Name: A Microfluidic System for Modeling Endothelial Dysfunction under Combined Physiological Pulsatile Shear Stress and Oscillatory Hyperglycemia (Video) | JoVE Journal
Uploaded: 2026-05-12T13:30:00.000+00:00
Duration: 14 min 10 s
Description: Explore a programmable microfluidic platform modeling endothelial dysfunction under synchronized oscillatory hyperglycemia and pulsatile shear stress. This method enables mechanistic studies and drug screening for diabetic vascular disease.

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5 minutes

This work was supported by the National Natural Science Foundation of China (grant numbers 12372304, 12172081) and the Fundamental Research Funds for the Central Universities (DUT25YG272).

Ethics statement:
NOTE: All reagents, devices, and software used in this protocol are listed in the Table of Materials.
1. Microfluidic chip fabrication
<ol>
	<li>Use computer-aided design (CAD) software to design the structure of the microfluidic chip (W x L x H = 2 mm x 30 mm x 0.1 mm, Figure 1A) and make the SU-8 master mold through a commercial company.</li>
	<li>Mix PDMS curing agent and prepolymer at a 10:1 ratio by weight in a clean mixing cup and vigorously stir manually for 5 min.</li>
	<li>Place the container in a vacuum chamber under -0.08 MPa for a minimum of 15 min to remove air bubbles trapped during the mixing process. 
	NOTE: To expedite bubble removal and prevent overflow, temporarily vent the chamber to atmospheric pressure to burst the large bubbles, and then immediately reapply the vacuum. Repeat this venting cycle 2&#8211;3 times until the bulk mixture becomes completely transparent and free of macroscopic bubbles.</li>
	<li>Pour the PDMS onto the wafer placed in a glass Petri dish.</li>
	<li>Degas the PDMS poured onto the wafer in a desiccator for an additional 15 min to remove any bubbles that might have formed during casting. 
	NOTE: If any stubborn micro-bubbles remain trapped near or within the SU-8 master mold structures, gently blow them to the edge or burst them using a manual rubber air blower. Complete removal of air bubbles is crucial to prevent structural defects in the microchannels.</li>
	<li>Place the petri dish containing mold and polymer in a hot oven set to 80 &#176;C for 2 h. 
	CAUTION: Use heat-resistant gloves when handling hot materials</li>
	<li>Carefully peel the solidified polymer slab from the master mold using a scalpel.</li>
	<li>Use a core punch tool (e.g., 1.2 mm diameter) to create inlet and outlet ports at the designated channel ends on the polymer slab.</li>
	<li>Clean the glass coverslip and the patterned face of the polymer slab with adhesive tape. 
	PAUSE POINT: The cured and punched PDMS slabs can be stored in a clean container (e.g., a sealed Petri dish) at room temperature for several weeks before plasma treatment and bonding.</li>
	<li>Treat both the polymer slab and the glass coverslip with a plasma cleaner at 65W power under room air for 60 s.</li>
	<li>Immediately align the polymer channel face to the treated side of the glass coverslip.</li>
	<li>Press gently to create a permanent, closed microfluidic channel.</li>
</ol>
2. Endothelial cell preparation and seeding
<ol>
	<li>Place the bonded microfluidic chip in a boronized glass bottle filled with deionized water and sterilize by autoclaving at 121 &#176;C and 15 psi for 20 min. 
	NOTE: Submerge the chip in water during autoclaving to equalize pressure and prevent deformation of PDMS microchannels. This also helps remove trapped air bubbles and primes the channels for subsequent fluid introduction. 
	PAUSE POINT: The sterilized microfluidic chips can be safely stored in the sealed glass bottle at room temperature for up to several weeks until they are needed for cell seeding.</li>
	<li>Place the sterilized chip into a 60 mm culture dish filled with a 1:1 mixture of phosphate buffer saline (PBS) and low glucose Dulbecco&#8217;s Modified Eagle Medium (DMEM).</li>
	<li>Introduce 100 &#181;g&#183;mL-1 fibronectin into the channel using a 1 mL syringe and incubate at 37 &#176;C for 4 h. 
	NOTE: Ensure that the microfluidic chip is placed below the liquid level. Connect the adapter hose to the syringe needle, fill the hose with liquid and insert it below the liquid level, connect the inlet and then inject to ensure that no bubbles enter.</li>
	<li>Add 24.5 mM D-mannitol to low glucose DMEM (5.5mM glucose) to elevate osmotic pressure. Add 5 mM D-glucose to high glucose DMEM (25mM glucose) to a final 30 mM glucose. Make normal glucose (NG) culture medium and high-glucose (HG) culture medium supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), respectively.</li>
