A human colonic epithelial cell line, T84, derived from a colonic carcinoma, has been examined both morphologically and functionally. The cells grew to confluence as a monolayer with the basolateral membrane attached to the surface of the culture dish and a microvillus-studded apical membrane facing the media. Tight junctions and desmosomes were demonstrated between adjacent cells. Confluent monolayer cultures conducted vectorial electrolyte transport that could be altered by a variety of secretagogues and antisecretagogues similar to isolated intestinal tissues. This cell line will serve as an excellent model system for the study of electrolyte transport processes and their regulation by peptide hormones and neurotransmitters.

Invest. 1995Invest. . 95:1897Invest. -1905

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Clinical trials with anti-EGF receptor MAbs are being conducted, and trials with anti-EGF receptor MAbs combined with doxorubicin are planned.

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We have used a well-differentiated human colonic cell line, the T4 cell line, as a model system to study the pathways of cellular ion transport involved in vasoactive intestinal polypeptide (VIP)-induced chloride secretion. A modified Ussing chamber was used to study transepithelial Na' and Cl-fluxes across confluent monolayer cultures of the T84 cells grown on permeable supports. In a manner analogous to isolated intestine, the Addition of VIP caused an increase of net Cl-secretion which accounted for the increase in short circuit current (1). The effect of VIP on I,, was dose dependent with a threshold stimulation at 10-10 M VIP, and a maximal effect at 10-8 M.Bumetanide prevented or reversed the response to VIP. Inhibition by bumetanide occurred promptly when it was added to the serosal, but not to the mucosal bathing media. Ion replacement studies demonstrated that the response to VIP required the simultaneous presence of Na', K+, and CF-in the serosal media. Utilizing cellular ion uptake techniques, we describe an interdependence of bumetanide-sensitive 22Na', "Rb+, and MCl-uptake, which is indicative of a Na+,K+iCl-cotransport system in this cell line. This transport pathway was localized to the basolateral membrane. Extrapolated initial velocities of uptake for each of the three ions was consistent with the electroneutral cotransport of 1 Na+:1 K+ (Rb+):2 Cl-. Our findings indicate that VIP-induced Cl-secretion intimately involves a bumetanide-sensitive Na+,K+,CF cotransport system which is functionally localized to the basolateral membrane.

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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...