Literature Review

Optimization of chemically defined cell culture media-Replacing fetal bovine serum in mammalian in vitro methods

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DOI: 10.1016/j.tiv.2010.03.016 · Source: PubMed
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Abstract
Quality assurance is becoming increasingly important. Good laboratory practice (GLP) and good manufacturing practice (GMP) are now established standards. The biomedical field aims at an increasing reliance 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 chemically defined media is part of the GCCP. This will decrease the dependence on animal serum, a supplement 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 limited 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 Copenhagen 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.
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Review
Optimization of chemically defined cell culture media – Replacing fetal
bovine serum in mammalian in vitro methods
J. van der Valk
a,*
, D. Brunner
b
, K. De Smet
c
, Å. Fex Svenningsen
d
, P. Honegger
e
, L.E. Knudsen
f
,
T. Lindl
g
, J. Noraberg
d
, A. Price
h
, M.L. Scarino
i
, G. Gstraunthaler
j
a
NCA, DWM, Fac. Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht, The Netherlands
b
zet-Life Science Laboratorium, zet – Centre for Alternative and Complementary Methods to Animal Testing, Industriezeile 36/VII, 4020 Linz, Austria
c
Federal Agency for Medicines and Health Products, DG PRE Authorisation, Victor Hortaplein 40, Bus 40, B-1060 Brussels, Belgium
d
Institute of Molecular Medicine, Department of Neurobiology Research, University of Southern Denmark, J.B. Winslows Vej 21, DK-5000 Odense C, Denmark
e
Department of Physiology, University of Lausanne, CH-1005 Lausanne, Switzerland
f
Department of Public Health, Faculty of Health Sciences, University of Copenhagen, Denmark
g
Institut für angewandte Zellkultur, München, Germany
h
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
i
INRAN, National Research Institute on Food and Nutrition, Via Ardeatina 546, 00178 Rome, Italy
j
Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria
article info
Article history:
Received 10 March 2010
Accepted 25 March 2010
Available online 31 March 2010
Keywords:
In vitro methods
Fetal bovine serum
Serum-free
Good cell culture practice
Tissue culture
3Rs
abstract
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.
Contents
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.
doi:10.1016/j.tiv.2010.03.016
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
experimental animals.
*Corresponding author. Tel.: +31 30 253 2163; fax: +31 30 253 7997.
E-mail address: j.vandervalk@uu.nl (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
1. Introduction
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
2
and 95% O
2
. 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
lines.
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
Levings, 1999).
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
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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
(GMP).
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.,
2004).
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,
2009).
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).
2.2. Supplements
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
below.
2.2.1. Hormones
All hormones of mammalian organisms are physiological con-
stituents in blood circulation and are thus also present in serum
Box 1
Culture media
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
antibodies), respectively.
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
Author's personal copy
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
3
), and
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
FBS are
a
1
-antitrypsin and
a
2
-macroglobulin (Gstraunthaler,
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
concentration.
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
cell cultures.
2.2.6. Proteins
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).
2.2.7. Vitamins
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.
2.2.9. Glutamine
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
(Ham, 1965).
2.2.11. Lipids
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-
ia formulations.
2.2.12. Antibiotics
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-
L
(or
D
)-ly-
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
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collagenous matrices (Kleinman et al., 1981) further facilitates the
adhesion of anchorage-dependent cells.
2.2.14. Osmolarity
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. ‘‘Buildinga 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 (
a
-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
(see above).
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
medium.
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
before.
increased
specificity
in serum-free
culture media
composition
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
growth factors:
EGF, FGF, NGF,
IGF-1, PDGF,
VEGF, TGF-
β
hormones:
glucocorticoids,
thyroid hormones, cell-
specific agonists that
signal via cAMP (ADH,
PTH, PGE2, glucagon)
lipids:
fatty acids,
cholesterol,
ethanolamine
vitamins acting
as anti-oxidants:
α-tocopherol
ascorbic acid,
vitamins:
retinoic acid
chemically defined,
serum-free media:
β
-ME
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
3.2).
