Toshiko Takaoka

Long-term cultivation of mammalian cell strains in protein- and lipid-free chemically defined synthetic media.

Abstract Ten substrains were established which grow indefinitely in protein- and lipid-free synthetic media: 5 substrains from rat liver parenchymal cells, and 1 substrain each from rat thymus reticulum cells, cynomolgous monkey kidney cells and rat ascites hepatoma AH-7974 cells. In addition to these 8 substrains of which original strains were established in our laboratory, 1 substrain was also achieved from L-929 and 1 from HeLa. In the mode of chromosome numbers, the substrains showed some shift from the original strain, so far as examined. The substrata obtained from a rat hepatoma maintained the capacity of back-transplantability into animals, and the tumor cells produced in the animals readily proliferated when re-cultured in a synthetic medium. The probability was strongly suggested that many other cell strains may grow in synthetic media.

Heme-biosynthetic enzyme activities and porphyrin accumulation in normal liver and hepatoma cell lines of rat.

The activities of four heme-biosynthetic enzymes, δ-aminolevulinic acid (ALA) synthase, ALA dehydratase, porphobilogen (PBG) dearninase, and ferrochelatase, were studied in five epithelial cell lines of normal rat liver origin (Re, REC-10, RLC-24, M, Culb-TC) and five cell lines derived from Yoshida ascites hepatoma (JTC-1, JTC-2, JTC-15, JTC-16, JTC-24). The JTC series of hepatoma-derived cell lines exhibited decreased ALA synthase activity and increased ALA dehydratase activity, although the activities of all four enzymes and the Km values for their respective substrates varied widely from one cell line to another, a finding suggesting that specific regulatory mechanisms for porphyrin metabolism might operate in each cell type. M cells, which were transformed by 4-dimethylaminoazobenzene in vitro, gave the most abnormal Km values of heme-biosynthetic enzymes among all the cell lines studies, and were found to accumu2ate hematoporphyrin derivative (HpD).

Cultivation of cells in protein- and lipid-free synthetic media.

Publisher Summary This chapter discusses the cultivation of cells in protein- and lipid-free synthetic media. Protein-free, chemically defined synthetic media are of great use in analyzing the chemical nature of cultured cells and of the metabolites excreted from them as well as in examining the effect of certain substances on cells, especially in cases where the substances may first interact with serum proteins in the medium. A number of studies have been carried out along these lines, as discussed. However, a very limited number of kinds of cell lines have been grown indefinitely in protein-free media. These are among the reasons why descriptions of so many mixtures have been published and not every kind of cell has as yet been serially grown in such media, especially in the primary culture.

Collagen fiber formation as a common property of epithelial liver cell lines in culture.

Abstract Collagen fiber formation in vitro was morphologically investigated on 22 epithelial-like cell lines originating from normal rat livers mainly by using specific histochemical stainings. All the cell lines, eight of which were cloned formed typical “collagenous” and/or “reticulin” fibers in the histological sense when they had been left as a full sheet for more than 3 weeks without subculture. Only one of five fibroblastic cell lines serving as control formed fibers to a lesser degree. Different patterns were recognized in fibers appearing between the liver cell cultures and the fibroblast cultures.

Chapter 13 Improved Synthetic Media Suitable for Tissue Culture of Various Mammalian Cells

Publisher Summary After examination of nutritional requirements, published or unpublished, of mammalian cells in tissue culture, various mixtures of synthetic media were designed, and 20 kinds of cell strains have been established in our laboratory that are capable of serially growing in protein- and lipid-free chemically defined synthetic media, for example , the mixtures DM-120 or DM-145. These mixtures, however, were found to be not necessarily suitable for the culture of every kind of cell, even when combined with sera. This chapter focuses on mixtures of multipurpose synthetic media—that is, media in which the growth of various kinds of cells is obtainable in combination with or without serum and in a closed system as well as in an open system of culture.

