---I~------"J~ ~ Alternatives to the Use of Fetal Bovine Serum: Serum-free Cell Culture Gerhard Gstraunthaler Department of Physiology, University of Innsbruck, A-Innsbruck

Summary Zusammenfassung: Alternativen zur Verwendung von fötalem Serum is commonly used as a supplement to cell culture media. Kälberserum: die serum-freie Zellkultur lt provides a broad spectrum of macromolecules, carrier Serum ist ein weitverbreiteter Zusatz zu Zellkulturmedien. Es for lipoid substances and trace elements, attachment liefert ein breites Spektrum an Makromolekülen, Transport- and spreading factors, low molecular weight nutrients, and proteine für lipoide Substanzen und Spurenelemente, hormones and growth factors. The most widely used animal Bindungsproteine und Anheftungsfaktoren, niedermolekulare serum supplement is fetal bovine serum, FBS. Since serum in Nährstoffe, sowie Hormone und Wachstumsfaktoren. Das general is an ill-defined component in cell culture media, a am häufigsten verwendete Serumsupplement ist fötales number of chemically defined serum-free media formulations Kälberserum, FKS. Da Serum im Allgemeinen eine nur sehr un- have been developed in the last two decades. Besides modern klar definierte Komponente in Kulturmedien darstellt, wurden cell biological advances in cell and tissue culture and efforts in den letzten zwei Jahrzehnten eine Reihe von chemisch towards a standardisation of cell culture protocols in Good Cell definierten, serum-freien Medien entwickelt. Neben den Culture Practice, in addition, considerable ethical concerns modernen zellbiologischen Fortschritten in der Zell- und Ge- were raised recently about the harvest and collection of fetal webekultur und den Bemühungen um eine Standardisierung bovine serum. Thus, in order to decrease the annual need for von Zellkulturprotokolten im Sinne einer Good Cell Culture bovine fetuses in terms of the 3Rs through any reduction in the Practice, sind in den letzten Jahren auch sehr Ernst zu use or partial replacement of serum, as well as in terms of an nehmende ethische Bedenken über die Gewinnung und Pro- improvement of cell and tissue culture methodology, serum-free duktion von fötalem Kälberserum geäußert worden. Um im cell culture represents a modern, valuable and scientifically Sinne der berühmten 3Rs die jährlichen Verbrauchszahlen von well accepted alternative to the use of FBS in cell and tissue Rinderfoten durch eine Reduktion im Verbrauch brw. dem culture. Ersatz von Serum zu senken, wie auch im Sinne einer Verbesserung von Methodologien in der Zell- und Gewebe- kultur, stellt die serum-freie Zellkultur eine moderne, wissenschaftlich voll akzeptierte und abgesicherte Alternative zur Verwendung von FKS in der Zellkultur dar.

Keywords: cell and tissue culture, serum-free cell culture, in vitro, chemically defined media, alternatives to animal-derived prod- ucts, replacement, 3R-principles, fetal bovine (calf) serum, vegetal serum, animal welfare

1 Introduction Burch (Balls et al., 1995), decreasing the The amount of FBS produced for the number of experimental animals. In addi- world market is estimated at approxi- Cell and tissue culture, i.e. the propaga- tion, during the past decade, invertebrate mately 500.000 litres per year. This tion and cultivation of cells in vitro (for cell and tissue culture has gained an in- means, that more than 1.000.000 bovine definitions, see Tab. 1) (Schaeffer, 1990), creasingly prominent role in basic cell fetuses have to be harvested, and it is ex- has become an indispensable tool in bio- and molecular biological research, since pected, that these numbers will continue medical sciences. The use of cell culture is cultured insect cells are widely used as to increase annually (Jochems et al., increasing exponentially, and new in vitro eukaryotic in vitro expression systems 2002; Shah, 1999). Recently, serious alternatives are constantly being developed. using baculovirus expression vectors ethical concerns were raised with regard The increased use of in vitro method- (Fraser, 1992; Jarvis, 1991). to the welfare of the donor fetuses in the ologies in basic research and applied However, in most cases, routine cell harvest, production and processing of biomedical sciences - in cell biology, and tissue culture demands the use of FBS (Shailer and Corrin, 1999; van der physiology, pharmacology, and toxico- animal-derived products, mainly fetal Valk et al., 2003). The concerns focused logy - is indeed a beneficial development bovine serum (FBS), as culture medium mainly on the current methods of collect- in terms of the 3R concept of Russel and constituents (see below). ing FBS that may cause suffering to the animals, in particular to fetuses. In brief, Received 9 Octobre 2003; received in final form and accepted for publication 27 Octobre 2003 the bovine fetuses, from which blood is

