Khraiche Massoud Louis, Zhou Anhong, Muthuswamy Jit
Harrington Department of Bioengineering, ECG 334, College of Engineering and Applied Science, Arizona State University, P.O. Box 879709, Tempe, AZ 85287-9709, USA.
J Neurosci Methods. 2005 May 15;144(1):1-10. doi: 10.1016/j.jneumeth.2004.09.029.
Neuronal adhesion plays a fundamental role in growth, migration, regeneration and plasticity of neurons. However, current methods for studying neuronal adhesion cannot monitor this phenomenon quantitatively in real-time. In this work, we demonstrate the use of an acoustic sensor to measure adhesion of neuro-blastoma cells (Neuro-2A) in real-time. An acoustic sensor consisting of a quartz crystal sandwiched between gold electrodes was placed in a flow cell and filled with 600 microl of phosphate buffered saline (PBS). Two sets of in vitro experiments were performed using sensors that had uncoated gold electrodes and sensors that were coated with a known neuronal adhesion promoter (poly-l-lysine or PLL). The instantaneous resonant frequency and the equivalent motional resistance of the acoustic sensor were monitored every second. Cell Tracker was used to confirm neuronal adhesion to the surface. Addition of 10 microl of media and Neuro-2A cells into the above set-up elicited exponential changes in the resonant frequency and motional resistance of the quartz crystal with time to reach steady state in the range of 2-11 h. The steady-state change in resonant frequency in response to addition of neurons was linearly related to the number of Neuro-2A cells added (R2=0.94). Acoustic sensors coated with the adhesion promoter, PLL showed a much higher change in resonant frequency for approximately the same number of neurons. We conclude that the acoustic sensor has sufficient sensitivity to monitor neuronal adhesion in real-time. This has potential applications in the study of mechanisms of neuron-substrate interactions and the effect of molecular modulators in the extra cellular matrix.
神经元黏附在神经元的生长、迁移、再生和可塑性方面发挥着基础性作用。然而，目前用于研究神经元黏附的方法无法实时定量监测这一现象。在这项工作中，我们展示了使用声学传感器实时测量神经母细胞瘤细胞（Neuro-2A）的黏附情况。将一个由夹在金电极之间的石英晶体组成的声学传感器放置在流动池中，并填充600微升磷酸盐缓冲盐水（PBS）。使用未涂覆金电极的传感器和涂覆有已知神经元黏附促进剂（聚-L-赖氨酸或PLL）的传感器进行了两组体外实验。每秒监测声学传感器的瞬时共振频率和等效运动电阻。使用细胞追踪器来确认神经元与表面的黏附。向上述装置中加入10微升培养基和Neuro-2A细胞会引起石英晶体的共振频率和运动电阻随时间呈指数变化，在2 - 11小时内达到稳态。响应神经元添加的共振频率稳态变化与添加的Neuro-2A细胞数量呈线性相关（R2 = 0.94）。涂覆有黏附促进剂PLL的声学传感器对于大致相同数量的神经元显示出更高的共振频率变化。我们得出结论，声学传感器具有足够的灵敏度来实时监测神经元黏附。这在神经元 - 底物相互作用机制以及细胞外基质中分子调节剂作用的研究中具有潜在应用。
