Insulin-transferrin-selenium

HITS hydrocortisone/insulin/transferrin/ selenium supplemented Homog tissue discruption in sterile tissue homogenizer... 

Kisiday JD, Kurz B, DiMicco MA, Grodzinsky AJ. Evaluation of medium supplemented with insulin-transferrin-selenium for culture of primary bovine calf chondrocytes in three-dimensional hydrogel scaffolds. Tissue Eng 2005 11 141-51. 

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. 

HAU HECS Hep cells HPRT HSV HTC cells IAA ITES haemagglutinin unit human endothelial cell supernatant human epithelial cells hypoxanthine phosphoribosyl transferase herpes simplex virus hepatoma tissue culture cells indole acetic acid medium supplement containing insulin, transferrin, ethanolamine and selenium... 

The most frequently used additions for serum-free growth are insulin, transferrin and selenium. Recommended starting concentrations for these are 10 mg mH, 10 mg mH and 5 x 10 M, respectively. Many other additions are described in the literature for specific cell lines. 

Whilst growth factors in the serum provide specific proliferative stimuli, studies with cultured cells have indicated other important components for cell proliferation [18]. For example, insulin is required to facilitate glucose and amino-acid uptake, and transferrin, which binds iron, makes it available to the cell. Serum is also believed to supply trace elements such as selenium, copper and zinc as well as fatty acids important for cell growth. Some serum components such as ascorbate, a-tocopherol, caeruloplasmin and albumin may serve important antioxidant functions [19]. 

Cell lines differ in the defined proteins they require for optimal cell growth. Usually, addition of insulin (I) and transferrin (T), along with the trace element selenium (S), is sufficient to enable most cell lines to grow in rich balanced nutrient media, such as the ones listed above. However, several categories of additives may enhance or be required for cell growth and/or production of the desired... 

Selenium, pyruvate, insulin, and transferrin (SPIT) solution (Sigma). 

For further work, serum has been replaced by insulin and transferrin, and a routine assay for retinoids in serum-free medium is performed as follows (Breitman et al., 1980a) HL-60 cells are seeded at a density of 2 x 10 cells per ml in defined medium, which is a 1 1 mixture of Dulbecco s modified Eagle s minimum essential medium and Ham s F-12 medium supplemented with 3 x 10 M selenium dioxide, insulin (5 xg per ml), and transferrin (5 ig per ml). Retinoids are added to the HL-60 cells at the start of the assay, using ethanol as the vehicle final concentration of ethanol does not exceed 0.1%. The cells are incubated for 4 or 5 days, and differentiation is then measured by NBT reduction (Collins et al., 1979) of cytospin slide preparations. Results are expressed as percentage of NBT positive cells. Approximately 4-8% of the cells will differentiate spontaneously in the absence of added retinoid. As with the F9 system, dose-response curves have been determined for a large number of retinoids, and some of the more significant results are shown in Table III. 

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