Advanced Microfluidics Flow Analysis Made Easy

Organ-on-a-chip systems are microfluidic cell culture systems with controlled conditions that are designed to replicate the complex environment and cell context of human organs in vitro (Leung et al., 2022; Figure 1). They have great potential to study organ function in vitro, establish disease modeling experiments, and offer insightful experimental designs for drug discovery and personalized medicine. Consequently, they are becoming increasingly popular. Various parameters can be controlled in such a system, including temperature and mechanical forces. Additionally, flow is an important aspect in such systems as it helps to mimic human physiological environments. The flow of media over cells removes waste and brings essential nutrients, or it might deliver drug substances. In addition, flow can be used to stimulate shear stress that can be critical for the behavior of endothelial cells or might influence stem cell differentiation (Campinho et al., 2020).

Three sets of experiments were performed: 1) varying flow rates, 2) varying positions along the y-axis, and 3) varying positions along the z-axis. The Automated Sequential Injection System Aria by Fluigent was used to pump a rhodamine 110 solution through a microfluidic flow cell (1000 µm width, 200 µm depth, 55.8 mm length) (Figure 2). Three sets of ZEISS Dynamics Profiler measurements were performed with the ZEISS LSM 900 laser scanning confocal with Airyscan 2 detector using a 63x/1.2 W FCS objective. The reference image helped to position the spots and to orient within the sample (Figure 3). 

The first data set was acquired to compare flow speed and flow angle at varying flow rates. For this, flow speed and flow angle were measured for two spots within the microfluidics channel in three consecutive measurements, with increasing flow rates each (40 µl/min, 160 µl/min, 320 µl/min). Spot positions inside the channel were not changed in-between measurements. The second data set was acquired (at 200 µl/min) to compare flow speed and angle at different positions across the channel along the y-axis (channel width). For this, eight spots were positioned; the stage moved twice by 50 µm to obtain the second and third set of eight measurement points. The “reuse Spot” option of ZEISS Dynamics Profiler facilitated spot positioning and ensured spacing between spots remained the same between the first, second, and third measurements. The third and last data set was acquired (at 230 µl/min) to compare flow speed and angle at different positions across the channel along the z-axis (channel height). For this, several rounds of 2 spot measurements were performed, starting at the bottom of the channel. In between the rounds, the stage was first moved up by 5 µm, and afterwards in 15 µm steps up to a total of 90 µm into the channel.

1) Varying flow rate

The first experiment was setup to confirm that measured flow speed correlates with the actual flow rate applied by the microfluidic system. Flow speed and flow angle were measured for two spots within the microfluidics channel in three consecutive measurements with increasing flow rates. The cross-correlation curves and their fits (not shown) along three different axes were calculated (Figure 5A). The shift to the right of one of the correlation curves of one axis pair indicates flow along this axis (here 60°-240° and 120°-300°). There is no clear curve shift along the 0°-180° axis. These shifts become more obvious the higher the flow rate. Higher flow speeds result in steeper cross-correlation curves with an overall shift to the left. The resulting flow speeds of the two spots with the same flow rate show minimal speed differences (Figure 5C). Average flow speed at 40 µl/min is ~4000 µm/s. Quadrupled flow rate of 160 µl/min results in a measured flow speed of around 18,000 µm/s, which corresponds to 4.5x the previous flow speed. Doubling the flow rate once more to 320 µl/min resulted in a flow speed of around 40,000 µm/s, 2.2x the flow speed as before. As expected, there is a linear correlation between the flow rate and flow speed. The average flow angle from all measurements was 84.8°. The resulting flow angle of the two spots with the same flow rate show minimal differences (Figure 5D). In summary, varying the flow rate resulted in a linear change of flow speed but had no influence on the flow angle, as expected.