U.S. patent application number 13/848587 was filed with the patent office on 2013-12-05 for defined systems for epithelial cell culture and use 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 Paul Battista, David JUDD.
Application Number | 20130323837 13/848587 |
Document ID | / |
Family ID | 31190482 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323837 |
Kind Code |
A1 |
JUDD; David ; et
al. |
December 5, 2013 |
DEFINED SYSTEMS FOR EPITHELIAL CELL CULTURE AND USE THEREOF
Abstract
The present invention provides cell culture media formulations
which support the in vitro cultivation of animal epithelial cells.
The media comprise at least one fibroblast growth factor (FGF) and
at least one agent that induces increased intracellular cAMP
levels, and optionally comprise ascorbic acid. The present
invention also provides methods of cultivating animal epithelial
cells in vitro using these cell culture media formulations, kits
comprising the media, cell culture compositions comprising the
culture media and an animal epithelial cell, and compositions that
may be used as replacements for organ or gland extracts in animal
cell culture media.
Inventors: |
JUDD; David; (Grand Island,
NY) ; Battista; Paul; (Eggertsville, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
31190482 |
Appl. No.: |
13/848587 |
Filed: |
March 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11348553 |
Feb 7, 2006 |
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13848587 |
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10694189 |
Oct 28, 2003 |
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11348553 |
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09695926 |
Oct 26, 2000 |
6692961 |
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10694189 |
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08948053 |
Oct 9, 1997 |
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09695926 |
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60028471 |
Oct 11, 1996 |
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Current U.S.
Class: |
435/375 |
Current CPC
Class: |
C12N 2501/01 20130101;
C12N 2500/32 20130101; C12N 2500/34 20130101; C12N 5/0629 20130101;
C12N 2500/38 20130101; C12N 2501/113 20130101; C12N 2500/35
20130101; C12N 2500/42 20130101; C12N 2500/30 20130101; C12N
2500/90 20130101 |
Class at
Publication: |
435/375 |
International
Class: |
C12N 5/071 20060101
C12N005/071 |
Claims
1. A serum-free cell culture medium comprising a fibroblast growth
factor (FGF) and an agent causing an increase in intracellular
levels of cyclic adenosine monophosphate (cAMP), wherein said
medium is capable of supporting the cultivation of an animal
epithelial cell in vitro.
2. The medium of claim 1, wherein said FGF is selected from the
group consisting of FGF-1 (aFGF), FGF-2 (bFGF) and FGF-7 (KGF).
3. The medium of claim 2, wherein said FGF is aFGF.
4. The medium of claim 1, wherein said agent causing an increase in
intracellular levels of cAMP functions through interaction with a
cellular G-protein.
5. The medium of claim 1, wherein said agent causing an increase in
intracellular levels of cAMP functions by directly increasing
intracellular cAMP levels.
6. The medium of claim 1, wherein said agent causing an increase in
intracellular levels of cAMP functions by inhibiting a cAMP
phosphodiesterase.
7. The medium of claim 1, wherein said agent causing an increase in
intracellular levels of cAMP is a .beta.-adrenergic receptor
agonist.
8. The medium of claim 4, wherein said agent is cholera toxin or
forskolin.
9. The medium of claim 5, wherein said agent is dibutyryl cAMP.
10. The medium of claim 6, wherein said agent is
isobutylmethylxanthine or theophylline.
11. The medium of claim 7, wherein said agent is isoproterenol.
12. The medium of claim 1, said medium further comprising ascorbic
acid.
13. The medium of claim 1, wherein said medium is a 1.times. medium
formulation.
14. The cell culture medium of claim 1, wherein said medium
formulation is a 10.times. concentrated medium formulation.
15. The cell culture medium of claim 1, said medium further
comprising one or more ingredients selected from the group of
ingredients consisting of an amino acid, a vitamin, an inorganic
salt, adenine, ethanolamine, D-glucose, epidermal growth factor
(EGF), heparin, N-[2-hydroxyethyl]-piperazine-N'-[2-ethanesulfonic
acid] (HEPES), hydrocortisone, insulin, lipoic acid, phenol red,
phosphoethanolamine, putrescine, sodium pyruvate, T3, thymidine and
transferrin.
16. The medium of claim 15, said medium further comprising ascorbic
acid.
17. The cell culture medium of claim 15, wherein said amino acid
ingredient 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.
18. The cell culture medium of claim 15, wherein said vitamin
ingredient 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.
19. The cell culture medium of claim 15, wherein said inorganic
salt ingredient comprises one or more inorganic salts selected from
the group consisting of a calcium salt, CuSO.sub.4, FeSO.sub.4,
KCl, a magnesium salt, a manganese salt, sodium acetate, NaCl,
NaHCO.sub.3, Na.sub.2HPO.sub.4, Na.sub.2SO.sub.4, a selenium salt,
a silicon salt, a molybdenum salt, a vanadium salt, a nickel salt,
a tin salt and a zinc salt.
20. A cell culture medium comprising the ingredients adenine,
ethanolamine, D-glucose,
N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES),
hydrocortisone, insulin, lipoic acid, phenol red,
phosphoethanolamine, putrescine, sodium pyruvate, T3, thymidine,
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-pantothenate, folic acid,
i-inositol, niacinamide, pyridoxine, riboflavin, thiamine, vitamin
B.sub.12, a calcium salt, CuSO.sub.4, FeSO.sub.4, KCl, a magnesium
salt, a manganese salt, sodium acetate, NaCl, NaHCO.sub.3,
NaHPO.sub.4, Na.sub.2SO.sub.4, a selenium salt, a silicon salt, a
molybdenum salt, a vanadium salt, a nickel salt, a tin salt, and a
zinc salt, wherein each ingredient is present in an amount which
supports the cultivation of an animal epithelial cell in vitro.
21-72. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional Patent
Application No. 60/028,471, filed Oct. 11, 1996, the contents of
which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to cell culture
medium formulations. Specifically, the present invention provides
systems comprising defined cell culture medium formulations that
facilitate the in vitro cultivation of epithelial cells,
particularly keratinocytes. The present invention also provides
methods for cultivation of animal cells using these systems.
[0004] 2. Related Art
Cell Culture Media
[0005] 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.
[0006] 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 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
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.
[0007] 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.
[0008] Typically, cell culture media formulations are supplemented
with a range of additives, including undefined components such as
fetal bovine serum (FES) (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.
[0009] 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 Pres New York, pp. 85-122 (1985)). For example, the
chemical composition of these supplements may 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 when
these contaminated supplements are used in cell culture media
formulations. Cell surface chemistry, which is a critical portion
of the in vitro microenvironment for many cell types, can be
adversely modified via adsorption or incorporation of serum or
extract proteins. 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
[0010] 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, mono
cytes/macrophages, fibroblasts, chondrocytes or hepatocytes which
are available from GIBCO/LTI (Gaithersburg, Md.). The distinction
between SFM and defined media, however, is that SFM are media
devoid of serum, 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); Wile, 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.
