produce
higher virus titers (e.g., 2-, 3-, 5-, 10-, 20-, 25-, 50-, 100-,
250-, 500-, or 1000-fold higher titers) than those cells not
cultivated in suspension in the media of the invention. These
methods may be used to produce a variety of mammalian viruses and
viral vectors, including but not limited to adenoviruses,
adeno-associated viruses, retroviruses and the like, and are most
preferably used to produce adenoviruses or adeno-associated
viruses. Following cultivation of the infected cells in the present
media, the used culture media comprising viruses, viral vectors,
viral particles or components thereof (proteins and/or nucleic
acids (DNA and/or RNA)) may be used for a variety of purposes,
including vaccine production, production of viral vectors for use
in cell transfection or gene therapy, infection of animals or cell
cultures, study of viral proteins and/or nucleic acids and the
like. Alternatively, viruses, viral vectors, viral particles or
components thereof may optionally be isolated from the used culture
medium according to techniques for protein and/or nucleic acid
isolation that will be familiar to one of ordinary skill in the
art.
[0170] Recombinant Protein Production
[0171] The present culture media may also be used in methods for
the production of recombinant proteins from mammalian cells,
particularly from mammalian cells grown in suspension. Cell lines
commonly used for recombinant protein production (e.g., CHO cells)
typically produce proteins that are abnormally glycosylated (Lao,
M.-S., et al., Cytotechnol. 22:43-52 (1996); Graner, M. J., et al.,
Biotechnol. 13:692-698 (1993); Graner, M. J., and Goochee, C. F.,
Biotechnol. Prog. 9:366-373 (1993)). However, the relatively low
.beta.-galactosidase and sialidase activities in 293 cells at
neutral pH, such as those provided by the present methods, may
facilitate the production of recombinant proteins that more closely
resemble their natural counterparts (Graner, M. J., and Goochee, C.
F., Biotechnol. Bioeng. 43:423-428 (1994)). Furthermore, since the
present culture media provide for rapid, high-density suspension
cultivation of mammalian cells, the present methods facilitate the
rapid production of recombinant proteins at higher concentrations
than has been possible heretofore.
[0172] Methods of producing a polypeptide according to the
invention comprise (a) obtaining a mammalian cell that has been
genetically engineered to produce a polypeptide; and (b)
cultivating the mammalian cell in the culture media of the present
invention under conditions favoring expression of the polypeptide
by the mammalian cell. Optimal methods for genetically engineering
a mammalian cell to express a polypeptide of interest are
well-known in the art and will therefore be familiar to one of
ordinary skill. Cells may be genetically engineered prior to
cultivation in the media of the invention, or they may be
transfected with one or more exogenous nucleic acid molecules after
being placed into culture in the media. According to the invention,
genetically engineered cells may be cultivated in the present
culture media either as monolayer cultures, or more preferably as
suspension cultures according to the methods described above.
Following cultivation of the cells, the polypeptide of interest may
optionally be purified from the cells and/or the used culture
medium according to techniques of protein isolation that will be
familiar to one of ordinary skill in the art.
[0173] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Materials and Methods
[0174] In each of the following examples, the following materials
and methods were generally used.
[0175] Unless otherwise indicated, all media and reagents were
obtained from Life Technologies, Inc. (Rockville, Md.).
Suspension Media Examples
[0176] Adenovirus type 5-transformed 293 human embryonic kidney
epithelial cells were obtained from ATCC(CRL 1573), and were
cultured as described in Example 4 by incubation at 37.degree. C.
in a humidified atmosphere consisting of 8% CO.sub.2/92% air.
Replacement Medium Examples
A. Media
[0177] CHO-S-SFM H (Life Technologies, Inc., Gaithersburg, Md.) is
a low protein (<100 .mu.g/ml) serum-free medium designed for
growth and recombinant protein expression by CHO cells in
suspension culture. CHO-S-SFM-II contains both insulin and
transferrin.
[0178] CHO III Prototype is an essentially protein-free formulation
which contains no animal-derived proteins and is designed for
growth and recombinant protein expression in suspension culture.
The terms "CHO III Prototype," CHO III PFM," and "CHO III" are
synonymous.
