B I O P R O C E S S TECHNICAL

Development and Optimization

of Cell Culture Media
Genomic and Proteomic Approaches
Daniel W. Allison, Kathryn A. Aboytes, Danny K. Fong, Stacy L. Leugers,
Terrell K. Johnson, Heather N. Loke, and Laurel M. Donahue

T
issue was first cultured in
the early 1900s, largely
from fragments of lower
vertebrate tissue that were
kept alive in dishes.
Occasionally, cells migrated from
the explant, but they rarely divided.
In the 1940s and 1950s it became
possible to take explants of avian or
mammalian tissue and derive either
normal cells or, in the case of
rodents, continuous cell lines. Those
cells were first cultured in a basal
medium (containing some of the
building blocks of the cell WWW.PHOTOS.COM
components, nutrients such as amino
acids, vitamins, and salts) including growth factors, cytokines, knowledge of cell nutritional
supplemented with mammalian hormones, attachment factors, and biochemistry and the identification
serum. Animal cells generally will other unknown components that of additional components, such as
not proliferate when cultured in only promote cell survival and growth factors, that have shown a
the basal medium components (1). proliferation in vitro. It can also positive effect on specific cell types.
Mammalian serum contains contain adventitious agents such as In addition, as resources permit, an
thousands of beneficial components, viruses and prions. To increase investigator will undertake some
reproducibility of this biologically amount of random screening of
PRODUCT FOCUS: GROWTH FACTORS, complex component and eliminate factors that may have a positive
HORMONES, CYTOKINES
the possibility of adventitious effect (for example, growth factors
agents, investigators began to described in the literature for an
PROCESS FOCUS: CELL CULTURE formulate serum-free media that unrelated cell type). Use of random
PRODUCTION AND PROCESS made use of defined components screening has benefited from the
DEVELOPMENT (2–6). It has been challenging to miniaturization of assay formats as
determine which serum components well as the introduction of statistical
WHO SHOULD READ: THOSE allow for cell-type–specific growth approaches to experimental design
INVOLVED IN PROCESS (MEDIA) and/or attachment. Concurrent that allow for examination of fewer
OPTIMIZATION AND ANALYSIS with the effort to eliminate serum, a test conditions (7–12). However, it
KEYWORDS: MEDIA, GENOMICS, long history of studying cell is still hit or miss, and hundreds of
PROTEOMICS, ANALYTICAL METHODS,
nutritional biochemistry has allowed factors could potentially aid growth.
MAMMALIAN CELL CULTURE, SERUM-FREE,
for better basal media formulations We have developed a method
MICROARRAY
(such as carbon source, amino acids, that allows for the targeted
vitamins, and trace metals). identification and testing of
LEVEL: INTERMEDIATE Serum-free media are currently components to which a cell of
formulated based on prior interest is poised to respond,
2 BioProcess International JANUARY 2005
thereby eliminating the need for
random testing. This method takes
advantage of select genomic
(microarray- or PCR-based
approaches) and proteomic
(antibody array analysis) tools to
identify the receptors for growth
factors, hormones, and cytokines,
cell adhesion molecules, and other
components of cell signaling
pathways expressed by a culture of
interest. It is presumed that the
molecules expressed will mediate
cellular functions such as
proliferation, cessation of
proliferation, apoptosis,
differentiation, adhesion, and
migration. Specific ligands for the
expressed receptors or components
that activate or block any of the
uncovered pathways can be added
to cell culture medium and tested
for effect. This allows investigators
to rationally test for a biological
response to components for which
the cells may be prepared to
respond.
MATERIALS AND METHODS
Cell Preparation: All materials were
from Sigma-Aldrich unless
otherwise noted. All cell types were
cultured in tissue-culture treated
T-flasks to 60–80% confluence to
maintain log-phase growth. Cells
were cultured in medium containing
the lowest amount of fetal bovine
serum (FBS) required to still allow
reasonable growth rates. Cells were
then scraped into RNAlater.
RNA Isolation, cDNA Labeling,
Microarray Hybridization and
Detection: Either total RNA or
mRNA was isolated using the
GenElute Mammalian Total RNA
Miniprep kit or GenElute Direct
mRNA Miniprep kit. Labeled cDNA
was prepared using dendrimer-based
3DNA Array 350 (or 900)
expression array kits using the Cy3
and Cy5 dyes (Genisphere Inc.,
www.genisphere.com). Separate Cy3
and Cy5 reverse transcription
reactions were prepared using the
appropriate oligo dT primer (each
containing a specific 3DNA capture
sequence) and RNA. Subsequently,
the reactions were pooled and run
over SigmaSpin post-reaction clean-
up columns to remove free primers, Figure 1: Increased proliferation due to
salts, and nucleotides. the addition of the ligand for four
expressed receptors
The cDNA was then concentrated
using a Microcon YM-30 centrifugal Epidermal Growth Factor
filter device (Millipore,
www.millipore.com) prewashed with
Tris-EDTA buffer. The cDNA
hybridization solution was prepared
by adding concentrated cDNA,
water, LNA dT blocker, human
repetitive sequence DNA (ID Labs,
www.idlabs.com) and 2

