Patterning axonal guidance molecules using a novel strategy for microcontact printing.

We present here a two-step strategy for micropatterning proteins on a substrate to control neurite growth in culture. First, conventional microcontact printing is used to prepare a micropattern of protein A, which binds the Fc fragment of immunoglobulins. Then, a chimeric protein, consisting of the extracellular domain of a guidance protein recombinantly linked to the Fc fragment of IgG (prepared using conventional molecular techniques), is applied from solution. The chimeric protein binds to the patterned protein A, taking on its geometric pattern. Using this method, we have micropatterned the extracellular domain of the cell adhesion molecule, L1 (as an L1-Fc chimera) and demonstrated that it retains its ability to selectively guide axonal growth. L1-Fc micropatterned on a background of poly-L-lysine resulted in selective growth of the axons on the micropattern, whereas the somata and dendrites were unresponsive. Substrates bearing simultaneous micropatterns of L1-Fc and poly-L-lysine on a background of untreated glass were also created. Using this approach, cell body position was controlled by manipulating the dimensions of the poly-L-lysine pattern, and the dendrites were constrained to the poly-L-lysine pattern, while the axons grew preferentially on L1-Fc. The two-step microcontact printing method allows preparation of substrates that contain guidance proteins in geometric patterns with resolution of approximately 1 microm. This method should be broadly applicable to many classes of proteins.