Sera Supplements

FGF), and hydrocortisone to grow serum free (see Table 4) (Hutchings and Sato, 1978). Both of these cell types can grow serum free with appropriate supplements at the same rate as that obtained in serum-supplemented medium. Both cell types can also survive over extended culture periods in hormonally defined serum-free medium. 

Figure 4c shows that the amount of adsorbed proteins is rapidly saturated within several minutes of exposing serum-containing medium to a surface. Albumin, the most abundant serum protein, was expected to preferentially adsorb onto the surfaces during early time points. Then, adsorbed albumin was expected to be displaced by cell adhesion proteins. To investigate the effect of preadsorbed albumin displacement on cell adhesion, SAMs were first exposed to albumin then, HUVECs suspended in a serum-supplemented medium were added [21, 42]. Very few cells adhered to hydrophobic SAMs that had been pretreated with albumin, due to the large interfacial tension between water and the hydrophobic surfactant-like surface. Albumin was infrequently displaced by the cell adhesive proteins Fn and Vn. One the other hand, HUVECs adhered well to hydrophilic SAM surfaces that had been preadsorbed with albumin. In that case, the preadsorbed albumin was readily displaced by cell adhesive proteins. 

The transfection efficiency and stability of some peptide/ siRNA complexes may be affected by the presence of serum. It is suggested to prepare the complexes in the absence of serum initially. Serum-supplemented culture medium can be used with complexes that show good transfection efficiency in the absence of serum. 

Mercille et al. [141] compared TFF and VFF for the perfusion cultivation of a hybridoma cell line producing an IgM, either using serum-supplemented or protein-free medium, with and without the addition of DNAse. They observed... 

The serum in the medium is not only expensive but also can be the source of virus or mycoplasma contamination. Since the chemical nature of serum is not well defined, its contents may vary batch after batch, which can affect the result of culture. The presence of many different proteins in serum can also complicate the downstream separation processes. For these reasons, many attempts have been made to formulate serum-free media. These formulations contain purified hormones and growth factors which can substitute for serum supplements (Butler, p.ll, 1987). 

It is also unlikely that the increased GSL in these cells is related to the uptake of GSL from serum supplemented medium, since the medium has previously been shown not to be the primary source of cellular GSL in cultured fibroblasts (29) (see also above). The effect of serum and lipoproteins on GSL biosynthesis will be the subject of future studies in this laboratory. 

Indirect patterning A protein repellent background is locally opened, rendering areas of the surface prone to protein adsorption. The patterning of cell-attractive areas is indirectly achieved by a subsequent deposition of proteins either by preincubation with a solution of proteins (most prominent are fi-bronectin, vitronectin, and laminin) or by adsorption from serum-supplemented cell culture media during cell seeding. 

The regulatory mechanism of cellular uptake of fatty acids appears to be limited and so the composition of the intracellular lipids is likely to reflect the availability of the fatty acids in the medium. This was shown for the CC9C10 hybridoma (Butler et al., 1997) and for BHK and CHO cells (Schmid et al., 1991). Thus, cells growing in serum-supplemented cultures are likely to attain a fatty acid composition reflecting that of serum, in which the predominant fatty acids are palmitic, stearic, oleic, and linoleic acids at a ratio 2 1 3 1, respectively. 

For insect cells the following basal media can be used Grace s, TC 100, TNM-FH, D22, Schneider, and M3. These media normally require supplementation with fetal bovine serum. Alternatively, different serum-free media are available for insect cells, such as Sf900II, Ex-Cell 400, 405, and 420, Express Five SFM, Insect-XPRESS , HyQ SFX-Insect , and IPL 41. These have the advantage of higher reproducibility and lower cost when compared with serum-supplemented basal media (Ikonomou et al., 2003). 

Any new formulation may be evaluated against the properties of a serum-substituted medium for growth and productivity. As well as growth promotion, a serum supplement in medium may also lead to a reduction in cell death by apoptosis and this allows extended culture longevity. This can enable a substantial increase in recombinant protein productivity. 

Protein hydrolysates may provide an alternative to a serum supplement. Such hydrolysates contain oligopeptides, peptides, and amino acids ob-... 

Several different cell lines have already been investigated in perfusion cultures using the acoustic cell filter. For instance, Shirgaonkar et al. (2004) used NS0, HEK-293, SP2-derived hybridoma, and insect cells in different serum-supplemented and serum-free media at different perfusion rates and acoustic chamber volumes. According to these authors, an adequate operation of the filter depends on optimization of operational... 