formamide-based hybridization
solution. The solution was then
applied to a prewarmed human
cytokine microarray (TaKaRa Mirus Stromal Cell-Derived Factor 1
Bio Inc., www.takaramirusbio.com)
under a 22
25I standard or
mSeries LifterSlip (Erie Scientific,
www.eriemicroarray.com).
Hybridizations were performed
overnight for 16–24 hours.
Following hybridization, the
arrays were washed extensively and
dried. The Cy3 and Cy5 capture
reagents were combined in the
formamide-based hybridization
buffer prepared with Anti-Fade
Basic Fibroblast Growth Factor
reagent. The 3DNA hybridization
mixture was added to a prewarmed
array under a LifterSlip. Following a
thorough wash, the arrays were dried
and then scanned using a
PerkinElmer ScanArray Express
(PerkinElmer, www.perkinelmer.com)
using the Cy3 and Cy5 channels at
Laser and PMT settings that yielded
the maximum signal with minimal
saturated pixels.
Resazurin Assay: Cells were plated
in wells of a 24-well tissue culture Oncostatin M
treated plate containing 1 mL of a
base medium. That medium
contained the lowest amount of FBS
required to maintain approximately
half-maximal growth of the given
cell type. Test conditions were
performed in triplicate, with each
test compound added at three
different concentrations.
The cells were cultured until they
reached approximately 33%
confluency. At this point, 100 µL of
the resazurin solution from the HTS 7000 Plus BioAssay reader
in vitro toxicology assay kit (product (Perkin-Elmer). Readings were
code TOX-8) was added to each taken once a day until the culture
well. After sufficient incubation time was confluent (typically four days).
to convert the resazurin, A plate with base medium only (no
fluorescence was measured on an
JANUARY 2005 BioProcess International 3
cells) was used as a blank and
subtracted from the test condition
relative fluorescence unit (RFU)
readings to establish the final RFU
values.
Adhesion Assay: Cells were plated
in the base medium at low density
on various substrates using BD
BioCoat extracellular matrix coated
plates (BD BioSciences,
www.bdbiosciences.com). Starting
24 hours postplating, the cells were
observed for both number of cells
attached and morphology (degree of
cell spreading).
RT-PCR
Polymerase chain reactions were
assembled using cDNA from WI-38
cultures. Reactions were carried out
in 96-well PCR plates (Z37, 490-3)
with typical amplification
conditions. Following cycling, 5-µL
samples were analyzed by horizontal
agarose gel electrophoresis and
visualized by staining with ethidium
bromide.
Real-time Quantitative RT-PCR: This
PCR reaction was assembled using
cDNA from WI-38 cultures.
Reactions were carried out in
96-well PCR plates using the DNA
Engine Opticon 2 continuous
fluorescence detection system (MJ
Research, Inc., www.mjr.com).
Typical amplification conditions
were performed, followed by a melt
curve analysis.
A fluorescence value for each well
was recorded during every cycle and
represents the amount of product
amplified to that point in the
amplification reaction. The
threshold cycle (Ct) is the point at
which the fluorescent signal
becomes statistically significant
above background. A higher
concentration of template in the
reaction requires a lower number of
cycles to reach its Ct value. The
melt curve analysis performed for
each sample was useful to
distinguish between the desired
amplicon and primer dimer based
on their differential melt curves.
RESULTS
Identification of Key Pathways Involved
in Proliferation: The power of our
method is the ability to identify key
pathways that can be manipulated in
cells in culture. We have chosen to
focus on the impact of various
ligands (growth factors, cytokines,
and hormones) on their receptors.
That was done solely because of the
relative ease in testing those models.
Importantly, this method is used as
a tool to highlight key pathways,
enabling us to home in on them on
a molecular basis. Much is already
known about many such pathways
in a variety of systems. Once we
have identified a pathway as
important, we can look at ways to
stimulate, inhibit, or modify that
pathway to obtain a desired output.
Probably the most common output
desired for cell culture is an increase
in the number of cells obtained,
which is a measure of cell
proliferation.
To test whether it was possible to
use information about the
expression profile of a cell culture to
determine factors that the cells
would respond to, we chose to
study the proliferation of HEK-293,
an adherent human epithelial cell
line that is typically grown in a base
medium containing anywhere from
1 to 10% fetal bovine serum (FBS).
Using standard media development
tools, we were able to reduce the
FBS levels to 1% while maintaining a
reasonable level of growth, but we
could not reduce the serum
requirement any further. Our
objective was to get the cells to