[0011] 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.
[0012] 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)).
Epithelial Cells
[0013] Overview
[0014] 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)).
[0015] 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.).
[0016] Keratinocytes
[0017] The specialized epithelial cells found in the epidermis of
the skin are known as keratinocytes. In the upper, cornified layers
of the skin (those exposed to the environment), the cytoplasm of
the keratinocytes is completely replaced with keratin and the cells
are dead. The keratinocytes located in the lower layers, however,
particularly in the basal epidermis (stratum basale), actively
divide and ultimately migrate up through the more superficial
layers to replace those cells being sloughed off at the external
surface. Accordingly, the skin can be thought of as a dynamic organ
comprising keratinocytes that are constantly dividing, maturing and
ultimately dying.
[0018] Cultures of human keratinocytes are increasingly being used
in examinations of skin stricture and disease, and as in vitro
models of human skin in toxicology studies (Boyce, S. T., and Ham,
R. G., in: In Vitro Models for Cancer Research, vol. III, Webber,
M. M., et al., eds., Boca Raton, Fla.: CRC Press, Inc., pp. 245-274
(1985)). Successful culture of keratinocytes has proven, however,
to be somewhat difficult, owing primarily to their nutritional
fastidiousness (Gilchrest, B. A., et al., J. Cell. Physiol.
120:377-383 (1984)). For example, in most early studies using
traditional serum-supplemented culture media, keratinocytes from
skin explants were rapidly overgrown by less fastidious and
faster-growing fibroblasts that were also resident in the tissue
(Freshney, Id.). Thus, there has been substantial work expended in
the attempt to formulate culture media favoring the selection and
successful in vitro cultivation of human keratinocytes.
Keratinocyte Culture Medium Formulations and Systems
[0019] A variety of systems have been developed to culture human
keratinocytes. Early work in this area used specialized culture
media such as Medium 199 (Marcelo, C. L., et al, J. Cell Biol.
79:356 (1978)) and NCTC 168 (Price, F. M., et al., In Vitro 16:147
(1980)) supplemented with serum. Alternatively, keratinocyte growth
and colony formation have been shown to be improved by plating
cells on lethally irradiated 3T3 fibroblasts and by adding
epidermal growth factor (EGF) and hydrocortisone to the medium
(Rheinwald, J. G., and Green, H., Cell 6:331 (1975)). One of the
first serum-free medium formulations developed for keratinocyte
culture was based on Medium 199 and included a growth factor
cocktail comprising bovine brain extract (Gilchrest, B. A., et al.,
J. Cell. Physiol. 112:197 (1982)), and serum-free culture of human
keratinocytes without the use of 3T3 fibroblast feeder layers
became widely accepted upon the development of a more specialized
basal medium, MCDB-153 (Boyce, S. T., and Ham, R. G., J. Invest.
Dermatol. 81:33 (1983); U.S. Pat. Nos. 4,673,649 and 4,940,666).
Serum-free MCDB-153 includes trace elements, ethanolamine,
phosphoethanolamine, hydrocortisone, EGF, and bovine pituitary
extract (BPE). This medium and several enhanced versions have been
used widely for human keratinocyte cultivation (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); Daley, J. P., et al., FOCUS
(GIBCO/LTI) 12:68 (1990)). The use of BPE is also common to many
commercially available media for keratinocyte cultivation,
including KGM (Clonetics Corporation; San Diego, Calif.), CS-2.0
Keratinocyte Cell Growth Medium (Cell Systems, Inc.; Kirkland,
Wash.), M154 (Cascade Biologicals, Inc.; Portland, Oreg.) and
Keratinocyte-SFM (GIBCO/LTI; Gaithersburg, Md.).
[0020] Serum-free medium containing BPE as the primary mitogen,
however, has several drawbacks, as generally described above. For
example, the undefined composition of BPE complicates experimental
models and interpretation of results, and may either stimulate or
inhibit the growth or differentiation of keratinocyte cultures,
depending on the concentrations of other components in the medium
(Wille, J. J., et al, J. Cell. Physiol. 121:31 (1984)). In
addition, BPE requires titration in different cell systems, and its
stability in medium is limited to about four weeks under normal use
and storage conditions. There has been at least one report of a
fully defined medium for the culture of epidermal cells, wherein
BPE is replaced with epidermal growth factor (EGF), insulin-like
growth factor 1 (IGF-1) and increased quantities of six specific
amino acids (U.S. Pat. No. 5,292,655). However, this medium was
designed for the specific purpose of in vitro formation of a skin
substitute comprising differentiated keratinocytes, and may not be
ideal for supporting continuous cultures of actively growing
cells.
[0021] Thus, a need remains for defined culture media, that are
serum- and organ/gland extract-free, for the cultivation of animal
epithelial cells including keratinocytes. 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 purification of biological substances produced by
cultured animal cells in the biotechnology industry, and will
provide more consistent results in methods employing the
cultivation of animal epithelial cells. The current invention
provides such defined media.
SUMMARY OF THE INVENTION
[0022] The present invention provides defined culture media that
replace BPE with growth-promoting additives such as insulin, EGF
and other additives. Specifically, the invention provides a cell
culture medium, capable of supporting the cultivation of an animal
epithelial cell in vitro, comprising insulin, EGF, and at least two
additional additives from the group consisting of FGF, an agent
that increases intracellular levels of cyclic adenosine
monophosphate (cAMP) and ascorbic acid. The medium provided by the
present invention may be a 1.times. formulation, or may be
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 an animal epithelial cell
in vitro. The medium may be used to culture a variety of animal
epithelial cells, including primary cells (e.g., keratinocytes or
cervical epithelial cells) and established cell lines (e.g., HeLa
cells). Cells supported by the medium of the present invention may
be derived from any animal, preferably a mammal, and most
preferably a human. The present invention also provides methods of
culturing animal epithelial cells using the culture medium
formulations disclosed herein, comprising the steps of (a)
contacting an animal cell with the cell culture medium of the
present invention; and (b) cultivating the animal cell under
conditions suitable to support its cultivation in vitro. The
invention also provides kits for use in the cultivation of an
animal epithelial cell. Kits according to the present invention
comprise a carrier means having in close confinement therein one or
more container means, wherein a first container means contains a
basal culture medium as described above, a second carrier means
contains a insulin, a third container means contains EGF, a fourth
container means contains FGF, a fifth container means contains at
least one agent that increases intracellular levels of cAMP, a
sixth container means contains heparin and a seventh container
means contains ascorbic acid. In a preferred embodiment, the second
container means of the kits contains insulin, EGF, FGF, at least
one agent that increases intracellular levels of cAMP, heparin and
ascorbic acid together in admixture. The invention further provides
cell culture compositions comprising the culture media of the
present invention and an animal epithelial cell. The invention also
provides compositions comprising heparin, EGF, FGF, at least one
agent that increases intracellular levels of cAMP, and optionally
ascorbic acid, which compositions may be used to replace organ or
gland extracts in serum-free animal cell culture media. The culture
media of the present invention are suitable for use in the
isolation and initiation of primary epithelial cell cultures, as
well as for the expansion of established epithelial cell cultures.
