Culture Techniques 31 Media and supplements

The media which have been used for human VSMC culture include Medium 199 and DMEM buffered with 3.7 g/L NaHCO3 and 5% CO2. Antibiotic supplements recommended are 100 IU/ml penicillin, 100 |g/ml streptomycin and 250 ng/ml amphotericin B (the latter is important to avoid fungal contamination in the early stages of culture). The medium is also supplemented with fresh 4 mmol/l of L-glutamine. The sera used for supplementing the medium commonly derive from fetal bovine sources, although some researchers may be able to use human or autologous serum if the VSMCs are derived from a surgically removed artery, for example during bypass grafting. Serum is used at concentrations of either 10% (for passaged cells) or 20% (for explant cultures) and should be heat-inactivated at 56°C to destroy complement. Since the concentration of some components of FBS differ drastically between different batches, serum testing is essential. Growth factors are not commonly required in addition to serum. A limited number of serum-free systems have been tested where it is necessary to use defined supplements, including endothelial cell growth factor (ECGF), to maintain cell viability [17]. However, cultures must first be established in the presence of serum, which is then gradually replaced with serum-free medium.

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