	<li>Warm 1&#215; PBS, trypsin, NG and HG culture media in a water bath at 37 &#176;C. Harvest the Human Umbilical Vein Endothelial Cells (HUVECs) from the culture dish at 90% confluency. Detach the cells using 0.25% Trypsin-EDTA solution and incubate at 37 &#176;C for 30 s. Count the harvested cells and adjust the concentration to 1 x 106 cells/mL in NG medium and HG medium, respectively.</li>
	<li>Inject 100 &#181;L of the cell suspension into the inlet port of a microfluidic chip using a 1 mL syringe, utilizing NG medium for the control group and HG medium for the experimental group. Place microfluidic chips in a humidified incubator at 37 &#176;C with 5% CO2 for 48 h. 
	NOTE: Allow cells to adhere for at least 4 h before further processing. Manually alternate the two culture media every 2 h to stimulate oscillatory hyperglycemia during the static culture phase. Keep the cells at 5.5 mM glucose overnight to mimic the in vivo situation (Figure 1B).</li>
</ol>
3. Dynamic stimulation setup
<ol>
	<li>Fill two 100 mL reservoirs with NG and HG culture media, respectively, and connect them to the corresponding channels of the programmable pressure control system.</li>
	<li>Link the reservoirs, the two-position three-way magnetic valve, and the microfluidic chip using silicone tubing.</li>
	<li>Use 1.3 mm diameter 90&#176; angled stainless-steel connectors to attach the inlet and outlet ports of the PDMS chip to the tubing. 
	NOTE: Sterilize the 100 mL reservoirs, silicone tubing and stainless-steel connectors in a high-pressure, high-temperature autoclave beforehand.</li>
	<li>Calculate the flow rate (Q) required to achieve the target PSS (&#964;). Use the formula &#964; = (6&#181;Q)/(wh2), where &#181; the viscosity, w is the channel width, and h is the channel height.</li>
	<li>Set a target physiological PSS (&#964; = 10 &#177; 2 dyn&#183;cm-2, 1 Hz), which corresponds to a calculated flow-rate of 200 &#177; 40 &#181;L&#183;min-1.</li>
	<li>Program the programmable air pump to generate a sine wave function to maintain the PSS. Program the valve controller to switch channels every 5 min to generate a square-wave glucose oscillation during the dynamic stimulation.</li>
	<li>Start the programmable pump system to deliver the combined OG and PSS for a total of 30 min.</li>
</ol>
4. Micro-PIV for flow field verification
<ol>
	<li>Prepare the NG and HG media containing a 0.05% volume concentration of fluorescent microparticles (1 &#956;m). Ensure the particles are evenly dispersed and free of aggregation.</li>
	<li>Place a clean, cell-free microfluidic chip (identical to the one used in the cell experiments) onto the stage of the inverted fluorescence microscope.</li>
	<li>Connect all components in the system (Figure 1C).
	<ol>
		<li>Connect the pressure source to the pressure controller.</li>
		<li>Connect the pressure controller to the valve controller, which is linked to the computer for automated control of pressure waveforms.</li>
		<li>Connect the pressure controller outlets to the HG and NG reservoirs via separate tubing lines.</li>
		<li>Connect both reservoirs to the 3/2 pressure valve, which switches between HG and NG media according to the programmed oscillatory glucose protocol.</li>
		<li>Connect the outlet of the 3/2 pressure valve to the inlet of the microfluidic chip via tubing.</li>
		<li>Connect the outlet of the microfluidic chip to the waste reservoir.</li>
		<li>Place the microfluidic chip on the microscope stage for live-cell imaging throughout the experiment. 
		NOTE: Ensure all tubing connections are secure and free of leaks before initiating flow.</li>
	</ol>
	</li>
	<li>Trigger the high-speed camera (operating at 15 Hz in double-frame mode) after the flow is steady to capture sequential image pairs of the flowing microparticles. Set the precise time delay (&#916;t) between the two frames of each pair to 80 &#956;s.</li>
	<li>Capture images for Region of Interest (ROI) within the channel. Select the ROI at the center of the straight channel section (Figure 2A), equidistant from both the inlet and outlet, to avoid end effects and ensure representative flow measurements.</li>
	<li>Process the acquired image pairs using PIV analysis software. Use an adaptive multi-pass cross-correlation algorithm, starting with an initial interrogation area of 64 &#215; 64 pixels and refining to a final area of 16 &#215; 16 pixels with an 8 &#215; 8 pixel grid step size, to determine the displacement vector (&#916;x) of the particles between the two frames.</li>
	<li>Calculate the velocity vector (v) for each interrogation area using the formula v = &#916;x / &#916;t.</li>
</ol>
5. Data acquisition
