Gradients generation and characterization inside the microfluidic chip
For the generation of a stable gradient through the Matrigel core compartment, a source-sink mechanism was created, with a solution of FITC-labeled nanoparticles (0.5 mg/ml) in PBS perfused through one side channel, and PBS perfused through other using a Nexus 3000 syringe pump (Chemyx). Three-milliliter syringes were used to introduce PBS with or without the nanoparticles at a rate of 8 μl/hour. The experiment was performed under a Nikon Eclipse Ti-E fluorescence microscope (Nikon Instruments, Japan) for time-lapse imaging of the gradient formation.
Profile and stability of the dynamic gradient were analyzed using NIS-Elements AR (Nikon) software. The chips were analyzed at different time points between 0 and 12 hours with continuous perfusion. Built-in “plot profile” module was used for assessing the gradient intensity and stability over time. The intensity profile corresponds to distance versus intensity. A 3D projection was also reconstructed using software.
