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Author's personal copy
Optimization of chemically deﬁned cell culture media – Replacing fetal
bovine serum in mammalian in vitro methods
J. van der Valk
, D. Brunner
, K. De Smet
, Å. Fex Svenningsen
, P. Honegger
, L.E. Knudsen
, J. Noraberg
, A. Price
, M.L. Scarino
, G. Gstraunthaler
NCA, DWM, Fac. Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands
zet-Life Science Laboratorium, zet – Centre for Alternative and Complementary Methods to Animal Testing, Industriezeile 36/VII, 4020 Linz, Austria
Federal Agency for Medicines and Health Products, DG PRE Authorisation, Victor Hortaplein 40, Bus 40, B-1060 Brussels, Belgium
Institute of Molecular Medicine, Department of Neurobiology Research, University of Southern Denmark, J.B. Winslows Vej 21, DK-5000 Odense C, Denmark
Department of Physiology, University of Lausanne, CH-1005 Lausanne, Switzerland
Department of Public Health, Faculty of Health Sciences, University of Copenhagen, Denmark
Institut für angewandte Zellkultur, München, Germany
In-Vitro Methods Unit/European Centre for the Validation of Alternative Methods, Institute of Health and Consumer Protection, European Commission Joint Research Centre,
Ispra (VA), Italy
INRAN, National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy
Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria
Received 10 March 2010
Accepted 25 March 2010
Available online 31 March 2010
In vitro methods
Fetal bovine serum
Good cell culture practice
Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manu-
facturing practice (GMP) are now established standards. The biomedical ﬁeld aims at an increasing reli-
ance on the use of in vitro methods. Cell and tissue culture methods are generally fast, cheap,
reproducible and reduce the use of experimental animals. Good cell culture practice (GCCP) is an
attempt to develop a common standard for in vitro methods. The implementation of the use of chemi-
cally deﬁned media is part of the GCCP. This will decrease the dependence on animal serum, a supple-
ment with an undeﬁned and variable composition. Deﬁned media supplements are commercially
available for some cell types. However, information on the formulation by the companies is often lim-
ited and such supplements can therefore not be regarded as completely deﬁned. The development of
deﬁned media is difﬁcult and often takes place in isolation. A workshop was organised in 2009 in Copen-
hagen to discuss strategies to improve the development and use of serum-free deﬁned media. In this
report, the results from the meeting are discussed and the formulation of a basic serum-free medium
is suggested. Furthermore, recommendations are provided to improve information exchange on newly
developed serum-free media.
Ó2010 Elsevier Ltd. All rights reserved.
1. Introduction . . . ..................................................................................................... 1054
2. Development of a serum-free medium . .................................................................................. 1055
2.1. Basal medium . . . . . . . . . ................................... ..................................................... 1055
2.2. Supplements . . . . . . . . . . .......................... .............................................................. 1055
0887-2333/$ - see front matter Ó2010 Elsevier Ltd. All rights reserved.
Abbreviations: ATCC, The American Type Culture Collection; ADCF, animal-derived component-free; BSA, bovine serum albumin; CD, chemically deﬁned; DMEM, Dulbecco
minimal essential medium; DSMZ, German Collection of Microorganisms and Cell Cultures; ECACC, European Collection of Animal Cell Cultures; ECOPA, European Concensus
Platform for Alternatives; ECVAM, European Centre for the Validation of Alternative Methods; EGF, epidermal growth factor; ESAC, ECVAM Scientiﬁc Advisory Committee;
ESTIV, European Society of Toxicology in vitro; FBS, fetal bovine serum; GCCP, good cell culture practice; GLP, good laboratory practice; GMP, good manufacturing practice;
INVITROM, the Dutch-Belgian Society for in vitro Methods; ITS, Insulin transferrin and selenium; MEM, minimal essential medium; NGF, nerve growth factor; PET,
polyethyleneterephthalate; PL, platelet lysates; SFM, serum-free medium; TEER, trans-epithelial electrical resistance; 3Rs, replacement, reﬁnement reduction of use of
*Corresponding author. Tel.: +31 30 253 2163; fax: +31 30 253 7997.
E-mail address: firstname.lastname@example.org (J. van der Valk).
Toxicology in Vitro 24 (2010) 1053–1063
Contents lists available at ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
Author's personal copy
2.2.1. Hormones . . . . . . . . . . . . . ................................................................................ 1055
2.2.2. Growth factors . . . . . . . . . ................................................................................ 1056
2.2.3. Protease inhibitors . . . . . . ................................................................................ 1056
2.2.4. Protein hydrolysates. . . . . ................................................................................ 1056
2.2.5. Shear force protectors . . . ................................................................................ 1056
2.2.6. Proteins . . . . . . . . . . . . . . . ................................................................................ 1056
2.2.7. Vitamins . . . . . . . . . . . . . . ................................................................................ 1056
2.2.8. Amino acids . . . . . . . . . . . ................................................................................ 1056
2.2.9. Glutamine . . . . . . . . . . . . . ................................................................................ 1056
2.2.10. Trace elements . . . . . . . . ................................................................................ 1056
2.2.11. Lipids. . . . . . . . . . . . . . . . ................................................................................ 1056
2.2.12. Antibiotics. . . . . . . . . . . . ................................................................................ 1056
2.2.13. Attachment factors . . . . . ................................................................................ 1056
2.2.14. Osmolarity . . . . . . . . . . . ................................................................................ 1057
2.3. ‘‘Building a serum-free medium. . . . . . . . ....................... .................................................... 1057
2.4. Adaptation of cell lines to serum-free medium. . . . . . . . . . . . ............................................. .............. 1057
2.4.1. Reduction of serum content. . . . . . . . . . . . . . . ................................................................ 1057
2.4.2. Sequential adaptation. . . . ................................................................................ 1057
2.4.3. Adaptation with conditioned medium . . . . . . ................................................................ 1057
2.4.4. Inside adaptation . . . . . . . ................................................................................ 1058
3. Promoting the development and use of serum-free media . . . . . . . . . . . . . . . . . .................................................. 1058
3.1. Information sources . . . . . . . . . . . . . . . . . ............................. .............................................. 1058
3.2. The serum-free media interactive online database (D. Brunner) . . . . . . . . . . . . . . . . . ....................... ................. 1058
3.3. Validating new media and adapted cells . .................... ....................................................... 1058
3.4. Other activities . . ....................... ....................................................................... 1059
4. Examples of serum-free studies ........................................................................................ 1059
4.1. Human platelet lysates as a serum substitute in cell culture media (G. Gstraunthaler) . . . . . . . . . . . . . . . . . ....... .............. 1059
4.2. Serum-free aggregating brain cell cultures (P. Honegger) . . . ....... .................................................... 1059
4.3. Organotypic brain slice cultures and defined serum-free medium Neurobasal with B27 (J. Noraberg) . . . . . ..................... 1059
4.4. Defined medium and serum-containing medium occasionally induce cells to use different signal transduction pathways to proliferate
(Å. Fex Svenningsen) . . . . . . . . . . . . . . . . . ................................................ ........................... 1060
4.5. Optimization of culture conditions for human intestinal Caco-2 cells to improve functional differentiation in serum-free media
(M.L. Scarino). . . . .... ............................................................................ .............. 1060
5. Conclusions. ........................................................................................................ 1061
6. General recommendations. . . . . ........................................................................................ 1061
7. Recommendations for developing serum-free cell culture media. . . . . . . . . . . . .................................................. 1061
Acknowledgements . . . . . . . . . . ........................................................................................ 1061
References . ........................................................................................................ 1061
In vitro methods are widely used tools to study physiological,
biological and pharmacological activities at the cell and tissue le-
vel. In addition, in vitro methods are also becoming increasingly
important in the production of biological components, such as
hormones and vaccines. Mammalian cells are generally grown un-
der well-established conditions in incubators, where the temper-
ature is typically kept at 37 °C with a controlled humidiﬁed gas
mixture of 5% CO
and 95% O
. To achieve good experimental
reproducibility, the composition of the cell culture medium is
essential. The simplest medium is the classical Ringer’s solution
(Ringer and Buxton, 1887), which was developed as a solution
with optimal concentrations of different salts to preserve frog
heart muscle tissue. To maintain cells and tissues for longer peri-
ods of time, the medium should also contain components like
nutrients and pH buffering substances. This type of medium was
formulated by Harry Eagle, who developed Eagle’s minimal essen-
tial medium (Eagle’s MEM or MEM). MEM also contained amino
acids, glucose and vitamins (Eagle, 1955). A similar basal medium,
MEM modiﬁed by Dulbecco (Dulbecco’s Modiﬁed Eagle’s Medium,
DMEM), is still used to maintain primary cell cultures and cell
To keep cells alive for longer periods of time and to evaluate
proliferation, migration and differentiation a basal medium must
be supplemented with several factors. Serum, from animals or
humans, is most commonly used to maintain and proliferate cells.