Table 1: Growth factor/cytokine receptors expressed by HEK-293

Receptor Ligand Receptor Ligand

AXL receptor tyrosine kinase gas6 Neuropilin 1 VEGF*
EGF receptor EGF* Macrophage stimulating 1 receptor MSP
Chemokine (C-X3-C) receptor 1 Fractalkine PDGF receptor,  polypeptide PDGF AB*
PDGF receptor,  polypeptide PDGF AB* Nerve growth factor receptor NGF*
Interleukin 15 receptor,  IL-15 Interleukin 11 receptor,  IL-11*
Interleukin 2 receptor,  IL-2* Interleukin 10 receptor,  IL-10*
Interleukin 2 receptor,  IL-2* FGF receptor 4 aFGF*
Chemokine (C-C motif) receptor 2 MCP1 Bone morphogenetic protein receptor, type II BMP-2*
Interleukin 2 receptor,  IL-2* TGF,  receptor II TGF*
Interleukin 18 receptor 1 IL-18 FGF receptor 1 bFGF*
Colony stimulating factor 1 receptor CSF-1 Chemokine (C-X-C motif), receptor 4 SDF1 *
Oncostatin M receptor OSM* Interferon  receptor 1 IFN *
Interleukin 4 receptor IL-4* Interferon  receptor 2 IFN *
Vitamin D3 receptor Vitamin D3*