Additionally, the media of the present invention provide superior
growth, and maintenance of morphological and physiological markers,
of primary animal epithelial cells.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1. Photomicrographs of phase contrast microscopy of
human keratinocytes. Cells were cultured in the defined
keratinocyte SFM of the present invention (panel A) or in a
BPE-containing keratinocyte SFM (panel B). Photographs are
100.times..
[0024] FIG. 2. Photomicrograph of fluorescence microscopy of human
keratinocytes cultured in the defined keratinocyte SFM of the
present invention and stained with fluorescent antibodies directed
against keratin 14.
[0025] FIG. 3. Bar graph demonstrating growth of primary human
keratinocytes in the defined keratinocyte SFM of the present
invention ("Defined Keratinocyte-SFM"), in a BPE-containing
keratinocyte SFM ("Keratinocyte-SFM") or in a keratinocyte SFM
obtained from Hyclone Laboratories (Logan, Utah) ("Supplier A").
Growth was determined six days after seeding, and values represent
means.+-.SEM, n=7.
[0026] FIG. 4. Bar graph demonstrating growth of secondary human
keratinocytes in the defined keratinocyte SFM of the present
invention ("Defined Keratinocyte-SFM"), in a BPE-containing
keratinocyte SFM ("Keratinocyte-SFM") or in a keratinocyte SFM from
Hyclone Laboratories ("Supplier A"). Growth was determined 72 hours
after seeding, and values represent means.+-.SEM, n=7.
[0027] FIG. 5. Bar graph demonstrating growth kinetic analysis of
human keratinocytes. Cells were cultured in the defined
keratinocyte SFM of the present invention (.box-solid.) or in a
BPE-containing keratinocyte SFM (.quadrature.). Values represent
the mean.+-.SD, n=2.
[0028] FIG. 6. Line graph demonstrating an evaluation of media
shelf life using primary human keratinocytes. Cells were cultured
in the defined keratinocyte SFM of the present invention (solid
line) or in a BPE-containing keratinocyte SFM (dashed line) over a
15-week period. Cells were counted after 6 days in medium stored
for given times and compared to control cells cultured in fresh
medium.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0029] 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.
[0030] 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.
[0031] 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."
[0032] 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.
[0033] By "culture vessel" is meant a glass, plastic, or metal
container that can provide an aseptic environment for culturing
cells.
[0034] 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.
[0035] 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.
[0036] The term "combining" refers to the mixing or admixing of
ingredients in a cell culture medium formulation.
[0037] 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, RPM-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 Allen R. Liss, N. Y.
(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.
[0038] 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.
Formulation of Culture Media
[0039] Basal Media
[0040] The cell culture media of the present invention are
aqueous-based, comprising a number of ingredients in a solution of
deionized, distilled water to form "basal media." Ingredients which
the basal media of the present invention may include are amino
acids, vitamins, inorganic salts, adenine, ethanolamine, D-glucose,
heparin, N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]
(HEPES), hydrocortisone, insulin, lipoic acid, phenol red,
phosphoethanolamine, putrescine, sodium pyruvate, triiodothyronine
(T3), thymidine and transferrin. Alternatively, insulin and
transferrin may be replaced by ferric citrate or ferrous sulfate
chelates. Each of these ingredients may be obtained commercially,
for example from Sigma (Saint Louis, Mo.).
[0041] 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.).
[0042] 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.).
[0043] Inorganic salt ingredients which may be used in the media of
the present invention include a calcium salt (e.g., CaCl.sub.2),
CuSO.sub.4, FeSO.sub.4, KCl, a magnesium salt (e.g., MgCl.sub.2), a
manganese salt (e.g., MnCl.sub.2), Sodium acetate, NaCl;
NaHCO.sub.3, Na.sub.2HPO.sub.4, Na.sub.2SO.sub.4, and ions of the
trace elements selenium, silicon, molybdenum, vanadium, nickel, tin
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,
Na.sub.2SiO.sub.3, (NH.sub.4)6Mo.sub.7O.sub.24, NH.sub.4VO.sub.3,
NiSO.sub.4, SnCl and ZnSO. These inorganic salts and trace elements
may be obtained commercially, for example from Sigma (Saint Louis,
Mo.).
[0044] The specific combinations of the above ingredients, their
concentration ranges and preferred concentrations in the basal
media are shown in Table 1.
