Development of a serum-free medium for a human immortalized fibroblast cell line (KMST-6/TNF) producing tumor necrosis factor-α (TNF-α) and growth inhibitory effects of its conditioned medium on malignant cells in culture

Dear Editor: By transfection of genes into mammalian cells, therapeutically useful proteins such as immunoglobulins and cytokines have been produced (Kaufman and Sharp, 1983; Hayter et al., 1991; Trill et al., 1995). For production of these proteins, serum-free culture systems are desirable because of the economical and technical advantages in subsequent processings such as purification. In addition, the risk of contamination by pathogenic agents such as viruses and mycoplasmata can be eliminated. For large-scale production of proteins, it is also convenient to use a serum-free medium without biological macromoleeular components that can cause problems in the subsequent processings (Glassy et al., 1988; Keen and Rapson, 1995). However, since there is no universal serum-free medium applicable to all cell lines, specific media suitable for each cell line need to be developed (Hayter et al., 1991; Keen and Rapson, 1995; Zang et al., 1995). Tumor necrosis factor-c~ (TNF-c 0 was originally described as a tumoricidal protein that induced necrosis in inethylcholanthreneinduced tumors in mice (Carswell et al., 1975). The eytotoxic effects of TNF-ct cover a broad range of animal and human tumors in vitro and in vivo (for review s e e Fiers, 1991). Moreover, TNF-ct can activate immunocompetent ceils such as lymphokine-aetivated killer cells (0stensen et al., 1987; Pfizenmaier et al., 1987; Chouaib et al., 1988; Wtilfel et al., 1993; Fujii et al., 1994) and maerophages, both in vitro and in vivo (Tahnage et al., 1988). We previously established a TNF-c~-producing cell line, KMST-6/TNK by transfection of an expression vector of human TNF-et into a human immortalized fibroblast cell line, KMST-6 (Fushimi et al., 1998). In this case, the conditioned medium (CM) containing serum inhibited the cell growth of several types of human tumor cells (Fushimi et al., 1998). In the present study, we attempted to develop serum-free medium that can allow proliferation of KMST-6/TNF cells and their production of TNF-c~. The cells used were as follows: KMS-6, normal human fibroblasts; KMST-6, KMS-6 cells immortalized with 6~ gamma rays; KMST6/RAS, neoplastically transformed KMST-6 cells with e-H-ras; and KMST-6/TNK KMST-6 cells transfeeted with the human TNF-R gene. The characteristics of these cell lines, KMST-6, KMST-6/RAS, and KMST-6/TNF, have been reported elsewhere (Namba et al., 1985; Jahan et al., 1993; Fushimi et al., 1998). Each cell line was ordinarily maintained with Eagle's minimal essential medium (Nissui Pharmaceutical Co., Tokyo) supplemented with 10% fetal bovine serum (FBS, Intergen Co., Purchase, NY) and 2 mM L-glutamine in a CO2 incubator at 37 ~ C. The serum-free basal medium (DF/ITS) used consisted of a 1:1 mixture of Dulbeceo's modified E@e's medium and Ham's F12 (Nissui) supplemented with 10 ~g/ml insulin (Sigma Chemical Co., St. Louis, MO), 10 ~g/ml transferrin (Sigma), and 10 8 M selenium. Amount of TNF-a in the serum-containing or serum-free CM was determined using an ELISA kit (R & D System Inc., Minneapolis, MN). We first examined the effects of soybean peptone on the growth of KMST-6/TNF cells in the serum-free medium DF/ITS. Peptone at 0.12% induced a maximum cell growth comparable to that obtained with the medium supplemented with 5% FBS (Table 1). The cells could proliferate in this 0.12% peptone-containing DF/ITS medium to 9.81 _+ 0.52 x 105 cells/35-mm dish, and in 5% FBScontaining medium to 12.29 X 105 cells/35-mm dish. These results show that supplementation of serum-free medium DF/ITS with peptone can sustain proliferation of KMST-6/TNF cells. When cholesterol (1-10 ~g/ml), oleic acid (1-10 txg/ml), or 2mercaptoethanol (1-100 txM) was added at various concentrations to this medium, none of them showed any stimulative effects on growth of the cells but, rather, inhibited the growth at high concentrations (Table 2). Ethanolamine (5-100 p.M), however, stimulated growth of KMST-6/TNF cells (Table 2). Thus, 5 gM ethanolamine was routinely added to the serum-free medium, DF/ITS, containing 0.12% peptone. Addition of peptone and ethanolamine allowed a long-term culture of at least 34 passages and TNF productivity (data not shown). This new serum-free medium for KMST-6/TNF cells was named DF/ITS/EE As shown in Fig. 1, the serum-free CM dose dependently inhibited the growth of the neoplastieally transformed KMST-6/RAS cells. Its inhibition was significantly effective even at the low concentration of 0.3%. In addition, the serum-free CM was more effective in growth inhibition of the cells than the 10% FBS-containing CM, when each CM was tested at a concentration of 10% (Fig. 2). These results indicate that TNF-c~ activity may be higher in the serum-free CM than in the 10% FBS-eontaining CM. However, the amount of TNF-c~ in the serum-free CM was about 75% of that in the 10% FBS-eontaining CM (Table 3). Thus, serum may contain growth stimulatory factor(s) or protect cell damage by TNF-a. Cell therapy is one of the choices for development of a new therapy model for cancers. We therefore established a TNF-c~-produeing cell line (KMST-6/TNF) by transfection of an expression vector of human TNF-a into a human immm'tal fibroblast cell line, KMST-6 (Fushimi et al., 1998). In the present study, we attempted to find suitable serum-free medium conditions for KMST-6/TNF cells. Interestingly, the parent KMST-6 cells of KMST-6/TNF could not grow in the serum-free DF/ITS/EP medium (data not shown). Meat peptone (A1-Rubeai and Spier, 1988; Jan et al., 1994) and proteose peptone (Hasegawa et al., 1988; Jan et ah, 1994) have also been proved to be effective for growth of hybridoma cell lines and for monoelonal antibody production. However, there is a possibility that these animal-derived materials are contaminated with pathogenic