Evaluation system of cardiac cells and tissues with combined micro-electrode array based electrical mapping and optical mapping system

为了心脏病学再生医学的进展，例如基于培养心脏组织的生物起搏器用于心律失常治疗，我们需要一个系统来评估心脏细胞、心脏组织和植入再生组织的整个心脏的功能。本研究开发了体外培养心脏组织和全心制备的光电联合测量系统。1)研制了具有动作电位传播动态实时反馈的心脏电刺激系统。该系统采用高时空分辨率的光学成像方法，实现了心电传播的可视化。实时确定传播激励波前，并根据传播方向控制电刺激的位置和时间。它可以根据心脏组织的相（去极化、再极化和静息相），通过控制时间和位置来启动Make/Break激励。用兰根多夫灌注兔心脏制剂验证了该系统的功能。2)我们还开发了用于培养心脏细胞和干细胞的测量系统，其中通过光刻技术制造的集成微电极阵列（64通道，30 μm X 30 μm，电极间距：130 μm）测量时间和空间电激活模式，并通过冷却CCD相机测量细胞内Ca的荧光图像。将新生大鼠心肌细胞分离培养。体外3天后观察机械收缩。它生长为培养细胞的扩展区域的收缩。电活动和机械收缩的同步也被确定。同时用荧光染料（fluo-4）测定细胞内钙浓度。确定了钙动力学和电活动的同步性。

For progress of regenerative medicine in cardiology, such as bio-pacemaker based on cultured cardiac tissue for arrhythmia therapy, we need a system to evaluate function of cardiac cell, cardiac tissue, and a whole heart where the regenerated tissue is implanted. In this study, combined optical and electrical measurement system of cultured cardiac tissue and whole heart preparation has been developed.1) We have developed a cardiac electric stimulation system with real time feedback of action potential propagation dynamics. In this system, cardiac electrical propagation is visualized by optical mapping method with high spatial and temporal resolution. Propagated excitation wave front is determined in real time, and the location and timing of electrical stimulation is controlled depending on the propagation pattern. It can initiate Make/Break excitation by controlling the timing and location considering the phase (depolarized, repolarized, and resting phase) of the cardiac tissue. The function of the system was confirmed by experiments with Langendorff perfused rabbit heart preparations.2) We have also developed measurement system for cultured cardiac cells and stem cells where temporal and spatial electrical activation patterns are measured by an integrated micro-electrode arrays fabricated with photo lithography (64 channels, 30 μm X 30 μm, electrode spacing: 130 μm), and a fluorescence image of intracellular Ca is measured by a cooled CCD camera. Cardiac cells were separated from neonatal rats and cultured. mechanical contraction was observed after three days in vitro. It grows to contraction of extended areas of cultured cells. Synchronization of the electrical activities and mechanical contraction were also identified. Intracellular calcium concentration was measured by fluorescent dye (fluo-4) simultaneously. Synchronization of calcium dynamics and electrical activities were identified.