TABLE-US-00001 TABLE 1 ANIMAL EPITHELIAL CELL CULTURE BASAL 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 1-250 9 9.00 L-Arginine 10-500
425 421.40 L-Asparagine 5-150 12 12.20 L-Aspartic Acid 1-100 5 4.00
L-Cysteine 2-250 42 42.00 L-Glutamic Acid 5-250 15 14.80
L-Glutamine 10-2500 1025 1020.00 Glycine 1-200 8 7.60 L-Histidine
5-250 50 50.40 L-Isoleucine 1-100 6 6.00 L-Leucine 25-250 130
131.20 L-Lysine 10-250 55 54.90 L-Methionine 5-200 15 13.50
L-Phenylalanine 1-150 10 10.03 L-Proline 1-250 35 34.60 L-Serine
5-250 126 126.20 L-Threonine 5-100 25 23.80 L-Tryptophan 2-100 10
9.30 L-Tyrosine 5-100 12 11.68 L-Valine 5-250 70 70.20 Other
Components Adenine 1-100 24 24.00 Ethanolamine 0.5-5 0.6 0.60
D-Glucose 500-5000 1500 1500.00 HEPES 1000-5000 3350 3336.20
Hydrocortisone 0.01-5 0.1 0.074 Insulin 0.5-25 5 5.00 Lipoic Acid
0.05-10 0.2 0.20 Phenol Red 0.5-15 1 1.20 Phospho- 0.05-5 0.2 0.141
ethanolamine Putrescine 0.01-1 0.2 0.20 Sodium Pyruvate 10-200 55
55.0 Triiodothyronine 0.001-1 0.01 0.0067 (T3) Thymidine 0.05-25
0.7 0.73 Transferrin 1-50 11 11.11 Vitamins Biotin 0.005-1 0.02
0.02 Choline Chloride 1-150 14 14.00 D-Ca.sup.++- 0.05-10 0.3 0.30
Pantothenate Folic Acid 0.1-10 1 0.80 i-Inositol 1-75 18 18.00
Niacinamide 0.01-5 0.05 0.04 Pyridoxine 0.005-10 0.06 0.06
Riboflavin 0.01-5 0.05 0.04 Thiamine 0.05-5 0.3 0.30 Vitamin B12
0.01-5 0.5 0.50 Inorganic Salts calcium salt 1-25 13 12.98 (e.g.,
CaCl.sub.2 CuSO.sub.4 0.001-0.1 0.002 0.002 FeSO.sub.4 0.1-5 0.4
0.403 KCl 1-500 112 112.00 magnesium salt 1-500 185 182.48 (e.g.,
MgCl.sub.2) manganese salt 0.000005-0.005 0.00002 0.00002 (e.g.,
MnCl.sub.2) Sodium acetate 50-500 300 301.00 NaCl 3000-9000 6800
6790.0 NaHCO.sub.3 100-4000 1160 1160.0 Na.sub.2HPO.sub.4 1-500 285
284.00 Na.sub.2SO.sub.4 0.5-10 4 3.38 selenium salt 0.001-0.1 0.005
0.00496 (e.g., Na.sub.2SeO.sub.3) silicon salt 0.05-0.5 0.15 0.137
(e.g., Na.sub.2SiO.sub.3) molybdenum salt 0.0001-0.1 0.001 0.00120
(e.g., (NH.sub.4).sub.6Mo.sub.7O.sub.24) vanadium salt 0.001-0.01
0.0005 0.00057 (e.g., NH.sub.4VO.sub.3) nickel salt 0.0005-0.001
0.0001 0.00013 (e.g., NiSO.sub.4) tin salt 0.0001-0.001 0.0001
0.00011 (e.g., SnCl.sub.2) zinc salt 0.01-5 0.15 0.133 (e.g.,
ZnSO.sub.4)
[0045] Complete Media
[0046] The above ingredients, when admixed together in solution,
form a "basal medium." To this basal medium, heparin, epidermal
growth factor (EGF), at least one agent increasing intracellular
cyclic adenosine monophosphate (cAMP) levels, and at least one
fibroblast growth factor (FGF), are added to formulate the complete
culture media of the present invention. Heparin, EGF, the
cAMP-increasing agent(s) and FGF(s) may be added to freshly
formulated basal medium, or they may be admixed as described in
detail in Example 1 in a solution of Dulbecco's Phosphate Buffered
Saline (DPBS) and stored frozen, preferably at about -20.degree. C.
to about -70.degree. C., until being added to basal medium to
formulate the complete medium of the present invention. This
admixture of heparin, EGF, the cAMP-increasing agent(s) and FGF(s)
may be used as a replacement for BPE or other organ/gland extracts
in animal cell culture media. The admixture may also be prepared as
a 1.times.-1000.times. formulation, most preferably as a 1.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.
[0047] Heparin may be obtained commercially, for example from Sigma
(Saint Louis, Mo.), and is preferably derived from porcine mucosa.
Heparin is added to the present media primarily to stabilize the
activity of the growth factor components, especially FGF
(Gospodarowicz, D., and Cheng, J., J. Cell. Physiol. 128:475-484
(1986); EP 0 408 146). To formulate the medium of the present
invention, heparin is added to the basal medium shown in Table 1 at
a concentration of about 1-500 U.S.P. units/liter, preferably about
5-50 U.S.P. units/liter, and most preferably about 10 U.S.P.
units/liter.
[0048] EGF may be natural or recombinant and may be human or
rodent. EGF is available commercially (e.g., from GIBCO/LTI,
Gaithersburg, Md.), or may be isolated from natural sources or
produced by recombinant DNA techniques (U.S. Pat. No. 4,743,679)
according to methodologies that are routine in the art. To
formulate the medium of the present invention, EGF should be added
to the basal medium shown in Table 1 at a concentration of about
0.00001-10 mg/L, preferably about 0.0001-0.1 mg/L, and most
preferably about 0.0002 mg/L.
[0049] A variety of agents that increase intracellular cAMP levels
may be used in formulating the media of the present invention.
Included are agents which induce a direct increase in intracellular
cAMP levels (e.g., dibutyryl cAMP), agents which cause an increase
in intracellular cAMP levels by an interaction with a cellular
G-protein (e.g., cholera toxin and forskolin), agents which cause
an increase in intracellular cAMP levels by acting as agonists of
.beta.-adrenergic receptors (e.g., isoproterenol) and agents which
cause an increase in intracellular cAMP levels by inhibiting the
activities of cAMP phosphodiesterases (e.g., isobutylmethylxanthine
(IBMX) and theophylline). Most preferable for use in formulating
the media of the present invention is isoproterenol. These
cAMP-increasing agents are available commercially, e.g. from Sigma
(St. Louis, Mo.), and are used at concentrations approximating
those described in Green (Proc. Natl. Acad. Sci. USA 15:801-811
(1978)). For example, cholera toxin is added to the basal medium
described above at a concentration of about 0.000005-1 mg/L,
preferably about 0.0007-0.1 mg/L, and most preferably about 0.08
mg/L. Dibutyryl cAMP is added to the basal media at a concentration
of about 25-750 mg/L, preferably about 45-500 mg/L, and most
preferably about 148 mg/L. IBMX may be added to the basal media at
a concentration of about 0.2-25 mg/L, preferably about 2-10 mg/L,
and most preferably about 7 mg/L. Most preferably, isoproterenol is
the agent used to increase intracellular cAMP levels, and is
formulated into the basal media at a concentration of about 0.01-10
mg/L, preferably about 0.1-5 mg/L, and most preferably about 0.25
mg/L
[0050] The FGF used in formulating the media of the present
invention may be any member of the FGF family of growth factors,
including FGF-1 (acidic FGF or aFGF), FGF-2 (basic FGF or bFGF),
FGF-3 (int-2), FGF-4 (K-FGF), FGF-5 (hst-1), FGF-6 (hst-2) and
FGF-7 (keratinocyte growth factor or KGF). Preferable are aFGF,
bFGF and KGF, and most preferable is aFGF. Natural or recombinant
FGF may be used, which may be of human, bovine, porcine or rodent
origin. Most preferably, recombinant human aFGF is used in
formulating the present media. aFGF, bFGF and KGF are available
commercially (e.g., from GIBCO/LTI, Gaithersburg, Md. and R&D
Systems, Inc., Minneapolis, Minn.), or may be isolated from natural
sources or produced by recombinant DNA techniques (EP 0 408 146 and
U.S. Pat. No. 5,395,756 for aFGF; U.S. Pat. No. 5,189,148 for bFGF;
WO 90/08771 and WO 95/01434 for KGF) according to methodologies
that are routine in the art. To formulate the medium of the present
invention, FGF should be added to the basal medium shown in Table 1
at a concentration of about 0.0001-10 mg/L, preferably about
0.001-0.1 mg/L, and most preferably about 0.005 mg/L.
