Gradient micropattern immobilization of EGF to investigate the effect of artificial juxtacrine stimulation

To investigate the concentration effects of immobilized biosignal molecules by microscopic observation, a gradient micropattern immobilization technique using a photomask was devised. Epidermal growth factor (EGF) was chosen as the biosignal molecule and was conjugated with photoreactive polyallylamine. The EGF derivative was immobilized onto a polystyrene plate by UV irradiation in the presence of a gradient-micropattern photomask. Gradient micropattern immobilization of the EGF derivative on the polystyrene plate was confirmed by immunostaining with anti-EGF antibody. Chinese hamster ovary cells overexpressing EGF receptors were cultured on the micropatterned plate. Growth enhancement was observed only in dense EGF derivative immobilized regions. The present technique is useful for the investigation of concentration-dependent effects of immobilized biosignal molecules.