Cell culture amphotericin

Resistance Variants with reduced susceptibility to amphotericin B have been isolated from several fungal species after serial passage in cell culture media containing the drug and from some patients receiving prolonged therapy with amphotericin B desoxycholate. The relevance of drug resistance to clinical outcome has not been established. 

Insect cell culture at 1-2 x 10 cells/ml EX-CEL 405 serum-free medium for Insect cells containing gentamycin sulphate (50 iJig/ml), amphotericin B (2.5 iJig/ml), and fetal calf serum (10%)... 

Sf9 cell culture 150-mm tissue culture plates High-titre recombinant viral stock EX-CEL 405 serum-free medium containing 50 xg/ml gentamycin sulphate, 2.5 i.g/ml amphotericin B, and 10% fetal calf serum 27°C incubator Microscope Haemocytometer... 

For adherent cell cultures, the following protocol for HeLa cells may serve as an example. Cervical adenocarcinoma (HeLa) cells (purchased from the American Tissue and Cell Culture Corp. ATCC Manhasset, VA, USA), cell line CCL-2, were seeded in 75 cm sterile plastic cell culture flasks (Fisher Scientific) at a concentration of approximately 2 X 10 cells cm l Typically, the growth medium consisted of 20ml Dulbecco s Modified Eagle s Medium (DMEM ATCC), supplemented with 10% fetal bovine serum (FBS ATCC). To prevent bacterial contamination, 2.5ggmT of amphotericin B (ATCC) and lOOIUmT penicillin/streptomycin (ATCC) was added to the medium. The cells were incubated at 37 °C in an atmosphere containing 5% COj. 

Lamb, K.A., Washington, C., Davis, S.S., and Denyer, S.P. (1991) Toxicity of amphotericin B emulsion to cultured canine kidney cell monolayetfePharm. Pharmacol., 43 522-524. 

Endothelial cells are maintained in a basal culture medium, such as DMEM or a proprietary medium supplied with the cells, which is supplemented with hydrocortisone (lug/mL). epidermal growth factor (EGF 100 ug/mL) bovine fibroblast growth factor (FGF 1 ng/mL), antibiotics (gentamicin and amphotericin, at 50 ug/mL) and fetal calf serum (2-10%). Alternatively, bovine brain extract (3 ug/mL) can be used instead of EGF and FGF. The cells are cultured in either standard tissue culture flasks directly on plastic or on flasks coated with collagen. The cells are grown to confluence and subcultured and reseeded in a ratio of 1 3. For all experiments primary endothelial cells should be used between passages 3 and 12. 

Sabra R and Branch RA. Effect of amphotericin B on intracellular calcium levels in cultured glomerular mesangial cells. Eur ] Pharmacol 226 79-85,1992. 

Joly V, Saint-Julien L, Carbon C, Yeni P. Interactions of free and liposomal amphotericin B with renal proximal tubular cells in primary culture. J Pharmacol ExpTher 1990 255 17-22. 

Many mammalian tissue culture cells are sufficiently resistant to polyenes to allow low concentrations of polyenes, usually amphotericin B or nystatin, to be included in tissue culture medium. Not all cells are resistant. It has been... 

Detach H5 insect cells grown to confluence in six (75 cm ) or three (150 cm ) culture flasks (approximately 1.8 x 10 cells) and seed into a Fembach culture flask in 150 mL F15 Insect-Xpress medium supplemented with 0.2% (v/v) Pluronic F-68, 50 pg/mL gentamicin, and 2.5 i /mL amphotericin B. 

The use of penicillin and Streptomycin is recommended in the culture media to maintain sterility. If gross bacterial, yeast, or fungal contamination occurs, destroy the cultures. If slight contamination occurs and it is desired to keep the cell Unes, treatment with gentamycin (50 JLg/ml) or amphotericin B (2.5 p-g/ml) can be attempted with caution. 

Chang s human conjunctival cells with a population of lOVml were cultured in Eagle s minimum essential medium supplemented with 10% fetal calf serum for 48hrs in 5% CO2 in a 37 C incubator. Cell sheets were exposed to various concentrations of amphotericin B (Bristol-Myers Squibb Co., purity 100%), miconazole (Janssen Pharmaceutica Co., purity 100%), and fluconazole (Pfizer Pharmaceuticals Inc. Co., purity 100%) for 2 or 4 min. Test drug concentrations were adjusted to clinical use. Cells were prepared, immediately after exposure to the test solutions, for scanning electron microscopy and flow cytometry or cultured for a further 24 hrs to examine cell growth. 

Orunsi, N. A., Trinci, A. P. J. Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B. Microbiol 1985,43,17-30. 

Human embryonic palatal mesenchymal cells (HEPM, ATCC CRL-1486) were seeded onto the coated surfaces in multiple 24-well tissue culture plates (50,000 cells/well). The cultures were maintained in Dulbecco s modified eagles medium (DMEM) supplemented with fetal bovine serum (FBS, 10%), L-glutamine (2 pmol/ml), penicillin G (100 U/ml), streptomycin sulfate (100 pg/ml) and amphotericin B (0.25 pg/ml). Replicate cultures were incubated at 37 C for 6, 24 and 72 hours and then harvested for analysis. 

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