Growth of hybridoma cells in serum-free medium: ethanolamine is an essential component.

Abstract

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...

Animal‐cell culture media: History, characteristics, and current issues

“…In addition, Hiroki Murakami et al. found that ethanolamine is essential for the serum‐free cultivation of hybridomas and developed the supplement, ITES, which consists of ITS plus ethanolamine 89. Various other supplements were designed for different cell types and cultivation purposes, with the aim of the addition of various trace elements to protein‐free culture media.…”

Immunostimulatoryin Vitroandin VivoEffects of a Water-Soluble Extract from Kale

“…7) HB4C5 cells were cultured in an ERDF medium supplemented with 100 units/mL of penicillin, 10 mg/mL of streptomycin, 10 mg/mL of insulin, 20 mg/mL of transferrin, 20 mM ethanolamine, and 25 nM sodium selenite (ITES-ERDF) at 37 C under 5% CO 2 in humidified air. 8) Human peripheral blood lymphocytes (PBLs) were obtained from peripheral blood of a healthy female donor as described in a previous report. 9) Briefly, human peripheral blood diluted with an equal volume of PBS was centrifuged at 800 Â g for 20 min on a lymphocyte separation medium (Nycomed Pharma, Oslo, Norway).…”

The mode of actions of glyceraldehyde‐3‐phosphate dehydrogenase identified as an immunoglobulin production stimulating factor

“…HB4C5 cells, which produce human lung cancer specific monoclonal IgM, were fusion products of a human lymphocyte and a human fusion partner NAT-30 cells [6]. HB4C5 cells were maintained in ERDF medium (Kyokuto Pharmaceutical, Japan) supplemented with 10/.tg/ml of insulin, 20/lg/ml of transferrin, 20/IM ethanolamine and 25 nM sodium selenite (ITES-ERDF) at 37°C under humidified atmosphere of 5% COJ95% air [7].…”