*ligands assayed for a proliferative response, as described in the text

4 BioProcess International JANUARY 2005

attach and grow in serum-free
conditions.
The first step was to identify a
method of screening the expression
of certain genes of interest. Because
of the presence of a large number of
genes of interest and the
commercial availability of the array,
we selected the human cytokine
chip from TaKaRa to perform our
screening. This microarray contains
approximately 700 cDNAs, many of
which represent growth
factors/cytokines and their
receptors. In our estimation, those
genes represent a population of
proteins that would at least initially
be the easiest to manipulate in a cell
culture assay.
RNA was prepared from cells in
log-phase growth, having grown in
reduced serum conditions (base
medium + 1% FBS). This RNA was
labeled with two fluorophores (Cy3
and Cy5) before hybridization to
the cytokine array. To interpret the
data obtained, we had to determine
which spots were positive and which
were negative in the microarrays.
We developed a selection strategy
using standard techniques to
identify positive signals (comparison
of signals to background and
negative controls). Using that
approach, we identified 27 receptors
that met our criteria (Table 1).
Of the initial 27 receptors
identified from the microarray, 16
ligands were selected (based on
signal intensity, as well as based on
Figure 2: Mimicry of receptor activation
with downstream activators
Arachidonic Acid
DAG/IP3
knowledge of the various factors),
added to a base medium (using a
broad concentration range based on
literature reviews), and assayed for a
proliferative response (marked with
an asterisk in Table 1). This was
performed in a “medium-
throughput” assay using the
metabolic dye resazurin. Resazurin
is converted to a fluorescent
product in the presence of
metabolically active cells. Therefore,
higher RFUs correlate to higher
numbers of cells.
When compared with the base
medium (1% FBS), four of the
chosen factors exhibited a positive
effect on the proliferation of HEK-
293 (Figure 1). These four factors
— basic fibroblast growth factor
(bFGF), epidermal growth factor
(EGF), stromal cell-derived growth
factor 1a (SDF1), and oncostatin
M (OSM) — all gave significant
increases in proliferation, whereas
the other 12 factors had an either
neutral or negative impact on
proliferation. Various combinations
of the positive effectors seemed to
have no additive or synergistic
effects, possibly indicating a
convergence of the pathways
involved in proliferation of this cell
line. Because SDF1 and oncostatin
M are factors we would not have
tested in HEK-293, the use of the
expression profiling was
indispensable to identify the
components of key pathways that
influence cell proliferation.
Beyond Ligand:Receptor
Interactions: Because of the high cost
of adding various growth factors
and cytokines to cell culture media
in sufficient quantities, as well as the
potential for those proteins to
interfere with downstream
processing, we looked for alternative
methods to stimulate the pathways.
Protein kinase C (PKC) is a
common downstream target of
many receptors, including the
receptors for EGF and SDF1. The
addition of arachidonic acid, which
has been found to stimulate PKC in
some systems, had a positive effect

Table 2: Growth factor/cytokine receptors expressed by WI-38

Receptor Ligand Receptor Ligand

Interferon  receptor 2 IFN * Insulin-like growth factor 1 receptor IGF1*
GDNF family receptor  3 Artemin* Interleukin 13 receptor,  1 IL-13*
Interleukin-1 receptor-associated kinase 1 IL-1* Bone morphogenetic protein receptor, type II BMP-2*
Chemokine (C-X-C motif), receptor 4 (fusin) SDF1* fms-related tyrosine kinase 1 VEGF*
Transforming growth factor,  receptor II (70-80kD) TGF * Fibroblast growth factor receptor 4 aFGF
Interferon  receptor 1 IFN * Interleukin 2 receptor,  IL-2
Platelet-derived growth factor receptor,  polypeptide PDGF AB* Interleukin 7 receptor IL-7*
Fibroblast growth factor receptor 1 bFGF* Interleukin 2 receptor,  IL-2
AXL receptor tyrosine kinase gas6