[0051] Together, the basal medium, heparin, EGF, cAMP-increasing
agent(s) and FGF(s) formulate complete culture media according to
the present invention. These complete media are suitable for use in
the culture of a variety of animal epithelial cells, as described
in more detail below. It may be preferable, however, 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 animal epithelial cells. To accomplish such
enrichment, ascorbic acid may be added to the complete media.
Ascorbic acid is available commercially in several forms.
Preferable for use in formulating the present media is L-ascorbic
acid phosphate, magnesium salt, available from Wako Pure Chemical
Industries, which is added to the media at a concentration of about
0.001-10 mg/L, preferably about 0.01-5 mg/L, and most preferably
about 0.1 mg/L.
[0052] The medium ingredients 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 275-350 mOsm, preferably about 285-325 mOsm, and most
preferably about 280-310 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.
[0053] 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, which is directed to methods of solubilizing
culture media components at high concentrations.
[0054] 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.
[0055] 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 0.1-1.0 .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.
[0056] The optimal concentration ranges for the basal medium
ingredients are listed in Table 1. These ingredients can be
combined to form the basal animal cell culture medium which is then
supplemented as described above with heparin, EGF, at least one
agent increasing intracellular cAMP levels, at least one FGF and
optionally with ascorbic acid, 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 heparin, EGF, aFGF,
isoproterenol and ascorbic acid as described above.
[0057] 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
which forms after these ingredients are combined.
[0058] 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.
Use of Culture Media
[0059] Cells which can be grown in the medium 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 of
human origin, which may be primary cells derived from a tissue
sample such as keratinocytes, cervical epithelial cells, bronchial
epithelial cells or tracheal epithelial cells, or transformed cells
or established cell lines (e.g., the HCAT human keratinocyte or
HeLa cervical epithelial cell lines). These cells 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 cultivation in the present media. Particularly
preferred are primary or secondary human keratinocytes derived from
a sample of normal or abnormal human skin. 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.
Isolation of Cells
[0060] Animal cells for culturing by the present invention may be
obtained commercially, for example from ATCC (Rockville, Md.), Cell
Systems, Inc. (Kirkland, Wash.), Clonetics Corporation (San Diego,
Calif.), BioWhittaker (Walkersville, Md.), or Cascade Biologicals
(Portland, Oreg.). Alternatively, cells may be isolated directly
from samples of animal tissue obtained via biopsy, autopsy,
donation or other surgical or medical procedure.
[0061] 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 GIBCO/LTI, Gaithersburg, Md.) to
promote dissociation of cells from the tissue matrix.
[0062] 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 hemocytometer.
Plating of Cells
[0063] The isolated cells can be plated 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. 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 culture conditions, using 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 or fibronectin, or natural or synthetic fragments
thereof). 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. Use of
attachment factors or a support matrix with the medium of the
present invention will enhance cultivation of many
attachment-dependent cells in the absence of serum
supplementation.
[0064] The cell seeding densities for each experimental condition
can be optimized for the specific culture conditions being used.
For routine culture in plastic culture vessels, an initial seeding
density of 1-5.times.10.sup.6 cells per cm.sup.2 is preferable.
[0065] 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,
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.
[0066] Cells in closed or batch culture should undergo complete
medium exchange (i.e., replacing spent media with fresh media)
about every 1-2 days, or more or less frequently as required by the
specific cell type. Cells in perfusion culture (e.g., in
bioreactors or fermenters) will receive fresh media on a
continuously recirculating basis.
Cell Culture Compositions
[0067] The cell culture media of the present invention may also be
used to produce cell culture compositions comprising the present
media and an animal epithelial cell. Animal epithelial cells which
may be used to formulate the cell culture compositions of the
present invention are those of animal origin, including but not
limited to cells obtained from mammals. Mammalian cells
particularly suitable for use in formulating the present cell
culture compositions 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 or tracheal epithelial cells, or transformed cells or
established cell lines (e.g., the HCAT human keratinocyte or HeLa
cervical epithelial cell lines), or derivatives thereof. These
cells 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. Particularly preferred are
primary or secondary human keratinocytes derived from a sample of
normal or abnormal human skin. 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 cell culture compositions of the present invention. These cell
culture compositions may be used in a variety of medical (including
diagnostic and therapeutic), industrial, forensic and research
applications requiring ready-to-use cultures of animal epithelial
cells in serum-free media.
[0068] 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
[0069] In each of the following examples, the following materials
and methods were generally used.
[0070] Isolation and Culture of Human Keratinocytes
[0071] Unless otherwise indicated, all media and reagents were
obtained from GIBCO/LTI (Gaithersburg, Md.). Human neonatal
foreskins were placed in serum-free medium (SFM) without growth
factors containing 5 .mu.g/ml gentamycin and were stored at
4.degree. C. Foreskins can be stored in this manner for about five
days without significant loss of cell viability. Foreskins were
briefly rinsed in 70% isopropanol and then placed into Dulbecco's
phosphate-buffered saline (DPBS), without Ca.sup.++ and Mg.sup.++,
containing 20 .mu.g/ml gentamycin for 60 minutes. Foreskins were
then cut into halves or quarters, depending upon the size of the
tissue, and the pieces were transferred, dermis side down, to a
petri dish containing 25 units/ml dispase, and were incubated 18-24
hours at 4.degree. C. Epidermal sheets were separated from the
full-thickness skin with forceps, pooled in 60 mm culture dishes
containing 5-7 ml of 0.05% trypsin/0.53 mM EDTA, and were incubated
at 37.degree. C. for 15-20 minutes with gentle pipetting to aid in
tissue dissociation. Pooling of the tissue specimens is performed
to reduce the effects of donor-to-donor growth variation. Trypsin
activity was terminated by addition of soybean trypsin inhibitor
(10 mg/ml in DPBS). Any remaining pieces of epidermal sheets were
carefully removed and discarded. The cell suspension was
transferred to a sterile centrifuge tube and the cells pelleted by
centrifugation at 40.times.g for 5 minutes at 22.degree. C., and
washed once with SFM. The supernatant was discarded, the cell
pellet resuspended in the appropriate medium, and cell densities
determined using a hemacytometer. Cells were plated in culture
flasks or dishes.