Fetal bovine serum (FBS) serves most purposes and is the present
standard. FBS is a complex mixture of different factors and contains
a large number of components, like growth factors, proteins, vita-
mins, trace elements, hormones, etc., essential for the growth and
maintenance of cells.
However, the use of FBS is controversial for a number of rea-
sons. First of all, the collection of serum causes unnecessary suf-
fering for the unborn calf (van der Valk et al., 2004). Secondly,
seasonal and continental variations in the serum composition,
produces batch-to-batch variations. This, in turn, causes pheno-
typical differences in the cell cultures, resulting in variations of
the results. Additionally, due to the likelihood of contamination
(e.g., BSE), the use of animal products is strongly discouraged
for production of new biological medicinal products (Anon,
1993; Schiff, 2005; van der Valk et al., 2004). In fact, as much
as 20–50% of commercial FBS is virus-positive (Wessman and
Since in vitro methods are among the most favoured methods
to replace animal methods (Hartung, 2007), there is a demand
for reliable and scientiﬁcally better deﬁned cell and tissue
culture methods including quality assurance (Gupta et al.,
2005). Guidelines for good cell culture practice (GCCP), involving
recommendations with respect to the use of serum-free media,
1054 J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063
Author's personal copy
have previously been published (Coecke et al., 2005; Hartung
et al., 2002). The ECVAM Scientiﬁc Advisory Committee (ESAC)
has also published a statement that strongly recommends the
use of serum-free substitutes for current and new in vitro
methods (ESAC, 2008). Although, there is no legal basis for
applying GCCP, it is recommended that GCCP becomes part of
good laboratory practice (GLP) and good manufacturing practice
A workshop, to discuss the possibilities to reduce the use of
FBS in cell and tissue culture was organised in 2003 (van der
Valk et al., 2004). The report from this meeting provides clear
recommendations to reduce or stop the suffering of live unborn
bovine calfs from which blood is drawn for the production of
FBS. Ethical, safety and scientiﬁc grounds were also given for
the replacement of FBS and other animal components in cell
and tissue culture methods. In 2009, a follow-up workshop
was organised to discuss current in vitro methods devoid of
FBS, or other animal components. The workshop, held in Copen-
hagen, Denmark, was organised under auspices of the European
Society of Toxicology In Vitro (ESTIV), the Dutch-Belgian Society
for In Vitro Methods (INVITROM) and the Danish in vitro
Toxicology Network. The results from this workshop clearly
demonstrate the possibilities to grow a number of different
primary cell and tissue cultures as well as cell lines without
the use of animal products. Furthermore, directions were
provided on how to develop a serum-free, chemically deﬁned,
culture media for mammalian cell and tissue cultures in basic
and applied research.
This report aims at discussing the advantages of deﬁned cell
culture media and to give directions for the development of a basic
deﬁned media for a wider audience.
2. Development of a serum-free medium
The attempts to grow cells date back for at least 50 years
(Pumper, 1958; Waymouth, 1955). Early attempts to grow cells
in serum-free, hormone-supplemented media were performed to
understand the role of serum in cell culture media. The efforts to
identify all the serum components that are physiologically relevant
to maintain proliferation of cells in culture, and the attempts to re-
place the serum with its deﬁned components, were not successful
(Taub, 1990). Since then, several different serum-free formulations
have been developed where the media are supplemented with
approximately 10 essential components (Pazos et al., 2004). About
10–20% of these strategies appeared to be successful (Pazos et al.,
The pioneering work by Hayashi and Sato (1976) replacing
serum by the addition of selected hormones, promoting
growth and stimulating differentiation of speciﬁc cells, led to the
development of a good chemically deﬁned, serum-free media
(see Box 1) (Barnes and Sato, 1980a,b; Bjare, 1992; Grillberger
et al., 2009; Gstraunthaler, 2003; Taub, 1990). In the last 10 years,
investigations into cell function have led to the identiﬁcation of a
growing number of components which have been useful in the
development of modern serum-free cell culture media. Many
transformed or newly transfected cell lines can successfully be
maintained in these enriched serum-free media without adapta-
tion and the number of cell-speciﬁc media is growing steadily.
Now, more than 100 different available serum-free media formula-
tions have been developed and can be readily used without great
investments in time and money to develop one’s own (Zähringer,
2.1. Basal medium
With time, it has become clear that almost every cell type has
its own requirements concerning medium supplements. Therefore,
a universal (serum-free) cell and tissue culture medium may not be
feasible. Different cell types have different receptors involved in
cell survival, growth and differentiation and release different fac-
tors to their environment.
The threshold for developing or using a new (deﬁned) medium
when the current FBS containing medium works well, is high for
obvious reasons. In order to aid in this process, a strategy for the
development of new media will be discussed below.
It is recommended to start a new formulation with a 50:50 (v/v)
mixture of DMEM and Ham’s nutrient mixture F-12 (Ham, 1965).
This medium formulation combines the high amino acid content
of DMEM with the highly enriched Ham’s F-12 (Barnes and Sato,
1980a,b; Jayme et al., 1997). Furthermore, the basal medium must
contain an essential, so called, ITS supplement (insulin, transferrin
and selenium). Insulin, the ﬁrst of the components of the ITS sup-
plement, has been known to be essential in cell culture from 1924
and is now the most commonly used hormone in culture media
(Gey and Thalhimer, 1924). Transferrin is also an essential protein
in culture medium where the main action is to transfer iron into
the cells (Bjare, 1992). Selenium is an essential trace element and
acts in particular in selenoproteins which protect cells against oxi-
dative stress (Helmy et al., 2000).
Although some cell types can be maintained in the basal medium
(Bettger and McKeehan, 1986; Butler and Jenkins, 1989), most cells
need additional supplements to survive, proliferate and/or differen-
tiate. The most commonly supplied components will be discussed
All hormones of mammalian organisms are physiological con-
stituents in blood circulation and are thus also present in serum
Serum-free media: serum-free media do not require sup-
plementation with serum, but may contain discrete pro-
teins or bulk protein fractions (e.g., animal tissue or
plant extracts) and are thus regarded as chemically unde-
ﬁned (see: chemically deﬁned media).
Protein-free media: protein-free media do not contain high
molecular weight proteins or protein fractions, but may
contain peptide fractions (protein hydrolysates), and are
thus not chemically deﬁned. Protein-free media facilitate
the down-stream processing of recombinant proteins
and the isolation of cellular products (e.g., monoclonal
Animal-derived component-free media: media containing
no components of animal or human origin. These media
are not necessarily chemically deﬁned (e.g., when they con-
tain bacterial or yeast hydrolysates, or plant extracts).
Chemically deﬁned media: chemically deﬁned media do
not contain proteins, hydrolysates or any other compo-
nents of unknown composition. Highly puriﬁed hormones
or growth factors added can be of either animal or plant
origin, or are supplemented as recombinant products
(see: animal-derived component-free media).
J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063 1055
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in varying amounts (Lindl and Gstraunthaler, 2008; Price and Greg-
ory, 1982). Supplementation with hormones was therefore a ﬁrst
step in the development of serum-free media (Barnes and Sato,
1980a,b; Hayashi and Sato, 1976). Insulin has been shown to be
obligatory in all serum-free media formulations. Other hormones
most widely used in serum-free cell culture are glucocorticoids
(dexamethasone and hydrocortisone), triiodothyronine (T
hormones that cell-speciﬁcally act by increasing intracellular
cAMP levels (see Section 2.3). Water-soluble complexes of steroids
are commercially available.
2.2.2. Growth factors
Growth factors are generally added to the basal medium to
increase cell proliferation and to stimulate speciﬁc cell functions.
Traditionally, growth factors and other supplements are added as
bulk in the form of fetal bovine serum (FBS).
Most growth factors are highly cell type speciﬁc. Others are of
more general use and can also have positive effects on several
different cell types. Fibroblast growth factor-2, for example, has a
positive effect on the phenotype of chondrocytes cultured in
serum-free medium (Mandl et al., 2004). Some cells in culture may
release growth factors thereby stimulating their own proliferation
and that of other cells (Gospodarowicz and Moran, 1976).
