Transferrin serum supplement

Serum ferritin, transferrin saturation supplemental iron recommended if ferritin <100 mcg/L or transferrin saturation <20%... 

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.)...

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... 

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.)...

Although EPO deficiency is the primary cause of CKD anemia, iron deficiency is often present, and it is essential to assess and monitor the CKD patient s iron status (NKF-K/DOQI guidelines). Iron stores in patients with CKD should be maintained so that transferrin saturation (TSAT) is greater than 20% and serum ferritin is greater than 100 ng/mL (100 mcg/L or 225 pmol/L). If iron stores are not maintained appropriately, epoetin or darbepoetin will not be effective, and most CKD patients will require iron supplementation. Oral iron therapy can be used, but it is often ineffective, particularly in CKD patients on dialysis. Therefore, intravenous iron therapy is used extensively in these patients. Details of the pharmacology, pharmacokinetics, adverse effects, interactions, dose, and administration of erythropoietin and iron products have been discussed previously. 

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.

Pretherapy iron evaluation - Prior to and during therapy, evaluate the patient s iron stores, including transferrin saturation and serum ferritin. Transferrin saturation should be at least 20% and ferritin should be at least 100 ng/mL. Virtually all patients will eventually require supplemental iron (see Precautions). Adequately... 

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. 

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. 

Iron status should be evaluated prior to and during treatment and iron supplementation administered if necessary. Patients receiving ESA maintenance therapy should be given iron supplements to keep their serum ferritin between 200 and 500 micrograms/L in both haemodialysis patients and non-haemodialysis patients, and either the transferrin saturation level above 20% (unless ferritin >800 micrograms /L) or percentage hypochromic red cells (%HRC) less than 6% (unless ferritin >800 micrograms/L). In practice it is likely this will require intravenous iron. 

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]. 

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. 

Intravenous iron therapy is an effective means to prevent iron deficiency and maintain adequate iron status for erythropoiesis. Parenteral iron improves the responsiveness to erythropoietic therapy and reduces the dose required to achieve and maintain the target Hgb/Hct. Iron administration in patients with what is known as a functional iron deficiency is more questionable. Functional iron deficiency is characterized by a low TSat (<20%) in the presence of a normal or elevated serum ferritin. In other words, there may appear to be adequate storage iron, but iron is not being carried by transferrin to the bone marrow for red blood cell production. If the Hgb is less than the target of II g/dL, under these conditions a trial of IV iron therapy may be warranted. Iron supplementation alone may improve Hgb/Hct and may... 

Most patients tolerate EPO therapy well. Iron deficiency can occur in patients treated with EPO and close monitoring of iron levels is necessary. Oral iron supplementation should be given if transferrin saturation drops to 20% or the serum ferritin level drops below 100 ng/mL. Some patients develop functional iron deficiency, in which the iron stores are normal, but the supply of iron to the erythroid marrow is less than that necessary to support the demand for RBC production. Therefore many practitioners routinely supplement EPO therapy with oral iron therapy. The hypertension commonly seen in end-stage renal disease patients on EPO is far less common in AIDS patients. More common toxicities of EPO administration include nausea, headache, fever, bone pain, and fatigue. Other adverse effects to monitor include seizures, thrombotic events, and allergic reactions such as rash or local reactions at the injection site. 

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. 

During erythropoietin therapy, absolute or functional iron deficiency may develop. Functional iron deficiency (i.e., normal ferritin levels but low transferrin saturation) presumably results from the inability to mobilize iron stores rapidly enough to support the increased erythropoiesis. Virtually all patients eventually will require supplemental iron to increase or maintain transferrin saturation to levels that will adequately support stimulated erythropoiesis. Supplemental iron therapy is recommended for all patients whose serum ferritin is below 100 pg/L or whose serum transferrin saturation is less than 20%. 

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