## 4 Results and Discussion
### Alignment of Cells  
Preliminary tests have been carried out to verify the capability of the device to generate acoustic waves that lead to alignment of a cellular suspension. Cells to be aligned were detached from the culture dish, dispersed in PBS as single cells, stained with the live dye Neutral Red, and put in the chamber of the device. Figure 6 shows the experimental setup with the device placed under the optical microscope and the tailored electronic interface used to drive the piezoelectric actuators.  
Initially, different concentrations of cells were tested in order to identify the amount required to appreciate the alignment in the volume of 500 \(\mu\)L, starting from \(2\times 10^{5}\) cells/mL and reaching \(4\times 10^{6}\) cells/mL. From these experiments, \(2\times 10^{6}\) cells/mL was identified as the optimal concentration for the formation of distinct lines of single cells, and therefore this amount was used for all the following experiments.  
Since the survival of L6 rat myoblasts despite acoustic wave exposition was critical, this cell line was used to verify that the actuation was not hampering cell viability. The experiment was set up by exposing different samples with the same cell concentrations to FPWs for different lengths of time. Cell viability was then measured by trypan blue cell exclusion (Table 1). Since cell viability remained high even at long times, a 5-min actuation period was chosen for all the following experiments.  
Figure 5: Block diagram of the tailored electronic interface used to drive the actuators.  
Figure 6: Experimental setup with the device placed under the optical microscope and the tailored electronic interface used to drive the piezoelectric actuators.  
Different wavelengths were tested to verify the effects on the alignment of human fibroblasts (HFF) by exciting a single actuator with a sinusoidal signal applied to A and B (C was floating). To demonstrate the alignment of cells induced by the acoustic waves, HFF dispersed in PBS were used, since their viability is unaffected by the acoustic field.  
The distance \(d\) between the different pressure nodes in the fluid, in which the cells are expected to be confined, corresponds to half the wavelength of the acoustic waves, resulting \(d=\lambda/2=v/2f_{\rm e}\), where \(v=1500\) m/s is the expected propagation velocity of the acoustic waves into PBS, and \(f_{\rm e}\) is the excitation frequency. Figure 7a,b show the results obtained with \(f_{\rm e1}=6.7\) MHz and \(f_{\rm e2}=8.0\) MHz, for which distances \(d_{\rm 1}=v/2f_{\rm e1}=112\)\(\upmu\)m and \(d_{\rm 2}=v/2f_{\rm e2}=93.8\)\(\upmu\)m are expected, respectively.  
As it can be observed, an increase of the excitation frequency, i.e., decrease of the wavelength, corresponds to a decrease in the distance between lines. The mean distances between the cell alignments \(d_{\rm 1,mean}=121\)\(\upmu\)m and \(d_{\rm 2,mean}=103\)\(\upmu\)m, which are in good agreement with predictions, have been estimated from the gray-scale intensity plots derived from the insets. Similar results have been obtained by exciting a single actuator with a sinusoidal signal applied to A and B (shorted) and C with frequencies of 6.0 and 7.2 MHz (not shown).