2.2.3. Protease inhibitors
The protease inhibitors that are introduced by the addition of
2003). The inhibitors terminate the trypsination process and act
beneﬁcially by inhibiting lysosomal peptidases that may occasion-
ally be released during cell turnover. Protease inhibitors thus have
a protective effect on cells, but are not essential. When no protease
inhibitors are supplied, one should carefully assess the trypsin
2.2.4. Protein hydrolysates
Protein hydrolysates are used to deliver amino acids and small
peptides. These are not essential in cell culture and the effect is
somewhat controversial. In fact, some studies report a beneﬁcial
effect in cell cultures (Burteau et al., 2003; Schlaeger, 1996), while
other studies demonstrated that protein hydrolysates do not sup-
port cell growth and that higher concentrations actually reduce cell
growth (Keay, 2004). Protein hydrolysates are chemically not de-
ﬁned (see Box 1) and may cause problems in reproducibility and
comparability of experiments.
2.2.5. Shear force protectors
Turbulence in bioreactors and perfusion cultures cause shear
stress in cells. Serum protects cells from this shear force (Elias
et al., 1995; van der Pol and Tramper, 1998). Pluronic F68 has a
similar effect (Zhang et al., 1992), but is not essential for ordinary
Proteins are carriers for different low molecular weight compo-
nents and may facilitate cell adhesion (Taub, 1990). Bovine serum
albumin (BSA) is often used as a lipid carrier. However, BSA is de-
rived from animals and may either be contaminated or may con-
tain impurities (Taub, 1990). Nowadays, recombinant proteins,
including albumin, are available for animal component-free cell
culture (Keenan et al., 2006).
Vitamins are provided by the basal medium. At least seven vita-
mins were found to be essential for cell growth and proliferation:
choline, folic acid, nicotinamide, pantothenate, pyridoxal, riboﬂa-
vin, and thiamine (Bjare, 1992; Butler and Jenkins, 1989; Taub,
1990). B-vitamins are necessary for cell biochemistry, and are also
present in DMEM as well as in Ham-F-12.
2.2.8. Amino acids
All 13 essential amino acids are necessary for culturing mam-
malian cells (Arg, Cys, Gln, His, Ile, Leu, Lys, Met, Phe, Thr, Trp,
Tyr, and Val) and are present in high concentrations (0.5–4 mM)
in DMEM. The seven non-essential amino acids (Ala, Asn, Asp,
Glu, Gly, Pro, and Ser) are provided by Ham’s F-12.
Glutamine is an essential precursor for the synthesis of proteins
and ribonucleotides. It is also important respiratory fuel for rapidly
dividing cells and cells that use glucose inefﬁciently (Glacken,
1988; Reitzer et al., 1979; Zielke et al., 1984). However, glutamine
also has its drawbacks: it is unstable in solution, and glutamine
breakdown and metabolism result in the production and accumu-
lation of ammonia, which is toxic to cells (Schneider et al., 1996),
since it is not absorbed by serum proteins in serum-free and/or
protein-free media. To overcome these disadvantages, alternatives
for the use of glutamine in culture media were developed. Gluta-
mate, for example, can replace glutamine in cell cultures that ex-
press sufﬁcient glutamine synthetase activity. A more recent
invention is the use of glutamine-containing dipeptides, alanyl-
glutamine and glycyl-glutamine, commercially available under
the trade name GLUTAMAX™ (Christie and Butler, 1994). These
dipeptides are more stable and heat resistant, which even makes
it possible to autoclave the media that contain these molecules.
The dipeptides are intra- or extracellularly cleaved by peptidases,
thereby releasing glutamine and either alanine or glycine. The
availability of glutamine is therefore dependent on the peptidase
activity, which results in lower rates of glutamine consumption
and ammonia production. GLUTAMAX™ can be substituted for
glutamine on a 1:1 M basis.
2.2.10. Trace elements
Most trace elements are available in the basal medium since
Ham’s F-12 is qualitatively rich in necessary trace elements
The role of fatty acids and lipids in cell culture has long been ne-
glected. Lipids serve as energy stores, as structural constituents of
cellular membranes, and in transport and signalling systems. Some
lipids are available in the basal medium. However, essential fatty
acids and ethanolamine are recommended as supplements.
Water-soluble supplements are commercially available. Serum
albumin is a carrier of fatty acids and lipids (see Section 2.2.6).
Essential fatty acids are components of several serum-free med-
Wherever possible, the use of antibiotics should be avoided
(Kuhlmann, 1996). Antibiotic-resistant microorganism may
develop, and antibiotics may also have adverse effects on cell
growth and function.
2.2.13. Attachment factors
Most mammalian cells need a special culture substratum for
cell attachment in order to survive and grow in vitro. The plastic
culture dish, that is speciﬁcally treated to introduce charge and
hydrophilicity into the polystyrol surface, e.g., with poly-
sine or ornithine, is the most commonly used substrate for cell
attachment. Coating the plastic dishes with other substrates
like extracellular matrix components (Kleinman et al., 1987)or
1056 J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063
Author's personal copy
collagenous matrices (Kleinman et al., 1981) further facilitates the
adhesion of anchorage-dependent cells.
Although mammalian cells express a reasonable wide tolerance
to osmolarity, osmolarity should always be carefully checked and
compensated for when adapting to a new cell culture formulations.
2.3. ‘‘Building”a serum-free medium
As shown above, to exclude FBS from a cell and tissue culture
medium, and still maintain cell adhesion, growth and proliferation
it is important to include a large number of several components in
the cell culture medium. In Fig. 1, a schematic modular approach
for the development of serum-free media is shown as a ‘‘media
pyramid”. The bottom of the pyramid contains the basal medium,
which includes DMEM/Ham’s F-12 (50:50, v/v), supplemented
with insulin–transferrin–selenium (ITS). To make adherent cells
stick to the bottom of the culture vessel, coating with components
of the extracellular matrix should be considered. Cell attachment
factors are often required for serum-free culture.
The next step in media formulation development is the addition
of speciﬁc hormones and growth factors. Epidermal growth factor
(EGF) and glucocorticoids (hydrocortisone and dexamethasone),
for example, are present in most media. Depending on the cell
type, additional cell-speciﬁc growth factors may also be needed,
like nerve growth factor (NGF) for neurons. It has been demon-
strated that cultures of epithelial cells need a supplementation
with agonists, that speciﬁcally elevates cellular cAMP levels
(Gstraunthaler, 2003). In this respect, also forskolin and cholera
toxin, although acting as strong pharmacological agents, were used
as in vitro mitogens.
The tip of the pyramid represents increased speciﬁcity in ser-
um-free media composition: the addition of lipids, antioxidants
and/or speciﬁc vitamins. Retinoic acid (vitamin A) is an additive re-
quired in cell culture media for a number of epithelial cell types.
Vitamin E (
-tocopherol) and ascorbate (vitamin C) are presum-
ably acting as antioxidants. Other antioxidants found in serum-free
media formulations are b-mercaptoethanol (b-ME) and selenium
2.4. Adaptation of cell lines to serum-free medium
There are several approaches to adapt cultured cells to a serum-
free medium (Fig. 2). Typically, a cell culture has to undergo a
gradual weaning process which involves progressive adaptation to
lower serum concentrations until serum-free conditions are
reached. The cultures to be adapted should be in the logarithmic
phase of growth and should have viability over 90%. However, one
should also keep in mind that an unwanted selection of a change
in the population of cells, during the adaptation process, by
indirectly selecting cells capable to grow in serum-free media, may
occur. Therefore, it is necessary to check the performance of cultures
and to monitor cellular morphology and function during weaning.
In order to aid the process of weaning several adaption proto-
cols are listed below (and in Fig. 2):
2.4.1. Reduction of serum content
In this protocol, serum content is reduced at each passage until
0.1% serum is reached. After cultivation in normal medium con-
taining 10% FBS, the consequent serum reduction steps (from 5%
to 0.1% FBS) are carried out in serum-free, hormone-supplemented
2.4.2. Sequential adaptation
Similar to protocol 1, cells are passaged into mixtures of
serum-containing and serum-free media, until complete serum-
free condition is reached. If the last step, changing from 75% to
100% serum-free, is too stressful for the cells, it is recommended
to keep the cell culture in a 10% serum-containing and 90% ser-
um-free medium mixture for 2–3 passages, before switching to a
complete serum-free medium.