ALTEX 20, 4/03 275 GSTRAUNTHALER ------~~~~ -----

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larity within acceptable limits. In addi- Tab. 1: The definitions of cell and tissue culture according to Schaeffer, 1990. tion, animal serum is frequently added to chemically defined basal media.

Cell culture: ... the maintenance or cultivation of human or animal cells in vitra including the culture of 3 The role of serum in cell culture single cells. In cell cultures, the celis are no longer organised into tissues . media

Tissue culture: The maintenance or growth of tissues , in The supplementation of basal culture vitro, in a way that may allow differentiation media with animal serum of different and preservation of their architecture origin is essential for cell growth and for and/or function. the stimulation of proliferation ("mito- genic effect"). The sera used most wide- ly are from adult or newbom animals, drawn for FBS production, are obtained environment), and by the culture medi- or of fetal bovine origin. Whole animal from pregnant cows sent to slaughter. In um, which represents the diffuse environ- serum as well as fetal bovine serum huge meat cattle herds, bulls and cows ment. The latter comprises all types of (FBS), in general, is an extremely com- roam freely together, and as a result, soluble molecules - nutrients and salts, plex mixture of a large number of con- many cows are pregnant at the time of hormones and growth factors (Freshney, stituents, low and high molecular weight slaughter. When a pregnant cow is dis- 1994). biomolecules with different, physiologi- covered in the slaughterline, the fetus is Thus it is obvious, that the culture cally balanced growth-promoting and separated at the abattoir, and fetal blood medium is by far one of the most impor- growth-inhibiting activities. The role of is collected under aseptic conditions. tant single factors in cell and tissue serum in cell culture media is sum- This is usually performed by cardiac culture. The culture medium must supply marised in Tab. 2. puncture. Altematively, fetal blood may all essential nutrients for cell meta- Contemporary knowledge in modem be harvested by means of umbilical vein bolism, growth and proliferation (Bettger cell biology and biochemistry allowed puncture or puncture of the jugular vein and McKeehan, 1986; Butler and Jenk- the identification of the (growth) factors (Jochems et a1.,2002). ins, 1989; Harn and McKeehan, 1979; involved in in vivo processes, like cell These aspects were the topic of a re- Levintow and Eagle, 1961; Nardone, proliferation and tissue repair (e.g. in cent workshop entitled "Towards Better 1987). These inc1ude biosynthetic pre- wound healing), and cell maturation and In Vitro Methods: The Replacement of cursors for cell anabolism, catabolic sub- differentiation (e.g. embryonie matura- Fetal Bovine Serum", and the reader is strates for energy metabolism, vitamins tion, stern cell lineage, epithelial differ- referred to the comprehensive workshop and trace elements whose function is entiation, etc.). The major functions of report (van der Valk et a1., 2003). Thus, primarily catalytic, and bulk inorganic serum in culture media are to provide (i) any efforts to decrease the global de- ions (electrolytes) whose functions are hormonal factors stimulating cell growth mands for FBS and thus to decrease the both catalytic and physiological, e.g. to and proliferation, and promoting differ- number of bovine fetuses required maintain culture medium pH and osmo- entiated functions, (ii) transport proteins should be welcomed and supported.

