The incorporation of L-[1-3-H]= fucose into non-collagenous glycoproteins secreted by human fibroblasts in culture.

Studies on the biosynthesis and secretion of macromolecules by cultured fibroblasts have concentrated mainly on collagen and proteoglycan-related species, but little is known of the nature and role of non-collagenous glycoproteins secreted by these cells. In this communication we describe the determination of optimum conditions of culture for the analysis of glycoprotein biosynthesis by human skin fibroblasts, and the preliminary characterization of secreted macromolecules labelled with ~-[l-~H]fucose. Investigations of glycoprotein synthesis and secretion are best performed when the maximum number of cells are present in the culture, i.e. at confluency. Serum-containing media are required for the satisfactory attainment of confluency, but their continued use for the maintenance of the confluent monolayer present certain difficulties when secreted glycoproteins are to be studied. The major problem concerns the presence of serum proteins in vast excess of the secreted species which makes characterization and isolation of the latter extremely difficult. Also there exists the possibility that serum contains degradative enzymes such as glycosidases, and there is some evidence that serum may induce or activate the cellular production and secretion of proteinases (Bzuikowski & Mitchell, 1973). To avoid these difficulties some workers have employed serum-depleted growth medium (Goldberg et al., 1972; Dell’Orco & Nash, 1973 : Bankowski & Mitchell, 1973) but these conditions, if used for extended periods, lead to appreciable cell detachment from the substrate and loss of cellular protein (Dell’Orco et al., 1973). To minimize the above problems we have examined the ability of various media to maintain confluent fibroblast cultures by using the parameters of cell viability, cell detachment and cellular protein as indices of cell integrity. Fibroblasts were grown from human skin biopsy material by using the explant technique, and cultured on plastic in Dulbecco’s modification of Eagle’s medium supplemented with 10% (v/v) foetal bovine serum (DMEM/10% FBS). Confluent monolayers were divided into four groups and maintained on the following four media: DMEM/10 % FBS, DMEM/O.S % FBS, DMEM alone and the completely chemically defined medium MAB 87/3 described by Gorham & Waymouth (1965). At 24h intervals over a 3-day period the following measurements were made in duplicate. (i) Cells detached from the substrate were collected by centrifugation of the medium at 500g for 4min, stained with Methylene Blue (0.2%, w/v) and counted. (ii) Cell viability was assessed with Trypan Blue (0.067 %, w/v) after trypsin treatment of the cell monolayers into a known volume of Hanks basal salts solution. (iii) Protein content of the trypsin-treated cells was determined by the method of Lowry et al. (1951). Cell viability was high (97-99%) in all four media studied over the whole 3-day period. Determination of cell detachment indicated that monolayers maintained with DMEM/lO% FBS or MAB 87/3 media lost less than 1 % of the total cell population after 3 days, compared with much higher values for the other two media (Fig. 1). The total cell protein determinations demonstrated decreases of approx. 30% and 20% with DMEM and DMEM/O.S% FBS media respectively after 3 days, but no significant decrease with either DMEM/10% FBS or MAB 8713 media. Waymouth’s MAB 8713 medium is thus shown to be superior to DMEM/O.S% FBS or DMEM for the maintenance of confluent fibroblast monolayers, and makes possible the study of glycoprotein secretion in such cultures in the absence of serum. The glycoprotein-specific marker, L-[ l-3H]fucose, was used to detect the secreted species of interest. Fibroblast cultures were grown to confluency in DMEM/10% FBS medium, and subsequently maintained on medium MAB 87/3 in the presence of 13H]fucose. Incorporation of the radioactivity into non-diffusible macromolecules in the medium was linear over a 3-day period. The location of the 3H in the secreted material