[0179] CD CHO is a particularly preferred embodiment of the
replacement medium of the present invention (Table 1, the
particularly preferred embodiment column), to which a Fe.sup.2+
and/or Fe.sup.3+ chelate has been added, and is a chemically
defined formulation designed for suspension culture applications.
Where applicable, glutamine is also added.
[0180] All three of the above media were formulated without
hypoxanthine and thymidine for DHFR amplified rCHO cultures.
B. CHO Cells
[0181] Wild-type CHO DG44 cells were obtained from Dr. Lawrence
Chasin (Columbia University) and were adapted to suspension culture
in CHO-S-SFM II supplemented with hypoxanthine and thymidine. Cells
were maintained in CHO-S-SFM II+HT Supplement or CHO III Prototype
(Life Technologies, Inc.).
[0182] To establish a recombinant bovine growth hormone (rbGH) CHO
cell line, wild-type CHO-K1 cells were transfected with two
plasmids: pRSVneo, containing a neomycin resistance gene, and a bGH
cassette using the protocol supplied with LipofectAMINE.TM. Reagent
(Life Technologies). Selection was conducted in the presence of the
neomycin analogue, G418, at a concentration of 1.2 mg/ml. Stock
cultures of rCHO cells were maintained in either CHO-S-SFM II or
CHO III Prototype supplemented with 0.6 mg/ml G418 (all products
from Life Technologies, Inc.).
[0183] To establish a recombinant .beta.-galactosidase
(r.beta.-Gal) CHO cell line, CHO cells deficient in dihydrofolate
reductase (DHFR.sup.-) were obtained from ATCC (CRL-9096,
Rockville, Md.) and transfected with two plasmids: pSV2dhfr (ATCC
37146), containing a gene for methotrexate (MTX) resistance, and
pCMV.beta.gal, which contains the lacZ cDNA. The transfection was
conducted using LipofectAMINE.TM. Reagent, and selection was
accomplished with 1.2 .mu.M methotrexate (Sigma Chemical Co., St
Louis, Mo.). Stock cultures were maintained in CHO-S-SFM II or CHO
III Prototype supplemented with 0.3 .mu.M MTX.
C. Assays
[0184] 1. Recombinant bGH Quantitation by ELISA
[0185] rbGH production was quantitated using the Non-Isotopic
Immunoassay System for bGH Transfection Protein.TM. (Life
Technologies Inc., Gaithersburg, Md.) following manufacturer's
instructions.
[0186] 2. r.beta.-Gal Assay
[0187] r.beta.-Galactosidase (r.beta.-gal or r.beta.eta-Gal) was
measured in cell lysates using a modification of the method
described by Hall et al., J. Mol. Appl. Gen. 2:101-109 (1983) and
Miller, Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory (1972). Briefly, a 2.0 ml sample of cell suspension was
collected from each sample and subjected to two freeze-thaw cycles,
centrifuged at 100.times.g for 4 minutes, and the supernatant was
saved for r.beta.-Galactosidase quantitation. 100 .mu.l of each
supernatant sample was added to 1.0 ml Z buffer (0.06 M
Na.sub.2HPO.sub.4, 0.04M NaH.sub.2PO.sub.4, 0.01M KCl, 0.01M
MgSO.sub.4.7H.sub.2O, 0.05M .beta.-mercaptoethanol, pH 7.0),
followed by 200 .mu.l of o-nitrophenyl-.beta.-D-galactoside (ONPG,
4.0 mg/ml in dH.sub.20) and incubated at 37.degree. C. for 120
minutes. The reaction was halted by addition of 500 .mu.l stop
buffer (1M Na.sub.2CO.sub.3) and absorbance at 420 nm was read
against a blank of the appropriate medium. Activity was calculated
using the following formulae:
1. 1000 .times. A 120 120 min .times. 0.1 ml = Units / r .beta. -
galactosidase 2. Units r .beta. - galactosidase / # of cells =
Activity / cell ##EQU00001##
[0188] r.beta.-Gal was detected in cells using the staining method
of Sanes et al., EMBO J. 5:3133-3142 (1986).