2 The basics in cell and tissue cuIture

For successful growth and maintenance of human or animal cells in vitro, either primary cultures or continuous cell lines, appropriate culture conditions are re- quired that mimic the physiological [ conditions in vivo et situ with respect to temperature, pH, osmolarity, and oxygen supply (Davis, 2002; Masters, 2000). 2 The microenvironment of acelI, depicted schematically in Fig. 1, must therefore be provided: by the culture substratum, Fig. 1. Parts of the microenvironment of the marnmalian cell regulating its behaviour i.e. the appropriate culture dish or specifi- in vivo and in vitro. (1) The diffuse environment, which in cell cultures is solely provided cally coated surfaces, that allow attach- by the culture medium, (2) the contact environment (cell-matrix adhesion), and (3) the ment and spreading of cells (the contact junctional connections between neighbouring cells (cell-cell adhesion).

276 ALTEX 20, 4/03 ~J~;~ ------GSTRAUNTHALER ~

different amounts of endotoxins, Tab. 2: The role of serum in cell culture media (Davis, 2002; Freshney, 1994; Lindl, haemoglobin, and other adverse factors, 2002; Masters, 2000). and (iv) serum can be a potential source of microbial contaminants, such as fungi, bacteria, viruses or prions (Dormont, Role of Serum in Cell Culture Media 1999; Eliot, 1999; Wessman and Lev- ings, 1999). Serum provides Thus, serum introduces several un- known variables into the tissue culture > growth factors and hormones system. In addition, serum-supplemented > binding. and transport proteins > attachment- and spreading factors media may be unable to support growth > additional amino acids, vitamins and trace elements of specific cell types, and in primary cul- > fatty acids and lipids tures of highly differentiated epithelial > protease-inhibitors celIs, serum may be unable to prevent the > 'detoxifieation' (due to binding and inaetivation) overgrowth of the culture by fibroblasts. > (eolloid)osmotic pressure (serum-free vs. -free) Therefore, in terms of standardised pro- tocols in Good Cell Culture Practice (Hartung et al., 2002), it might be worth- carrying hormones (e.g. ), polypeptide growth factors which are while to omit the supplementation of cul- minerals and trace elements (e.g. trans- present in animal serum and activate ture media with animal sera. ferrin), and lipids (e.g. lipoproteins), (iii) cultured cells with varying degrees of attachment and spreading factors (i.e. specificity are fibroblast growth factor components of the extracellular matrix), (FGF) (Gospodarowicz et al., 1987), 4 Alternatives to fetal bovine serum and (iv) stabilising and detoxifying fac- nerve growth factor (NGF) (Levi-Mon- in cell and tissue culture tors, needed to maintain pH or to inhibit talcini, 1987), endothelial growth factor, proteases either directly, such as a-anti- or insulin-like growth factors IGF-l and The removal of serum from the cell trypsin or a2-, or indirect- IGF-2 (Deuel, 1987). culture medium and/or the replacement ly, by acting as an unspecific sink for Because of its rich content of growth with other complex biological fluids and proteases and other (toxic) molecules factors and its low gamma- extracts initiates multifarious variations (Tab. 2). content, FBS has become the standard in the interactive nature of the cell It is a weIl known fact that natural clot supplement of cell culture media. In most culture system that are introduced in a serum is more effective than plasma cases, FBS is used at concentrations of in stimulating cell proliferation. This 10% (v/v), although this may vary for appears to be due to the release of certain specific applications (e.g. low serum Tab. 3: Variation profile of fetal bovine polypetides from activated platelets media). Other advantages in the use of sera (Price and Gregory, 1982). during the clotting process. FBS include the following: (i) FBS is a In recent decades, a number of growth cocktail of most of the factors required Composition of factors, hormones, transport proteins, co- for cell proliferation and maintenance Fetal Bovine Serum factors, and essential minerals and trace (see above), (ii) it is thus an almost elements have been identified, that all universal growth supplement effective in Components Cone. Range drive specific gene expression, initiate most types of human and animal (includ- total protein 3.8 g/100 ml 3.2-7.0 and control the cell cycle and thus cell ing insect) cells. (iii) Using serum- 2.3 g/100 ml 2.0 - 3.6 division, and program specific cell differ- supplemented medium therefore reduces endotoxin 0.36 ng/ml 0.01 -10.0 entiation (HilI and Treisman, 1995). We the need to spend time and effort devel- haemoglobin 11.3 mg/100 ml 2.4 - 18.1 know today, for example, that a specific oping a specific, optimised medium for- cholesterol 31 mg/100 ml 12- 63 transcriptional activation pro gram is mulation for each cell type. fattyacids phospholipids mg/ml needed to initiate cell growth and pro- However, the use of animal serum in triglycerides } liferation in vitro, e.g. the specific acti- cell culture also bears a number of disad- vation of mitogen-activated pro tein vantages (Lindl, 2002; van der Valk et al., glucose 125 mg/100 ml 85 - 247 (MAP) kinases, like the extracellular 2003): (i) serum is an ill-defined medium insulin 10 ~U/ml 6-14 signal-regulated kinase (ERK) cascade supplement, and thus an ambiguous cortisone 0.5 ~g/1 00 ml <0.1 - 2.3 trijodo-thyronine 119 ng/100 ml 56 - 223 (Denhart, 1996; Kyriakis and Avruch, factor in cell culture (Bjare, 1992), (ii) thyroxine 12.1 ng/100 ml 7.8-15.6 2001; Widmann et al., 1999), by epider- serum batches display quantitative and PTH 1718 pg/ml 85 - 6180 mal growth factor (EGF) (Carpenter and qualitative variations in their composi- PGE 5.91 nglml 0.5 - 30.5 Cohen, 1979; Cohen, 1987) or platelet- tion, and thus introduce a significant PGF 12.33 ng/ml 3.8 - 42.0 derived growth factor (PDGF) (HeIdin et lot-to-lot variability (Price and Gregory, al., 1985; Ross et al., 1986). Other 1982) (Tab. 3), (iii) serum may contain

