25 28

pyruvate, Hepes, and potassium nitrate instead of ferric nitrate. This medium has proved useful as a basis for serum-free formulations (28) and has been widely used for the growth of lymphocytes and hybridomas.

• MCDB 104 was developed from Ham's F12 as a medium for the clonal growth of human diploid fibroblasts.

• MCDB 110 was developed from MCDB 104 for the growth of human diploid fibroblasts (29). The modifications included the addition of fatty acids, phospholipids, growth factors, hormones, and other supplements to simulate the action of serum (30).

• MCDB 153 was developed as a basal medium for the serum-free growth of human epidermal keratinocytes and other epithelial cell types (31-33).

• Medium 199 was formulated in an attempt to produce growth medium free from added protein, although for the growth of some cell lines serum is still added. This in an extremely complex medium based on Earle's salt solution and containing an extensive range of amino acids, vitamins, nucleic acid derivatives, growth factors and lipids (15,25,34).

• NCTC 135 medium was originally formulated for the serum-free growth of mammalian cells over long periods. The medium is a modification of the original NCTC 109 medium (35,36).

• Waymouth's medium MB 752j 1 was originally designed for the growth of L929 cells under serum-free conditions. However, it has been used for the growth of many cell types, usually with serum supplementation (15,37).

Standard media formulations such as those outlined previously can be purchased from commercial suppliers (e.g., Sigma, Life Technologies, etc.), either as a liquid or as a powder. Liquid forms can come as 1 x or 10 x concentrates, the latter requiring dilution with sterile deionized distilled water. Alternatively, powdered media may be preferred, especially if large quantities are needed. In this case, the powder should be dissolved in distilled water and sterilized by filtration through a 0.22 mm filter under positive pressure. Sterilized supplements of sodium bicarbonate, antibiotics, and glutamine are often added after sterilization of the bulk of the medium. This is essential if the medium is autoclaved, as these supplements are unstable.

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