Yutaka Matsuya

An autoclavable powdered culture medium for mammalian cells.

Summary An autoclavable powdered tissue culture medium could be successfully prepared by applying its thermostability in acidic pH. The formula of the medium is modified from Eagle MEM so as to be autoclaved at pH 4-4.5. The medium can be used not only for the cloning culture of established cell lines but also for the primary cultures of various mammalian cells.

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

SummaryA newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.

Role of iron chelators in growth-promoting effect on mouse hybridoma cells in a chemically defined medium

SummaryThe role of various iron chelators on the multiplication of mouse hybridoma cells in an albumin-free, transferrin-deficient defined medium was investigated. Fe(III)-dihydroxyethylglycine, Fe(III)-glycylglycine, Fe(III)-ethylenediamine-N,N′-dipropionic acid, or Fe(III)-iminodiacetic acid supported the excellent growth of the cells. In addition, the growth of the iron-starved cells, which had been preincubated in a protein-, iron- and chelator-free defined medium, restored rapidly when the medium was supplemented with holotransfeerrin, ferric iron, and chelator compared to that when supplemented with holotransferin, but without iron and chelator. The results suggest that such chelators modulate a progression of transferrn cycle in the presence of transferin and ferric iron. An alternative explantation is that there is a decrease in generation of iron-catalyzed free radicals.

Tumor‐promoting Phorbol Ester Induces Alterations of Sialidase and Sialyltransferase Activities of JB6 Cells

Sialidase and sialyltransferase activities were studied in JB6 mouse epidermal cells before and after exposure to phorbol ester, 12‐O‐tetradecanoyl phorbol‐13‐acetate (TPA), which irreversibly induces anchorage‐independent growth and tumorigenicity. JB6 cells exhibited sialidase activities toward 4‐methylumbelliferyl‐α‐d‐N‐acetylneuraminic acid (4MU‐NeuAc) and gangliosides at pH 4.5 in the particulate fraction but apparently not in the cytosol at pH 4.5 or 6.0. In JB6 cells exposed to TPA and in the anchorage‐independent transformants, the sialidase activity toward 4MU‐NeuAc was decreased and the activity toward gangliosides was increased compared with those in untreated JB6 cells. Immunological analysis with antisera against membrane‐associated sialidases I and II revealed that plasma membrane‐associated sialidase I was increased and lysosomal membrane‐associated sialidase II was decreased under these conditions. TPA treatment also affected the sialyltransferase activities of JB6 cells: an elevation of the transfer activities toward asialo‐orosomucoid and asialo‐porcine submaxillary mucin but a reduction of GM3 and GD3 synthase activities were observed on exposure to TPA and in cells transformed by TPA to retain anchorage‐independency. These results suggest that an increase in sialic acid bound to glycoproteins and a decrease in that bound to glycolipids may occur in JB6 cells exposed to TPA and in the anchorage‐independent transformants.

Reduced tumorigenicity by addition in vitro of Sendai virus

Abstract Treatment of Ehrlich ascites tumor (EAT) with Sendai virus produced polyploid variants that could grow on selective media, whereas no cell growth occurred in untreated control cultures. All of these variants isolated had more chromosomes and showed less transplantability than EAT. The banding patterns of each chromosome were analyzed. The karyotypes of the variants appeared to be more heterogenous than EAT. Other biological properties of cultured control and its variants, agglutinability by concanavalin A and the anchorage dependency of cell growth proved to be unrelated to tumorigenicity. Similar variants were isolated by treating EAT with another cell-fusion inducers, lysolecithin or polyethylene glycol. Like the variants induced by Sendai virus treatment, they had more chromosomes and were less tumorigenic than EAT. However, the frequency of occurrence of these variants ( −7 ) was far less than that ( 10 −5 ) of the variants induced by Sendai virus. It is probable that variants pre-existing in EAT begin to propagate in vitro after the treatment with fusion-inducing agents.

Cell Fusion and Cell Agglutination: Enhancing Effect by a Combined Use of Lectin and Polycation

The technique efficient for hybridization of mammalian cells was improved by combining a procedure of cell agglutination of cells pretreated with a combination of lectin and polycation and the procedure of conventional polyethylene glycol (PEG) -induced cell fusion. The agglutinability of cells treated with lectin, polycation, and both of them was tested. The effects of these agglutinogens on the hybridization frequencies of cells were also compared. The appearance rate of hybrid colonies was found to be correlated to the extent of cell agglutination. The pretreatment with a combination of lectin and polycation induced the highest degree of cell agglutination and the highest frequency of resulting hybridization. The enhancing effect by the pretreatment with a combination of these agglutinogens on the hybridization frequency was confirmed through experiments on the crosses of several cell types including nuclear fraction (karyoplast).

Confirmation of the human thymidine kinase locus, 17q21 → 17qter, by means of a man-mouse somatic cell hybrid, D98/AH-2 X LMTK−Cl-1D

SummaryA large metacentric marker chromosome, m20, in a line of human D98/AH-2 cells was identified by Q bands as being a translocation (1;17)(p36;q21). This was confirmed by means of somatic cell hybridization between D98/AH-2 and thymidine kinase (TK) deficient mouse cells. The hybrid clones by HAT selective system retained m20, indicating the presence of TK locus on this chromosome. The results also provide evidence that TK gene is located on the distal region of the breakpoint in 17q21 but not on 17q21 → 17pter.

Establishment of Syrian Hamster Fibroblast Culture in Albumin Fortified Medium

Summary A successful routine method of developing cell lines from diploid hamster cells has been established. Bovine serum albumin fraction V was essential for serial long-term culture. Stimulation of growth was optimal at albumin concentrations between 0.5 and 1%. In the presence of albumin, hamster embryonic and neonatal lung cells and whole hamster embryonic cells developed into established lines.