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
Passage 1:
75% normal medium
25% serum-free medium
Passage 2:
50% normal medium
50% serum-free medium
Passage 3:
25% normal medium
75% serum-free medium
Passage 4:
100% serum-free
3. adaptation with
conditioned medium
cultivation of cells
in normal medium with 10% FBS
Passage 1:
50% conditioned medium
50% serum-free medium
Passage 2:
50% conditioned medium
from passage 1, 50% SFM
Passage 3:
25% conditioned medium
from passage 2, 75% SFM
Passage 4:
100% serum-free
4. „inside“ adaptation
cultivation of cells
in normal medium with 10% FBS
to confluence
change to serum-free medium
continued culture in
confluent state
trypsinization of confluent
monolayer culture
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
medium).
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
(G. Gstraunthaler)
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
1
(porcine kidney)
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
1
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
DNA technology.
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.
Scarino)
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
media.
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.
5. Conclusions
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
formulations.
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
formulations.
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-
ture medium.
(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
subject.
7. Recommendations for developing serum-free cell culture
media
(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
numerous studies.
(2) When glutamine is used, it should be added at a concentra-
tion of 2–4 mM. Also Glutamax I™ (
L
-Ala–
L
-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
necessary.
(4) Pay attention to osmolarity.
(5) For some studies or cell types, specific proteins may be
added.
(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
affected.
(12) When successful, share your formulation with colleagues,
and through existing cell culture databases.
Acknowledgements
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.
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Literature Review
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    The search for alternatives to fetal bovine serum (FBS) has become a major goal in the field of cell and tissue culture research. Although the supplementation of culture media with FBS is routine practice, FBS bears a number of disadvantages: unknown composition, high lot-to-lot variablity, ethical concerns about the harvest from bovine fetuses, and possible shortage in global supply. Several strategies have been developed to reduce or replace FBS in cell culture media (Bjare 1992; Even et al. 2006; Gstraunthaler 2003; van der Valk et al. 2004, 2010). Here we report on the use of human platelet lysates (PL) as a serum replacement (Alden et al. 2007; Bernardo et al. 2007; Bieback et al. 2009; Doucet et al. 2005; Johansson et al. 2003; Kocaoemer et al. 2007; Müller et al. 2009; Schallmoser et al. 2009). PL in DMEM support growth, proliferation and differentiation, as assessed by dome formation, of proximal tubule-like LLC-PK1 (porcine kidney) and HK-2 (human kidney) cells, as well as PL-supplemented DMEM/Ham F-12 for distal tubule-like MDCK (dog kidney) cells. 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 RPMI-1640 medium in suspension. In order to biochemically determine the proliferative potential of PL, the stimulation of extracellular signal-regulated MAP kinase (ERK1/2) was determined. Addition of PL to quiescent LLC-PK1 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.
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    Mise au point sur les besoins nutritifs de cellules animales mises en culture: source d'energie, acides amines, lipides, inhibiteurs de croissance, besoins en oxygene, supplementation en serum. La culture a grande echelle dans un bioreacteur permettant d'obtenir des produits commerciaux, l'optimisation des rendements de ces productions est indispensable, d'ou la necessite de bien connaitre les besoins des cultures cellulaires
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    A summary of the effects on the growth of tissue in vitro noted when insulin had been added to the culture medium is presented because of its potential value in this line of investigation.Dr. Thalhimer suggested the addition of insulin as a possible means to augment the growth of tissue and to disclose the nature of its action.Chick fibroblasts were cultivated from 9 to 10 day old embryos, the Carrel flask method being used. The medium contained glucose in percentages from 0.1 to2.5. Two sets of cultures were maintained. To one set, from 1 to 2 per cent. of insulin was added; to the second, otherwise identical, no insulin was added.The sugar content was determined in duplicate cultures by the Folin-Wu method, and the H-ion concentration was estimated by the drop calorimetric procedure. The perimeter of the growth was compared by using the planimeter method of Carrel