ALTEX 20, 4/03 277 ------GSTRAUNTHALER 0:~;~ -- ~~ single step. A poorly defined medium supplement (FBS) is replaced by another Tab. 4: Cell physiological arguments for serum-free cell culture. ill-defined product. Nevertheless, meth- ods to reduce the requirements for FBS in culture media as weIl as alternative Cell Physiological Arguments animal serum substitutions were recently for Serum-free Cell Culture described (Jayme et al., 1988). The latter include substitution with newborn or • defined and controlled culture conditions in vitra: adult calf or the use of sera from sera, > delined synthetic or recombinant media constituents other animal species (horse, pig, goat, > exact control 01 physiological action etc.). Other animal-derived alternatives (synergistic, antagonistic effects of hormones and factors) are tissue extracts (e.g. pituitary extracts, > studies on hormonal and nutritional requirements 01 cells chicken embryo extracts), bovine milk (autocrine, paracrine loops) fractions, or bovine colostrum (Belford > isolation (selection) 01 specilic cell types et al., 1995; Klagsbrun, 1980; Pakkanen and Neutra, 1994; Steimer et al., 1981). Most recently, protein fractions from plant extracts, called vegetal serum, were isolation of cell culture products), and (v) a 50:50 (v/v) mixture of Dulbecco's successfully introduced to grow and most of all, ethical aspects, i.e. reduction Modified Eagle's Medium (DMEM), that maintain epithelial ceIls in culture (Har- of the worldwide use of FBS and thus the supplies all essential nutrients in suffi- tung et al., 2003). Additional methods of numbers of bovine fetuses used as source cient quantities, and the highly enriched choice to reduce FBS requirement in- of FBS (Jochems et al., 2002; van der Ham's F-12 Nutrient Mixture. Other clude (i) the optimisation of existing Valk et al., 2003). highly enriched media which have been culture media, (ii) the use of reduced Serum-free media are generally more developed for serum-free culture are serum media, or (iii) the design of serum- cell-specific. Thus, for basic research in media of the MCDB (Molecular, Cellular free, chemicaIly defined media (Barnes cell physiology, additional benefits in the and Developmental Biology, University and Sato, 1982b; Froud, 1999; Gstraun- use of serum-free media can be defined of Colorado) series, CMRL 1066 and thaler, 2001). (Tab. 4) (Barnes et al., 1987). CMRL 1415 (Connaught Medical Serum-free media formulations for the Research Laboratories) media, or NTCT initiation of primary cultures of specific 135 (National Cancer Institute, Tissue 5 Serum-free cell culture cell types, as weIl as for culturing contin- Culture Section) medium (Harn and uous cell lines, are published in increas- McKeehan, 1979; Morton, 1970). Today, the importance of culturing mam- ing number (Barnes and Sato, 1980a; The basic medium (DMEM/Ham malian cells in medium without serum sup- Taub, 1990) and their commercial F-12) is then supplemented with specific plementation is widely recognised (Bjare, availability is increasing steadily (De- factors, like hormones and growth fac- 1992; McKeehan et al., 1990). With the francesco, 1998). Therefore, one key step tors, fatty acids and lipids, and vitamins identification, cloning, and recombinant towards the use or the development of a and trace elements (Taub, 1990). In ad- production of essential growth factors and serum-free formulation for a desired cell dition, due to the lack of attachment and nutrients required by different cell types type and/or cell line may include a re- spreading factors in serum-free medium (Bames et al., 1987), a broad selection of view of the published literature, scanning formulations, a precoating of culture ves- chemically defined, serum-free media for of commercial catalogues, and contact- sels with components of the extracellular continuous celllines as well as for specific ing other laboratories working in similar matrix is required for some cells (Klein- cell types in primary culture has been de- fields in order to determine whether a man et al., 1987). During the last decades signed and is available (Bames and Sato, suitable media formulation for a specific it turned out that for most cell lines the 1980a, 1980b; Bottenstein et al., 1979; cell line might already exist. Most re- supplementation of DMEMlHam F-12 Froud, 1999; Merten, 1999; Taub, 1990). cently, important initiatives were started with insulin, and selenium is From the disadvantages in the use of aiming to establish searchable data banks an obligatory requirement. Insulin is FBS in culture media, as listed above, the containing all serum-free media formula- essential for glucose transport into cul- advantages and benefits of serum-free tions that have been published for the ini- tured cells. It is added at relatively high media are easily deducible: (i) chemical- tiation of specific primary cultures and concentrations (1-10 ug/ml) due to its ly defined and controlled culture condi- the maintenance of continuous cell lines, rapid loss in bioactivity. Insulin and its tions in vitro, (ii) reduced variability in respectively (Falkner et al., 2003; Fischer degradation products may also mimic the qualitative and quantitative culture medi- et al., 2001; Strebel and Fischer, 2003). growth-stimulatory activity of related um cornposition, (iii) elimination of a po- Based on the pioneering work of Sato peptides, like insulin-like growth factors tential source of microbial contamination and coworkers (Bames and Sato, 1980a, (IGF) and somatomedins. Transferrin is (Froud, 1999; Merten, 1999), (iv) advan- 1980b), a basal medium for serum-free aserum protein that is responsible for tages in down-stream processing (i.e. the cell culture was established, consisting of cellular iron transport. Among the trace

