Plasma membrane glycoproteins covalently bound to silica beads as a model for molecular studies of cell-cell interactions in culture

In previous studies, we have shown that plasma membrane glycoproteins are of major importance in the density-dependent regulation of growth of normal diploid fibroblasts. Due to the hydrophobic portions of these molecules, functional studies in cell culture are often difficult to perform and to interpret. Specifically, the addition of these molecules in soluble form to cell culture, after depletion of detergents needed for their solubilization, leads to aggregation and internalization. Therefore, we developed a method for the covalent immobilization of the solubilized plasma membrane proteins to derivatized silica beads for further investigations on the molecular nature of the active molecules. The addition of immobilized plasma membrane glycoproteins to sparsely seeded human fibroblasts resulted in cellular reactions similar to those found in confluent cell cultures (strongly reduced cell proliferation; high collagen type III synthesis). The method consists in the derivatization of silica beads (Lichrosphere Si 500, 10 microns) with isothiocyanatopropyltriethoxysilane. Amino-groups react with the SCN group under physiological conditions, resulting in a stable linkage of amino-group bearing molecules with the silica beads. Due to the easy handling of the silica beads (e.g. washing by short centrifugation steps), the mild coupling conditions, and the stable bondings this system is highly suited for functional studies of molecules involved in cell-cell interactions.