Transferrin cell culture

Cell Culture-Derived Media-Derived Protein Impurities. Immunoassays can detect low impurity levels (<1 ppm).4 The ELISA is probably one of the most sensitive analytical methods. If bovine serum is used as a media component, then testing should include ELISAs for bovine serum albumin (BSA), bovine transferrin, bovine fetuin, and bovine IgG. Often hormones and growth factors, such as insulin or insulinlike growth factor, are used as media components. ELISAs should be used to detect and quantitate these residuals in the various production steps as well as in the final product. There are commercially available antibodies to most commonly used media components. If proprietary media components are used, then the same investment in time and effort is required for the production of specific antibodies, as described above for host cell impurities. 

Takeda A, Devenyi A, Connor JR (1998) Evidence for non-transferrin-mediated uptake and release of iron and manganese in glial cell cultures from hypotransferrinemic mice. J Neurosci Res 51 454-462... 

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. 

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]. 

Sievertsson, H., Fryklund, L., Uthne, K., Hall, K., and Westermark, B., Isolation and chemistry of human somatomedins A and B. Ado. Metah. Disord. 8, 47-60 (1975). Skinner, M. K., and Griswold, M. D., Multiplication stimulating activity (MSA) can substitute for insulin to stimulate the secretion of testicular transferrin by cultured Sertoli cells. Cell Biol. Int. Bep. 7, 441-446 (1983). 

Since the original description of Detrisac et al. [46] a 1 1 mixture of DMEM and HAMF12, supplemented with hydrocortisone, triiodothyronine, insulin, transferrin and selenate (currently known as K1 medium) [51], has been the most widely used medium in human (proximal) tubular epithelial cell culture. Several authors add serum because of its growth promoting effect. 

Suarez N, Walum E, Eriksson H. 1995. Cellular neurotoxicity of trivalent manganese bound to transferrin or pyrophosphate studied in human neuroblastoma (SH-SY5Y) cell cultures. Toxicol in Vitro 9 717-721. 

Darlington (1977) obtained hybrids of somatic cells which were isolated from hepatic and other cell cultures of mice (i.e., of hepatic and nonhepatic origin). The hepatic cells HePa la and HH synthesize albumin, transferrin and a-fetoprotein. Most somatic Hybrids between these cells and L-cells did not demonstrate albumin and a-fetoprotein synthesis, but continued to synthesize transferrin. This type of expression was similar for hybrids with one and two doses of the hepatic genes. In hybrid cells between hepatic cells and embryonic hepatic cells, 3 of 17 cells synthesized embryonic a-fetoprotein. The investigators suggested that albumin and a-fetoprotein production in the hybrid cells depends upon the epigenetic status of the nonhepatic parent. 

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.)...

While I fully understand concern about animal welfare, it is difficult to see how our understanding of iron metabolism, not to mention the development of new drugs, can evolve without carrying out animal studies. It is difficult to see how one can extrapolate from results obtained using cultured cell lines which typically have very large numbers of transferrin receptors when compared with the normal cells from which they are derived. 

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.

QDs recognized specific antigens/antibodies -DNA immobilization to QDs surfaces and possibility of hybrid assemblies [35] -Coupled to transferrin, QDs underwent receptor-mediated endocytosis in cultured HeLa cells... 

Bacterial hosts are inappropriate choices for expression of proteins such as the blue copper proteins stellacyanin, laccase, and ceruloplasmin which are extensively glycosylated. In these cases, it may be necessary to employ tissue cultures of appropriate origin to obtain the native protein. In this regard, the amino-terminal half of human serum transferrin, which lacks carbohydrate, has been expressed in high yield in baby hamster kidney cells by Funk et al. [13], while the glycosylated carboxyl-terminus has proved to be more problematic [103]. 

Ogris, M., Steinlein, P., Kursa, M., Mechtler, R. and Wagner, E. (1998) The size of DNA/ transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther., 5, 1425-1433. 

Yeh, C. J., and Faulk, W. P. Killing of human tumor cells in culture with adriamycin conjugates of human transferrin. Clin. Immunol. Immunopathol. 32 1—11, 1984. 

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. 

---