[0189] 3. Amino Acid, Ammonia, Glucose and Lactate Measurements
[0190] Amino acid and ammonia levels in culture supernatants were
measured by HPLC using the Waters AccQ-Tag method (Millipore Corp.,
Milford, Pa.). Glucose and Lactate concentrations were determined
with the YSI Select Biochemistry Analyzer (Model 2700, YSI, Yellow
Springs, Ohio).
D. CHO Cell Culture
[0191] Unless indicated otherwise below, CHO cell culture
conditions were as follows for the following Examples. 25-35 mL of
CHO cells were cultured in 125 mL shake flasks in humidified air
containing 5-10% carbon dioxide. The shake flasks were shaken on an
orbital shaking platform at 125-135 rpm. Temperature was maintained
at 37.degree. C. Cells were subcultured every three to four days to
a density of 2-3.times.10.sup.5 cells/mL.
Example 1
Formulation of Complete Suspension Medium
[0192] Formulation of Basal Cell Culture Medium.
[0193] To formulate the basal cell culture medium, the following
were blended as powders: L-arginine.HCl (430.00 mg/L; 355.6 mg/L
L-arginine free base), L-asparagine (anhydrous) (26.40 mg/L),
L-aspartic acid (75.00 mg/L), L-cysteine (57.6 mg/L), L-glutamic
acid (29.40 mg/L), L-glutamine (585.00 mg/L), L-histidine (42.15
mg/L), L-isoleucine (190.00 mg/L), L-leucine (280.00 mg/L),
L-lysine (204 mg/L), L-methionine (115.00 mg/L), L-phenylalanine
(70.00 mg/L), L-serine (250.00 mg/L), L-threonine (60.00 mg/L),
L-tryptophan (20.00 mg/L), L-tyrosine (69.2 mg/L), L-valine (190.00
mg/L), biotin (0.097 mg/L), D-Ca.sup.++-pantothenate (1.19 mg/L),
choline chloride (14.00 mg/L), folic acid (5.00 mg/L), i-inositol
(18.00 mg/L), niacinamide (1.22 mg/L), pyridoxine.HCl (1.03 mg/L;
0.85 mg/L pyridoxine free base), riboflavin (0.22 mg/L), thiamine
(0.99 mg/L), vitamin B.sub.12 (1.03 mg/L), putrescine (0.087 mg/L),
D-glucose (4500.00 mg/L), KCl (276.30 mg/L), NaCl (4410.00 mg/L),
HEPES (2980.00 mg/L), linoleic acid (0.06 mg/L), D,L-lipoic acid
(2.00 mg/L), phenol red (1.00 mg/L), PLURONIC F68 (300.00 mg/L),
sodium pyruvate (110.0 mg/L), Na.sub.2HPO.sub.4 (125.00 mg/L),
insulin (zinc human recombinant) (10.00 mg/L), transferrin (human
hobo-, heat-treated) (5.00 mg/L), ethanolamine.HCl (5 mg/L; 3.2
mg/L ethanolamine), Fe(NO.sub.3).sub.3.9H.sub.2O (0.810 mg/L),
MgCl.sub.2 (76.20 mg/L), MgSO.sub.4 (24.10 mg/L), CaCl.sub.2 (11.10
mg/L), ZnSO.sub.4.H.sub.2O (0.0874 mg/L), Na.sub.2SeO.sub.3
(0.0000067 mg/L), MnCl.sub.2 (0.0001 mg/L) and NH.sub.4VO.sub.3
(0.0006 mg/L).
[0194] NaHCO.sub.3 (2400.00 mg/L) was added to the medium solution,
and the pH of the solution was then adjusted with HCl to
7.2.+-.0.05 and the volume adjusted to the full desired volume with
ddH.sub.2O. The osmolality was determined to be 265-275 mOsm.
[0195] To formulate the complete culture medium, 100 mg/L dextran
sulfate (average molecular weight=5,000 daltons) were added to the
basal medium, and the complete medium was filtered through a low
protein-binding filter and used immediately or stored at 4.degree.