2.4.3. Adaptation with conditioned medium
This adaptation follows protocol 2, however, cells are passaged
into decreasing mixtures of conditioned media from the passage
pre-coating of culture vessels with:
collagen type I, type IV, laminin, fibronectin,
Basement Membrane Matrigel™
basal medium: DMEM / Ham F-12 (50:50, v/v) + ITS
EGF, FGF, NGF,
thyroid hormones, cell-
specific agonists that
signal via cAMP (ADH,
PTH, PGE2, glucagon)
Fig. 1. Media pyramid: a modular approach for the development of serum-free media (for details see Section 2.3). Abbreviations: ADH, antidiuretic hormone; EGF, epidermal
growth factor; FGF, ﬁbroblast growth factor; IGF-1, insulin-like growth factor 1; ITS, insulin–transferrin–sodium selenite supplement; b-ME, b-mercaptoethanol; NGF, nerve
growth factor; PDGF, platelet-derived growth factor; PGE2, prostaglandin E2; PTH, parathyroid hormone; TGF-b, transforming growth factor-b; and VEGF, vascular
endothelial growth factor.
J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063 1057
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2.4.4. Inside adaptation
In this protocol, freshly seeded cells are weaned in serum-free
medium, and cultures are grown to conﬂuence. The conﬂuent
monolayer is then passaged into serum-free medium.
3. Promoting the development and use of serum-free media
3.1. Information sources
Before using the experimental approach to set up a serum-free
medium for a given cell type, cell lines or tissue culture, a search
for already existing media formulations should be performed This
can be done by a thorough literature survey, or by a search in a re-
cently established serum-free media online database (see Section
There are several databases that contain information on com-
mercially available serum-free media formulations and supple-
ments (Anon, 2009a,b,c). Approximately 450 different serum-free
cell culture media formulations are now commercially available,
but only for a limited number of cell types (Anon, 2009a,b,c).
Regrettably, the formulations of speciﬁc supplements for the com-
mercially available media are generally not available, and those
can therefore not be considered as fully deﬁned media. Such for-
mulations have also been changed without informing the users
(Chen et al., 2008; Cressey, 2009), and supplements with the same
name may differ in formulation between suppliers.
Today, the information on available serum-free media formula-
tions, particularly when these are not commercially available, is
unfortunately scarce. Nevertheless, development of serum-free
media and cell adaptation protocols are ongoing processes in sev-
eral laboratories, often without knowledge about research pro-
cesses, experiences and results of other laboratories regarding
this topic. This is partly due to a lack in communication between
labs and particularly the lack of a common forum where such for-
mulations could be posted. It is recommended that these obstacles
must be overcome in order to encourage future use and develop-
ment of serum-free media.
It is further recommended to collect formulations of ‘‘in lab
developed” media in databases, where access to reliable protocols
including detailed formulations, should be free. It is also recom-
mended to publish established protocols in dedicated online
databases like Springer Protocols and Nature Protocols. When pub-
lishing studies with newly developed serum-free formulations spe-
ciﬁc keywords should be used in the publication to enable easy
retrieval of the publications. Key words like 3R, serum-free media
or deﬁned media are recommended.
3.2. The serum-free media interactive online database (D. Brunner)
To make the search for serum-free media easier, a new collec-
tion of commercially available serum-free media has been devel-
oped in a free accessible unique interactive online database
(Brunner et al., 2010; Falkner et al., 2006).
Speciﬁcations of serum-free media (i.e., ability to maintain cells
of speciﬁc organism, organs, tissue, cell type and disease) were col-
lected and systematically arranged with respect to speciﬁc stan-
dards (ICD 2007 of WHO and ITIS). Additional commercially
available cell lines, hybridoma and primary cells from ATCC, ECACC
and DSMZ are included in the database to allow a ‘‘reverse search”
by specifying the used cells to gain a serum-free medium. This
search modus is based upon comparison of speciﬁcations and can
also be used to ﬁnd most similar serum-free media.
Furthermore, the degree of chemical deﬁnition, e.g., serum-free
(SFM), animal-derived component-free (ADCF) or chemically de-
ﬁned (CD), and the kind of medium, e.g., basal media, media sup-
plements, or full replacement media can be selected. Presently,
452 serum-free media and 4817 continuous cell lines, hybridoma
lines and primary cultures from ATCC, ECACC and DSMZ that are
commercially available are included in the database. Despite
extensive search for serum-free media and adapted cell lines, there
is still a lack of detailed information from companies and suppliers.
It is intended to open the database for interactive exchange of
information and guidelines from experts in the ﬁeld in order to
continuously improve and extend the serum-free online database.
The database is accessible at http://www.goodcellculture.com.
3.3. Validating new media and adapted cells
In a statement on the use of FBS and other animal-derived
supplements (ESAC, 2008), the ECVAM Scientiﬁc Advisory Commit-
tee strongly argues for the development of new serum-free in vitro
culture methods. Furthermore, when an in vitro method using
serum-containing media is presented to ECVAM for validation, a
justiﬁcation for using serum must be provided. To promote the
use of serum-free media, ECVAM (European Centre for the Valida-
tion of Alternative Methods) will encourage the submitters of new
tests systems for validation studies to make their protocols public
if their model is designed under serum-free conditions. Existing
culture methods where animal components are being replaced,
should be validated against serum-containing media to ensure that
1. reduction of serum content
cultivation of cells
in normal medium with 10% FBS
subcultivation of cells
in serum-free medium with 5% FBS
subcultivation of cells
in serum-free medium with 1% FBS
further reduction of serum content
to 0.1% FBS
cultivation and maintenance
in serum-free medium
2. sequential adaptation
cultivation of cells
in normal medium with 10% FBS
75% normal medium
25% serum-free medium
50% normal medium
50% serum-free medium
25% normal medium
75% serum-free medium
3. adaptation with
cultivation of cells
in normal medium with 10% FBS
50% conditioned medium
50% serum-free medium
50% conditioned medium
from passage 1, 50% SFM
25% conditioned medium
from passage 2, 75% SFM
4. „inside“ adaptation
cultivation of cells
in normal medium with 10% FBS
change to serum-free medium
continued culture in
trypsinization of confluent
passage of cells
in 2 - 4x hi gher seeding density
with serum-free medium
Fig. 2. Adaptation of cultures to serum-free medium. A comparison of the most
common adaptation protocols (FBS: fetal bovine serum and SFM: serum-free
1058 J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063
Author's personal copy
the original endpoints are not affected. When serum is being re-
placed in an already validated in vitro method, the new method
should be validated in a catch-up study (Hartung et al., 2004).
On the ECVAM web site (http://ecvam.jrc.ec.europa.eu) a spe-
cial discussion forum will be opened in the near future for the shar-
ing of information which is relevant to serum-free culturing,
particularly in the context of validation studies.
3.4. Other activities
Other incentives to promote the substitution of FBS could be
provided by funding institutions requesting a priori use of ser-
um-free media. Workshops and conferences on in vitro culturing
and processing should also be encouraged to host speciﬁc seminars
and poster sections with emphasis on exchange of experiences re-
lated to serum-free media. Seminars could be prepared by working
groups within the scientiﬁc cell and tissue cultures societies as e.g.,
ESTIV. In addition, the value of serum-free cell and tissue culture
should be educated and be part of basic culture techniques educa-
tion and training (Coecke et al., 2005; Hartung et al., 2002; Lindl
and Gstraunthaler, 2008).
Support for coordinated actions, promoting exchange of infor-
mation related to speciﬁc cell and tissue cultures should be
encouraged at national, Europeans well as international, levels by
the announcement of project calls, etc.
4. Examples of serum-free studies
In the following sections, participants of the workshop describe
ways in which serum substitutes are developed and how speciﬁc
cells are cultured in serum-free media.
4.1. Human platelet lysates as a serum substitute in cell culture media
Below, the use of human platelet lysates (PL) as a serum
replacement is reported. PL was prepared from outdated human
donor thrombocyte concentrates. Maximum activation of throm-
bocytes was achieved by freeze–thawing in hypo-osmolar saline.