[0072] Secondary cultures were established by removing the spent
medium, briefly washing the cell monolayer with Versene (1:5000
dilution), and adding an appropriate volume of 0.05% trypsin/0.53
mM EDTA. Cells were incubated at 37.degree. C. until they became
round (about 5 minutes), trypsin was removed, and the cells were
incubated at 37.degree. C. until they detached from the culture
surface with gentle tapping (about 5 minutes). Trypsin activity was
inactivated by addition of 10 mg/ml soybean trypsin inhibitor
solution; cells were pelleted by centrifugation at 40.times.g for 5
minutes at 22.degree. C., washed once with SFM, and resuspended in
the appropriate medium. Secondary cell cultures were also
established from primary keratinocytes obtained from Cell Systems
Corporation (Kirkland, Wash.) with results comparable to those
found with cultures established from neonatal foreskins.
[0073] Trypsinization times are critical to the performance of any
keratinocyte medium. Human keratinocytes that remain in trypsin too
long have lower plating efficiencies and may be induced to
differentiate.
[0074] Cultures were incubated at 37.degree. C. in a humidified
atmosphere consisting of 5% CO.sub.2/95% air. Stock cultures were
maintained at a split ratio of 1:2 to 1:3 and subcultured at 70% to
80% confluence. Keratinocytes at passage 0 through passage 4 were
used for experimental evaluation.
[0075] Morphology and Growth Assays
[0076] Morphological analysis and immunostaining of cells were
performed in 8-chamber glass culture slides. Keratinocytes were
plated at 2.times.10.sup.4 cells/cm.sup.2 in a total volume of 400
.mu.l/0.8 cm.sup.2 chamber. Cells were incubated for 24 hours, then
fixed with 3.7% formaldehyde, permeabilized with 0.5% TRITON X-100
in DPBS, and allowed to react with rabbit anti-cytokeratin 14
antibody (1:200 dilution). Cells labeled with antibodies were
visualized using goat anti-rabbit F(ab').sub.2 FITC conjugate (1:50
dilution).
[0077] Human keratinocyte growth assays were performed in 24-well
culture dishes (2 cm2 growth area) utilizing a seeding density of
1.times.10.sup.4 cells/cm.sup.2. Endpoint growth assays were
assessed at 6 days postseeding for primary cells and 72 hours for
secondary cells. Growth kinetic assays were counted at 24 hour
intervals over 96 hours without medium replacement. Single-cell
cloning assays were performed in 96-well tissue culture-treated
plates by serial dilution of cell suspensions to 5 cells/ml in the
appropriate medium and plating 100 .mu.l/well. Plates were
incubated for 5 days before observation. In comparison assays,
media of the present invention were examined for their
growth-promoting abilities relative to a defined human keratinocyte
medium derived from Supplier A and to a BPE-containing formulation
(Keratinocyte-SFM; GIBCO/LTI, Gaithersburg, Md.).
Example 1
Formulation of Complete Medium
[0078] Formulation of Basal Cell Culture Medium. Distilled,
deionized water (hereinafter "ddH.sub.2O") was measured out to 80%
of the total desired volume. While gently stirring this water with
a magnetic stirrer, the following were added: L-alanine (9.00
mg/L), L-arginine.HCl (421.40 mg/L), L-asparagine-HCl (12.20 mg/L),
L-aspartic acid (4.00 mg/L), L-cysteine.HCl.H.sub.2O (42.00 mg/L),
L-glutamic acid (14.80 mg/L), L-glutamine (1020.00 mg/L), glycine
(7.60 mg/L), L-histidine.HCl.H.sub.2O (50.40 mg/L), L-isoleucine
(6.00 mg/L), L-leucine (131.20 mg/L), L-lysine.HCl (54.90 mg/L),
L-methionine (13.50 mg/L), L-phenylalanine (10.03 mg/L), L-proline
(34.60 mg/L), L-serine (126.20 mg/L), L-threonine (23.80 mg/L),
L-tryptophan (9.30 mg/L), L-tyrosine-disodium salt (11.68 mg/L),
L-valine (70.20 mg/L), biotin (0.02 mg/L), D-Ca.sup.++-pantothenate
(0.30 mg/L), choline chloride (14.00 mg/L), folic acid (0.8 mg/L),
i-inositol (18.00 mg/L), niacinamide (0.04 mg/L), pyridoxine.HCl
(0.06 mg/L), riboflavin (0.04 mg/L), thiamine.HCl (0.30 mg/L),
vitamin B12 (0.50 mg/L), putrescine.2HCl (0.20 mg/L), D-glucose
(1500.0 mg/L), KCl (112.0 mg/L), NaCl (6790.0 mg/L), thymidine
(0.73 mg/L), adenine (24.00 mg/L), HEPES (3336.20 mg/L), lipoic
acid (0.20 mg/L), phenol red (1.20 mg/L), sodium pyruvate (55.0 g),
sodium acetate (301.00 mg/L), Na.sub.2HPO.sub.4 (284.00 mg/L),
Na.sub.2SO.sub.4 (3.39 mg/L), human insulin (5.00 mg/L) and human
transferrin (11.11 mg/L).
[0079] A stock solution of ethanolamine.HCl was prepared in
ddH.sub.2O at 976.00 mg/L/L and 0.615 ml/L of this stock was added
to the medium solution, to give a final concentration of
ethanolamine.HCl of 0.60 mg/L.
[0080] A stock solution of phosphoethanolamine was prepared in
ddH2O at 1408.00 mg/L and 0.1001 ml/L of this stock was added to
the medium solution, to give a final concentration of
phosphoethanolamine of 0.141 mg/L.
[0081] A stock solution of FeSO.sub.4.7H.sub.2O (41.70 mg/L),
MgCl.sub.2.6H.sub.2O (18890 mg/L), and CaCl.sub.2.2H.sub.2O (1344
mg/L) was prepared in water containing 0.5 ml/L concentrated HCl,
and 9.660 ml of this stock solution was added to the medium
solution, to give final concentrations of 0.403 mg/L
FeSO.sub.4.7H.sub.2O, 182.48 mg/L MgCl.sub.2.6H.sub.20 and 12.98
mg/L CaCl.sub.2.2H.sub.2O.
[0082] A stock solution of ZnSO.sub.4.7H.sub.2O (137.68 mg/L) was
prepared in water, and 0.9660 ml of this solution was added to the
medium solution to give a final concentration of 0.133 mg/L
ZnSO.sub.4.7H.sub.2O.