278 ALTEX 20, 4/03 GSTRAUNTHALER -----~~I~ ------~, elements, selenium, added as sodium se- water-soluble form. The present "state- widely used renal epithelial cell lines lenite, seems to be important as cofactor of-the-art" -technologies in serum-free LLC-PKI, MDCK and OK (Gstraun- of selenium-dependent enzymes, like cell culture are summarised in Tabs. 5-8. thaler, 1988) are described (Chuman et glutathione reductase and glutathione Serum-free media are highly cell al., 1982; Leiderman et al., 1989; Taub et peroxidase, catalyzing glutathione specific. Therefore, for a certain ceIlline al., 1979). It was noted, that for optimal metabolism and turnover. Based on this serum-free medium formulations have to growth an adenylate cyclase-stimulating knowledge, ITS - insulin-transferrin- be developed on an individual basis. For hormone was required: vasopressin for selenite - is commercially available as example, serum-free media for the differ- the LLC-PK, cellline, and prostaglandin an universal medium supplement. Other ent cell types lining the mammalian EI in case of MDCK cells. The cAMP- hormones used in serum-free cell culture nephron - proximal tubular ceIls, thick generating hormones could be replaced are parathyroid hormone, glucocorti- ascending limb cells, distal tubular and by dibutyryl-cAMP, a ceIl-permeable coids (e.g. hydrocortisone, dexametha- collecting duct cells - have been elabo- derivative, or by the phosphodiesterase sone), thyroid hormones (triiodothyro- rated (Sens et al., 1999; Taub and Sato, inhibitor isobutyl methylxanthine nine), glandotropic factors, like 1980). Based upon the fact that renal (IBMX), inhibiting intracellular cAMP thyroid-stimulating hormone, adreno- tubular epithelial cells differ substantial- breakdown (Taub, 1990). Also cholera corticotropin, or follicle-stimulating ly in their metabolie properties (Wirthen- toxin was successfully introduced for hormone, and estrogens (e.g. estradiol). sohn and Guder, 1986) and hormone re- culturing rat proximal tubule cells under Most commonly used growth factor in- ceptor expression profile (Morel, 1981), serum-free conditions (Hatzinger and gredients are EGF, FGF, NGF, and nephron-specific, chemically defined, Stevens, 1989). Thus, elevated cellular PDGF, mostly available as recombinant serum-free media could be designed cAMP levels are essential for growth and peptides. Furthermore, small lipid (Chung et al., 1982; Taub, 1990; Wang proliferation. Obviously, cAMP-regulat- preparations as weIl as essential unsatu- and Taub, 1991; Wilson and Horster, ed protein kinase A, together with acti- rated fatty acids and ethanolamine have 1983). In addition, serum-free media for vated MAP kinases, may play a crucial to be added, which can be purchased in growth and maintenance of the most role in regulating G 1-cyclins and GI to S