The extent of the release of platelet granule growth factors was
determined by ELISA and Western blotting (Rauch et al., 2008;
Rauch et al., 2009). The growth promoting and mitogenic capacity
of PL was tested on a broad selection of continuous cell lines, for
which growth characteristics, phenotypes, and differentiation end-
points are well-established (Gstraunthaler, 1988). PL in DMEM
support growth, proliferation and differentiation, as assessed by
dome formation of proximal tubule-like LLC-PK
and HK-2 (human kidney) cells, whereas distal tubule-like MDCK
(dog kidney) cells grow well in serum-free DMEM/Ham-F-12 sup-
plemented with platelet extracts (Gstraunthaler et al., 1985). In
addition to adherent epithelial cell lines, anchorage-independent
Raji human lymphoma cells were investigated. PL fully supported
growth and proliferation of Raji cells in suspension. Proliferation
was monitored in all cell lines by determination of cell density of
epithelial cultures (cell number per growth area) (Pfaller et al.,
1990), and by resazurin or WST-8 assays. Rates of growth and pro-
liferation were comparable between media conditions with 10%
FBS or 5% PL, respectively. Serum-free media served as negative
controls. In order to biochemically determine the proliferative po-
tential of PL, the stimulation of extracellular signal-regulated MAP
kinase (ERK1/2) was determined (Feifel et al., 2002). Activation of
the MAP kinase signalling pathway by GR leads to speciﬁc phos-
phorylation of downstream kinases, like ERK1/2. Addition of PL
to quiescent LLC-PK
cultures resulted in speciﬁc phosphorylation,
and thus activation, of ERK1/2 within minutes. The time course is
identical with ERK1/2 activation upon addition of FBS.
The data show the high potential of PL as a valuable substitute
for FBS in mammalian cell and tissue culture.
4.2. Serum-free aggregating brain cell cultures (P. Honegger)
Thirty years ago, the original method for the preparation and
maintenance of aggregating brain cell cultures in serum-free chem-
ically deﬁned medium was published (Honegger et al., 1979). The
cultures derived from embryonic rat brain and maintained as sus-
pension culture under continuous gyratory agitation in a chemi-
cally deﬁned medium, reproduced critical morphogenic events
such as migration, proliferation, differentiation, synaptogenesis,
and myelination. This enabled reconstitution of highly differenti-
ated histotypic brain structures and functions (Honegger and
Monnet-Tschudi, 2001). This approach also enabled studies into
the role of growth factors and hormones in brain development
and offered a suitable model for neurotoxicological investigations,
including the study of developmental neurotoxicity and long-term
(chronic) toxicity of chemicals (Forsby et al., 2009; Monnet-Tschudi
et al., 2000; Zurich et al., 2003). The advantages of this 3D cell cul-
ture system include a high yield, robustness, easy handling, and
excellent reproducibility. Nevertheless, careful analyses showed
that despite their excellent features, serum-free aggregating brain
cell cultures did not reach the same level of maturation as their
counterparts grown in the presence of FBS. The most signiﬁcant dif-
ferences were found in the level of myelination and in the fre-
quency and intensity of spontaneous electrical activity. None of
the various commercialized serum substitutes could further im-
prove the maturation of aggregating brain cell cultures. Similar
observations were reported by others working with cell lines.
Although it was possible to adapt various cell lines to serum-free
medium, they showed increased fragility and altered growth char-
acteristics in comparison with the original cell lines grown in ser-
um-containing medium. New attempts were undertaken to
identify the factors, presumably present in serum, that were able
to further enhance brain cell maturation. The current working
hypothesis is that the missing factors are lipid-soluble constituents
such as cholesterol, which is difﬁcult to handle in aqueous solutions
and in isolated form. Interestingly, it was observed that the dialyzed
serum was as effective as complete serum, and it was thus assumed
that one or several macromolecular factor(s) have beneﬁcial effects.
The different lipoprotein fractions are currently isolated from ser-
um (i.e., fetal calf serum, newborn calf serum, and human plasma),
and their activity in aggregating brain cell cultures derived from 16-
day embryonic rat brain is investigated. The isolated fractions are
examined for their beneﬁcial effects on neuronal and glial cell mat-
uration in these cultures. If essential macromolecular serum com-
ponent(s) are identiﬁed which reproduce the developmental
effects of serum, these will be extensively tested in brain cultures
and in different cell lines. When they are proven successful, syn-
thetic production will be explored, e.g., by animal-free recombinant
4.3. Organotypic brain slice cultures and deﬁned serum-free medium
Neurobasal with B27 (J. Noraberg)
Organotypic brain slice cultures can be grown for weeks and
months, preserving their basal cellular structure, composition
and connections, and have been increasingly used as models to
study mechanisms and treatment for neurodegenerative disorders
(Noraberg, 2009; Noraberg et al., 2005). Organotypic brain slice
cultures have been grown since the early 1980s, ﬁrst as roller tube
cultures, with each culture embedded in a plasma clot on a
coverslip in a test tube placed in a roller drum (Gahwiler, 1981),
J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063 1059
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and later, in the early 90s mostly as interface cultures, where the
slices grow on a porous membrane in the interface between med-
ium and air (Stoppini et al., 1991). In the mid 1990s, the Serum
Optimem with 25% horse serum culture medium was replaced by
the deﬁned serum-free Neurobasal medium with B27 supplements
(Brewer et al., 1993). This medium is changed 2 days after culture
start up in Serum Optimem (Noraberg et al., 1999). The change to a
serum-free medium was made to avoid the effects of unknown
growth factors and the variability in the different batches of serum.
Neurobasal with B27 supplement worked well and produced
excellent cultures until the beginning of 2000 (Bonde et al.,
2003), when necrotic holes in the cultures were found. After a thor-
oughly evaluation it was decided to grow the cultures in Serum
Optimem and at the lower temperature of 33 °C (compared to
the earlier 36 °C) for the ﬁrst 2–3 weeks. After that, the cultures
were changed to Neurobasal with B27 and 36 °C 1 day before
experiments were performed. This has worked well until recently
(Montero et al., 2009), when it was discovered that just changing
to Neurobasal with B27, 24 h before studies of oxygen–glucose
deprivation (OGD), induced a high and signiﬁcant amount of cell
death compared to Serum Optimem in cultures just submersed
in control medium for the standard time of 30 min. Now, the cul-
tures are kept in Serum Optimem for the entire time of culture,
though good replacement for this serum-containing medium is
preferred. Other groups have experienced similar difﬁculties using
B27 (Cressey, 2009), and an American group has recently published
the recipe for a new supplement, N21, a re-deﬁned and modiﬁed
formulation of B27 (Chen et al., 2008). Sigma–Aldrich has just an-
nounced that they, in the beginning of 2010, will launch a series of
complete media for neuronal and stem cell cultures, Stemline™,
where all necessary supplements are included. Using Neuroba-
sal + N21 and the complete media from Sigma–Aldrich, cell viabil-
ity will be tested and compared with Serum Optimem in two types
of cell cultures present in our laboratory: organotypic brain slice
cultures and primary neuronal cultures (see 4.4).
4.4. Deﬁned medium and serum-containing medium occasionally
induce cells to use different signal transduction pathways to
proliferate (Å. Fex Svenningsen)
Dissociated cultures made from different parts of the rat or mice
fetal central and peripheral nervous system are useful and perhaps
necessary tools when investigating cell–cell interactions or re-
sponses to toxic agents. Such cell culture systems have been devel-
oped and used to study neuronal development as well as toxic
effects on neuronal development (Svenningsen et al., 2003). Some
of these cell cultures can be grown for up to 3 months, developing
synapses, robust myelination and astrocytic networks in deﬁned
medium. The different cell cultures were developed with the aim
to keep all cells normally present in the nervous system in the cul-
ture, to make the environment as ‘‘in vivo-like” as possible and to
use a deﬁned cell culture medium to be able to control the cellular
environment and avoid variations from various batches of serum.
Neurobasal medium supplemented with B27 (Brewer et al.,
1993) was chosen because, apart from being completely deﬁned,
it enables long-term culture without ﬁbroblast or astrocyte over-
growth. However, using a deﬁned medium instead of one contain-
ing serum to grow primary cultures sometimes produces disparate,
inconsistent results (Svenningsen and Kanje, 1998). e.g., our preli-
minary data show that some glial cells grown in deﬁned media, to-
gether with neurons, use other signal transduction pathways to
proliferate than cells grown in serum-containing media. Which sig-
nal transduction pathways these cells use for proliferation ‘‘in vivo”
is thus not yet clear.
In order for primary cell culture to be an useful tool, where cell
interactions in vitro can be investigated and results can be relied
on, it is necessary to compare in vitro with in vivo results, and to
adjust media and cells to what is most in vivo-like.
