Preparation and use of synthetic cell culture surfaces. A new reagent for the covalent immobilization of proteins and glycoproteins on a nonionic inert matrix.

To study cell interactions with external molecules immobilized on a chemically defined nonionic, inert matrix, we have prepared flat polyacrylamide matrices containing covalently attached carbohydrate or protein. A new acrylamide derivative, containing a terminal 1,2-dihydroxy group, was synthesized and then copolymerized with acrylamide and bisacrylamide to make 20% polyacrylamide matrices, which could be oxidized with NaIO4 to generate reactive aldehyde groups. Molecules containing a free amine (e.g. proteins or glycopeptides) can be coupled to the aldehyde-activated matrix by formation of a Schiff base and reduction with NaCNBH3 to form a stable -CH2-NH-bond. Unreacted aldehyde groups are reduced to hydroxyl groups with NaBH4. In order to immobilize polysaccharides on the activated surfaces, these molecules are first modified to contain a free amine. We have described a procedure to convert purified hyaluronic acid oligosaccharides to a reactive alkylamine derivative uniquely modified at the reducing end (Raja, R. H., LeBoeuf, R. D., Stone, G. W., and Weigel, P. H. (1984) Anal. Biochem. 139, 168-177). The covalent attachment of [3H]hyaluronate-amine, [14C]ethanolamine, or 125I-bovine serum albumin, to activated surfaces was complete within 5-24 h. The amount immobilized was directly proportional to the amine concentration and to the aldehyde content of the matrix and inversely proportional to the molecular weight of the amine. About 90% of the available aldehyde groups reacted with ethanolamine, whereas less than 0.01% reacted with albumin. Molecules larger than 3300 Da were excluded from the interior of the matrix and could therefore only be attached to the surface of the matrix. These synthetic surfaces can be used in long term culture experiments to study cellular interactions with virtually any type of immobilized molecule.