Isao Yamane

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.

Primary culture of human diploid cells and its long-term transfer in a serum-free medium☆

Abstract Using a serum-free culture medium, primary human embryo fibroblasts can be grown in long-term serial culture. The basal medium consists of the components of modified Eagles minimum essential medium (MEM) and non-essential amino acids, various growth factors and trace metals. Human fibronection (FN) and bovine serum albumin (BSA) were added. BSA was found to be essential for long-term serial culture in the presence of FN. Incubation in a gaseous environment of low oxygen (7% O 2 ) and low-temperature trypsinization at the time of transfer were also found to be important for growth in serum-free medium.

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.

Role of Bovine Albumin in a Serum-free Suspension Cell Culture Medium

Summary A serum-free culture medium, supplemented with 1% bovine serum albumin, supported the growth of both primary and continuous suspension-type cultures of various mammalian tumor cells. The role of albumin added to the medium was also studied. Defatted albumin failed to support cell growth, unless reconstituted with its lipid extract. Similarly, defatted albumin, when combined with oleic and linoleic acids, also supported cell growth. Therefore, albumin-bound fatty acids play an important growth-promoting role in serum-free medium. We thank the Nissui Saiyaku Company for supplying the fatty acids. This work was partly supported by a Grant-in-Aid from the Educational Ministry of Japan.

Long-term serial cultivation and growth requirements for human umbilical vein endothelial cells

SummaryHuman umbilical vein endothelial cells (HUV-EC) grew rapidly in vitro in medium supplemented with epidermal growth factor, fetal bovine serum (FBS) and human diploid fibroblast-conditioned medium. The effect of FBS could be replaced partially by bovine serum albumin, cholesterol, and vitamin E, and completely by further addition of serum dialysate or refeeding every other day. Among these components, fibroblast-conditioned medium is essential for HUV-EC growth. The HUV-EC were cultured serially for over 50 population doublings in the 10% FBS containing fibroblast-conditioned medium and for over 40 population doublings in the serum-free medium. Mitogenic factor(s) present in the medium conditioned by fibroblasts may be related to endothelial cell growth factor and play an important role angiogenesis and regeneration of vascular endothelium in vitro.

Role of Bovine Serum Albumin in Blastoid Transformation of Lymphocytes by Phytohemagglutinin

Summary Employing a serum-free medium (SF medium), blast transformation of human peripheral lymphocytes stimulated by PHA was investigated. A remarkable PHA response with extremely high stimulation indices was obtained in SF medium containing 0.25-0.5% BSA. However, lymphocytes were unresponsive to PHA in the medium containing defatted BSA, unless supplied with oleic and linoleic acids. It is concluded that the albumin-bound fatty acids play an important role in lymphocyte transformation in SF medium, as observed in the case of tumor cell cultivation. This work supported by a Cancer Research grant from the Ministry of Education, Science and Culture, Japan.

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.

Spontaneous interferon production and growth of lymphoblastoid cells in serum-free medium

Abstract Numerous lymphoblastoid cell lines were established from human adult peripheral blood and cord blood lymphocytes, using Epstein Barr virus, and most cell lines from cord blood lymphocytes spontaneously produced abundant interferon without induction with Sendai virus, whereas lymphoblastoid cells from adult peripheral blood lymphocytes did not. These potential cells grow well in a newly developed serum-free culture medium based on Dulbeccos modified Eagle medium supplemented with non-essential amino acid, vitamins, nucleic acid derivatives, metal compounds, human transferrin, insulin and bovine or human serum albumin (Chon Fr.V). In serum-free medium, as well as in serum-containing conventional medium (RPMI-1640), the cells could also spontaneously produce interferon. The cells in the serum-free, culture could produce about 10 000 U/ml of interferon every day, harvesting the culture fluid and refeeding the cells with the fresh medium at the saturation cell density (107 cells/ml). The interferon proved to be α-type interferon on the basis of its physico-chemical and antigenic properties.

EFFECTS OF FERROUS IRON AND TRANSFERRIN ON CELL PROLIFERATION OF HUMAN DIPLOID FIBROBLASTS IN SERUM-FREE CULTURE

SummaryIron-free RITC 80-7 defined medium was used to examine effects of ferrous iron and transferrin on cell proliferation of human diploid fibroblasts. Both ferrous iron and holotransferrin stimulated cell proliferation in the medium, but apotransferrin did not. When 5 g/l human serum albumin (HSA) was added to the defined medium, excellent growth was obtained under hypoxic conditions, whereas a reduction of cellular growth during the culture periods was observed under aerobic conditions. When ferrous iron was added to the HSA medium alone, the reduction in growth increased in proportion to the concentrations, whereas the addition of transferrin prevented this reduction in a concentration-dependent manner. This suggests that the ferrous iron concentration in media causes a reduction in growth under aerobic conditions and transferrin prevents this reduction because it decreases the ferrous iron concentration. Further, serum albumin seems to be a source of iron in media.