An improved chemically defined medium, CMRL-1415, has been devised by testing the response of trypsinized, newly explanted mouse embryo cells in stationary cultures to various modifications of an earlier medium, CMRL-1066. The improvements are attributed to changes in amino acid levels, in the vitamin-coenzyme composition, and to an enhanced buffering capacity resulting from the use of free base amino acids, galactose and pyruvate, together with greatly reduced levels of glucose and sodium bicarbonate and the inclusion of both monobasic and dibasic sodium phosphate in the ratio 1:4. When the medium is equilibrated with 5% CO2 in air, an initial pH of 7.2–7.4 is achieved, with excellent buffering capacity. CMRL-1415 contains 50 ingredients (9 fewer than CMRL-1066) and is prepared from six stable stock concentrates. By omitting sodium bicarbonate (to give CMRL-1415-ATM), the medium may be used in unsealed cultures in free gas exchange with air. CMRL-1415 and CMRL-1415-ATM are intended for use with and without serum protein and other supplements; and by preparing them double strength they may be combined with agar or other gelling agents to provide a semi-solid substrate.

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