[0083] A stock solution containing Na.sub.2SeO.sub.3 (0.513 mg/L),
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O (0.124 mg/L),
NaSiO.sub.3.9H.sub.2O (14.2 mg/L), NiSO.sub.4.6H.sub.2O (0.013
mg/L), MnCl.sub.2.4H.sub.2O (0.002 mg/L), SnCl.sub.2.2H.sub.2O
(0.011 mg/L) and NH.sub.4VO.sub.3 (0.059 mg/L) was prepared in
water with 0.5 ml/L concentrated HCl, and 9.660 ml of this stock
solution was added to the medium solution to give final
concentrations of 0.00496 mg/L Na.sub.2SeO.sub.3, 0.00120 mg/L
(NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O, 0.137 mg/L
NaSiO.sub.3.9H.sub.2O, 0.00013 mg/L NiSO.sub.4.6H.sub.2O, 0.00002
mg/L MnCl.sub.2.4H.sub.2O, 0.00011 mg/L SnCl.sub.2.2H.sub.20 and
0.00057 mg/L NH.sub.4VO.sub.3.
[0084] A stock solution of hydrocortisone was prepared at 370 mg/L
in 95% ethanol, and 0.2 ml of this stock was added to the medium
solution to give a final concentration of hydrocortisone of 0.074
mg/L.
[0085] A stock solution of triiodothyronine (T3) was Prepared at
67.00 mg/L in 70% ethanol, and 0.1 ml of this stock was added to
the medium solution to give a final concentration of T3 of 0.0067
mg/L.
[0086] NaHCO.sub.3 (1160 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 290.+-.15 mOsm.
[0087] This basal medium formulation was then filtered through a
low protein-binding filter, bottled and stored under diminished
light conditions at 4.degree. C. until use.
[0088] Formulation of the Growth Supplement.
[0089] To a solution of Dulbecco's Phosphate Buffered Saline (DPBS)
the following were added while gently stirring: ascorbic acid
phosphate, magnesium salt (50 mg/L), aFGF (2.5 mg/L), heparin (5000
units/L) and EGF (0.1 mg/L).
[0090] A stock solution of isoproterenol (100,000 mg/L) was
prepared in DPBS containing 50 mg/L ascorbic acid, and 1.25 ml/L of
this solution was added to the above, to form a 500.times.
formulation of the growth supplement.
[0091] This 500.times. solution was then filtered through a low
protein-binding filter, and added to the basal medium or aliquoted
and stored at -20 to -80.degree. C. until use in epithelial cell
culture medium as a replacement for an organ or gland extract such
as BPE.
[0092] Preparation of the Complete Medium.
[0093] One ml of the growth supplement was added to 500 ml of the
basal medium, and the complete medium was used immediately or
stored at 4.degree. C. under diminished light conditions until
use.
Example 2
[0094] Effects of aFGF
[0095] To examine the utility of the basal medium in supporting the
growth of human keratinocytes, and to determine the effects of FGF,
primary human keratinocytes were isolated as described above and
cultured in the basal medium from Example 1 supplemented with 10
U.S.P. units/L heparin and 0.0002 mg/L EGF, or in the basal medium
containing 10 U.S.P. units/L heparin, 0.0002 mg/L EGF and 0.005
mg/L aFGF. Representative results of five separate experiments,
comparing growth in the basal medium with and without aFGF to that
in a BPE-containing keratinocyte SFM ("control") are shown in Table
2.
TABLE-US-00002 TABLE 2 EFFECTS OF aFGF (CELLS/ML .times. 10.sup.5).
Defined SFM (Present Invention) Control -aFGF +aFGF 1.85 0.738
0.860 1.86 0.924 0.970 0.844 0.686 0.756 4.38 3.40 3.54 3.66 3.76
4.00
[0096] These results indicate that the basal medium supports the
growth of primary keratinocytes, albeit usually to a lesser extent
than BPE-containing control medium. Furthermore, the results
indicate that the addition of aFGF to the basal medium enhances its
ability to promote the growth of keratinocytes.
Example 3
Effects of Isoproterenol
[0097] To determine if the performance of the defined culture
medium could be further enhanced by inclusion of an agent that
raises intracellular cAMP levels, the basal medium containing
heparin, EGF and aFGF from Example 2 was examined with and without
the addition of 0.25 mg/L isoproterenol. Primary human
keratinocytes were isolated and cultured as described for Example
2. Representative results of five separate experiments, comparing
growth in the medium with and without isoproterenol to that in a
BPE-containing keratinocyte SFM ("control") are shown in Table
3.
TABLE-US-00003 TABLE 3 EFFECTS OF ISOPROTERENOL (CELLS/ML .times.
10.sup.5). Defined SFM (Present Invention) Control -isoproterenol
+isoproterenol 0.604 0.564 0.765 1.234 0.922 1.443 0.819 0.565
1.177 1.396 0.956 1.777 1.772 0.674 1.776
[0098] These results demonstrate that the addition of isoproterenol
to the aFGF-containing basal medium of the present invention
further enhances its ability to promote the growth of
keratinocytes. In fact, the medium containing aFGF and
isoproterenol promoted the growth of primary human keratinocytes
better than did the BPE-containing control. These findings thus
indicate that a defined medium comprising the basal medium,
heparin, EGF, aFGF and isoproterenol is an optimal formulation for
a fully defined, BPE-free SFM that supports cultivation and
promotes growth of human keratinocytes. Furthermore, these results
demonstrate that a composition comprising heparin, EGF, FGF and a
cAMP-activating agent such as isoproterenol may be used as a
replacement for an organ or gland extract such as BPE in SFM for
the culture of epithelial cells such as keratinocytes.
Example 4
Effects of Ascorbic Acid
[0099] Since some basal media used in the culture of keratinocytes
contain ascorbic acid (Gilchrest, B. A., et al., J. Cell. Physiol.
120:377-383 (1984)), the effect of the addition of 50.0 mg/L
ascorbic acid to the EGF/aFGF/isoproterenol-containing medium from
Example 3 was examined. Primary human keratinocytes were isolated
and cultured as described for Example 2. Representative results of
four separate experiments, comparing growth in the medium with and
without ascorbic acid to that in a BPE-containing keratinocyte SFM
("control") are shown in Table 4.
TABLE-US-00004 TABLE 4 EFFECTS OF ASCORBIC ACID (CELLS/ML .times.
10.sup.5). Defined SFM (Present Invention) Control -ascorbic acid
+ascorbic acid 1.107 1.088 1.221 6.258 11.61 14.10 0.801 1.475
1.492 1.860 2.290 2.860
[0100] These results demonstrate that the addition of ascorbic acid
to the aFGF/isoproterenol-containing medium of the present
invention further enhances its ability to promote the growth of
keratinocytes. The effects of ascorbic acid, however, were not as
dramatic as those observed for the addition of either aFGF (Example
2) or isoproterenol (Example 3); the defined medium containing
ascorbic acid performed only marginally better than that without
ascorbic acid, suggesting that the inclusion of ascorbic acid in
the defined medium of the present invention may be optional. Both
defined media, however, significantly outperformed the control
medium, confirming the results obtained in Example 3.