*ligands assayed for a proliferative response, as described in the text

JANUARY 2005 BioProcess International 5

Figure 3: Verification of microarray technique using RT-PCR
on the proliferation of the cells
(Figure 2). Endogenous
intermediates such as inositol 1,4,5-
triphosphate (IP3) and diacyl
glycerol (DAG, produced by the
activation of phospholipase C) act
through stimulation of PKC and
release of intracellular Ca++. This
combination of IP3 and DAG also
leads to increased proliferation.
Therefore, identifying growth factor
and/or cytokine receptors expressed
by a particular cell type can be used
to identify a pertinent pathway,
which can be affected in a variety of
ways, not necessarily only by the
addition of the ligand for the
receptor.
In the design of serum-free
media, the adhesion properties of
cells are also very important. As the
levels of serum in a medium
decrease, so does the ability of cells
6 BioProcess International JANUARY 2005
to attach. Adhesion is another type
of pathway we want to manipulate
with additions to the media. To that
end, we identified a list of proteins
expressed by HEK-293, including
many integrins (involved in
cell–substrate interactions) and
cadherins (involved in cell–cell
interactions). Those proteins, in
combination with enzymes such as
matrix metalloproteinases (MMPs),
regulate the interactions that
determine cell attachment. Based on
the expression profiles, we correctly
predicted that the cells would be
more adherent to collagen I than to
collagen IV (data not shown). That
result indicates that we can use this
technique for more than
identification of expressed growth
factor receptors.
Generation of a Serum-Free
Formulation: Using the methods
described above, we evaluated the
receptor expression of a different
cell line. The normal human
fibroblast, WI-38, typically requires
supplementation with 10% FBS in
culture. We were able to reduce its
serum requirement to 3% using
standard medium development
techniques, then used the
microarray technology to move
toward a serum-free formulation.
Seventeen receptors were expressed
based on microarray analysis (Table
2). The ligands for four of them had
a positive effect on proliferation
(data not shown). In this case, two
of the growth factors were
synergistic in their effects on
proliferation, which allowed us to
produce a serum-free formulation
for WI-38. To our knowledge, this
is the first example of a serum-free
medium formulation developed
using genomic tools.
Verification of Microarray and the
Use of Other Identification Methods:
To validate the microarray
technology we used to identify key
pathways, we have used a variety of
additional methods to ascertain the
expression profile of key molecules.
Microarray technology is an
accepted method for detecting the
presence of mRNA for a given
protein, but other methods can
detect both the mRNA message as
well as the protein itself. To test
various technologies, we chose one
receptor that was positive on the
microarray screen as well as one that
was negative on the microarray to
use in our method comparisons.
In Figure 3A, the Cy3 and Cy5
labeled spots from the microarray
represent positive expression of the
platelet-derived growth factor
receptor (PDGFR), stimulation of
which had a positive effect on
proliferation. Using standard RT-
PCR techniques, we amplified a band
corresponding to this receptor (see
the inset agarose gel with the positive
band and the primer dimer below it).
We were also able to verify the
presence of the transcript using
standard quantitative PCR (qPCR)
techniques (data not shown). The
cDNA from this cell line gave a low
CT value, which is indicative of a
significant level of expression.
 Figure 3B shows the same
experiments performed for a
receptor that was negative on the
microarray screen, chemokine
receptor 7 (CCR7). We saw little or
no labeling in the Cy3 and Cy5
channels, no bands on the RT-PCR
(primer dimer only), and a high CT
value from qPCR. Beta-actin, a
standard housekeeping gene, is
represented in Figure 3C. It showed
a very high level of Cy3 and Cy5
fluorescence on the microarray, a
bright band on RT-PCR, and a
correspondingly low CT value in
qPCR. All methods verified the
expression of the PDGF receptor in
WI-38 and the lack of expression of
the CCR7 receptor.
A NOVEL CONCEPT
The idea of using the expression
profile of a cell culture to identify
key pathways suitable for
manipulation is a novel concept in
the world of cell culture medium
development. We believe that this
technology represents a powerful
tool for medium design that speeds
development time and thereby
reduces cost. In addition, some
products now can be developed for
which the chance of success using
standard methods had been slim
because of our inability to identify
key components and pathways. We
are actively involved in the
generation of additional tools and
assays, both genomic and
proteomic, to better determine and
manipulate the physiologically
important pathways in a given
culture system.
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Corresponding author Daniel W.
Allison is senior R&D scientist, P.O.
Box 14508, St. Louis, MO 63178,
1-314-289-8496 x1341, fax 1-314-286-
7645, dallison@sial.com. His coauthors,
all at Sigma-Aldrich in St. Louis, are
Kathryn A. Aboytes and Danny K. Fong,
senior R&D scientists; Stacy L. Leugers,
R&D scientist; Terrell K. Johnson,
principal R&D scientist; Heather N.
Loke, associate R&D scientist; and
Laurel M. Donahue, manager.
JANUARY 2005 BioProcess International 7