Serum-free medium

Two major disadvantages in the use of serum (lack of reproducibility in quality and possible risk of contamination) are avoided by using serum-free medium. In addition, it is possible to culture a wider variety of differentiated cells using defined media. 

Growth with various commercial serum-free media and media containing serum 

Basal medium Factor added % Growth Cost ( /l)  

Mito+ is supplied by Collaborative Research Products through Flow Laboratories Limid., as a X1000 concentrate. 

Hybri-Clone SF is supplied by ICN as a Xl medium or as a X100 concentrate (SF-M) to be added to normal medium. 

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Andersen et al. (1996) and Andersen (1995) have studied the effect of temperature on the recombinant protein production using a baulovinis/insect cell expression system. In Tables 17.15, 17.16, 17.17, 17.18 and 17.19 we reproduce the growth data obtained in spinner flasks (batch cultures) using Bombyx mori (Bm5) cells adapted to serum-free media (Ex-Cell 400). The working volume was 125 ml and samples were taken twice daily. The cultures were carried out at six different incubation temperatures (22, 26,28, 30 and 32 TT). 

Induces SGs Requires serum-free media, works on all cells tested... 

A study of the potency of the antibiotic daptomycin cited plasma protein binding of 92%, but it claimed only a 2-fold shift in potency in serum (expected 12-fold) [68]. This type of discrepancy is relatively common and can often reflect substantial binding to components in the "serum-free" media. In the cases of HIV-directed non-nucleotide reverse transcriptase inhibitors, this has been dealt with by measuring the unbound drug concentration in the "serum-free" medium and using that data to calculate the intrinsic, serum-free potency [69]. 

Taub, M. (1984). Growth of primary and established kidney cell cultures in serum-free media. In Methods for Serum-Free Culture of Epithelial and Fibroblastic Cells (Barnes, D.W., Sirbasku, D.A.A. and Sato, G.H., Eds.). Alan R. Liss, New York, pp. 3-24. 

At SmithKline, the scientists in manufacturing had already expressed a preference for having every cell line grown in bioreactors (fed-bateh fermenfation tanks) and for having every cell line grown in serum-free media. (The fermenfation tanks used in the industry today are basically adapted beer vats, which is why so many of my old colleagues in... 

XTT stock solution (fresh) Dissolve XTT in serum-free media as a 1 mg/mL solution. XTT solution is warmed at 37°C for 5 min to dissolve the XTT completely (cloudiness will disappear). 

In Section 2, factors that could lead to particle assembly and secretion into the supernatant were discussed. At this point a deeper analysis of the factors affecting cell infection will be made. Optimisation of the production process should take into account virus-cell interactions, and more specifically viral attachment and internalisation into the cell. The impact of chemical modifications of the medium in baculovirus attachment-internalisation has not been carefully studied. It is widely known for example, that serum increases the infec-tivity of baculovirus. These reviewers have had one case where we were only able to succeed in infecting Sf9 cells adapted to growth in serum-free media [52], with a baculovirus produced by Sf9 cells (not adapted to grow in serum-free media), after adding serum to the culture (authors unpublished observations). However, since serum is not desirable for use in industrial production, its utilisation should be avoided as much as possible. 

Incubation temperature and medium pH are also important regarding proteolytic activity of baculovirus infected insect cell cultures. Cruz et al. [25] have shown that the highest proteolytic activity was obtained at the normal culture conditions, 27 °C and pH 6.5. This could then be considered a drawback when the production of protease sensitive particles Hke HIV-CLPs and HIV-VLPs is envisaged [5]. The pH of Sf9 cells has been reported to reach a minimum of 5.9 in serum-free media under uncontrolled pH conditions in stirred tank reactors... 

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). 

Helinski EH, Bielat KL, Ovak GM, Pauly JL. Long-term cultivation of functional human macrophages in Teflon dishes with serum-free media. Journal of Leukocyte Biology 1988, 44, 111-121. 

The need to deal with some of these differences demands enormous efforts for the development of culture media (chemical environment) or shear, mixing, viscosity and bubbling conditions (physical environment), which should be optimized to result in an industrial process that can be validated. Requirements to avoid contamination have led to the formulation of serum-free media or even of protein-free, chemically defined media for the production of biopharmaceuticals (Griffiths, 1988). 

