Aligned Multimask Patterning of Biomolecules and Cells

Surface engineering of cell culture substrates has developed into a powerful tool for controlling multicellular organization at the micrometer scale. This new capability has brought valuable in¬sight into the biological mechanisms by which the cellular microenvironment determines cell fate and function. However, studies requiring more complex tissue structures have been hindered by limitations in surface patterning. Typically, molecules that mediate cell attachment are patterned against a non-adhesive background, allowing arrays of a single cell type to be formed with control of cell positioning and relative spacing. Alternatively, patterns composed of two different adhesive regions can be employed to form patterned co-cultures of two different cell types, as long as one cell type selectively attaches to a specific region. However, there have been a few examples where multiple attachment chemistries have been successfully combined with non-adhesive surfaces in a multicomponent pattern. This has prevented the realization of configurations in which cell-cell contact and spacing between different cell types are controlled.

The use of photolithography with multiple aligned masks is well established for generalized multicomponent patterning, but it is often too harsh for biomolecules. We report a two-mask photo¬lithographic process that is tuned to preserve bioactivity in patterns composed of covalently coupled polyethylene glycol (PEG), adsorbed extracellular matrix protein (e.g., collagen I), and ad¬sorbed serum proteins (e.g., vitronectin). Thereby, we pattern two cell types-primary hepatocytes and 3T3 fibroblasts-demonstrating control over contact and spacing (20-200 µm) between the two cell types for over one week. This method is applicable to the study of intercellular communication in cell biology and tissue engineering.