## 4 Results and Discussion
### Entrapment of Cells in a Matrix  
Switching off the actuator lets the cells scatter in the fluid, thereby losing the alignment. Hence, to maintain the cell alignment and separation, actuated cells have been entrapped in a 3D matrix.  
To allow for wave propagation, a low-viscosity fluid capable of going through a sol-gel transition in a short time was needed, in which the polymerization had to start after the alignment. During the time needed for the sol-gel transition of the culture medium, i.e., five min in our experiments, the cell viability could be affected, given the impossibility of the cells, during this time, to make contact through the integrins with proteins of the cell matrix, a mechanism that has already been showed to regulate cell viability [29]. Relying on previous experience in the angiogenesis field, a fibrin gel, obtained by adding thrombin to a liquid fibrinogen solution, was used, rather than other biomaterials,  
\begin{table}
\begin{tabular}{c c} \hline
**Actual Time (min)** & **Cell Viability (\%)** \\ \hline
2 & 100 \\
4 & 98 \\
5 & 100 \\
7 & 98 \\ \hline \end{tabular}
\end{table}
Table 1: Cell viability as a function of the actuation time of the device.  
Figure 7: Effects of different wavelengths of acoustic waves on a human foreskin fibroblast (HFF) cell suspension. Cell accumulation in lines caused by the acoustic waves generated by exciting a single IDT at \(f_{\rm e1}=6.7\) MHz (**a**) and \(f_{\rm e2}=8.0\) MHz (**b**), respectively. The mean distances \(d_{\rm 1,mean}=121\)\(\upmu\)m and \(d_{\rm 2,mean}=103\)\(\upmu\)m have been estimated from the gray-scale intensity plots derived from the insets.  
such as inert substrates mixed or linked to extracellular matrix molecules [8; 30]. Besides, the survival of fibroblasts [31; 32], fibroblasts/myoblasts [33], and HUVECs [34; 35] in the fibrin gel has already been widely demonstrated. Cells were subsequently resuspended in a 10 mg/mL fibrinogen solution maintained at 4 \({}^{\circ}\)C, and thrombin 0.4 U was added just before transferring the cell suspension into the chamber. After five minutes, the fibrin clot was formed and aligned cells were entrapped in the matrix. For a long-term culture, the cloths containing the cells were transferred to a cell culture dish, covered with cell culture medium, and observed in the following days. A 6.7 MHz excitation frequency was chosen for the following experiments.  
The first experiments were done using rat myoblasts that, although totally alive after the actuation process, did not survive the following 24 hour culture, thereby suggesting that the stiffness of the fibrin matrix may not represent the correct environment for myoblast survival [36].  
The next set of experiments was therefore performed with HFF. These cells entrapped in the fibrin clot maintained the aligned geometry for several days, as evidenced in Figure 8a-c, respectively. Of note, the magnification box in Figure 8a shows that each line is composed of single cells. Starting from 48 h, cells showed cytoplasmic protrusions, reflecting cell health, that became more complex over time, as shown in Figure 8d by a histological section of the clot and by the higher magnification box.