HEK-293 cells, derived from human embryo kidney, were transformed with human type 5 adenovirus (Graham et al., 1977). These cells exhibit epithelial morphology and can be adapted to suspension growth in serum-free media. In addition, this cell line is easily transfected and has been explored for viral vector production for gene therapy and for obtaining human recombinant proteins with normal glycosylation profiles. 

High-Five cells (BTI-TN-5BI-4) are derived from Trichoplusia ni cells and are frequently employed due to their capacity to express high protein levels when compared with other insect cell lines, such as Sf-9 cells (Rhiel et al., 1997). This cell line shows high growth rates in adherent culture, and can easily be adapted to grow in suspension and in serum-free media. 

Many attempts have been made to develop culture media that do not need this type of supplementation, that is, serum-free media formulations. These serum-free media present several practical advantages. However, the formulations that have been developed are specific for certain cell types and a universal culture medium suitable for the culture of all animal cell types seems, so far, unreachable. 

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). 

Many cells require media supplemented with complex B vitamins, while other vitamins are presumably supplied by the addition of serum to culture media. Nevertheless, when serum-free media are employed, not only the water-soluble vitamins should be provided, but also the lipid-soluble ones, such as biotin, folic acid, niacin, panthotenic acid, thiamine, and ascorbic acid, as well as the vitamins B12, A, D, E, and K. 

Another fundamental compound for cellular growth is insulin, which presents weak affinity to IGF-1 receptors. Insulin can activate several mitogenic responses, through IGF-1 receptors when in large doses. However, insulin is also added to most serum-free media due to its ability to promote anabolic metabolism, such as oxidation processes, glycogen synthesis, and amino acids transport (Jenkins, 1991). 

Some cell lines, such as Namalwa, can grow satisfactorily in medium in which the only protein is albumin. Other cell lines show distinct protein requirements, such as albumin, transferrin, and insulin, or the addition of polypeptide growth factors, isolated from non-serum sources that have shown stimulation of many cell types in culture. Some cells have very fastidious growth requirements and their stability and productivity may be reduced significantly in serum-free media. 

Schlaeger EJ (1996), The protein hydrolysate, Primatone RL, is a cost-effective multiple growth promoter of mammalian cell culture in serum-containing and serum-free media and displays anti-apoptosis properties, J. Immunol. Methods 194 191-199. 

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... 

Traditionally, the production of mAbs uses complex culture media containing glucose and amino acids as the main sources of carbon for cell metabolism, as well as vitamins, micronutrients and sometimes animal serum, usually fetal bovine serum. Chapter 5 provides a discussion on composition of culture media and recent trends in the search for formulas that do not require the use of animal serum, or of proteins of animal origin. These serum-free formulations use substitutes such as peptones, epithelial and fibroblast growth factors, hydrolysates, yeast extract, choline, and inositol. For the production of mAbs, various serum-free formulas are available, some of these developed specifically for a given cell line (Chu and Robinson, 2001). The development of those media is easier for non-anchorage-dependent cells, such as those used for mAb production. Thus, approximately 50% of the antibodies for therapeutic use are already produced using serum-free media. In some circumstances, the elimination of serum should be accompanied by the addition of other substances with the same shear stress protective effect of serum proteins,... 

Glassy MC, Tharakan JP, Chau PC (1988), Serum-free media in hybridoma culture and monoclonal antibody production, Biotechnol. Bioeng. 32 1015-1028. 

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. 

The aim of most technologies is to generate a population of cells each of which secretes a desired product into the medium. For this to occur the cells do not need to be actively dividing and, in fact, this is sometimes counterproductive. Cells can remain viable at high cell densities using perfusion systems (Chapter 3) and the product purified from the spent medium (Spier, 1988). Furthermore, the simpler the growth medium (i.e. the fewer protein factors required) then the easier is the downstream processing and this is one reason for the development of serum-free media especially for the culture of hybridoma cells used for monoclonal antibody production ( 5.8). 

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