Cells interact with a spatially complex and highly dynamic extracellular matrix (ECM), which orchestrates their function. There is a growing interest among biomedical scientists to engineer in-vitro scaffolds that recapitulate certain important facets of the natural ECM to systematically probe and understand how extracellular signals alter various cellular functions, such as migration, differentiation, and proliferation. This chapter outlines methods and techniques to spatially pattern and dynamically alter biochemical cues in in-vitro cell culture scaffolds, and further highlights examples of their application in answering important questions about cell biology, tissue engineering, and regenerative medicine.