Protein-free cell culture media

Development of serum-free medium has great value for large-scale biopesticide production. The latest formulations are serum-free, such as SF900II (GIBCO /Invitrogen) and EX-CELL (JRH Biosciences). Cell culture medium supplementation using yeast extract (usually from alcoholic fermentation processes), milk, or soy protein concentrates, can also be an alternative to decrease cell culture medium costs (more details can be found in Chapter 5). 

A very important but often neglected aspect of in vitro toxicology is pharmacokinetics or toxicokinetics. Kinetics deals with how a test compound is altered by the system it is applied to. For an in vitro system, the available concentration of the compound can be decreased by binding to cell culture-ware such as a plastic cell culture dish, by binding to proteins in the cell culture medium, by evaporation and due to cellular uptake or cellular metabolism. The latter two points are critical in both in vitro and in vivo systems and are discussed in detail in Chap. 22. Knowing the actual concentration that cells can interact with, either by measurement as a free concentration in the cell medium or as a tissue concentration in the cell lysate, is crucial not only for experimental interpretation but also to extrapolate to the in vivo situation. Indeed, we must eventually extrapolate from in vitro to in vivo in order to establish safe exposure limits, which is after all the end goal of the exercise. These issues are dealt with in Chaps. 23 and 24. 

Cell culture medium Protein-free medium... 

ESF 921 Insect Cell Culture Medium, Protein-Free (Expression systems, 96-001-01)... 

Pushotten and Pineus (1961) reported that rabbit embryos of two and eight cells grew and developed for 4 days on a protein-free chemically defined medium containing amino acids. However, early blastocysts (72-hour embryos) required an additional medium supplement of 10 serum for successful culture. Daniel and Krishnan (1967), working with somewhat older rabbit blastocysts (5 days), observed that they expanded to a greater extent for the first 36 hours of culture when the defined... 

Although plant cell culture is not as cost effective as plant cultivation in the open field, it will become an economical process if higher protein yields can be achieved [58]. The cultivation medium of plants is chemically defined, consisting of a carbon source, minerals, vitamins and phytohormones [69]. Furthermore, it is protein-free and relatively inexpensive. In contrast, animal cells often require complex supplements such as fetal calf serum and/or expensive growth factors, although serum-free cultivation is possible in case of Chinese hamster ovary (CHO) cells [70]. 

Alice et al studied the turnover kinetics of Listeria OTonocytogenex-secreted p60 protein (a murein hydrolase) by host cell cytosolic proteasomes. J774 cells, seeded in flasks and incubated overnight in culture medium, were infected with log-phase cultures of E. monocytogenes for 30 min, washed, and incubated in culture medium for 3 h, with gentamicin (50 tg/ml) added after the first 30 min to inhibit extracellular bacterial growth. Cells then were washed and placed in methionine-free medium with spectinomycin, gentamicin, the eukaryotic protein synthesis inhibitors [cycloheximide (50 tg/mL) and anisomycin (30 tg/ml),] and 25 dVI calpain inhibitor I. After 30 min, [ S]methionine was added, and the cells were pulse-labeled for periods of 20 to 60 min. Cells... 

After cell cultivation, a cell separation step is generally needed to process the culture medium to purify the product. This step is performed either to obtain a cell-free medium for an extracellular product or to obtain a cell concentrate if the product is intracellular. In animal cell cultures, the product is normally secreted. Thus, after cell separation, the product is in solution and can be processed by chromatographic techniques or other protein purification methods, as presented in Chapter 12. 

Johnson M, Lantier S, Massie B, Lefebvre G, Kamen AA (1996), Use of the Centritech lab centrifuge for perfusion culture of hybridoma cells in protein-free medium, Biotechnol. Progr. 12 855-864. 

It is produced in recombinant CHO cells cultured in a medium free of animal-derived components. The BeneFix production process involves an ultrafiltration/diafiltration step, followed by four chromatographic steps ion exchange (Q resin), pseudo-affinity (Cellufine sulfate resin), hydroxyapatite, and affinity (immobilized Cu2+ ions). After these chromatographic steps, there are membrane processes (nanofiltration for viral clearance and diafiltration for solvent exchange), after which the purified protein is formulated (Edwards and Kirby, 1999). 

A serum-free medium supplemented with insulin, transferrin, ethanolamine and selenium (ITES) allows growth of certain hy-bridomas at 17-74% the rate found with 15% FBS (Wolpe, 1984) and Cleveland et al. (1983) devised a protein-free medium for growth of myeloma cells which, with addition of BSA at 2.5 mg/ml, forms the basis of Costar s SF-1 and SF-X supplemented media. Cloning is still very difficult in serum-free media, but feeder layers can be replaced by culture supernatants from human endothelial cells (HECS Astaldi, 1983) or Ewing s sarcoma cells (ESG Ley et al., 1980) — see 5.8.5. 

A 50 50 mixture of fresh growth medium with one of the above conditioned media is often recommended for growth of embryonic stem cell cultures. Otherwise the protein fraction can be concentrated for addition to serum-free medium. 

Cell growth during the time of the assay might influence the results, in the sense that the filling of the artificial wound could be contributed from both cell replication and movement. In order to avoid this problem, the assay should be conducted in serum-free or at least low-serum conditions, to limit cell growth. Alternatively, inhibitors of DNA synthesis (mitomycin-C or aphidicolin at 0.5 /Lig/ml), can be included in the culture medium, since it has been shown that active migration may depend on protein, but not DNA synthesis (Geimer and Bade, 1991 Chen et al., 1994). 

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