Moro AM, Rodrigues MTA, Gouveia MN, Silvestri MLZ, Kalil JE, Raw I (1994), Multi-parametric analyses of hybridoma growth on glass cylinders in a packed-bed bioreactor system with internal aeration. Serum-supplemented and serum-free media comparison for MAb production, J. Immunol. Methods 176 67-77. 

Fig. 5.2. Primary baby mouse kidney cultures were established at about 103 cells/cm2 in medium based on a 50 50 mixture of DMEM F12 supplemented with 10% FBS (a) or a mixture of 5 hormones (PGE1, hydrocortisone, triodothyronine, insulin and transferrin (b). Although over 99% of the attached cells were epithelial at day 1, by the time the photograph was taken (day 11), fibroblasts had completely overgrown the epithelial cells in the serum-supplemented medium. Only epithelial cells are present in the hormone-supplemented culture. (Reproduced from Taub et al., 1979, with thanks.)...

Figure 1. IR spectrum of freshly prepared RPMI 1630 cell culture medium (+5% calf serum supplement) with no cells present...

Walker et al. demonstrated that streptomycin (a usual antibiotic additive to cell culture media) has synergistic effects on CSA toxicity to LLC-PKj cells [185]. Since that publication most investigators have conducted CSA experiments without antibiotics. This represents a good example that the utmost of care must be taken to exclude the possibility of interfering substances such as growth hormones, serum supplements and antibiotics, when conducting toxicity studies in vitro. 

Normal animal tissues (e.g., skin, kidney, liver) or whole embryos are commonly used to establish primary cell cultures. To prepare tissue cells for a primary culture, the cell-cell and cell-matrix interactions must be broken. To do so, tissue fragments are treated with a combination of a protease (e.g., trypsin or the collagen-hydrolyzing enzyme collagenase or both) and a divalent cation chelator (e.g., EDTA) that depletes the medium of usable Ca or Mg. The released cells are then placed in dishes in a nutrient-rich, serum-supplemented medium, where they can adhere to the surface and one another. The same protease/chelator solution is used to remove adherent cells from a culture dish for biochemical studies or subculturing (transfer to another dish). 

Chung H A, Kato K, Itoh C, et al. (2004). Casual cell surface remodeling using biocompatible lipid-poly (ethylene glycol) (n) Development of steal cells and monitoring of cell membrane behavior in serum-supplemented conditions. J. Biomed. Mater. Res. 70A 179-185. 

Cell quiescence under neither serum supplemented medium nor analogous knockout replacement Quiescence Quiescence Non-quiescence... 

B. Difficulties When Employing Single Type Serum Supplemented Media... 

Recommended formulations employ commercially available components for Eagle s Minimal Essential Medium (MEM). Suggested supplements collectively enhance retention of diploidy, facilitate in vitro adaptation of malignant drivatives, minimize serum toxicity, and stabilize ionic and osmological relationships between cell and medium. These modifications are recommended when single-serum supplemented MEM Eagle s and... 

Coated dishes Make fresh a 0.1% gelatin solution (Sigma) in PBS by gentle heating and shaking. Filter-sterilize while still warm. Completely cover the base of tissue-culture dishes. Leave for 2 h at room tempoature, and then aspirate off and wash IX with sterile PBS. Plate cells or cover with fiesh PBS and store at d C (maximum 2 wk). Serum-free medium 10 mL media, 20 pL N3 (serum suppl ent—see item 6). N3 serum supplement 246 pL Hank s balanced salt solution (HBSS) without calcium and magnesium, 50pL 10 mg/mL bovine serum albumin in HBSS (store at 4°C), 100 pL 100 mg/mL human transferrin in HBSS (store at -20°C), 20 pL 80 mg/mL putrescine hydrochloride in HBSS (store at -20 C), 50 pL 10 mAf sodium selenate in... 

Most historical medium formulations were supplemented with serum, tissue extracts, or other humoral fluids. As these supplements were vital to the success of the technique they undoubtedly supplied nutritional factors that were absent from the nutrient media. Although serum fractions have been characterized, total biochemical definition is a complex challenge, as it has been reported that serum contains more than 1000 proteins (Lambert and Birch, 1985). Complete characterization must identify cytokines and transport and attachment factors, as well as address other serum functions, such as pH buffering capacity, toxin inactivation, and protease activity. Such unspecified growth promotional and nutritional serum properties are perceived by users as quality. Other contributors to quality, inherent to serum supplementation, include lot-to-lot variability, availability, cost, and absence of adventitious contaminants. 

Reduced serum medium A basal medium that is supplemented by growth additives. The growth additives may partially substitute for nutritional aspects normally supplied by serum, so that the required level of serum supplementation may be significantly diminished. 

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