4.5. Optimization of culture conditions for human intestinal Caco-2
cells to improve functional differentiation in serum-free media (M.L.
Although representing the best and most extensively used cell
culture model of absorptive enterocytes, the human Caco-2 cell
line displays a high degree of heterogeneity in the expression of
differentiated functions that is largely due to differences in culture
procedures (Sambuy et al., 2005). Fetal bovine serum (FBS) in the
culture medium is an important source of variability in the perfor-
mance of this differentiated cell model and several attempts have
been made over the years to cultivate these cells using serum-free
Halleux and Schneider developed, in 1991, a serum-free med-
ium for Caco-2 cells that was adapted from a nutritive medium
studied for hepatocytes (Basal Deﬁned Medium – Gibco) supple-
mented with insulin, epidermal growth factor, albumin–linolenic
acid, hydrocortisone and triiodothyronine (T3) (Halleux and
Schneider, 1991). Cells were grown on polyethyleneterephthalate
(PET) ﬁlters coated with collagen, as a model of the intestinal bar-
rier, and showed after 15 days in culture many differentiated func-
tions of enterocytes, correct polarization and a decrease of
paracellular permeability, indicating the establishment of func-
tional tight junctions. Shortly after, Jumarie and Malo obtained dif-
ferentiated Caco-2 seeded on plastic substrate using DMEM
supplemented with ITS (Jumarie and Malo, 1991). This deﬁned
medium allowed for normal differentiation of the cells after
3 weeks, as shown by morphological and functional characteristics.
Only sucrase activity was lower than in cells cultivated with ser-
um, but was increased by addition of T3. A serum-free medium
containing ITS and lipids (modiﬁed from Jumarie and Malo
(1991)) was used to differentiate the parental Caco-2 line and three
clonal lines cultivated on PET ﬁlter inserts and the permeability
characteristics were investigated. Medium composition did not af-
fect the establishment of trans-epithelial electrical resistance
(TEER) while an increase in paracellular permeability to the extra-
cellular marker mannitol was observed in cells grown in serum-
free conditions as compared to cells grown in serum supplemented
medium (Ranaldi et al., 2003). Increased paracellular permeability
was also observed by Gangloff et al. that used Caco-2 cells differen-
tiated in serum-free medium to investigate iron uptake (Gangloff
et al., 1996).
In the attempt to improve the performance of the ITS-lipid med-
ium, an investigation is in progress using Caco-2 cells grown for up
to 21 days on permeable polycarbonate cell culture inserts. Cells
were allowed to differentiate in media containing different supple-
ments: (A) insulin, transferrin, selenium (ITS) and lipid mixture
(oleate, palmitate, cholesterol and BSA as carrier); (B) a deﬁned
mixture of growth factors and hormones derived from ITS (MI-
TO + serum extender), and; (C) MITO + serum extender and lipid
mixture. Since the supplements were added only to the basolateral
compartment, the control conditions included medium supple-
mented with 10% FBS in both compartments (symmetrical control)
or in the basolateral compartment (asymmetrical control). The ef-
fects of the different serum substitutes on cell differentiation were
assessed by permeability assays, gene expression studies, apical
enzyme assays (alkaline phosphatase and sucrase) and transport
activity of P-glycoprotein. Preliminary results indicate that
Caco-2 differentiation occurred under all tested conditions,
although distinct endpoints responded differently to the various
serum substitutes (Ferruzza et al., 2009). Thus, a single recipe for
optimal Caco-2 differentiation in deﬁned medium cannot yet be
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Author's personal copy
recommended and further studies are required to deﬁne the role of
single supplements in this process.
The use of serum to enable cell and tissue cultures is problem-
atic, both for ethical and scientiﬁc reasons. It is therefore recom-
mended to change from serum supplemented cell and tissue
culture media to serum-free media, with, preferably, animal com-
ponent-free and chemically deﬁned supplements, by using already
existing formulations and developing formulations for cells and
tissues where SF media do not yet exist.
It is therefore recommended to apply the ‘‘No, unless...” princi-
ple: no supplementing with serum, unless a SF medium has not yet
developed. In which case, all efforts should be undertaken to devel-
op a SF medium for the particular cell or tissue culture. Information
on existing SF media formulations is readily available in databases,
which should be continuously fed with newly developed
Several components are essential, and others useful, when
developing SF media. Furthermore, several approaches are avail-
able to adapt cells to the new SF medium. Special care should be
taken to the validation of using the new medium so that the origi-
nal studied biomarkers are still expressed by the cells and tissues.
Successful approaches to develop SF media, also for speciﬁc cell
types and cultures, are described. The described models show that
the development of SF media is not an easy task and that several
hurdles, which may be dependent on the cell type and the culture
system used, have to be taken. Extensive information exchange,
through databases, meetings/workshops and electronic networks
will facilitate and stimulate the development of new SF media
6. General recommendations
(1) When considering supplementing cell and tissue culture
media with animal serum the ‘‘No, unless....” principle
should be applied. Preferentially, the medium should not
contain any animal-derived component, unless it was
proved to be an absolute requirement.
(2) For scientiﬁc arguments, cell and tissue culture medium
should be chemically deﬁned.
(3) In particular in vitro methods that are used in a regulatory
testing context should be based on a chemically deﬁned cul-
(4) To use deﬁned media is a strong recommendation from ESAC
and the GCCP. Since GCCP has no legal basis, it is strongly
recommended to make it part of GLP and/or GMP.
(5) The use of (fetal bovine) serum as supplement in in vitro
studies has to be justiﬁed.
(6) Many media supplements are commercially available, but
the formulation of these should be chemically deﬁned.
(7) For ethical reasons, FBS should be replaced by supplement-
ing with deﬁned chemicals (e.g., recombinant components)
or animal or plant extracts.
(8) For scientiﬁc reasons, FBS should be replaced by deﬁned sup-
plements, which should have a non-animal origin when
human safety is at stake.
(9) SOP’s of established SF and, preferably, chemically deﬁned
media should be made readily available through publica-
tions and databases.
(10) It is recommended that a platform is established for serum-
free media developers and users to discuss experiences, to
facilitate further progress and to organise meetings on this
7. Recommendations for developing serum-free cell culture
(1) When developing FBS-free media, start with an appropriate
basal medium. A 50:50 (v/v) mixture of DMEM and Ham’s-F-
12, supplemented with ITS has been successfully used in
(2) When glutamine is used, it should be added at a concentra-
tion of 2–4 mM. Also Glutamax I™ (
-Gln) can be sup-
plemented for some cell lines.
(3) Supplement with cell type speciﬁc growth factors, hor-
mones, vitamins, trace elements and lipids wherever
(4) Pay attention to osmolarity.
(5) For some studies or cell types, speciﬁc proteins may be
(6) Some essential fatty acids, not present in the basal medium,
may have to be added.
(7) Preferably, no antibiotics should be used.
(8) For some cell types and primary cultures a substrate for
attachment may be used. Many serum-free formulations
require a pre-coating of culture dishes.
(9) For bioreactors and perfusion cultures a shear force protec-
tor (Pluronics F68) may be added.
(10) Carefully adapt the cells to the new medium.
(11) Always check whether the performance of the cells has
changed and whether the endpoints of the study are
(12) When successful, share your formulation with colleagues,
and through existing cell culture databases.
This workshop and report would not have been possible with-
out the ﬁnancial support of the Doerenkamp-Zbinden Foundation
(CH), the Program committee on alternatives to animal experi-
ments of the Netherlands Organisation for Health Research and
Development, the Danish Forsøgsdyrenes Værn and the Danish In
Vitro Toxicology Network.
Anon, 1993. Points to consider in the characterization of cell lines used to produce
biologicals. Available from: <http://www.fda.gov/downloads/BiologicsBlood
forManufacturers/UCM062745.pdf> (accessed 16.02.10).
Anon, 2009a. Good cell culture. Available from: <http://www.goodcellculture.com/>
Anon, 2009b. Serum free cell lines. Available from: <http://www.sefrec.com>
Anon, 2009c. Serum free media for cell culture. Available from: <http://
Barnes, D., Sato, G., 1980a. Methods for growth of cultured cells in serum-free
medium. Analytical Biochemistry 102 (2), 255–270.
Barnes, D., Sato, G., 1980b. Serum-free cell culture: a unifying approach. Cell 22 (3),
Bettger, W.J., McKeehan, W.L., 1986. Mechanisms of cellular nutrition. Physiological
Reviews 66 (1), 1–35.
