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

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.