Insulin serum supplement

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.)...

Fetal bovine serum can be effectively replaced by a few known proteins such as albumin, transferrin, and insulin as supplement to basal cell culture medium. 

F9 embryonal carcinoma cells have a simple set of growth supplements which are required for growth in serum-free medium insulin, transferrin, and fibronectin (Rizzino and Sato, 1978). Fibronectin is a component of the extracellular matrix and facilitates the attachment of the cells to the culture dish. In addition, high density lipoprotein (HDL) has been observed to promote the growth of F9 cells serum-free. 

Figure 10. Primary cultures of mouse kidney cells. Primary cultures of kidney epithelial cells derived from 10-day-old mice were grown either in hormonally defined medium with five supplements (5 pg/ml insulin, 5 pg/ml transferrin, 25 ng/ml PCE, 5 X10" M hydrocortisone, and 5 x 10" M Tj), or in medium supplemented with 10% fetal calf serum. After 10 days, primary cultures still were epithelial in morphology serum free (a) but were overgrown with fibroblasts with serum (b). (Taub et al., 1979 with permission.)...

KHALIL D A, LUCAS E A, lUMA S, SMITH B J, PAYTON M E and ARJMANDI B H (2002) Soy prOteiu supplementation increases serum insulin-like growth factor-1 in young and old men but does not affect markers of bone metabolism. J Nutr 132, 2605-8. 

Fig. 7.3.1. Effect of estradiol on MCF7 cell proliferation. MCF7 cells were grown for 6 days in estrogen-depleted medium supplemented with 5% charcoal dextran-treated human serum (5% CDHuS) or with insulin (100 ng mP1) and transferrine (25 p,g ml-1) (ITDME). Estradiol was added to cultures at concentrations ranging from 0.1 pM to 1 nM. The maximal estrogenic response corresponded to 10 pM of estradiol and higher concentrations. Cell yields were six-fold (10 pM, 6.7 1.2) those of control. No proliferative response was observed in cells maintained in ITDME. Each point is the mean of three counts from four culture wells bars indicate SDs.

The second category includes mesenchymal cells, such as fibroblasts (BALB/c 3T3, Swiss 3T3), adipocytes, endothelial cells, smooth thin muscle cells, and neuroectodermic cells (such as glia cells). Most of these cells need maintenance factors. Some cells, such as the NIH-3T3, can grow in a serum-free medium containing minimal medium supplemented with transferrin (25 pg/ml), insulin (10 pg/ml), EGF (100 ng/ml), bFGF (100 ng/ml), and PDGF (0.5 U/ml). 

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. 

The requirements for epithelial cells are somewhat different (Reiss and Dibble, 1988). Mouse keratinocytes (MK-1 cells) enter a GO-phase within 24 h when confluent cultures are fed a serum-free, low Ca2+ (< 0.1 mM) medium supplemented with insulin, transferrin and sodium selenate (see 5.8). Addition of EGF (10 ng/ml) causes cells to enter S-phase after 10-12 h although the percentage of cells responding is not known. Insulin is not essential for this effect but apparently leads to a threefold increase on the rate of DNA synthesis measured 22-24 h after addition of EGF. TGF/ (100 pM) completely abolishes the effect of EGF. 

In monoclonal antibody purification, biological risks are primarily related to the host animal cells, but also to animal supplements for culture medium such as fetal bovine serum or pure proteins (e.g., bovine albumin, insulin, and transferrin). A special risk associated with production of antibodies with rodent cell lines is their high load of C-type particles. These particles are considered as incomplete retroviruses. The danger regarding infecting humans is not clear. Thus, the efficient separation of these particles must be guaranteed. These particles are quantified either by immunological techniques or electron microscopy. 

Amato P, Morales AJ, Yen SS. Effects of chromium picolinate supplementation on insulin sensitivity, serum lipids, and body composition in healthy, nonobese, older men and women. J Gerontol A Biol Sci Med Sci 2000 55(5) M260-3. 

The development and use of serum free hormonally supplemented medium is, however, a step in the right direction. The apphcation of defined medium allows a more standardized approach to cell culture delivering greater reproducibility and transferability. For renal tubular cells, defined medium supplements have been described as far back as 1982 [64], and we have successfully cultured human renal proximal tubular cells in defined medium containing EGF, hydrocortisone, insulin, transferrin and sodium selenite using DMEM-Hams F12 as the base medium [42]. 

Transfer a portion of the cells to 2% serum. At this serum level it is harder for the cells to adapt. If the cells do not grow, try transferring a new aliquot of cells from 5% to 4% or 3% serum and/or try adding defined supplements (e.g. insulin). For more information on medium supplements, refer back to Types of serum-free media . At each step, when the cells have become adapted, freeze some of the cells. Freeze a larger stock of vials when the cells are adapted to 2% serum. Should problems arise later on, this is a good point to return to. 

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