Serum-Free Media Supplement
ITSE (Insulin, transferrin selenium & ethanolamine) In order to achieve successful in vitro growth of cells and tissue, cell culturing methods typically attempt to mimic physiological conditions. One of the key factors of this technique is the selection of the culture medium. The most widely used media supplement is animal serum. The serum is a highly complex mixture of numerous components that exhibit a variety of biological activities. These include providing growth factors, hormones, and trace elements, as well as attachment and spreading factors.
The high albumin content assists in cell maintenance by preventing pH fluctuations, inhibiting proteases, and protecting from shear forces [1, 2].Despite the serum’s excellent support of cell and tissue growth, there are significant disadvantages to using serum as a supplement to culture media. These include a relatively high cost, a decline in availability, an increase in experimental variability, a possible obstruction to downstream processing, and the potential of being a source of contamination and adverse factors, as well as the raising awareness of animal welfare and suffering [2, 3].
The shortcomings of the use of serum in cell culturing has led inrecent years to a high demand for serum-freemedia formulations, both from the research andindustry groups[3]. Besides overcoming the disadvantages of using serum in culture media, serum-free media has two majorbenefits, which are the ability to create specificmedia for a particular cell type and the ability to exercise finer control of cellular proliferation and differentiation processes [2]. The basal serum-free media is highly enriched and was developed to supply the cells with all of their essential nutrients in sufficient quantities. Typically, the basic media is further supplemented with specific factors, such as growth factors, fatty acids, vitamins and trace elements. The experience that was accumulated during the years of using serum-free media indicated that for most cell lines it was obligatory to supplement the basal media (mostly DMEM/F12) with ITS (insulin, transferrin and selenium) [4], while for some cell lines it was essential to also add ethanolamine to the media [5]. Why ITSE? The use of the ITSE supplement instead of the common ITS has the advantage that the users are not always aware that the cell line they use is among those cell lines that require ethanolamine for optimal growth and the use of the ITSE supplement covers this option.
Insulin
Transferrin
Supports cell growth and regulates the cellular uptake and utilization of glucose, amino acids, and lipids.
Is a glycoprotein that is abundant in serum and can reversibly bind Fe3+. It has a central role in iron transport and metabolism and is an important extracellular antioxidant, and therefore, an essential supplement to serum-free media. Initially, transferrin was isolated from serum, however, due to consistency and safety issues, recombinant transferrin was developed with equivalent activity in a variety of systems, including plants [7].
Insulin also has anti-apoptotic properties. In mammalian cell culture, recombinant insulin is added in concentrations much higher than the physiological concentration. At this high concentration, insulin acts as a growth factor with mitogenic and anti-apoptotic effects through activation of the insulin-like growth factor (IGF-1) receptor, in addition to the metabolic and anti-apoptotic effects mediated the insulin receptor (IR) [6].
Selenium
Ethanolamine
Is a trace element and a critical component of several enzymes such as glutathione peroxidase, thioredoxin reductase, and other antioxidant enzymes. It is known to be an essential addition to serum-free medium [8].
Is an organic compound, which is both a primary amine and a primary alcohol, and serves as phospholipid precursor. It plays an important role in the proliferation of hybridoma cells and is frequently added to the medium used for culturing hybridoma cells, as well as for other cell types [5, 9].
References: 1. Brunner, D., Frank, J., Appl, H., et al. (2010). Serum-free cell culture: the serum-free media interactive online database. Altex, 27(1), 53. 2. Freshney, R. I. (2005). Serum‐Free Media. Chapter 10 in: Culture of animal cells, John Wiley & Sons. pp, 129-143. 3. Butler, M. (2013). Serum-free media: standardizing cell culture system. Pharmaceutical Bioprocessing, 1(4), 315-318. 4. Gstraunthaler, G. (2003). Alternatives to the use of fetal bovine serum: serum-free cell culture. Altex, 20(4), 275-281. 5. Murakami, H., Masui, H., Sato, G. H., et al. (1982). Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component. Proceedings of the National Academy of Sciences, 79(4), 1158-1162. 6. Kim, J. J., & Accili, D. (2002). Signalling through IGF-I and insulin receptors: where is the specificity?. Growth Hormone & IGF Research, 12(2), 84-90. 7. Brandsma, M. E., Jevnikar, A. M., & Ma, S. (2011). Recombinant human transferrin: beyond iron binding and transport. Biotechnology advances, 29(2), 230-238. 8. Saito, Y., Yoshida, Y., Akazawa, T., Takahashi, K., & Niki, E. (2003). Cell death caused by selenium deficiency and protective effect of antioxidants. Journal of Biological Chemistry, 278(41), 39428-39434. 9. Tsao, M. C., Walthall, B. J., & Ham, R. G. (1982). Clonal growth of normal human epidermal keratinocytes in a defined medium. Journal of cellular physiology, 110(2), 219-229.
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