Development of multiphoton laser fabrication system for tissue engineering

我们开发了一种三维激光加工系统，可用于培养条件下的活体组织结构建模。以锁模Ti：蓝宝石激光器（脉冲宽度80-100fs，波长750-900nm，重复频率82MHz）为光源，聚焦成光聚合材料。在聚焦点处，材料具有多光子吸收并发生聚合。由于聚合区域位于焦点体积内，对焦点的扫描使我们能够在三维空间中创建任何形状的结构。x-y方向的扫描激光是由一对振镜产生的，z方向的扫描是用压电级扫描样品进行的。在采用NA1.0物镜的情况下，该系统可制备的结构尺寸约为500μm x 500μm x 100μm，空间分辨率约为200nm。制备的三维结构被用作控制细胞在三维空间生长的支架。为了在结构上培养细胞，我们使用光固化明胶作为光聚合材料。对激光功率、曝光时间、脉冲宽度、激光波长、明胶浓度等实验条件进行了优化。我们还研究了可以制造的结构的最大尺寸，以确认系统的性能。通过对大鼠心肌细胞和人类癌细胞在结构上的培养和观察，我们证实了制备的微结构可以控制两种细胞的生长。

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.