Development of multiphoton laser fabrication system for tissue engineering

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We developed a three-dimensional laser fabrication system which can be applied for modeling living tissue structure in cultural condition. A mode-locked Ti : Sapphire laser (pulse width : 80-100fs, wavelength : 750-900nm, repetition rate : 82MHz) was used as a light source and was focused into a photopolymerizable material. At the focusing point, the material has multiphoton absorption and is polymerized. Since the polymerization area is localized within the focal volume, scanning of the focus enables us to create any shape of structures in three dimensions. Scanning laser in x-y directions was made by a pair of galvanometer mirrors, and scanning for z direction was done by scanning sample with using a piezoelectric stage. The size of structures which can be fabricated by the developed system is about 500μm x 500μm x 100μm with the spatial resolution of about 200nm when NA1.0 objective lens is used. The fabricated three-dimensional structure was used as a scaffold which control cell growth in three-dimensional space. For culturing cells on a structure, we used photocurable gelatin as a photopolymerizable material. We optimized experimental conditions for fabrication, such as laser power, exposure time, pulse width, wavelength of the laser, and concentration of gelatin. We also investigated the maximum size of structures that can be fabricated in order to confirm the performance of the system. By culturing and observing rat cardiomyocytes and human cancer cells on the structure, we confirmed that growth of both type of cells can be controlled with the fabricated microstructure.