ABSTRACT
The vocal folds (VFs) are constantly exposed to mechanical stimulation leading to changes in biomechanical properties, structure, and composition. The development of long-term strategies for VF treatment depends on the characterization of related cells, biomaterials, or engineered tissues in a controlled mechanical environment. Our aim was to design, develop, and characterize a scalable and high-throughput platform that mimics the mechanical microenvironment of the VFs in vitro. The platform consists of a 24-well plate fitted with a flexible membrane atop a waveguide equipped with piezoelectric speakers which allows for cells to be exposed to various phonatory stimuli. The displacements of the flexible membrane were characterized via Laser Doppler Vibrometry (LDV). Human VF fibroblasts and mesenchymal stem cells were seeded, exposed to various vibratory regimes, and the expression of pro-fibrotic and pro-inflammatory genes was analyzed. Compared to current bioreactor designs, the platform developed in this study can incorporate commercial assay formats ranging from 6- to 96-well plates which represents a significant improvement in scalability. This platform is modular and allows for tunable frequency regimes.
ABSTRACT
Neutrophils are the most abundant type of leukocytes in the blood, traditionally regarded as the first immune responders to infections and inflammations. In the context of tumors, neutrophils have been shown to possess both tumor-promoting and tumor-limiting properties. A better understanding of the inter-cellular dynamics between the neutrophils and aggregated tumors could possibly shed light on the different modalities of neutrophil involvement in tumor progression. To studyin-vitrothe interactional dynamics of neutrophils and growing tumor aggregates, in this work, we engineered a novel, microfluidics-integrated, three-dimensional (3D) tumor-immune microenvironment (TIME)-on-Chip device, and we investigated the effect of neutrophils on the inception of collective 3D invasion of ovarian tumor cells. Herein, tumor spheroids generated and cultured on hydrogel based multi-microwell plates, and embedded within collagen matrix of defined thickness, were magnetically hybrid-integrated with a 3D bioprinting enabled microfluidic system fabricated on a porous membrane and carrying neutrophils. This setting recreated a typical TIMEin-vitroto model dynamic neutrophil migration and 3D tumor invasion. Using this device, we observed that neutrophils respond to the growing tumor spheroids through both chemotaxis and generation of neutrophil extracellular traps (NETs). The formation of NETs stimulated the reciprocation of tumor cells from their aggregated state to collectively invade into the surrounding collagen matrix, in a manner more significant compared to their response to known tumor-derived stimulants such as transforming growth factor and Interleukin- 8. This effect was reversed by drug-induced inhibition of NETs formation, suggesting that induction of NETs by cancer cells could be a pro-migratory tumor behavior. Further, we additionally report a previously unidentified, location-dictated mechanism of NETosis, in which NETs formation within the stromal extracellular collagen matrix around the spheroids, and not tumor-contacted NETs, is important for the induction of collective invasion of the ovarian tumor cells, thus providing a rationale for new anti-tumor therapeutics research.