C. under diminished light conditions until use.
Example 2
Formulation of Lower Protein and Protein-Free Culture Media
[0196] To produce a culture medium that was lower in protein, a
basal medium was formulated as described in Example 1 except that
transferrin was omitted from the formulation. In place of
transferrin, either 40 .mu.M FeSO.sub.4-EDTA (Sigma; St. Louis,
Mo.) or 60 .mu.M FeCl.sub.3-sodium citrate (Sigma) were added to
the basal media, and this lower protein medium was then filtered
and stored as described in Example 1.
[0197] To formulate a culture medium that is completely free of
protein, a lower protein medium containing no transferrin is
produced as described above, except that insulin is also omitted
from the formulation and, instead, the final concentration of
ZnSO.sub.4 is increased to 0.354 mg/L as a substitute for insulin.
Protein-free culture media are then filtered and stored as
described in Example 1.
Example 3
Enrichment of Culture Medium
[0198] To provide a more enriched culture medium, the basal and/or
complete media described above were supplemented with additional
components. In one such enrichment, resulting in a culture medium
that was low in animal protein (or animal protein-free), a
formulation of hydrolyzed rice peptides (Hy-Pep Rice Extract; Quest
International, Norwich, N.Y.) was added to the complete media of
Example 1 or Example 2 (for animal protein-free media) at a
concentration of about 100 mg/L (concentrations of about 1-1000
mg/L were found to be acceptable as well). Enriched culture media
were then filter-sterilized and stored until use as described in
Example 1. In an alternative formulation providing an enriched
culture medium that is free of animal protein, a formulation of soy
peptides (Hy-Soy; Quest International) is added at similar
concentrations.
[0199] Other enriched culture media are prepared by adding one or
more cytokines, such as growth factors at the following optimal
concentrations: EGF (about 0.00001-10 mg/L), aFGF or bFGF (about
0.0001-10 mg/L), KGF (about 0.0001-10 mg/L) or HGF (about
0.00001-10 mg/L). Other cytokines are also optionally added at
concentrations that are easily optimized by routine experimentation
(e.g., dose-response curves). Cytokines are available commercially,
for example, from Life Technologies, Inc. (Rockville, Md.).
[0200] Other enriched culture media are prepared by adding one or
more animal peptides, such as bovine serum albumin (BSA), human
serum albumin (HSA) or casein. Animal peptides are available
commercially, for example from Sigma (St. Louis, Mo.), and are
added to basal media or complete media at concentration ranges of
about 1-30,000 mg/L; optimal concentrations for a particular
application are easily determined by routine experimentation.
[0201] Other enriched culture media are prepared by adding one or
more yeast peptides, such as yeast extract, yeastolate or yeast
extract ultrafiltrate. Yeast extract and yeastolate are available
commercially, for example from Difco (Norwell, Mass.), while yeast
extract ultrafiltrate is prepared as described in U.S. Application
No. 60/028,197, filed Oct. 10, 1996, the disclosure of which is
incorporated herein by reference in its entirety. Yeast peptides
are added to basal or complete media at concentration ranges of
about 1-30,000 mg/L; optimal concentrations for a particular
application are easily determined by routine experimentation.
[0202] Following preparation, enriched culture media are filter
sterilized and stored as described in Example 1 for complete
medium.
Example 4
Use of Culture Media for Suspension Culture of Epithelial Cells
[0203] To demonstrate the efficacy of the present media in
suspension culture of anchorage-dependent cells, 293 human
embryonic kidney cells transformed with adenovirus type 5 DNA were
cultivated. Cultures of 293 cells in serum-supplemented media were
weaned from serum by passage 2-3 times in OptiMEM medium (Life
Technologies, Inc.; Rockville, Md.) supplemented with 2% normal
horse serum in 165 cm.sup.2 culture flasks. After the second or
third passage, when cells reached 50-75% confluence, they were
dislodged from the growth surfaces by gently rapping the flasks
several times; trypsin or other proteolytic agents were not used,
since such agents often cause irreversible damage to cells
cultivated in low-serum or serum-free media. Cells were resuspended
in the present culture media and triturated until aggregates
dispersed into a single cell suspension, and cell concentration and
viability was determined by trypan blue exclusion counting on a
hemacytometer according to routine procedures.
