Chemically characterized concentrated corodies for continuous cell culture (the 7C's culture media)

Abstract

A series of concentrated chemically characterized media has been developed for continuous culture of mammalian cells and designated as 7C's media. They contain amino acids at levels from 27 to 162 m M/l comparable to the amino acid content of 5 to 30 per cent of serum or 0.3 to 1.5 per cent of lactalbumine hydrolysate, but about 5 to 30 times the amino acid concentrations contained in most of the chemically defined media currently in use. The 7C's media contain fat-soluble vitamins, the polyunsaturated fatty acids, and trace elements known to be nutritional essentials for the mammalian organism. The proportionality pattern of the amino acids in these media are, in general, similar to those found in animal tissues and food proteins of high biological values and, therefore, avoids gross imbalance. The levels of the vitamins were based on their known biological potency determined for whole organisms and taking into consideration the concentrations found in nonstorage human or animal tissues. The trace elements content are in proportion similar to those existing in plasma or body fluids. The buffering capacity of the 7C's media has been improved compared to most other CDM due to two factors: (1) substitution of the bicarbonate buffer with β-glycerophosphoric acid disodium corrects the tendency of pH to rise during incubation because of the loss of CO 2 and since the calcium salt of glycerophosphate is soluble in water it does not form precipitates with calcium like other inorganic phosphate buffers; (2) the high levels of amino acids in these media increase the buffering capacity due to the zwitterionic character of these substances [19]. Continuous culture studies with WRC-256 and MBIII lymphoblast cells demonstrate that the high amino acid levels of 7C's media AN-54 and AN-81 are able to support satisfactory growth of these cell lines without supplements of serum or any other tissue extracts. These two cell lines have been maintained in these serum-free media for five and two years, respectively.