Bjare, U., 1992. Serum-free cell culture. Pharmacology and Therapeutics 53 (3),
Bonde, C., Sarup, A., Schousboe, A., Gegelashvili, G., Noraberg, J., Zimmer, J., 2003.
GDNF pre-treatment aggravates neuronal cell loss in oxygen-glucose deprived
hippocampal slice cultures: a possible effect of glutamate transporter
upregulation. Neurochemistry International 43 (4–5), 381–388.
Brewer, G.J., Torricelli, J.R., Evege, E.K., Price, P.J., 1993. Optimized survival of
hippocampal neurons in B27-supplemented neurobasal, a new serum-free
medium combination. Journal of Neuroscience Research 35, 567.
Brunner, D., Frank, J., Appl, H., Schöfﬂ, H., Pfaller, W., Gstraunthaler, G., 2010. Serum-
free cell culture: the serum-free media interactive online database. ALTEX 27
J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063 1061
Author's personal copy
Burteau, C.C., Verhoeye, F.R., Mols, J.F., Ballez, J.S., Agathos, S.N., Schneider, Y.J., 2003.
Fortiﬁcation of a protein-free cell culture medium with plant peptones
improves cultivation and productivity of an interferon-gamma-producing
CHO cell line. In Vitro Cellular and Developmental Biology Animal 39 (7),
Butler, M., Jenkins, H., 1989. Nutritional aspects of the growth of animal-cells in
culture. Journal of Biotechnology 12 (2), 97–110.
Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B.A., Hell, J.W., 2008. NS21: re-
deﬁned and modiﬁed supplement B27 for neuronal cultures. Journal of
Neuroscience Methods 171 (2), 239–247.
Christie, A., Butler, M., 1994. Growth and metabolism of a murine hybridoma in
cultures containing glutamine-based dipeptides. GIBCO Focus 16 (1), 9–13.
Coecke, S., Balls, M., Bowe, G., Davis, J., Gstraunthaler, G., Hartung, T., Hay, R.,
Merten, O.W., Price, A., Schechtman, L., Stacey, G., Stokes, W., 2005. Guidance
on good cell culture practice. A report of the second ECVAM task force on good
cell culture practice. Alternatives to Laboratory Animals 33 (3), 261–287.
Cressey, D., 2009. Neuroscientists claim growing pains. Nature 459 (7243), 19.
Eagle, H., 1955. Nutrition needs of mammalian cells in tissue culture. Science 122
Elias, C.B., Desai, R.B., Patole, M.S., Joshi, J.B., Mashelkar, R.A., 1995. Turbulent shear
stress – effect on mammalian cell culture and measurement using laser Doppler
anemometer. Chemical Engineering Science 50 (15), 2431–2440.
ESAC, 2008. ESAC statement on the use of FCS and other animal-derived
supplements. Available from: <http://ecvam.jrc.it/publication/
ESAC28_statement_FCS_20080508.pdf> (accessed 16.02.10).
Falkner, E., Appl, H., Eder, C., Losert, U.M., Schofﬂ, H., Pfaller, W., 2006. Serum free
cell culture: the free access online database. Toxicology in Vitro 20 (3), 395–
Feifel, E., Obexer, P., Andratsch, M., Euler, S., Taylor, L., Tang, A., Wei, Y., Schramek,
H., Curthoys, N.P., Gstraunthaler, G., 2002. P38 MAPK mediates acid-induced
transcription of PEPCK in LLC-PK1-FBPase+ cells. American Journal of
Physiology – Renal Physiology 283, F678–F688.
Ferruzza, S., Rossi, C., Natoli, M., Sambuy, Y., Scarino, M.L., 2009. Optimization
of culture conditions for human intestinal Caco-2 cells to improve
functional differentiation: serum-free medium and substrate effects. ALTEX
Forsby, A., Bal-Price, A.K., Camins, A., Coecke, S., Fabre, N., Gustafsson, H., Honegger,
P., Kinsner-Ovaskainen, A., Pallas, M., Rimbau, V., Rodriguez-Farre, E., Sunol, C.,
Vericat, J.A., Zurich, M.G., 2009. Neuronal in vitro models for the estimation of
acute systemic toxicity. Toxicology in Vitro 23 (8), 1564–1569.
Gahwiler, B.H., 1981. Organotypic monolayer cultures of nervous tissue. Journal of
Neuroscience Methods 4 (4), 329–342.
Gangloff, M.B., Lai, C., Van Campen, D.R., Miller, D.D., Norvell, W.A., Glahn, R.P.,
1996. Ferrous iron uptake but not transfer is down-regulated in Caco-2 cells
grown in high iron serum-free medium. Journal of Nutrition 126 (12), 3118–
Gey, G.O., Thalhimer, W., 1924. Observations on the effects of insulin introduced
into the medium of tissue cultures. JAMA 82 (20), 1609.
Glacken, M.W., 1988. Catabolic control of mammalian cell culture. Bio/Technology
Gospodarowicz, D., Moran, J.S., 1976. Growth factors in mammalian cell culture.
Annual Review of Biochemistry 45, 531–558.
Grillberger, L., Kreil, T.R., Nasr, S., Reiter, M., 2009. Emerging trends in plasma-free
manufacturing of recombinant protein therapeutics expressed in mammalian
cells. Biotechnology Journal 4 (2), 186–201.
Gstraunthaler, G.J., 1988. Epithelial cells in tissue culture. Renal Physiology and
Biochemistry 11 (1–2), 1–42.
Gstraunthaler, G., 2003. Alternatives to the use of fetal bovine serum: serum-free
cell culture. ALTEX 20 (4), 275–281.
Gstraunthaler, G., Pfaller, W., Kotanko, P., 1985. Biochemical characterization of
renal epithelial cell cultures (LLC-PK1 and MDCK). American Journal of
Physiology 248 (4 Pt 2), F536–544.
Gupta, K., Rispin, A., Stitzel, K., Coecke, S., Harbell, J., 2005. Ensuring quality of
in vitro alternative test methods: issues and answers. Regulatory Toxicology
and Pharmacology 43 (3), 219–224.
Halleux, C., Schneider, Y.J., 1991. Iron-absorption by intestinal epithelial-cells. 1.
Caco2 cells cultivated in serum-free medium, on polyethyleneterephthalate
microporous membranes, as an invitro model. In Vitro Cellular and
Developmental Biology 27 (4), 293–302.
Ham, R.G., 1965. Clonal growth of mammalian cells in a chemically deﬁned,
synthetic medium. Proceedings of the National Academy of Sciences of the
United States of America 53, 288–293.
Hartung, T., 2007. Food for thought....on cell culture. ALTEX 24 (3),
Hartung, T., Balls, M., Bardouille, C., Blanck, O., Coecke, S., Gstraunthaler, G., Lewis,
D., 2002. Good cell culture practice. ECVAM good cell culture practice task force
report 1. Alternatives to Laboratory Animals 30 (4), 407–414.
Hartung, T., Bremer, S., Casati, S., Coecke, S., Corvi, R., Fortaner, S., Gribaldo, L.,
Halder, M.E., Hoffmann, S., Janusch Roi, A., Prieto, P., Sabbioni, E., Scott, L.,
Worth, A.P., Zuang, V., 2004. A modular approach to the ECVAM principles on
test validity. ATLA Alternatives to Laboratory Animals 32 (5), 467–472.
Hayashi, I., Sato, G.H., 1976. Replacement of serum by hormones permits growth of
cells in a deﬁned medium. Nature 259 (5539), 132–134.
Helmy, M.H., Ismail, S.S., Fayed, H., El-Bassiouni, E.A., 2000. Effect of selenium
supplementation on the activities of glutathione metabolizing enzymes in
human hepatoma Hep G2 cell line. Toxicology 144 (1–3), 57–61.
Honegger, P., Monnet-Tschudi, F., 2001. Aggregating neural cell cultures. In:
Fedoroff, S., Richardson, A. (Eds.), Protocols for Neural Cell Culture, third ed.
Humana Press, Totowa, NJ, pp. 199–218.
Honegger, P., Lenoir, D., Favrod, P., 1979. Growth and differentiation of aggregating
fetal brain cells in a serum-free deﬁned medium. Nature 282 (5736), 305–308.
Jayme, D., Watanabe, T., Shimada, T., 1997. Basal medium development for serum-
free culture: a historical perspective. Cytotechnology 23 (1–3), 95–101.