Two different activities of alkaline phosphatase in cultured mammalian cells

Alkaline phosphatase activities in various mammalian cell strains in culture, some of which had been grown in protein-free synthetic media, were investigated and characterized. Two distinctly different activities of alkaline phosphatase were found in crude extracts of various cell strains. The first activity (designated alkaline phosphatase I) was the highest around the range of pH 10.0 and inhibited by cyanide or β -mercapto-ethanol. This activity was mainly detected in particulate fractions of cells. The second was shown to be optimal at pH 8.6 and inhibited by p -chloromercuribenzoate (designated alkaline phosphatase II). Among various cell strains examined, the former activity was detected only in some of them, whereas the latter type was present in each strain. These two activities were separated from each other by DEAE-cellulose chromatography.

A new approach to the modification of cell membrane glycosphingolipids: Ganglioside composition of JTC-12 P3 cells altered by feeding with galactose as a sole carbohydrate source in protein- and lipid-free synthetic medium

A significant difference in the glycosphingolipid composition of JTC-12 P3 cells established from monkey kidney tissue was observed when cells cultured in a protein- and lipid-free synthetic medium containing glucose (DM-160) as a sole carbohydrate source were transferred and cultured in the same medium containing galactose and pyruvic acid (DM-170) in place of glucose. In particular, the amounts of gangliosides GM3, GM2, and GD3 in the cells cultured in DM-170 were 5.3-, 17.8-, and more than 8-fold those in the cells cultured in DM-160, respectively, indicating that anabolism of gangliosides is greatly enhanced in cells cultured in the presence of galactose and pyruvic acid, as compared with cells cultured in the presence of glucose. In fact, after cultivation of cells in the medium with N-acetyl-D-[14C]mannosamine for 96 h, the radioactivity incorporated into the gangliosides of the cells in DM-170 was 10-fold that of the cells in DM-160. Among the gangliosides of the cells in DM-170, highly sialylated molecules such as GD3, GD1a, GD1b, and GT1b were preferentially labeled, indicating that the sialyltransferases responsible for the synthesis of gangliosides are significantly more activated in cells cultured in DM-170 than in DM-160. These observations reveal that the glycosphingolipid composition of the plasma membrane can be modified epigenetically under well-defined conditions and provide important clues for clarifying the roles of glycosphingolipids associated with particular cell functions.

Survival of cultured cells in the cold: A kinetic study with special reference to the effect of serum in culture media

Abstract The survival at 4 °C of mouse fibroblasts (strain L-929) and rat liver cells (strain JTC-25·P5) was kinetically analysed after they had been pre-incubated at 37 °C in medium with or without supplement of serum. Both the composition of medium used for preincubation at 37 °C and that employed for storage at 4 °C had influence on the survival period.When the cells had been grown at 37 °C in Eagle minimal essential medium (MEM) alone, they rapidly lost their viability at 4 °C from the beginning. However, when grown at 37 °C in MEM supplemented with calf serum, they maintained viability at 4 °C for about 16 days and 8 days for L cells and JTC-25·P5 cells respectively, before the initiation of rapid loss of viability. The presence of macromolecular fraction of calf serum in the medium during preincubation was found to be responsible for the prolongation of survival at 4 °C.

A polyelectrolyte buffer system for bacterial and mammalian cell culture

Abstract A cross-linked polymethacrylic acid, Amberlite IRC 50, was examined for its buffering capacities to hydrogen ions and calcium ions in the physiological ranges of ionic strength and ion concentration of hydrogen and calcium. Titration experiments demonstrated fairly strong buffering action of IRC 50. After pretreatment with medium components, IRC 50 was added to cultures of E. coli cells and mouse fibroblasts of strain L. The resin accelerated and maintained the growth of E. coli cells continuously, at least for 15 h which otherwise entered stationary phase after the 5th hour. The effect on L cells was examined by the use of cell counting for 12 days. In a medium consisting of calf serum, lactalbumin hydrolysate and vitamins, the addition of the resin resulted in the acceleration and maintenance of cell proliferation, at least until the 12th day with no renewal of medium. Some of the general properties of the polyelectrolyte buffer system as well as the mode of action of IRC 50 on the growth of E. coli cells and L cells were discussed.