Transferrin cell culture medium

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. 

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. 

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]. 

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. 

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.)...

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.

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. 

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.)...

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. 

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... 

Consideration may have to be given to using a supplemented medium during the initial stages of culture to aid cell attachment and to offset the effects of low cell density, which will be more critical in microcarrier than stationary culture. The supplementation may be simple, e.g. non-essential amino acids, pyruvate (0.1 mg mH), adenine (10 jxg mH), hypoxanthine (3 xg ml" ) and thymidine (10 xg mH). Other supplements include tryptose phosphate broth (1 mg mH), HEPES (5 mM), transferrin (10 mg T ) and fibronectin (2 xg mH). Serum, unless serum factors are added, may have to be used at 5-10% initially before being reduced after 1-2 days of culture. 

Human transferrin receptor-positive target cells are cultured at 37°C with 5% C02 in tissue culture flasks containing RPMI-1640 medium with 10% fetal calf serum, L-glutamine, and penicillin-streptomycin (GIBCO, Grand Island, NY). Adherent cells are released from the culture flask using 0.05% trypsin, and 0.02% EDTA for 10 min at 37°C, and then medium plus serum is added to inactivate the trypsin. 

The way to create a serum-free culture is to adapt the cells to the serum-free medium. In our laboratory, we tried to adapt a human lymphoblastoid cell line, Namalwa, from a medium containing 10% serum to serum-free. We were able to adapt Namalwa cell to a ITPSG serum-free medium which contained insulin, transferrin, sodium pyruvate, selenious acid and galactose in RPMI-1640. In the case of cell adaptation for production of autocrine growth factor, we were able to grow the cell line in serum- and protein-free media as well as in K5 62-K1 (T1) which produces an autocrine growth factor, LGF-1 (leukemia derived growth factor-1). 

The specific standard methods of a new perfusion culture will now be described for growth and maintenance of mammalian cells in suspension cultures at high density. The biofermenter was used for high density culture of Namalwa cells with serum-free medium as the model. In 1980, the parent Namalwa cells were obtained fi-om Mr. F. Klein of Frederick Cancer Research Center, Frederick, Maryland, U. S. A. In our laboratories, we were able to adapt the cells to a serum and albumin-fi ee medium and named the cells KJM-1. ITPSG and ITPSG+F68 used a serum-free medium containing insulin, 3 g/ml Transferrin, 5 g/ml sodium pyruvate, 5 mM seienious acid,... 

Using cultured mammalian sarcoma cells, it has been found that transferrin is necessary in the growth medium for galllum-67 uptake to occur (95,96.97). A "transferrin receptor" on EMT-6 sarcoma cells for 25i iabeled transferrin was characterized by Scatchard analysis to have an average association constant K = 4.54 X 10 1/mole and approximately (with variation) 500,000 receptors per cell ( ). It was proposed that tumor accumulation of galllum-67 can occur only if the metal is complexed with transferrin so that it can interact with the receptors of tumor, as well as non-mallgnant cells (.33). The complex then enters the cell via an "adsorptive endocytosis" process (95.96.97.98.99) similar to the manner in which iron is taken up by reticulocytes and bone marrow cells (100.101). These transferrin receptors are saturable (that is, a plot of 125i transferrin uptake versus extracellular transferrin concentration reaches a peak ( at about 200 u g/ml) as more carrier transferrin is added to the medium) (95). Since uptake is also proportional to the fraction of... 

Coated dishes Make fresh a 0.1% gelatin solution (Sigma) in PBS by gentle heating and shaking. Filter-sterilize while still warm. Completely cover the base of tissue-culture dishes. Leave for 2 h at room tempoature, and then aspirate off and wash IX with sterile PBS. Plate cells or cover with fiesh PBS and store at d C (maximum 2 wk). Serum-free medium 10 mL media, 20 pL N3 (serum suppl ent—see item 6). N3 serum supplement 246 pL Hank s balanced salt solution (HBSS) without calcium and magnesium, 50pL 10 mg/mL bovine serum albumin in HBSS (store at 4°C), 100 pL 100 mg/mL human transferrin in HBSS (store at -20°C), 20 pL 80 mg/mL putrescine hydrochloride in HBSS (store at -20 C), 50 pL 10 mAf sodium selenate in... 

For the cultivation of airway epithelial cells, the cells are usually plated onto tissue culture plates coated with collagen or extracellular matrix components. Hormonally defined Ham s F12 medium contained 1 % penicillin-streptomycin, 5 ig/ml insulin, 5 ig/ml transferrin, 25 ng/ml epidermal growth factor, 15 ig/ml epithehal growth factor supplement, 2 X 10" M triiodothyronin, and 10" M hydrocortisone. 

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