(646e) Immobilized Gradients for Presentation of Growth Factors

Interactions between cells and their environment are extremely complex. One of the major difficulties in studying these interactions is the ability to create a defined, controlled surface with which to present selected ligands. The system described here allows presentation of multiple ligands, each spatially controlled. We fabricate gradients of immobilized growth factors utilizing protein-protein interactions and microfluidics. Our immobilization strategy takes advantage of leucine zipper coiled-coil interactions which form heterodimers. One leucine zipper is UV crosslinked to a solid surface. The partner leucine zipper is cloned as a fusion protein to either fibroblast growth factor 4 (FGF4) or to the RGD-containing domain of fibronectin (FN). Both fusion proteins maintain their biological activity towards cells. FGF4 and FN fusion zippers flow over the surface zippers in laminar flow micro-channels and become immobilized due to the leucine zipper heterodimer association. By adjusting the ratios of the fusion proteins, gradients of FGF4 can be formed on either a background of constant FN or a gradient of FN.

The method described here allows for the creation of controlled surfaces presenting both growth factor gradients and cell binding proteins. Immobilization of multiple ligands allows us to independently change the cell attachment to the surface, governed by FN, and the concentration of growth factor. These surfaces are promising tools for investigation of growth factor presentation to cells, in particular, the response of embryonic stem cells which rely on immobilized FGF4 for differentiation signaling.