NOTE: Quantify cell viability and function using the Calcein-AM/propidium iodide (PI) Kit and live-cell oxidative stress fluorescent probe to verify the damage caused by glucose fluctuations and evaluate the effect of co-stimulation.
<ol>
	<li>Cell viability
	<ol>
		<li>Prepare the Calcein-AM/PI working solution according to the manufacturer&#8217;s instructions at the experimental endpoint. 
		CAUTION: PI is a known mutagen and a suspected carcinogen that intercalates into DNA. Handle with extreme care. Wear gloves and handle within a designated safe workspace.</li>
		<li>Rinse the channel one time with PBS and introduce the Calcein-AM/PI working solution into the channel. Incubate the device at 37 &#176;C with 5% CO2 for 30 min in the dark.</li>
		<li>Immediately capture fluorescent images of the stained cells using an inverted fluorescence microscope equipped with a 10&#215; objective lens. Use the FITC (Green) filter for Calcein-AM and the TRITC or Rhodamine (Red) filter for PI.</li>
	</ol>
	</li>
	<li>Measure Intracellular ROS
	<ol>
		<li>Prepare the live-cell oxidative stress fluorescent probe (see Table of Materials) working solution at the concentration specified by the manufacturer (5 &#956;M) in PBS. 
		CAUTION: Fluorescent probes and their common stock solvents (such as DMSO) can be toxic and may facilitate the absorption of hazardous substances through the skin. Wear gloves and handle within a designated safe workspace.</li>
		<li>Flush the channel three times with PBS and introduce the live-cell oxidative stress fluorescent probe solution into channel. Incubate the chip at 37 &#176;C with 5% CO2 for 30 min in the dark.</li>
		<li>Flush the channel three times with PBS to remove the unbound probe. Immediately capture fluorescent images using the Far-Red filter set (excitation 640 nm, emission 665 nm) through a 20&#215; objective lens, maintaining the exact same exposure parameters for all groups. 
		NOTE: Increased fluorescence intensity indicates higher ROS levels.</li>
	</ol>
	</li>
</ol>
6. Data analysis
PAUSE POINT: Once all raw fluorescent images and high-speed camera images are acquired and securely saved to a hard drive, the subsequent image processing, quantitative analysis, and statistical comparisons can be paused and performed at any later time.
<ol>
	<li>Quantify cell viability
	<ol>
		<li>Open the acquired Calcein-AM (green) and PI (red) images in ImageJ (see Table of Materials). Manually adjust the threshold for each channel to distinguish fluorescent signals from background. Use the cell counter function to count fluorescent cells, adjusting the minimum cell size according to the observed cell size, and count the number of live (NLive) and dead (NDead) cells in multiple fields of view. 
		NOTE: NLive refers to the number of Calcein-AM-positive (green) cells, NDead refers to the number of PI-positive (red) cells.</li>
		<li>Calculate the percentage of live cells (NLive / (NLive + NDead) x 100%) for each experimental condition using spreadsheet software. Calculate the mean and standard deviation across multiple fields of view using statistical analysis software for comparison between groups.</li>
	</ol>
	</li>
	<li>Quantify intracellular ROS
	<ol>
		<li>Drag image of choice in ImageJ. Set the scale by calibrating the pixel size using the microscope&#39;s metadata (or a known reference grid): Analyze | Set Scale.</li>
		<li>Remove a non-specific uniform background signal from the entire image using the background subtraction function: Process | Subtract Background.</li>
		<li>Manually adjust the threshold slider to segment and define the intracellular ROS signal: Image | Adjust | Threshold. Once the threshold is set, use Analyze | Set Measurements to choose Area and Mean gray value. Set the minimum cell size according to the observed cell size and measure the mean intensity of the segmented signal within the cell monolayer ROI: Analyze | Analyze Particles.</li>
		<li>Report the ROS level for each experimental condition as the means of the measured values (e.g., mean intensity) derived from at least three randomly selected fields of view per chip.</li>
	</ol>
	</li>
	<li>Performing statistical comparisons
	<ol>
		<li>Input the quantitative results (viability percentage and ROS mean intensity) into data analysis and graphing software (see Table of Materials).</li>
		<li>Determine statistical significance by comparing the experimental groups with the control group using two-way Analysis of Variance (ANOVA) followed by Tukey&#8217;s post-hoc test, with a significance level of p = 0.05. Use the software to perform the analysis and generate plots with error bars representing standard deviation (SD).</li>
	</ol>
	</li>
</ol>

