(I-330) Acoustofluidic fabrication of functional neuromuscular tissue construct

Introduction:: For biological research and practical biomedical applications, it is crucial to construct artificial biological tissues exhibiting their own functions. During embryonic development, the expression of tissue functions necessitates the geometric arrangement and aggregation of cells by geometric, mechanical, and chemical cues. In this study, we present the high-resolution acoustofluidic cell patterning technique in which the cells can be precisely arranged into an in vivo-like structure by the acoustofluidic cue. The developed technique is advantageous in constructing tissues in varied shapes, sizes, and complex cellular alignments. We demonstrated that the fabricated tissue exhibited an enhanced structure maturity and therapeutic potential.

Materials and Methods:: The acoustofluidic device comprised a piezoelectric device, temperature controller, printed circuit board, and tissue mold. The solution of hydrogel containing skeletal muscle cells and nerve cells was injected into the tissue mold. Application of the acoustic wave resulted in a three-dimensional arrangement of cells in the solution. After the hydrogel solution was solidified, the tissue with arranged cells was detached from the acoustofluidic device. The fabricated tissues were then characterized by immunocytochemistry, live-dead assay, quantitative real-time polymerase chain reaction analysis, calcium assay, and particle image velocimetry. The therapeutic efficacy of tissues fabricated by the acoustofluidic technique was investigated through transplantation to a mouse model with volumetric muscle loss.

Results, Conclusions, and Discussions:: The acoustofluidic device generates the acoustofluidic cues, including acoustic radiation forces and fluidic drag forces, on the cells in the liquid. The acoustic radiation forces caused cells to migrate toward the pressure nodes and form a three-dimensional geometric pattern. The aggregation of the cells by the acoustofluidic cues significantly enhanced the direct physical contact between cells, which might promote the direct cell-cell interaction. The optimization of the acoustic properties of the components of the acoustofluidic device was conducted to obtain the acoustic potential fields at which cells were located in cell patterning. We demonstrated that the developed acoustofluidic device is able to fabricate tissues on a large scale of several centimeters. The acoustofluidic technique could upregulate the structural and functional maturation of the neuromuscular tissues. The animal tests found that the cellular arrangement by the acoustofluidic cues improved the restoration of the muscle tissues suffered by the volumetric loss.
The acoustofluidic technique is able to fabricate functional tissues in a volumetric and label-free manner. This technique is advantageous in fabricating tissues with high cell density and precise cellular patterns. It is also compatible with various types of hydrogels and cells. The acoustofluidic cell patterning technique can be combined with other biofabrication methods, such as extrusion bioprinting, and might contribute to the in vitro replication of the in vivo organs.
In this study, we developed an acoustofluidic cell patterning technique for the scalable and user-defined fabrication of functional tissues. The acoustofluidic technique was able to fabricate a tissue construct with a geometric alignment and aggregation of cells. The neuromuscular tissue fabricated by the acoustofluidic technique exhibited high functionality and therapeutic efficacy. The technique and results can be beneficial in developing a drug screening method, disease model, and transplantable tissue construct.

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