[0204] Cells were then seeded into the present culture medium in
Erlenmeyer flasks at a density of about 2.5-3.0.times.10.sup.5
cells/ml. To minimize aggregation of the cells, the seed volume in
the culture flasks was kept below about 20% of the total volume of
the flask (e.g., for 125 ml flasks, total volume after dilution of
cells was no more than about 21-22 ml; for 250 ml flasks, no more
than about 40-45 ml). To maintain cells in suspension, flasks were
then placed on a rotary shaker platform in an incubator (37.degree.
C., 8% CO.sub.2/92% air) and shaken at 125 rpm. Cell densities and
viabilities were determined at least every other day, and cells
were subcultured when the density approached about
7.5-10.0.times.10.sup.5 cells/ml by diluting cultures with fresh
culture medium to a density of about 2.5-3.0.times.10.sup.5
cells/ml. Subculturing was continued until aggregation of cells
during cultivation appeared minimal.
[0205] Once cells were adapted to cultivation in suspension in the
present culture media, the cultures were scaled up into
larger-volume spinner flasks or bioreactors. Cells were
concentrated from flask cultures by centrifugation at about 400 g
for five minutes, pellets were resuspended by gentle trituration in
the present media followed by vortex mixing for 45 seconds, and
cells seeded into spinner flasks or bioreactors in the present
media at a density of about 2.5-3.0.times.10.sup.5 cells/ml. To
minimize shearing of cells while maintaining the cells in
suspension, for spinner cultures the spinner speed was set to about
150 rpm while for bioreactor cultures the impeller speed was set to
about 70 rpm.
[0206] In Erlenmeyer flask cultures of 293 cells, viable cell
densities of about 2.5-3.0.times.10.sup.6 cells/ml were obtained
(data not shown). As shown in FIG. 1, cell densities of up to
3.5-4.0.times.10.sup.6 cells/ml, with nearly 100% viability, were
obtained in bioreactors and spinner cultures of 293 cells in the
complete media of the invention within 2-3 days after initiation of
the cultures. Similar results were obtained with suspension
cultures of HeLaS3 cervical epithelial cells (not shown).
Lower-protein culture media of the invention, in which transferrin
had been replaced by either FeSO.sub.4-EDTA or FeCl.sub.3-sodium
citrate chelates, performed equivalently to complete media in
supporting 293 cell growth (FIG. 2).
[0207] Together, these results demonstrate that the present culture
media promote high-density suspension culture of
anchorage-dependent epithelial cells and 293 cells in a serum-free,
low-protein or protein-free environment.
Example 5
Production of Viruses by Suspension Cultures of 293 Cells
[0208] To examine the utility of the present media in virus
production protocols, suspension cultures of 293 cells were
prepared as described in Example 4 and infected with adenovirus.
Infection of cells was performed directly in the suspension
cultures by adding adenovirus at a MOI of about 5-50, and cultures
were then maintained as in Example 4 for about 48-96 hours.
Adenovirus was then harvested with the culture medium and titered
on the permissive host cell line A549.
[0209] As shown in FIG. 3, adenovirus-infected 293 cells cultured
in the media of the invention produced high titers of adenovirus-5
beginning at approximately day 2 post-infection and continuing
through day 4. These results demonstrate that the present culture
media facilitate the rapid, large-scale production of active
viruses, such as adenovirus, in suspension cultures of 293
cells.
Example 6
Expression of Exogenous Genes by Suspension Cultures of 293
Cells
[0210] The utility of the present media in facilitating the
production of recombinant polypeptides was also examined. Line 293
cells were plated in RPMI-1640 containing 10% FBS (growth medium)
in six-well culture plates at 3.times.10.sup.5 cells/ml, and one
day later were transfected with 1.5 .mu.g of
pCMV.SPORT-.beta.-galactosidase and 0.5 .mu.g of pSV2neo plasmid
DNAs (LTI; Rockville, Md.) in the presence of 12 .mu.l of
LipofectAMINE (LTI) using the standard LipofectAMINE protocol. At
24 hours post-transfection, cells in each well were subcultured
into growth medium in a 10 cm.sup.2 plate at final dilutions of
1:50, 1:200 and 1:500, and at 48 hours post-transfection the growth
medium was replaced with selection medium (growth medium containing
500 .mu.g/ml genetecin (LTI)). Medium was changed as necessary to
remove nonviable cells, and resistant clones were selected with
cloning cylinders and transferred to 24-well plates for adaptation
to suspension culture.
