Foetal bovine serum

Tissue culture, more frequently used as cell culture, enables animal and plant cells to be cultured in large numbers by techniques comparable to those used in microbiology but, because of the fragile nature of the cells, does require special cultural conditions. The culture media used must supply all the essential factors for growth, such as a wide range of amino acids, nucleotides, enzyme co-factors as well as indeterminate factors that can only be supplied in special products, e.g. foetal bovine serum. The environmental conditions must be carefully controlled, particularly pH, and this is frequently maintained by culturing in a bicarbonate buffer system and a carbon dioxide saturated atmosphere. 

The growth requirements and properties of the host cell. Cells requiring foetal bovine serum will be more expensive to use than cells for which a cheaper serum source is adequate. Cells which grow in suspension are much easier to scale up to large cultures than cells which are anchorage dependent. 

In order to reduce costs the concentration of foetal bovine serum (FBS) can be reduced in growth media. Figure 5.3 shows the effect of lowering the serum concentration on the growth of a rat hy-bridoma cell line. Initially cells can be plated in 5% FBS and later this concentration can be reduced by using a combination of FBS (1.5%) and new bom calf serum. This leads to yields of diploid fibroblasts on Cytodex-1 microcarriers of 80% those obtained with 10% FBS (Clarke, 1983) ( 3.6). 

Ham s medium F12 has been designed specifically for cloning cells (Ham, 1965) and should be supplemented with 10-30% foetal bovine serum. Great care must be taken to prevent the medium becoming too alkaline by loss of C02. 

Exponentially growing cells are used and it is important to maintain constant pH and temperature throughout the selection procedure. Klevecz et al. (1974, 1975) have used CHO cells growing in McCoy s medium with 20% foetal bovine serum and Hepes buffer. They maintain the cells throughout in this medium at 37°C and, of the cells they select, 98-99% are in mitosis. The viability of these selected cells approaches 100% and on reseeding half the cells attach within 1 h of selection and maximum attachment is found by 4 h (Klevecz, 1975). However, they select a very small proportion of the original cells. 

Seed with 2 X 106 (9-cm dish) or 105 (TC well) cells in 10 ml or 0.5 ml Eagle s medium containing non-essential amino acids, penicillin-streptomycin and 10% foetal bovine serum (Appendix 1). 

Incubate at 37°C for 60-90 min to allow time for virus adsorption, overlay with Eagle s medium containing 5% foetal bovine serum (8 ml/dish 0.4 ml/well). 

FBS FGF FITC GO-phase G1-phase G2-phase GMP, GDP, GTP foetal bovine serum fibroblast growth factor fluoroscein isothiocyanate the resting stage of the cell cycle the first gap in the cell cycle (between M and S) the second gap in the cell cycle (between S and M) guanosine monophosphate, diphosphate, triphosphate... 

Suspension cultures can be achieved using F12 medium with 10% foetal bovine serum (FBS) and gassing the culture vessels continuously with 5% CO2. The doubling time under these conditions is typically 18 h versus 14 h for monolayer cultures. The growth requirements of CHO dhfr cells are hypoxanthine (or adenine), glycine and thymidine in addition to the usual requirement of CHO cells for proline. 

Low serum-containing medium Iscove s modified Dulbecco s medium (IMDM) (Sigma Chemical Company, St Louis, MO, USA) supplemented with 2% foetal bovine serum and 5 x 1(L M p-mercaptoethanol... 

Dulbecco s modified Eagle s medium (DMEM) with 10% foetal bovine serum (FBS)... 

Eagle s minimum essential medium (MEM) (or equivalent alternative) with 10% foetal bovine serum (FBS), Eagle s non-essential amino acids and other supplements as necessary (see General Principles , point 4). 

The conditions of cell culture are critical for chemical testing. Particular attention must be given to the medium used for the cultivation of cells. While the use of serum, usually foetal bovine serum (FBS), has long been customary to provide isolated cells with a pool of nutrients, attachment factors, and hormones, several issues are encouraging scientists to move towards serum-free hormonally defined medium [53]. In particular, the composition of FBS varies from batch to batch, rendering highly discriminative analyses such... 

AR42J and HT-29 cells were cultured in Ham s F12K nutrient medium supplemented with 5% foetal bovine serum, 100 lU/mL penicillin and 50 pg/ mL streptomycin. MCF-7 cells were cultured in Dulbecco s modified Eagle s medium (DMEM) containing 10% foetal bovine serum, 100 lU/mL penicillin, 50 pg/mL streptomycin and lOmM glutamine. All cell cultures were grown to confluence in a humidified atmosphere with 5% CO, at 37°C. Trypsin-EDTA solution was used for subculturing and cell isolation. 

The AR42J cell line expressing somatostatin receptor subtype 2 was obtained from ATCC (Manassas, VA, USA). The cells were maintained in Kaighn s modification of Ham s F12 medium (F12K) with 2mM L-glutamine, 10% foetal bovine serum and 1.5 g/L of sodium bicarbonate in a humidified atmosphere at 5% CO2 and 37°C. 

