A Microfluidic System for Modeling Endothelial Dysfunction under Combined Physiological Pulsatile Shear Stress and Oscillatory Hyperglycemia

This study investigates how synchronized oscillatory hyperglycemia and the pulsatile shear stress together drive endothelial dysfunction in diabetic vascular disease. Existing models use starting high glucose or steady laminar flow. We investigate how pulsatile shear stress counters oscillatory glucose injury.

To begin, use computer-aided design software to design the microfluidic chip structure. Make the SU-8 master mold through a commercial company. In a clean mixing cup, mix the polydimethylsiloxane curing agent and pre-polymer at a 10:1 ratio by weight and stir the mixture vigorously by hand for five minutes.

Then place the container in a vacuum chamber at a pressure of 0.08 megapascal for at least 15 minutes to remove air bubbles trapped during mixing. Pour the polydimethylsiloxane onto the wafer placed in a glass Petri dish. Degas the polydimethylsiloxane poured onto the wafer in a desiccator for an additional 15 minutes to remove any bubbles formed during casting.

Place the Petri dish containing the mold and polymer in a hot oven set to 80 degrees Celsius for two hours. Using a scalpel, carefully peel the solidified polymer slab from the master mold. Using a core punch tool, create inlet and outlet ports at the designated channel ends on the polymer slab.

Clean the glass cover slip and the patterned face of the polymer slab with adhesive tape. Next, treat both the polymer slab and the glass cover slip with a plasma cleaner at 65 watts under room air for 60 seconds. Immediately align the polymer channel face with the treated side of the glass cover slip.

Then press gently to create a permanently closed microfluidic channel. Place the bonded microfluidic chip in a boronized glass bottle filled with deionized water. Sterilize by autoclaving at 121 degrees Celsius and 15 pounds per square inch for 20 minutes.

Place the sterilized chip into a 60 millimeter culture dish filled with an equimolar mixture of PBS and low glucose DMEM. Then using a one milliliter syringe, introduce 100 micrograms per milliliter fibronectin into the channel and incubate at 37 degrees Celsius for four hours. Next, add 24.5 millimolar demanitol to low glucose DMEM to elevate osmotic pressure.

Add five millimolar D-glucose to high glucose DMEM to obtain a final concentration of 30 millimolar glucose. Prepare normal glucose culture medium and high glucose culture medium supplemented with 20%FBS and 1%penicillin streptomycin. Then warm PBS, trypsin, normal glucose culture medium, and high glucose culture medium in a water bath at 37 degrees Celsius.

Harvest the human umbilical vein endothelial cells from the culture dish at 90%confluency. Detach the cells using 0.25%trypsin-EDTA solution. After incubating the cells at 37 degrees Celsius for 30 seconds, count the harvested cells and re-suspend them separately in normal glucose and high glucose media at a concentration of 1 times 10 to the power of 6 cells per milliliter.

Using a one milliliter syringe, inject 100 microliters of the cell suspension into the inlet port of the microfluidic chip with normal glucose medium for the control group and high glucose medium for the experimental group. Place the microfluidic chips in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for 48 hours. Calculate the flow rate required to achieve the target pulsatile shear stress using the presented equation.

Connect the pressure source to the pressure controller. Then link the pressure controller to the valve controller connected to the computer for automated control of pressure wave forms. Fill two 100 milliliter reservoirs, one with normal glucose culture medium and the other with high glucose culture medium.

Connect the reservoirs to the corresponding channels of the pressure controller. Then link the reservoirs and the two position three-way magnetic valve using silicone tubing. Set the target physiological pulsatile shear stress corresponding to a flow rate of approximately 200 microliters per minute.

Program the programmable air pump to generate a sine wave function to maintain the pulsatile shear stress. Next, program the valve controller to switch channels every five minutes to generate a square wave glucose oscillation during dynamic stimulation. Link the two position three-way magnetic valve and the microfluidic chip using silicone tubing.

Then initiate the programmable pump system to prime the tubing. Next, using the 1.3 millimeter diameter 90 degree angled stainless steel connectors, attach the inlet and outlet ports of the microfluidic chip to the tubing. Connect the microfluidic chip outlet to the waste reservoir, then start the pump again to deliver combined oscillatory glucose and pulsatile shear stress for a total of 30 minutes.