[0211] Clones expressing .beta.-galactosidase were adapted to
suspension culture in the present culture media according to the
procedures described in detail in Example 4. For all cultures, to
maintain selective pressure on the cells, genetecin was included in
the present culture media at concentrations of up to 50 .mu.g/ml.
Higher concentrations of geneticin are discouraged, since they may
be toxic in serum-free, low-protein media such as those of the
present invention.
[0212] As shown in FIG. 4, significant quantities of
.beta.-galactosidase were produced by transfected 293 cells
cultured in the present media, beginning at day 1 post-transfection
and continuing throughout the six day course of the culture. These
results demonstrate that the present culture media facilitate
stable transfection of epithelial cells with exogenous genes, and
the rapid, large-scale production of recombinant polypeptides by
suspension cultures of 293 cells.
Example 7
[0213] r.beta.-gal CHO cells were planted at 3.times.10.sup.5
cells/ml in CHO-S-SFM II medium (without hypoxanthine and
thymidine), CD CHO medium, or CHO III Prototype medium. Samples
were saved at various time points for determination of cell density
and .beta.-galactosidase expression levels. As shown in FIG. 5A,
the highest cell density was obtained in CD CHO medium, which is
the medium of the present invention. At day 4 of culturing, the
highest level of .beta.-galactosidase expression was observed in
CHO III medium (FIG. 5B). By day 6 of culturing, whereas the level
of .beta.-galactosidase expression in cells grown in CHO III medium
had declined, the level of .beta.-galactosidase expression in cells
grown in the replacement medium of the present invention (CD CHO
medium) had increased. Indeed, the level of .beta.-galactosidase
expression in cells grown in CD CHO medium continued to increase at
day 7 of culturing.
Example 8
[0214] r.beta.-gal CHO cells were planted at 2.times.10.sup.5
cells/ml in either CHO-S-SFM II medium (without hypoxanthine and
thymidine), CD CHO medium, or CHO III medium. On day 3
post-planting, when the CD CHO cultures had reached approximately
1.2.times.10.sup.6 cells/ml, the CD CHO cultures were centrifuged
and resuspended in fresh CD CHO or CHO III media. By day 8, the CD
CHO culture that had been changed to CHO III medium had reached a
lower peak cell density than the culture kept in CD CHO medium
(FIG. 6A).
[0215] At day 7 of culturing, the level of r.beta.-galactosidase
expression in cells grown in the replacement medium of the present
invention (CD CHO medium) was comparable to the level in cells that
were switched to CHO III medium (FIG. 6B).
Example 9
[0216] r.beta.-gal CHO cells were planted at 1.8.times.10.sup.5
cells/ml in CD CHO medium supplemented with increasing
concentrations of methotrexate (MTX). Concentrations indicated are
final concentrations. Samples were taken daily for determination of
cell density and r.beta.-galactosidase expression. Although MTX
concentration was inversely proportional to cell density (FIG. 7A),
MTX concentration was proportional to r.beta.-galactosidase
specific activity (FIG. 7B). Thus, the medium of the present
invention, when supplemented with methotrexate, can be used to grow
recombinant DHFR amplified CHO cells which express high levels of
recombinant protein. Because the medium of the present invention is
protein-free, the recombinant protein product can be easily
purified.
Example 10
[0217] rbGH CHO cells were planted at 2.times.10.sup.5/ml in 125 ml
shake flasks (35 ml volume) in either CHO III medium or CD CHO
medium. Daily samples were taken for determination of viable cell
density and rbGH levels. The CD CHO cultures reached a higher peak
cell density (FIG. 8A) and expressed higher levels of rbGH (FIG.
8B).
Example 11
[0218] r.beta.-gal CHO cells were planted at 3.times.10.sup.5/ml in
125 ml shake flasks (20-35 ml volume) in either CHO-S-SFM II medium
or CD CHO medium. Daily samples were taken for determination of
viable cell density and r.beta.-gal levels. Total cell density
continued to rise in CD CHO cultures (days 3 through 8, FIG. 9A),
while total cell density remained constant in CHO-S-SFM II culture.
r.beta.-gal levels continued to rise in both culture conditions,
but CD CHO cultures expressed more total r.beta.-gal product (FIG.
