The ability to design and create biologically relevant patterns via microfluidic patterning on surfaces provides new capabilities for cell biology, the production of biosensors and tissue engineering. However, cellular patterns, defined by microfluidic methods, often lose integrity over time due to cell growth and migration immediately upon removal of the PDMS stamp. In this study, biopolymer matrices were used in conjunction with cellular micropatterning to control cell attachment, growth, and long-term maintenance of these patterns. The incorporation of appropriate matrix materials with microfluidic cell patterning methods results in highly compliant patterns of adherent human endothelial cells (HUVECs) and fibroblasts after several day in vitro. Furthermore, cell type and chemical components in these biopolymer matrices influence the ability of the biopolymer matrices to control cell growth, proliferation and compliance to the patterns. Cell growth and migration in micropatterned biopolymers such as agarose, collagen, collagen-GAG mimics, and collagen-fibronectin are quantitatively measured and compared, and cell-matrix interactions are also examined over time. Results suggest that the use of an appropriate biopolymer matrix helps to control cell growth and maintain pattern integrity for long periods of time. This is essential for conducting stable biological experiments, as well as achieving control over tissue engineering constructs with multiple cell types.