After performing flow field verification, prepare the calcein AM and propidium iodide working solution according to the manufacturer's instructions at the experimental endpoint. Once the channel is rinsed one time with PBS, introduce the calcein AM and propidium iodide working solution into the channel and incubate at 37 degrees Celsius with 5%carbon dioxide for 30 minutes in the dark. Immediately capture fluorescent images of the stained cells using an inverted fluorescence microscope equipped with a 10 times objective lens.

Immediately capture fluorescent images of the stained cells using an inverted fluorescence microscope equipped with a 10x objective lens. Use the fluorescein isothiocyanate green filter for calcein AM and the tetramethylrhodamine or rhodamine red filter for propidium iodide. After flushing the channel three times with PBS, introduce the live cell oxidative stress fluorescent probe solution into the channel and incubate as demonstrated earlier.

Flush the channel three times with PBS to remove the unbound probe. Immediately capture fluorescent images using the far red filter set through a 20x objective lens while maintaining identical exposure parameters across all groups. Open the acquired calcein AM green and propidium iodide red images in ImageJ.

Manually adjust the threshold for each channel to distinguish fluorescent signals from background. Use the cell counter to quantify fluorescent cells, adjusting the minimum cell size based on observations to determine live and dead cell counts across multiple fields of view. Calculate the percentage of live cells using spreadsheet software for each experimental condition.

Then calculate the mean and standard deviation across multiple fields of view using statistical analysis software to compare groups. Drag the image of choice into ImageJ. Select Analyze and choose Set Scale to calibrate the pixel size using the microscope metadata.

Then remove the non-specific uniform background signal from the entire image using the background subtraction function by selecting Process and choosing Subtract Background. Next, select Image and choose Adjust followed by Threshold to manually adjust the threshold slider and define the intracellular reactive oxygen species signal. Set the measurements by selecting Analyze and choosing Set Measurements to include Area and Mean gray value.

Select Analyze and Analyze Particles to determine the minimum cell size based on observed cell dimensions and measure the mean intensity of the segmented signal within the cell monolayer ROI. Report the reactive oxygen species level for each experimental condition as the mean of measured values derived from at least three randomly selected fields of view per chip. Input the quantitative results into data analysis and graphing software.

Determine statistical significance by comparing experimental groups with the control group using two-way analysis of variance followed by Tukey post-hoc test with a significance level of 0.05. Use the software to perform the analysis and generate plots with error bars representing standard deviation. Representative fluorescence images showed a high proportion of viable cells across all groups with negligible cell death observed in the normal glucose and normal glucose with pulsatile shear stress groups, while the oscillatory glucose and oscillatory glucose with pulsatile shear stress groups exhibited a visible increase in dead cells.

Quantitative analysis showed that oscillatory glucose exposure resulted in a significant reduction in cell viability compared to the control group, and pulsatile shear stress application under oscillatory glucose conditions significantly increased viability compared to oscillatory glucose alone. Under normal glucose conditions, intracellular reactive oxygen species levels were low and were further reduced following the application of shear stress. Whereas oscillatory glucose conditions caused a marked increase in red fluorescence intensity, indicating elevated reactive oxygen species.

The oscillatory glucose-induced increase in intracellular reactive oxygen species was reduced following the application of shear stress. Quantitative analysis revealed that shear stress significantly reduced levels of reactive oxygen species under normal glucose conditions. In contrast, oscillatory glucose conditions led to a significant increase in reactive oxygen species levels compared to normal glucose.

Furthermore, shear stress significantly decreased reactive oxygen species levels, even under oscillatory glucose conditions. This protocol enables quantifying ROS levels and the cell availability to study endothelial dysfunction under oscillatory hyperglycemia and the pulsatile shear stress. Following this procedure, molecular analysis, such as the NRF2 pathway or iNOS signaling can be performed to enucleate endothelial protection mechanisms.

Future study could use this system to explore mechanobiological pathways, incorporate disturb the flow geometries or smooth muscle cell co-culture.