9B). Thus, compared to a medium that contains insulin and
transferrin, the medium of the present invention supports increased
cell growth and level of protein expression.
Example 12
[0219] r.beta.-gal CHO cells were planted at 3.times.10.sup.5/ml in
125 ml shake flasks (20-35 ml volume) in CD CHO, CHO III PFM, or
FMX-8 media. FMX-8 medium is disclosed in Zang, M. et al.,
Bio/Technology 13:389-392 (1995).
[0220] As shown in FIG. 10, after seven days of culturing, cells
grown in CHO III PFM medium grew to a density about two-fold that
of cells grown in FMX-8 medium. As shown in FIG. 10, cells grown in
CD CHO medium grew to a density about three-fold that of cells
grown in CD CHO medium and about six-fold that of cells grown in
FMX-8 medium.
[0221] As shown in FIG. 11, cells grown in CHO III PFM medium
expressed r.beta.-gal at a level about three-fold that of cells
grown in FMX-8 medium. As shown in FIG. 11, cells grown in CD CHO
medium expressed r.beta.-gal at a level about 1.6-fold that of
cells grown in CD CHO medium and about five-fold that of cells
grown in FMX-8 medium. Thus, compared to the FMX-8 medium, the
medium of the present invention supports increased levels of cell
growth and protein expression.
Example 13
[0222] The CD CHO medium supports scaled-up cultures of mammalian
cells as well. r.beta.-gal CHO cells were planted at
1-3.times.10.sup.5/ml in 250 ml shake flasks (75 ml working volume)
in CD CHO medium and cultured at pH 7.40, 50% air saturation,
37.degree. C., while shaking at 125-135 r.p.m. For bioreactor
experiments, r.beta.-gal cells were planted at
1-3.times.10.sup.5/ml in a 5 L stirred tank Celligen bioreactor
(3.8 L working volume) in CD CHO medium and cultured at pH 7.40,
50% air saturation, 37.degree. C., while stirring at 90 r.p.m. The
arrow indicates supplementation with 3 g/L glucose (final
concentration) and 1 g/L glutamine (final concentration) at day
nine of culturing.
[0223] As shown in FIG. 12A, the growth kinetics of rCHO cells
cultured in the bioreactor were similar to those observed in the
shake flask. As shown in FIG. 12B, the level of r.beta.-gal
expression was higher in cells cultured in the bioreactor.
Supplementation with glucose and glutamine did not boost cell
growth over the level reached on day nine. These results indicate
that the CD CHO medium can be used successfully in scaled up cell
culture.
Example 14
[0224] To determine the effect of the anticlumping agent dextran
sulfate on cell growth and protein expression, recombinant cells
were cultured in the presence or absence of dextran sulfate
(molecular weight of either 5,000 or 500,000). r.beta.-gal CHO
cells were planted at 3.times.10.sup.5/ml in 125 ml shake flasks
(20-35 ml volume) in CD CHO medium. Dextran sulfate was added to
the medium, at the time of cell planting, to a final concentration
of 25 .mu.g/mL. Results are shown in FIGS. 13 and 14. In FIGS. 13
and 14, "A" is dextran sulfate (m.w. 5,000) and "C" is dextran
sulfate (m.w. 500,000). CD CHO Control cells are cells to which
dextran sulfate was not added. As shown in FIG. 13, cells grown
medium containing dextran sulfate (m.w. 5,000) displayed increased
cell growth and viability at days 5, 7, and 9. As shown in FIG. 13,
cells grown in dextran sulfate (m.w. 500,000) displayed increased
cell growth and viability at days 5 and 7, but not at day 9. As
shown in FIG. 14, cells grown in dextran sulfate (m.w. 5,000)
displayed an increase in the level of r.beta.-gal expression at
days 5, 7, and 9. As shown in FIG. 14, cells grown in medium
supplemented with dextran sulfate (m.w. 500,000) did not display
enhanced expression.
[0225] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0226] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *