Flow-dependent TGF-β1 secretion from HCT116 cells following constriction-based microfluidic passage

Transforming growth factor β1 (TGF-β1) is a multifunctional cytokine involved in tissue remodeling, mechanobiological signaling, and cartilage-related cellular responses. In this proof-of-concept study, we developed and evaluated a constriction-based microfluidic platform for rapidly modulating TGF-β1 secretion from HCT116 cells through short-duration mechanical perturbation. Suspended cells were passed through repeated constrictions under different flow conditions, and a simplified droplet-based numerical model was used as a device-level framework to compare apparent deformation behavior, pressure amplification, transit time, and simulation-derived shear exposure descriptors. The model was used to describe relative passage trends and was not intended to represent living-cell viscoelastic mechanics, intracellular stress transmission, or mechanotransduction pathways. Experimentally, constriction-based microfluidic passage produced a clear flow-condition-dependent TGF-β1 response. Among the tested conditions, the intermediate flow condition, Case 3, showed the highest net TGF-β1 concentration in the culture supernatant, with TGF-β1 secretion increasing by up to approximately 250% compared with the negative control. Consistently, TGF-β1 mRNA expression increased by approximately 3.5-fold in Case 3. Lower-flow/high-resistance conditions were associated with reduced cell viability, indicating that cytokine output depends on a balance between microfluidic stimulation and post-processing cell survival. Conditioned medium collected from Case 3 enhanced COL2A1 and SOX9 protein expression in a simplified two-dimensional AD-MSC bioactivity assay, supporting the biological activity of the microfluidic-conditioned medium and its ability to promote early chondrogenic marker expression under the tested culture conditions. Overall, this study presents a compact constriction-based microfluidic approach for rapidly modulating TGF-β1 secretion and generating biologically active conditioned medium, while establishing a foundation for future studies incorporating straight-channel controls, time-resolved HCT116 imaging, improved cell-mechanics modeling, and three-dimensional AD-MSC assays.