Jay C. Bryant

CHEMICALLY DEFINED MEDIA FOR CULTIVATION OF LONG-TERM CELL STRAINS FROM FOUR MAMMALIAN SPECIES.

Abstract A number of new cell lines have been grown in chemically defined medium NCTC 109 free of any exogenous protein. The list in this laboratory now includes fibroblast-like and epithelial-like cells, normal and malignant, and in origin from the mouse, hamster, monkey and human. The lines growing in medium NCTC 109 were then tested for their growth in simpler media of the NCTC series: 117, 126, 131, 132 and 133. Medium 117 lacks the six coenzymes and deoxyguanosine, deoxyadenosine, 5-methyl-cytosine, and sodium acetate present in medium 109. Media 126, 131, 132 and 133 lack all of these except sodium acetate; nine vitamins, glutathione, cysteine and sodium glucuronate were also omitted. Medium 131 is identical with medium 126 except that the former contains vitamin B 12 . Media 132 and 133 are identical with medium 126 except that 132 contains inositol and 133 contains both inositol and vitamin B 12 . Two human lines grew readily in media 117 and 126, as did also the monkey kidney cells and some of the mouse cell strains. All mouse cells tested grew in medium 117 except the mouse liver cells. Hela and mouse liver cells showed anomalous growth behavior in media 117, 126, 131 and 132; these two strains are still under test. When mouse cells did not growin media 126 and 131 it was usually because they required inositol. Of all of the cell strains conclusively tested only one strain of mouse cells and the one Chinese hamster strain required vitamin B 12 for continuous growth at least when thymidine and deoxycytidine were included. This report, timely because of the increased significance and more widespread use of various chemically defined media, also describes improvements in quality control and simplified preparation methods for the NCTC series of media. Our experience strongly emphasizes the necessity for high quality in the chemicals and for the meticulous preparation and storage of such media when used without any protein supplements.

Growth of cell suspensions in tissue culture.

In earlier publications,’preliminary methods and concepts were described by which it had been possible to demonstrate thc proliferation of strain 1,-929 cells as a free-floating cell suspension maintained in a rapidly agitated fluid culture medium. I n these exploratory studies several factors were given special attention. These were: (1) It was obviously necessary to keep the cells in suspension in the nutrient fluid. To accomplish this, rapid agitation of the nutrient fluid was used. To assist in this, viscosity of the culture fluid was slightly increased by addition of 0.1 per cent methyl cellulose CPS 4000. This rapid agitation served also to maintain circulation of the nutrient fluid over the cell surface and to maintain adequate oxygenation of the cell and culture fluid. I n the later study,? a Brunswick type shaker was used. This shaker could handle culture flasks up to 1.5 1. in size. (2) ,4s an extension of earlier studies on the influence of cell population densities on proliferation, it seemed probable that it would be necessary to start the culture with a high population cell density. In exploratory experiments, cells of clone strain L-929 showed rapid cell proliferation under these experimental conditions. The present study gives additional data and development of the concepts and equipment on the growth of certain strains of fixed tissue cells in this type of agitated fluid suspension culture.

MAMMALIAN CELLS IN CHEMICALLY DEFINED MEDIA IN SUSPENSION CULTURES

This paper is concerned with the application and development of batch-type suspension culture methods to the cultivation of mammalian cells. It deals primarily with research in our tissue culture laboratory. Major emphasis is on certain biophysical or biochemical parameters, especially chemically defined media, with less emphasis on the instrumentation involved in controlling the in vitro environment. For the defenseless mammalian cell no longer in its normal milieu, the strictly axenic culture is the only kind ordinarily possible, in marked contrast to cultures of some free-living microorganisms, which can tolerate at least limited competition. When the mammalian cell culture is no longer axenic the cells are either dead or no longer present. An exception to this generalization is the troublesome PPLO (Mycoplasma) , which may coexist in the same culture with viable mammalian cells.