Microcontact printing was used to pattern a mixture of poly-d-lysine and extracellular matrix (ECM) gel on an agarose-coated coverslip. PDMS stamps were fabricated according to the protocol described previously (13). The ratio of prepolymer and curing agent of PDMS (SYLGARD 184, Dow Corning) was optimized for each micropattern design, which was 10:1 for the no-bond and single-bond patterns and 7.5:1 for the triple-bond and merged patterns. The three-dimensional topography of the stamp surfaces was analyzed using confocal microscopy (VK-X260, KEYENCE).

To print proteins, a glass coverslip (12-545-84, Thermo Fisher Scientific) was first cleaned by sonication in 100% ethanol and subsequent treatment in air plasma for 60 s (PM-100, Yamato). Four dots of paraffin wax (P3558, Sigma-Aldrich) were then placed at the periphery of the coverslip (approximately 0.5 mm in height), and the surface was then coated with 0.2% agarose (A9918, Sigma-Aldrich). After drying the agarose overnight, the coverslips were sterilized by ultraviolet irradiation for 30 min, and protein ink [ECM gel (E1270, Sigma-Aldrich; 1:100 dilution) + poly-d-lysine (50 μg ml−1; P0899, Sigma-Aldrich)] was stamped using a single-axis micromanipulator. The coverslip was dried overnight in a fume hood and was subsequently immersed in the neuronal plating medium [minimum essential medium (MEM; 11095-080, Gibco) + 5% fetal bovine serum + 0.6% d-glucose] a day before cell plating.

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