Selective silane removal: a technique for micropatterning of neurons in culture

The authors have developed a new photoresist based technique for micrometer resolution patterning of covalently bound silanes on glassy substrates in order to pattern the growth of primary hippocampal neurons in low density culture. The technique is named Selective Silane Removal because the substrate is initially reacted with a cytophilic aminosilane (trimethoxy-silylpropyl-diethylenetriamine or DETA), which, is then aggressively removed by plasma etching through a masking layer of patterned photoresist. The process is completed by reacting the cleaned surface areas with a cytophobic arylsilane (phenyltrichlorosilane or PTCS) followed by removal of the photoresist, rinsing and sterilization. Embryonic rat hippocampal neurons are cultured on the substrates at very low density (20000 cells/cm²) using serum free B27/Neurobasal(TM) defined medium. Up to 90% of cell bodies grown on these substrates complied to a grid pattern of 3, 5 or 10 μm wide lines. An average of 76% of the background squares were free of stray neurites or cells connected to the pattern