Monitoring and optimization of the microenvironment in a gravity-driven microfluidic system placed on a slow-tilting...

Gravity-driven microfluidic chips offer portability and flexibility in different settings because pumps and connecting tubes are unnecessary for driving fluid flow. In a previous study, human induced pluripotent stem cells were cultured using gravity-driven microfluidics, with the liquid flow rate regulated by a tilting table. However, instability in cell culture has been observed, occasionally leading to cell death owing to unknown causes. This study measured the ability of a gravity-driven microfluidic system to maintain essential microenvironments, specifically the flow rate, CO₂ levels, temperature, and humidity. The incubation procedure was improved to stabilize the parameters at target values. Improvements in the incubation process reduced the time required to reach the stabilized value for CO₂, temperature, and humidity by 85, 67, and 5 %, respectively, compared to previous methods. The system demonstrated a precise flow rate, confirmed by a consistent increase in the downstream tank's medium volume after 4 h of perfusion. In addition, the adjustment of the tilting table maintained a steady angle and effectively regulated the flow rate, with the measured flow rate consistent with the theoretical value. The gravity-driven microfluidic system effectively facilitated the culture and differentiation of human iPSCs into the mesodermal lineage after bone morphogenetic protein 4 induction, as indicated by positive SSEA1 immunostaining, demonstrating its potential for stem cell research. Gravity-driven microfluidic systems satisfy these requirements and are suitable for stem cell culture experiments.