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<li>Han, Y., Huang, K., Yao, Q. P., Jiang, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanobiology+in+vascular+remodeling%5BTitle%5D)+AND+%22National+Science+Review%22%5BJournal%5D)">Mechanobiology in vascular remodeling.</a> National Science Review. 5 (6), 933-946 (2018).</li>
<li>Cheng, C. K., Wang, N., Wang, L., Huang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biophysical+and+biochemical+roles+of+shear+stress+on+endothelium%3A+A+revisit+and+new+insights%5BTitle%5D)+AND+%22Circ+Res%22%5BJournal%5D)">Biophysical and biochemical roles of shear stress on endothelium: A revisit and new insights.</a> Circ Res. 136 (7), 752-772 (2025).</li>
<li>Tovar-Lopez, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+microfluidic+system+for+studying+the+effects+of+disturbed+flow+on+endothelial+cells%5BTitle%5D)+AND+%22Front+Bioeng+Biotechnol%22%5BJournal%5D)">A microfluidic system for studying the effects of disturbed flow on endothelial cells.</a> Front Bioeng Biotechnol. 7, 81(2019).</li>
<li>Estrada, R., Giridharan, G. A., Nguyen, M. D., Prabhu, S. D., Sethu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+endothelial+cell+culture+model+to+replicate+disturbed+flow+conditions+seen+in+atherosclerosis+susceptible+regions%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Microfluidic endothelial cell culture model to replicate disturbed flow conditions seen in atherosclerosis susceptible regions.</a> Biomicrofluidics. 5 (3), 032006(2011).</li>
<li>Van Engeland, N. C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+biomimetic+microfluidic+model+to+study+signalling+between+endothelial+and+vascular+smooth+muscle+cells+under+hemodynamic+conditions%5BTitle%5D)+AND+%22Lab+Chip%22%5BJournal%5D)">A biomimetic microfluidic model to study signalling between endothelial and vascular smooth muscle cells under hemodynamic conditions.</a> Lab Chip. 18 (11), 1607-1620 (2018).</li>
<li>Ma, Q., Ma, H., Xu, F., Wang, X., Sun, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidics+in+cardiovascular+disease+research%3A+State+of+the+art+and+future+outlook%5BTitle%5D)+AND+%22Microsyst+Nanoeng%22%5BJournal%5D)">Microfluidics in cardiovascular disease research: State of the art and future outlook.</a> Microsyst Nanoeng. 7, 19(2021).</li>
</ol>

