Synthesis of protein-coated gelatin microspheres and their use as microcarriers for cell culture. Part I. Derivatization with native collagen

Abstract

A novel technique for the synthesis of native collagen (type I)-coated gelatin microspheres was developed using the emulsion-polymerization principle, which is realized in four steps: emulsification, separation, stabilization, and protein modification. A 20% gelatin solution was emulsified in sunflower oil and the resulting beads were polymerized with glutaraldehyde. The novelty of our technique consists in the protein modification step, where we derivatized the beads by saturation of the free aldehyde groups on the cross-linked gelatin in the native collagen solution (0.3 mg/ml in 0.05 M Tris, pH 8.6) and further stabilized the beads with sodium borohydride. The resulting microspheres exhibited excellent properties as microcarriers for in vitro cell culturing using Vero cells as the model system. In comparison with pure gelatin beads, the cells attached more rapidly and grew faster on native collagen-coated beads. Approximately 90% of the Vero cells attached to the microcarrier after 1 h. After a lag period of nearly 24 h, the cells began to grow rapidly and reached confluence for 4-5 days, whereas the cells on pure gelatin beads reached the same density about 1 or 2 days later.