Cell seeding. Human fetal osteoblasts (hFOB 1.19, CRL-11372, American Type Culture Collection) were seeded at an initial density of 1 x 104 cells cm 2 in a medium containing a 1 1 mixture of Dulbecco s Modified Eagle s Medium (DMEM) without phenol red and Ham s F12 medium and supplemented with 0.3 mg/ml G418, antibiotics (100 U/ml penicillin and 100 pg/ml streptomycin) and 10% foetal bovine serum. Cells were grown for 48 h at 37°C in an humidified atmosphere of 5% C02 on the cell culture plastic supports (control), and sucrose thin films deposited on glass substrates (SI substrate). Prior to cell culture, the samples were sterilized by UV light exposure. 

There have recently been reports (Lowenberg et al, 19941 Callen et al, 1995) indicating that passivation of Ti-6A1-4V in HNO3 increases the release of all three constituent elements in a culture medium (a-Minimal Essential Medium with 15% foetal bovine serum and 10% antibiotics). Table 9.17 exemplifies the results obtained for titanium ions, for three periods of immersion of three days each. The level of Ti is significantly reduced throughout the 9-day experimental period. 

In these investigations, we have used A549 cells, derived from a human lung carcinoma, grown as adherent cultures in RPMl 1640 medium supplemented with 10% foetal bovine serum. The effect of either BLM or BCNU on cell proliferation was assessed by measuring the increase in the number of cells in cultures 3 days after drug treatment. This is expressed as percentage cell number increase , defined as... 

Apart from the concentrafion of sericin, its extraction method has been shown to influence cell viability, also. If compared to heat, acid, or alkaline extraction methods, urea-extracted silk sericin is linked to the lowest cell viability of L929 mouse fibroblast (Aramwit et al., 2010). Sericin can also be used to replace bovine semm in cell freezing media. It cryopreserves cells as effectively as standard freeze medium containing foetal bovine serum. This effect has been demonstrated for various cell lines including P3U1 myeloma cell line, Chinese hamster ovary CHO cells, human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC 12 and Sf9 insect cells (Sasaki et al., 2005), human primary hepatocytes (Miyamoto et al., 2010), rat pancreatic... 

Sericin is mainly used as a cell culture ingredient to replace foetal bovine serum. 

Terbutaline (10 M) after 2 days significantly (P<0.05) increased the expression of a-rENaC mRNA in rat alveolar type 11 cells isolated by elas-tase digestion, purified by a differential adherence technique, and cultured in minimum essential medium containing 10 % foetal bovine serum (Mina-KATA et al. 1996). Rat type II cells plated on Transwell membrane (but not on plastic dishes) incubated with 10 M terbutaline showed an increased binding of surfactant protein A (Chen et al. 1996). 10 M terbutaline increased Na -K -ATPase activity in cultured rat alveolar type 11 cells in an exposure time-dependent manner over 7 days in culture (Minakata et al. 1998). 

Arachidonic acid metabolism of THP-1 monocytic cells grown in RPMI supplemented with 10 % foetal bovine serum, 100 U penidlUn, 0.1 mg/ml streptomycin, and 2 mM L-glutamine was activated by purified lipopolysaccharide R595 from S. tninne-sota (Pfau et al. 1998). 

When B-lymphocytes from healthy volunteers were transformed by Epstein-Barr virus and maintained in RPMI 1640 medium with 10% foetal bovine serum, herbimycin A, a specific inhibitor of tyrosine kinase, significantly decreased 12-0-tetradecanoylphorbol-13-acetate-stimulated superoxide production in a dose-dependent manner (Yang et al. 2000). The amount of p91, the catalytic subunit of NADPH oxidase, was decreased in the cellular membrane of herbimycin A treated cells compared to untreated controls. Similar results were obtained for the movement of a regulatory subunit of the NADPH oxidase complex, p47. 

Osteoblastic cells were obtained from new bom rat calvariae, isolated and sub-cultured as described by Has awa et al. [17] and Chehroudi et al. [18]. Frontal, parietal and occipital bone were dissected, rinsed in phosphate-buffered saline (PBS, Fluka Chemicals, Buchs, Switzerland), placed in Dulbecco s modified Eagles medium (a-DMEM, Invitrogen, Basel, Switzerland), supplemented with 1% antibiotics (Penstrep, Invitrogen, Basel, Switzerland) and 10% foetal bovine serum (Invitrogen, Basel, Switzerland). The minced tissue was digested with a mixture of clostridial collagenase and trypsin (both from Sigma-Aldrich, Buchs, Switzerland) and then placed in tissue-culture flasks. 

Human gingival fibroblasts (HGF) were maintained according to the method of Elvin and Evans [19]. Stock cultures were recovered from liquid nitrogen and plated at 200,000 cells per 25 cm tissue-culture flask in a-DMEM with 10% foetal bovine serum and 1% antibiotics. 

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