The authors declare no conflict of interest.

The microfluidic system described herein provides a robust and physiologically relevant platform to investigate the integrated effects of OG and PSS on endothelial cell function. The primary advantage of this method lies in its capability to precisely control and decouple mechanical and biochemical stimuli within a defined microenvironment. Unlike conventional static cultures that lack hemodynamic forces or animal models that often obscure cellular level signaling4,17<...

Endothelial dysfunction is a critical early event in the development of diabetic vascular complications, driven by both metabolic disturbances and abnormal hemodynamic forces1,2. OG levels, rather than sustained hyperglycemia alone, are increasingly recognized as potent inducers of oxidative stress and inflammatory signaling in endothelial cells, leading to vascular injury and impaired homeostasis3,4,5. Concurrently, hemodynamic shear stress is a major determinant of endothelial phenotype, with PSS under physiological exercise conditions promoting nitric oxide production, antioxidant defense, and vascular protection6,7,8. Therefore, understanding the integrated effects of oscillatory hyperglycemia and hemodynamic stimulation is essential for elucidating mechanisms of endothelial dysfunction.
Conventional in vitro models, which often rely on static high-glucose conditions or simplified laminar flow, fail to replicate the complex, dynamic interplay between metabolic and mechanical cues present in vivo9,10,11. While animal models capture systemic aspects of diabetes, they lack the cellular-level resolution needed to dissect signaling under controlled microenvironmental conditions12. Microfluidic technology offers an effective alternative, providing precise spatiotemporal control of biochemical and biomechanical stimuli in physiologically relevant geometries13, and has driven significant recent advancements across various innovative biomedical applications14,15. For example, Chen et al.16 designed a microfluidic device with spatial and temporal wall shear stress and ATP signaling for studying endothelial cell intracellular Ca2+ dynamics. Yu et al.11 developed a hemodynamic microfluidic chip system for detecting the cellular responses under different combinations of physiological PSS and high glucose concentrations. Although these systems have been used to study endothelial responses to shear stress or single-factor metabolic perturbations, few have successfully integrated both glucose oscillations and PSS within a single system. Therefore, a standardized, reproducible protocol that synchronizes both factors in a controlled microfluidic setting remains lacking.
This method addresses this gap by establishing a reproducible microfluidic system capable of delivering coupled OG and PSS. The primary goal of this approach is to model oxidative stress, impaired cell viability and mechanobiological responses underlying endothelial dysfunction in a physiologically relevant manner. Unlike static culture or conventional perfusion chambers, this approach improves upon existing models by enabling simultaneous and controlled modulation of both metabolic and mechanical stimuli, more closely approximating the diabetic vascular microenvironment. Furthermore, the system is compatible with live-cell imaging, real-time monitoring, and molecular analysis, making it suitable for a wide range of applications including mechanistic studies, disease modeling, and drug screening for endothelial function and injury.

