Rapid and efficient acoustofluidic device for 3D stem cell spheroid assembly with enhanced therapeutic potential

Three-dimensional (3D) spheroids of human adipose-derived stem cells (ADSCs) offer distinct advantages for regenerative medicine applications, such as chronic wound healing, owing to their enhanced angiogenic and immunomodulatory paracrine factors, improved survival after transplantation, and superior integration with host tissue. However, conventional spheroid-formation methods, including ultra-low attachment (ULA) plates, are often time-consuming, typically requiring several days to produce spheroids of consistent size. In this study, we introduce and validate an acoustofluidic-device for the rapid and remote fabrication of ADSC spheroids. Using a 2-MHz focused ultrasound (FUS) transducer integrated with a microfluidic device, we hypothesized that ADSCs could be assembled into 3D spheroids through FUS-induced local convective flow. Acoustic parameters were systematically optimized to achieve efficient spheroid assembly. The cell viability, stemness characteristics, and proangiogenic potential of FUS-assembled spheroids were then compared with those generated by conventional monolayer culture and the ULA method. The results showed that the proposed acoustofluidic successfully produced uniform ADSC spheroids within 300 s, approximately 1,100 times faster than the conventional ULA method, which typically requires about three days—without compromising cell viability. The spheroid diameter and stability could be precisely tuned by adjusting acoustic parameters. In addition, quantitative RT-PCR, immunofluorescence, and cytokine-array analyses showed that FUS-assembled spheroids significantly upregulated key proangiogenic factors, notably vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF), compared with monolayer and ULA cultures. At one day of spheroid formation, the acoustofluidic-device group showed significantly higher VEGF expression (3.2 ± 0.9-fold) than both monolayer and ULA groups. HGF expression was also significantly elevated compared with monolayer culture (4.6 ± 0.5-fold), although no significant difference was observed between the acoustofluidic and ULA groups for this marker. After three days of spheroid formation, qPCR analysis revealed further upregulation of both HGF (2.9 ± 0.6-fold) and VEGF (3.3 ± 0.8-fold) in the acoustofluidic group compared with both control groups. Taken together, these results indicate that the proposed acoustofluidic device provides a rapid, efficient, and scalable approach for generating ADSCs spheroids with superior proangiogenic properties. This ultrasound-based strategy offers a powerful platform for advancing cell-based therapies and holds strong potential for tissue engineering and regenerative medicine applications.