Neuronal networks in vitro: formation and organization on biofunctionalized surfaces

Receptor-mediated recognition of substrate molecules is a prerequisite for nerve cells in order to find their target structures in vivo and leads to formation of neuronal connections and networks. In order to study these mechanisms under in vitro conditions, we cultured embryonic hippocampal neurons or neuronal cell lines, SH-SY5Y and PCC7-PCC7-Mz1, onto biofunctionalized surfaces. Micropatterning on polymer surfaces, glass- and silicone-oxide-based chip materials was performed in a micrometer range by microcontact printing using polydimethylsiloxane (PDMS) stamps. Hippocampal neurons were found to form networks on chip surfaces under serum-free conditions and remained functional for more than a week. Human neuroblastoma cells SH-SY5Y as well as PCC7-Mz1 stem cells were found to follow microcontact printed pattern on polystyrene surfaces. Both cell lines showed neuronal marker expression and were cultured for up to 7 days with serum containing culture medium. Widths of 3-5 microm of coating lines were found to enhance single cell spreading along the pattern. The techniques described in this study may be useful in promoting nerve cell regeneration and organization following transection due to trauma or surgery. The neuronal alignment and network formation in vitro may furthermore serve as a model system in the field of biosensors.