Bioprinting could spatially align various cells in high accuracy to simulate complex and highly organized native tissues. However, the uniform suspension and low concentration of cells in the bioink and subsequently printed construct usually results in weak cell-cell interaction and slow proliferation. Acoustic manipulation of biological cells during the extrusion-based bioprinting by a specific structural vibration mode was proposed and evaluated. Both C2C12 cells and human umbilical vein endothelial cells (HUVECs) could be effectively and quickly accumulated at the center of the cylindrical tube and consequently the middle of the printed construct with acoustic excitation at the driving frequency of 871 kHz. The full width at half maximum (FWHM) of cell distributions fitted with a Gaussian curve showed a significant reduction by about 2.2 fold in the printed construct. The viability, morphology, and differentiation of these cells were monitored and compared. C2C12 cells that were undergone the acoustic excitation had nuclei oriented densely within ±30° and decreased circularity index by 1.91 fold or significant cell elongation in the printing direction. In addition, the formation of the capillary-like structure in the HUVECs construct was found. The number of nodes, junctions, meshes, and branches of HUVECs on day 14 was significantly greater with acoustic excitation for the enhanced neovascularization. Altogether, the proposed acoustic technology can satisfactorily accumulate/pattern biological cells in the printed construct at high biocompatibility. The enhanced cell interaction and differentiation could subsequently improve the performance and functionalities of the engineered tissue samples.

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3D bioprinting becomes one of the popular approaches in the tissue engineering. In this emerging application, bioink is crucial for fabrication and functionality of constructed tissue. The use of cell spheroids as bioink can enhance the cell-cell interaction and subsequently the growth and differentiation of cells in the 3D printed construct with the minimum amount of other biomaterials. However, the conventional methods of preparing the cell spheroids have several limitations, such as long culture time, low-throughput, and medium modification. In this study, the formation of cell spheroids by SSAW was evaluated both numerically and experimentally in order to overcome the aforementioned limitations. The effects of excitation frequencies on the cell accumulation time, diameter of the formed cell spheroids, and subsequently, the growth and viability of cell spheroids in the culture medium over time were studied. Using the high-frequency (23.8 MHz) excitation, cell accumulation time to the pressure nodes could be reduced in comparison to that of the low-frequency (10.4 MHz) excitation, but in a smaller spheroid size. SSAW excitation at both frequencies does not affect the cell viability up to 7 days, > 90% with no statistical difference compared with the control group. In summary, SSAW can effectively prepare the cell spheroids as bioink for the future 3D bioprinting and various biotechnology applications (e.g., pharmaceutical drug screening and tissue engineering).

Accumulation of particles in a high concentration on a microchannel wall is a common phenomenon in a colloidal fluid. Gradual accumulation/deposition of particles can eventually obstruct the fluid flow and lead to clogging, which seriously affects the accuracy and reliability of nozzle-based printing and causes damage to the nozzle. Particle accumulation in a 100 μm microchannel was investigated by light microscopy, and its area growth in an exponential format was used to quantify this phenomenon. The effects of the constriction angle and alginate concentration on particle accumulation were also studied. In order to reduce the clogging problem, an acoustic method was proposed and evaluated here. Numerical simulation was first conducted to predict the acoustic radiation force on the particles in the fluid with different viscosities. Interdigital transducers (IDTs) were fabricated on the LiNbO3 wafer to produce standing surface acoustic waves (SSAW) in the microchannel. It was found that the actuation of SSAW can reduce the accumulation area in the microchannel by 2 to 3.7-fold. In summary, the particle accumulation becomes significant with the increase of the constriction angle and fluid viscosity. The SSAW can effectively reduce the particle accumulation and postpone clogging.

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