Isolation of tumor-secreted products from human carcinoma cells maintained in a defined protein-free medium.

Abstract

A protein-free synthetic cell-growth medium has been defined that permits long-term survival (>120 days) of an established human colon tumor cell line, HT-29. Viability is dependent upon both the concentration of L-glutamine in the medium and the cell density at the time of initial transfer into it. Cell proliferation is minimal, thus obviating the necessity for subculturing. HT-29 adenocarcinoma cells maintained in large-scale culture with this medium continue to secrete the established colon tumor marker carcinoembryonic antigen as well as growth factors and lysozyme. These and, potentially, other important tumor-derived products can therefore be generated continuously in such cultures so that they can be isolated from a conditioned medium free of contaminating serum and protein supplements.The large-scale in vitro cultivation of malignant cells can potentially produce tumor-derived biomolecules in yields sufficient for both biological and chemical characterization. However, for this purpose exogenous serum and/or its growth factor components § are virtually compulsory additives. This has greatly complicated the procedure itself and, beyond that, the very objective, i.e., the identification and purification of tumor constituents which are usually present in vanishingly small amounts. Successful attempts to culture tumor cells under serum-free conditions have been reported, but, while eliminating addition of serum, they have substituted moieties obtained from it (1-3). Moreover, each cell line generally then requires a set of such molecular species unique for its survival and devised to bring about cellular proliferation rather than maintenance of long-term viability and secretory capacity. The latter has proven difficult if not impossible to achieve in this manner.The present studies, part of our effort to isolate and characterize tumor-secreted products, were aimed at maintaining long-term viability rather than proliferation of human tumor cells in serum-free media and in the absence of serum-generated factors. The medium that we have developed is devoid of exogenous proteins or other growth factors and supports the survival of at least two colon adenocarcinoma lines which remain physiologically active with respect to some functions. (4.5 mg/ml), gentamicin (50 Ag/ml), and Fungizone (0.5 ,.g/ml) (DME) and supplemented with 5% heat-inactivated fetal bovine serum and 2 mM L-glutamine (DME/5%). BALB/c 3T3 cells, clone A31-MK25, were provided by M. Klagsbrun and were maintained in DME supplemented with 2 mM L-glutanline and 10% heat-inactivated bovine serum. Cultures were incubated at 370C in humidified air maintained at 7% CO2. Cells were fed every 2-3 days as required and subcultured at confluence by standard trypsinization techniques. MATERIALS AND METHODSCell Growth Curves. To evaluate HT-29 growth in serumfree medium, cells maintained as above were trypsinized, resuspended in DME/5%, and seeded into 24-well (1.9-cm2) Nunc microplates (Vangard International, Neptune, NJ). At specified intervals, condi...

Differential purification of autocrine motility factor derived from a murine protein-free fibrosarcoma

“…In medium harvested by day 2, however, serum-derived factors are still expected to be present [51]. Alderman et al reported that serumderived albumin could not be detected immunochemically by day 7 [52]. The cultured medium presented here showed a distinct distribution to FCS on the SDS-PAGE, and contained a smaller number of peptides than FCS [37].…”

Biosynthesis and glycosylation of the carcinoembryonic antigen

“…The present results permit the elucidation of some steps in the biosynthetic pathway of CEA, an important tumour marker. This antigen, characterized in human carcinoma, has also been demonstrated to be present in some cultured tumour-cell lines (Alderman et al, 1985;Beauchemin et al, 1987;Philben et al, 1986;Tsao & Kim, 1978); we describe here a study on MKN-45, a CEA-producer-cell type from human gastric adenocarcinoma.…”

Human angiogenin, an organogenic protein

“…Therefore conditions were sought where tumour cells could be grown in the absence of foetal calf serum. Eventually it was found that tumour cells could be maintained in serum-free medium supplemented with 5mM glutamine (Alderman et al, 1985). Although they do not proliferate under these conditions, the cells continuously secrete proteins into the medium for periods of up to several months.…”