A serum-free medium supplemented with a few growth factors was devised to grow lymphocyte hybridomas. The medium was developed with the hybridoma line MPCll-BL, a fusion product between a mouse plasmacytoma cell line (MPCllTG70na3) and mouse (BALB/c) spleen cells. In the process of developing the-medium, ethanolamine was found to be an essential growth factor for the hybridoma. Phosphoethanolamine at 10-fold higher concentration could substitute for ethanolamine. Long-term cultivation of the cells was achieved in the defined medium supplemented with insulin, transferrin, ethanolamine, and selenium. The defined medium supported the growth of various other mouse hybridoma cell lines, mostly at a rate comparable to that observed in a serum-containing medium. After one-step ammonium sulfate precipitation of the spent medium, more than 95% of the protein recovered was immunoglobulin as shown by NaDodSO/polyacrylamide gel electrophoresis.Traditionally, tissue culture media have required serum supplementation in order to support long-term culture of cells. Serum is a complex mixture of components which may vary according to the age, sex, and species of its source. Sato and his co-workers have systematically investigated the hormone and growth factor requirements ofa large number ofcell lines. They described completely defined serum-free media that promote growth comparable to serum-containing media and in which cells maintain their differentiated characteristics (1, 2). Among the lines investigated 'are the mouse myeloma MPC11 (3), rat neuroblastoma B104 (4), and human colon carcinomas (5).Fusion of a plasmacytoma and a splenic plasma cell can produce a hybridoma that continues to secrete splenic antibody (6), and the monoclonal antibodies produced have been useful for immunology, molecular biology, and cell biology research (6, 7). Cultivation of these cells in a defined medium would facilitate such studies by eliminating serum, the major source of protein contamination.We have developed a hormone-and growth factor-supplemented, serum-free medium for culturing mouse plasmacytomas and hybridoma cell lines of MPC1l-BL and SP2/0-BL' origins. In the process of defining this medium we identified two compounds, ethanolamine and phosphoethanolamine, as necessary growth-promoting materials. These compounds, which had originally been found to be essential for growth of a rat mammary carcinoma (8, 9) have been required for growth of all hybridoma lines tested. Analyses of proteins precipitated at 50% ammonium sulfate saturation of the spent medium of hybridoma cell cultures indicated that in most cases >95% of the proteins were IgG or IgM. MATERIALS AND METHODSCells and Cell Culture. Most ofthe hybridoma cell lines used in these studies were obtained from W. Raschke (Salk Institute, La Jolla, CA). For NaDodSOipolyacrylamide gel electrophoresis of antibodies, hybridomas between SP2/0 and BALB/c splenocytes immunized against S100 protein were used. These cell lines were ordinarily maintained in a 1:1 mixture of Ham's F-1...
We have identified phosphoethanolamine as the pituitary-derived growth-promoting material that specifically stimulates the rat mammary carcinoma cell line 64-24. We have been studying the growth characteristics of the 64-24 cell line, which was isolated from a highly hormone-dependent tumor and which retains in culture many characteristics of the original tumor. Previously, crude bovine pituitar extract was shown to contain a significant amount of growth-stimulating activity for these cells, and a growth factor from this extract was purified to homogeneity. This report describes the identification of the growth factor as phosphoethanolamine. Further, the biological activity of phosphoethanolamine was found to be virtually identical to that of the purified growth factor. A possible role of phosphoethanolamine in the growth of mammary tumor cells as well as of normal mammary epithelial cells and other tissues is discussed.We have been studying growth characteristics in culture of a clonal line of hormone-dependent rat mammary carcinoma, 64-24. The MCCLX tumor, from which the 64-24 line was isolated, requires the functional pituitary for growth: hypophysectomy of the tumor-bearing animals caused the regression of the tumor (1). Injection of prolactin to the hypophysectomized animals, however, partially restored the growth of the tumor, indicating that the tumor requires prolactin for growth (2). We have previously shown that the cells proliferate well in the presence of fetal calf serum, but that they do not proliferate, or do so only slowly, in the presence of calf serum. Known pituitary hormones, including prolactin, when added to the calf serum-supplemented medium, have not been able to stimulate the growth of 64-24 cells, however (1). This led us to examine the growth-stimulatory activity of crude pituitary extract. Indeed, we found that the crude bovine pituitary extract contains the growth-stimulatory activity of the 64-24 cells (3); subsequently, this pituitary-derived growth factor for mammary cells (MGF) was purified to homogeneity (4). The factor is a ninhydrin-positive, acidic, hydrophilic material whose molecular weight is less than 1000. Now, we have identified MGF as phosphoethanolamine (PEtn).MATERIAL AND METHODS Materials. Bovine pituitary glands were purchased from Pel-Freez. Calf serum and horse serum were from Microbiological Associates (Walkerville, MD) and Flow Laboratories (Inglewood, CA), and fetal calf serum was obtained from KC Biologicals (Lenexa, KS). PEtn was synthesized by A. Oikawa, Tohoku University School of Medicine, and ethanolamine was purchased from Wako Pure Chemical institute, Tokyo, Japan. Cheng Chin polyamide layer sheets were from Accurate Chemical and Scientific, Hicksville, NY. Purification Procedure. A partial purification procedure of MGF has been published (3), and the complete purification procedure has also been described (4). Frozen bovine pituitaries were homogenized in a Waring Blendor with two vol of cold 0.075 M acetic acid for 5 min at 4VC. The homogenat...
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