Development of a method to measure viscoelasticity of cardiomyocytes using scanning probe microscope

我们开发了一种测量动物心肌细胞粘弹性的方法。大鼠用戊巴比妥钠麻醉，心脏逆行灌流胶原酶分离细胞。细胞在层粘连蛋白包被的培养皿中孵育4h(37℃，5%CC_2)，在此期间细胞牢固地附着在培养皿上。我们制作了一个电路，并对扫描探针显微镜进行了改装，使我们能够根据外部命令用压电器件控制探针的垂直位置。我们还开发了一个软件来执行命令，并测量压电器件的位置和悬臂梁的弯曲。当施加正弦振动时，悬臂梁的弯曲是同步的，表明超融合液的粘度可以忽略不计。我们通过对细胞进行电刺激来测量细胞在心动周期中弹性的变化，但结果表明没有显著变化。众所周知，渗透压确实会改变弹性，我们推测探头检测的是局部弹性，而探头大小的变化似乎是检测心肌细胞弹性变化的必要因素。缺乏预负荷似乎是造成弹性的微小变化的原因。无论是用胶原酶灌流，还是用超声波搅拌，或者用蛋白酶超融合，我们都不能收集足够的健康细胞。

We developed a method to measure viscoelasticity of cardiomyocytes from animals. Rats were anesthetized with pentobarbital, and the hearts were retrogradely perfused with collagenase to isolate cells. The cells were incubated for 4 hour (37℃, 5 %CC_2) in a laminin-coated dish, during which the cells firmly attached to the dish. We made a circuit and modified a scanning probe microscope so that we were able to control the vertical position of the probe with a piezoelectric device according to external command. We also developed a software to impose command and to measure the position of the piezoelectric device and the bending of the cantilever. When we imposed a sinusoidal vibration, the bending of the cantilever was in phase, indicating the viscosity of the superfusate is negligible.We measured the changes in elasticity of cells during cardiac cycle by subjecting the cell to electrical stimulation, but the results indicated no significant changes. As it is known osmotic stress do change elasticity, we conjectured that the probe detects the local elasticity, and the changes in probe size seem necessary to detect the myocardial cellular elasticity changes. The lack of preload seems to contribute the small changes in elasticity.We were not able to collect sufficient healthy cells either by superfusion with collagenase with or without ultrasonic agitation, or super fusion with protease.