Jumarie, C., Malo, C., 1991. Caco-2 cells cultured in serum-free medium as a model
for the study of enterocytic differentiation invitro. Journal of Cellular Physiology
149 (1), 24–33.
Keay, L., 2004. Autoclavable low cost serum-free cell culture media. The growth of L
cells and BHK cells on peptones. Biotechnology and Bioengineering 17 (5), 745–
Keenan, J., Pearson, D., Clynes, M., 2006. The role of recombinant proteins in the
development of serum-free media. Cytotechnology 50 (1–3), 49–56.
Kleinman, H.K., Klebe, R.J., Martin, G.R., 1981. Role of collagenous matrices in the
adhesion and growth of cells. Journal of Cell Biology 88 (3), 473–485.
Kleinman, H.K., Luckenbill-Edds, L., Cannon, F.W., Sephel, G.C., 1987. Use of
extracellular matrix components for cell culture. Analytical Biochemistry 166
Kuhlmann, I., 1996. The prophylactic use of antibiotics in cell culture.
Cytotechnology 19 (2), 95–105.
Lindl, T., Gstraunthaler, G., 2008. Zell- und Gewebekultur. Von den Grundlagen zur
Laborbank. Spektrum Akademischer Verlag, Heidelberg.
Mandl, E.W., Jahr, H., Koevoet, J.L., van Leeuwen, J.P., Weinans, H., Verhaar, J.A., van
Osch, G.J., 2004. Fibroblast growth factor-2 in serum-free medium is a potent
mitogen and reduces dedifferentiation of human ear chondrocytes in
monolayer culture. Matrix Biology 23 (4), 231–241.
Monnet-Tschudi, F., Zurich, M.G., Schilter, B., Costa, L.G., Honegger, P., 2000.
Maturation-dependent effects of chlorpyrifos and parathion and their oxygen
analogs on acetylcholinesterase and neuronal and glial markers in aggregating
brain cell cultures. Toxicology and Applied Pharmacology 165 (3), 175–183.
Montero, M., Gonzalez, B., Zimmer, J., 2009. Immunotoxic depletion of microglia in
mouse hippocampal slice cultures enhances ischemia-like neurodegeneration.
Brain Research 1291, 140–152.
Noraberg, J., 2009. Organotypic brain slice cultures. SciTopics. Avialable from:
Noraberg, J., Kristensen, B.W., Zimmer, J., 1999. Markers for neuronal degeneration
in organotypic slice cultures. Brain Research Protocols 3, 278–290.
Noraberg, J., Poulsen, F.R., Blaabjerg, M., Kristensen, B.W., Bonde, C., Montero, M.,
Meyer, M., Gramsbergen, J.B., Zimmer, J., 2005. Organotypic hippocampal slice
cultures for studies of brain damage, neuroprotection and neurorepair. Current
Drug Targets: CNS and Neurological Disorders 4 (4), 435–452.
Pazos, P., Boveri, M., Gennari, A., Casado, J., Fernandez, F., Prieto, P., 2004. Culturing
cells without serum: lessons learnt using molecules of plant origin. ALTEX 21
Pfaller, W., Gstraunthaler, G., Loidl, P., 1990. Morphology of the differentiation and
maturation of LLC-PK1 epithelia. Journal of Cellular Physiology 142 (2), 247–254.
Price, P.J., Gregory, E.A., 1982. Relationship between in vitro growth promotion and
biophysical and biochemical properties of the serum supplement. In Vitro 18
Pumper, R.W., 1958. Adaptation of tissue culture cells to a serum-free medium.
Science 128 (3320), 363.
Ranaldi, G., Consalvo, R., Sambuy, Y., Scarino, M.L., 2003. Permeability
characteristics of parental and clonal human intestinal Caco-2 cell lines
differentiated in serum-supplemented and serum-free media. Toxicology in
Vitro 17 (5–6), 761–767.
Rauch, C., Feifel, E., Schöfﬂ, H., Pfaller, W., Gstraunthaler, G., 2008. Alternatives to
the use of fetal bovine serum: platelet lysates as serum replacement in cell and
tissue culture. ALTEX 25 (Suppl. 1), 54–55.
Rauch, C., Feifel, E., Spötl, H.P., Amann, E.-M., Schennach, H., Schöfﬂ, H., Pfaller, W.,
Gstraunthaler, G., 2009. Alternatives to the use of fetal bovine serum: platelet
lysates as a serum substitute in cell culture media. ALTEX 26 (Special Issue), 119.
Reitzer, L.J., Wice, B.M., Kennell, D., 1979. Evidence that glutamine, not sugar, is the
major energy source for cultured HeLa cells. Journal of Biological Chemistry 254
Ringer, S., Buxton, D., 1887. Upon the similarity and dissimilarity of the behaviour of
cardiac and skeletal muscle when brought into relation with solutions
containing sodium, calcium and potassium salts. Journal of Physiology 8,
Sambuy, Y., Angelis, I., Ranaldi, G., Scarino, M.L., Stammati, A., Zucco, F., 2005. The
Caco-2 cell line as a model of the intestinal barrier: inﬂuence of cell and culture-
related factors on Caco-2 cell functional characteristics. Cell Biology and
Toxicology 21 (1), 1–26.
Schiff, L.J., 2005. Review: production, characterization, and testing of banked
mammalian cell substrates used to produce biological products. In Vitro
Cellular and Developmental Biology Animal 41 (3–4), 65–70.
Schlaeger, E.J., 1996. The protein hydrolysate, primatone RL, is a cost-effective
multiple growth promoter of mammalian cell culture in serum-containing and
serum-free media and displays anti-apoptosis properties. Journal of
Immunological Methods 194 (2), 191–199.
Schneider, M., Marison, I.W., vonStockar, U., 1996. The importance of ammonia in
mammalian cell culture. Journal of Biotechnology 46 (3), 161–185.
Stoppini, L., Buchs, P.A., Muller, D., 1991. A simple method for organotypic cultures
of nervous tissue. Journal of Neuroscience Methods 37 (2), 173–182.
1062 J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063
Author's personal copy
Svenningsen, A.F., Kanje, M., 1998. Regulation of Schwann cell proliferation in
cultured segments of the adult rat sciatic nerve. Journal of Neuroscience
Research 52 (5), 530–537.
Svenningsen, A.F., Shan, W.S., Colman, D.R., Pedraza, L., 2003. Rapid method for
culturing embryonic neuron-glial cell cocultures. Journal of Neuroscience
Research 72 (5), 565–573.
Taub, M., 1990. The use of deﬁned media in cell and tissue culture. Toxicology in
Vitro 4, 213–225.
van der Pol, L., Tramper, J., 1998. Shear sensitivity of animal cells from a culture-
medium perspective. Trends in Biotechnology 16 (8), 323–328.
van der Valk, J., Mellor, D., Brands, R., Fischer, R., Gruber, F., Gstraunthaler, G.,
Hellebrekers, L., Hyllner, J., Jonker, F.H., Prieto, P., Thalen, M., Baumans, V., 2004.
The humane collection of fetal bovine serum and possibilities for serum-free
cell and tissue culture. Toxicology in Vitro 18 (1), 1–12.
Waymouth, C., 1955. Simple nutrient solutions for animal cells. Texas Reports on
Biology and Medicine 13 (3), 522–536.
Wessman, S.J., Levings, R.L., 1999. Beneﬁts and risks due to animal serum used in
cell culture production. Developments in Biological Standardization 99, 3–8.
Zähringer, H., 2009. Leckerlis für die Zellen. Laborjournal 4, 74–81.
Zhang, Z., Al-Rubeai, M., Thomas, C.R., 1992. Effect of pluronic F-68 on the
mechanical properties of mammalian cells. Enzyme and Microbial Technology
14 (12), 980–983.
Zielke, H.R., Zielke, C.L., Ozand, P.T., 1984. Glutamine – a major energy-source for
cultured mammalian-cells. Federation Proceedings 43 (1), 121–125.
Zurich, M.-G., Monnet-Tschudi, F., Costa, L.G., Schilter, B., Honegger, P., 2003.
Aggregating brain cell cultures for neurotoxicological studies. In: Tiffany-
Castiglioni, E. (Ed.), In Vitro Neurotoxicology: Principles and Challenges.
Humana Press, Totowa, NJ.
J. van der Valk et al. / Toxicology in Vitro 24 (2010) 1053–1063 1063