Tabs. 5-8: Contemporary "state-of-the-art"-technologies in serum-free cell and tissue culture.

Serum-free Cell Culture Serum-free Cell Culture (2): "state-of-the-art"

• chemically defined, standardised culture media basal medium DMEM/Ham's F-12 (50:50)

adhesion factors coating of culture vessels basal medium supplemented with synthetic or recombinant with collagen (type I, type IV) products laminin etc > adhesion-, attachment- and spreading faetors > growth factors and hormones 9rowth factors epidermal growth ractor (EGF) > proteins (transport proteins, albumin) fibroblast growth factor(FGF) > vitamins und trace elements platelet-derived growth factor (PDGF) > fatty acids and lipids, ethanolamine nerve growth faetor (NGF) insulin-like growth factor (IGF) > protease-inhibitors (trypsin inhibitor) cytokines (interferons, interleukins)

Serum-free Cell Culture (3): "state-of-the-art" Serum-free Cell Culture (4): "state-of-the-art"

hormones insulin proteins bovine hydrocortisone, dexamethasone transferrin trijodo-thyronine, thyroxine progesterone, estradiol vitamins vitamin A-derivatives: retinoic acid prolactin retinol-acetate glucagon vasopressin trace elements sodium selenite PTH fatty acids and lipids linoleic acid, oleic acid prostaglandins cholesterol, phospholipids ethanolamine

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