Hajim Katsuta

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.

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.

Arrest of cultured rat liver cells in G2 phase by the treatment with dibutyryl cAMP

Abstract When rat liver cells which had been grown in a protein- and lipid-free synthetic medium were incubated in the presence of dibutyryl adenosine 3′:5′-cyclic monophosphate (But 2 cAMP) and theophylline, labeling index of cells with [ 3 H]-thymidine was decreased. Percentage of cells which had twice the amount of DNA per nucleus compared to the basic value increased during the cultivation of cells in the presence of the drugs. Mitotic index increased immediately after the removal of the drugs. From these results, it was concluded that But 2 cAMP arrested the cell cycle mainly at G2 phase.

Induction of alkaline phosphatase activity by dibutyryl adenosine 3′:5′-cyclic monophosphate in aneuploid rat liver cells☆

Abstract Alkaline phosphatase activity (ALP) in cultured rat liver cells, strain JTC-25 · P5, was found to increase more than tenfold when cells were incubated in the presence of dibutyryl cyclic adenosine 3′:5′-monophosphate (But2cAMP) and theophylline. Among six subclones of this cell strain, four were able to be induced for ALP activity, but two were not. However, the incorporation of 3H-But2cAMP into cells did not differ from each other significantly. The cellular growth was apparently inhibited in the presence of theophylline and But2cAMP, and extensive cytoplasmic elongation was observed in both types of subclones. The increase of ALP activity appeared to depend upon the de novo protein synthesis, since cycloheximide inhibited the induction.

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.

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.