[0101] Together, the findings of Examples 2-4 indicate that a
defined medium comprising the basal medium, heparin, EGF, aFGF and
isoproterenol, and optionally including ascorbic acid, is an
optimal formulation for a fully defined, BPE-free SFM that supports
cultivation and promotes growth of human keratinocytes. In
addition, these results demonstrate that a solution comprising
heparin, EGF, FGF, a cAMP-increasing agent such as isoproterenol
and ascorbic acid may be used as a replacement for an organ or
gland extract such as BPE in SFM for the culture of epithelial
cells such as keratinocytes.
Example 5
Growth of Primary Human Keratinocytes in Defined SFM
[0102] Primary human keratinocytes were isolated as described above
and were cultured in either the defined SFM of the present
invention containing EGF, aFGF, isoproterenol and ascorbic acid as
described in Example 4 ("Defined Keratinocyte-SFM"), or in a
BPE-containing keratinocyte SFM ("Keratinocyte-SFM"). After
multiple passages, cultures were isolated, plated and cultured for
24 hours, and or were stained with antibodies against keratin 14
and examined by fluorescence microscopy for the expression this
standard marker of basal human keratinocytes (FIG. 2).
[0103] As shown in FIG. 1, human keratinocytes cultured in the
defined medium of the present invention (FIG. 1A) exhibited the
same contact-inhibited, "crazy paving" pattern morphology, typical
of cultured primary keratinocytes (Daniels, J. T., et al, Exp.
Dermatol. 4:183 (1995)), observed for cells cultured in the
BPE-containing media (FIG. 1B). Monolayer cultures in both media
had distinct borders and prominent nuclei, indicating the cultures
were in general good health. As demonstrated in FIG. 2, the cells
in the defined keratinocyte medium of the present invention stained
positively for keratin 14, indicating that the medium of the
present invention allows the retention of keratinocyte-specific
markers by cultured primary cells. Similar results were obtained
with cells cultured in BPE-containing medium.
[0104] To examine the utility of the media in supporting growth of
primary keratinocytes, cells were incubated over 6 days in the
medium of the present invention ("Defined Keratinocyte-SFM"), in a
BPE-containing keratinocyte SFM ("Keratinocyte-SFM"), or in a
defined keratinocyte medium obtained from Hyclone Laboratories
("Supplier A"). As shown in FIG. 3, primary human keratinocytes
cultured in the medium of the present invention demonstrated
significantly enhanced growth (p.ltoreq.0.05) when compared to the
other keratinocyte media. Population doubling times for each medium
were: Defined Keratinocyte-SFM: 46.3.+-.5.9 hours;
Keratinocyte-SFM: 66.6.+-.12.8 hours; and Supplier A: 83.5.+-.19.1
hours.
[0105] Together, these results indicate that the defined serum-free
medium of the present invention supports the growth of primary
human keratinocytes, and outperforms even undefined, traditionally
used BPE-containing media.
Example 6
Growth of Secondary Human Keratinocytes in Defined SFM
[0106] To determine if the utility of the media of the present
invention extended to secondary cultures of keratinocytes, cells
derived from actively growing cultures or from isolates obtained
commercially were cultured in the three media described in Example
5 and examined for growth rate. As demonstrated in FIG. 4, the
growth of secondary cultures was similar in the medium of the
present invention and in the BPE-containing medium. However,
significantly better cell growth (p<0.05) was obtained in the
present medium than in the defined medium from Supplier A.
Population doubling times for these secondary keratinocytes were:
Defined Keratinocyte-SFM: 25.0.+-.1.1 hours; Keratinocyte-SFM:
29.0.+-.1.6 hours; and Supplier A: 35.4.+-.4.1 hours.
[0107] Daily growth kinetic experiments using secondary cultures of
keratinocytes confirmed that cells cultured in the medium of the
present invention proliferated at a rate comparable to that of
BPE-containing media (FIG. 5). Furthermore, cloning efficiencies of
about 40% have been achieved with human keratinocytes cultured in
the medium of the present invention in single-cell cloning
experiments, comparable to those achieved with cells grown in
BPE-containing media (data not shown). In the media of the present
invention, secondary cultures can be maintained for at least six
passages with split ratios of about 1:2 performed twice weekly.
[0108] Taken with those in Example 5, these results indicate that
the defined serum-free medium of the present invention supports the
growth of primary and secondary human keratinocytes, and
outperforms both undefined BPE-containing
Example 7
Stability of Defined SFM
[0109] To evaluate the shelf life of the medium of the present
invention, primary human keratinocytes were cultivated for six days
in the present media (Defined Keratinocyte-SFM) or in
BPE-containing media (Keratinocyte-SFM). Media were evaluated
weekly over a storage period of 15-weeks after formulation, and
cell counts were compared at each time point to those obtained for
freshly prepared media.
[0110] As shown in FIG. 6, the fully supplemented defined SFM of
the present invention had a shelf life of over 14 weeks, which was
considerably longer than the BPE-containing medium. These results
indicate that the medium of the present invention, when stored
properly as described above, demonstrates an extended shelf life
compared to more traditionally used BPE-containing media.
General Discussion
[0111] Culture systems designed to propagate human keratinocytes
have evolved to reduce the undefined components and to increase
culture longevity and cell yields. The results of the above
Examples demonstrate that BPE can be replaced in SFM by a solution
comprising heparin, EGF, at least one FGF, at least one agent that
increases intracellular cAMP levels, and that optionally comprises
ascorbic acid. Furthermore, this replacement of BPE may be effected
without adversely affecting cellular proliferation rates and the
general physiology of human keratinocytes. The removal of BPE as a
medium component while maintaining medium performance represents a
step forward in human keratinocyte culture by providing a more
standardized and controlled culture environment, as has also been
shown lacking for other highly used primary cell cultures (Watson,
C. A., et al., Science 268:447-448 (1995)).
[0112] Thus, taken in combination, the results in Examples 1-6
indicate that an optimal culture medium formulation for supporting
the cultivation of animal cells is the basal medium formulation
shown in Table 1, supplemented with EGF at about 5-10 mg/Liter,
aFGF at about 5 mg/Liter, isoproterenol at about 0.3 mg/Liter and
ascorbic acid at about 50 mg/Liter (although ascorbic acid may be
eliminated with only a slight diminution of growth promotion).
[0113] Having now fully described the present invention it will be
understood by those of ordinary skill in the art that the same can
be performed within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any embodiment thereof.
[0114] All publications, patents and patent applications cited
herein are indicative of the level of skill of those skilled in the
art to which this invention pertains, and are herein incorporated
by reference in their entirety.
* * * * *