Improved growth of mammalian and insect cells in media containing increased levels of choline.

Most cell growth media contain about 1 mg of choline per liter, consistent with concentrations usually employed for vitamins. One notable exception is the medium described by Waymouth (3) which contains 250 mg of choline per liter. For L cells growing in a chemically defined medium, I observed that increased choline permitted continuing growth of cells in the absence of medium replenishment over a longer period of time. Choline apparently became deficient when used at 1 mg/liter. This observation was tested further with cultures of HeLa (human), L (mouse), and Aedes aegypti (mosquito) cells. The mammalian cells were grown in suspension in a heat-stable, chemically defined medium described previously (1). Mosquito cells were grown in suspension in a medium modified from one described earlier (2). The modified insect cell medium, designated no. 35, contained the following (expressed in milligrams per liter): L-alanine, 100; L-arginine hydrochloride, 400; L-aspartic acid, 300; L-cysteine hydrochloride, 100; L-glutamic acid, 400; glycine, 400; L-histidine hydrochloride, 200; L-isoleucine, 150; L-leucine, 300; L-lysine hydrochloride, 300; L-methionine, 60; L-phenylalanine, 150; Lproline, 300; L-serine, 150; L-threonine, 75; L-tryptophan, 50; L-tyrosine, 100; L-valine, 150; NaCl, 4500; KCl, 2500; CaCl2-2H20, 400; MgCI2.6H20, 400; MgSO4-7H20, 400; NaH2PO4VH20, 200; glucose, 5000; D-biotin, 1.0; choline chloride, 1.0 (except where noted); folic acid, 1.0; niacinamide, 1.0; calcium pantothenate, 2.0; pyridoxal hydrochloride, 1.0; thiamine hydrochloride, 1.0; i-inositol, 1.0; riboflavine, 0.1; and B12, 0.002. The medium was adjusted to pH 6.5 with KOH and sterilized by autoclaving at 121 C for 15 min. After the medium was cooled, 15-centipoise methylcellulose (as a 2% sterile solution) was added to a final concentration of 0.05%; L-glutamine was added (500 mg/liter) as a dry powder after it was autoclaved in a stoppered, 15-ml serum bottle for 1 hr at 121 C; heated fetal bovine serum (15 min at 56 C) was added to a final concentration of 5%; and streptomycin and penicillin were added at final concentrations of 100 ,tg and 100 units per ml, respectively. Cultures of HeLa and L cells were grown at 34 to 36 C in rubber-stoppered, 100-ml serum bottles, each with 25 ml of medium, on a gyratory shaker (New Brunswick Scientific Co., Inc., New Brunswick, N.J.) at 124 to 130 rev/min. Mosquito cells were grown at 26 to 28 C in 250-ml plastic flasks (Falcon Plastic, Division of B-D Laboratories, Inc., Los Angeles, Calif.), also with 25 ml of medium, on a New Brunswick gyratory shaker operating at 60 rev/min. Cell numbers were determined by hemocytometer count. Viability of mammalian cells was routinely observed by counting only those cells with theability to exclude trypan blue. The trypan blue procedure was not used for mosquito cells; however, only apparently healthy, intact mosquito cells were counted. Results of growth of the three cell lines in their respective media containing graded levels ofcholine are given in Table 1. Three cultures were prepared for each concentration of choline for each cell type. The data represent the average values for at least two sets of triplicate cultures for each concentration, based on the highest counts obtained in 5, 6, and 7 days of incubation