<table><tbody><tr><td>0.25% Trypsin-EDTA</td><td>NEST Biotechnology Co., Ltd.</td><td>211052</td><td>Cell dissociation</td></tr><tr><td>1 mL syringe</td><td>Shanghai Zhengbang Medical Science Co., Ltd.</td><td>1mL</td><td>Inject fluid</td></tr><tr><td>60 mm culture dish</td><td>NEST Biotechnology Co., Ltd.</td><td>705001</td><td>Cell culture</td></tr><tr><td>90&amp;deg; angled stainless-steel connector</td><td>Luoyang Mayer Trading Co.,Ltd.</td><td>1.3 mm</td><td>Adapter sealing device</td></tr><tr><td>AutoCAD software</td><td>Autodesk</td><td>RRID:SCR_014981</td><td>Computer-aided design software</td></tr><tr><td>Boronized glass bottle</td><td>Hunan Aidete Scientific Instruments Co., Ltd.</td><td>Double-pass 100mL</td><td>reservoir</td></tr><tr><td>Calcein-AM/PI Kit</td><td>Beyotime</td><td>byt-c2015</td><td>Staining</td></tr><tr><td>Cell incubator</td><td>ESCO</td><td>Esco CelMate</td><td>Cell culture</td></tr><tr><td>CellROX Deep Red</td><td>Invitrogen</td><td>C10422</td><td>Staining</td></tr><tr><td>D-glucose</td><td>Sigma Aldrich</td><td>47249</td><td>Glucose</td></tr><tr><td>D-mannitol</td><td>Macklin</td><td>M813423</td><td>Balance osmotic pressure</td></tr><tr><td>DynamicStudio</td><td>Dantec Dynamics</td><td>V8.6</td><td>PIV analysis software</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>14190-094</td><td>Medium</td></tr><tr><td>Fibronectin</td><td>Sigma Aldrich</td><td>F-2006</td><td>Cell adhesion</td></tr><tr><td>Fluorescent microparticles</td><td>Thermo Scientific</td><td>G0100</td><td>Micro-PIV</td></tr><tr><td>Glass coverslip</td><td>Citotest Scientific Co.,Ltd.</td><td>#1</td><td>Microfluidic chip</td></tr><tr><td>High glucose DMEM</td><td>Solarbio</td><td>12100</td><td>Medium</td></tr><tr><td>High-speed camera</td><td>Dantec Dynamics</td><td>FlowSense EO</td><td>Micro-PIV</td></tr><tr><td>ImageJ</td><td>NIH</td><td>RRID:SCR_003070</td><td>Image processing software</td></tr><tr><td>Inverted fluorescence microscope</td><td>Olympus</td><td>IX73</td><td>Cell viability</td></tr><tr><td>Inverted fluorescence microscope</td><td>Olympus</td><td>IX83</td><td>Observation and acquisition</td></tr><tr><td>Low glucose DMEM</td><td>Solarbio</td><td>31600</td><td>Medium</td></tr><tr><td>Origin</td><td>OriginLab</td><td>RRID:SCR_014212</td><td>Data analysis and graphing software</td></tr><tr><td>PDMS</td><td>Dow lnc.</td><td>184</td><td>Microfluidic chip</td></tr><tr><td>Penicillin/streptomycin (P/S)</td><td>NEST Biotechnology Co., Ltd.</td><td>211092</td><td>Medium</td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>NEST Biotechnology Co., Ltd.</td><td>211031</td><td>Cell culture</td></tr><tr><td>Plasma cleaner</td><td>Jiarun Wanfeng Technology Co., Ltd.</td><td>PC-6D</td><td>Microfluidic chip</td></tr><tr><td>Programmable pressure control system</td><td>Elvesys</td><td>OB1 MK4</td><td>Flow control</td></tr><tr><td>Silicone tube</td><td>Nanjing Runze Fluid Control Equipment Co., Ltd.</td><td>96410</td><td>1mm*3mm</td></tr><tr><td>SU-8 master mold</td><td>Boao Biology Group Co. Ltd.</td><td>N/A</td><td>Microfluidic chip</td></tr><tr><td>Valve controller</td><td>Elvesys</td><td>MUX Wire V3</td><td>Flow control</td></tr></tbody></table>

a microfluidic system for modeling endothelial dysfunction under combined physiological pulsatile shear stress and oscillatory hyperglycemia

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

Endothelial dysfunction in diabetic vascular complications involves complex interactions between metabolic disturbances, specifically oscillatory glucose (OG) and pulsatile shear stress (PSS). Although these factors have been investigated in isolation, conventional models remain limited to static high-glucose or simplified laminar flow, lacking the capacity to mimic the integrated spatiotemporal coupling found in the diabetic vasculature. By utilizing a programmable microfluidic platform, this protocol provides a valuable platform to investigate the integrated effects of synchronized oscillatory hyperglycemia and physiological PSS on endothelial cells. This protocol aims to model endothelial dysfunction under physiologically relevant coupled metabolic and mechanical conditions. The use of a polydimethylsiloxane (PDMS) chip combined with a pressure-driven control system allows for the precise, independent modulation of flow waveforms and glucose concentration profiles. The hemodynamic fidelity of the system is validated by Micro-Particle Image Velocimetry (Micro-PIV), while the cellular response is quantitatively characterized by monitoring intracellular reactive oxygen species (ROS) levels and cell viability. Representative results demonstrate that physiological PSS effectively attenuates the oxidative injury induced by OG. This effect is demonstrated by reduced intracellular ROS levels and improved cell viability under combined stimulation conditions. Depending on the research question, parameters such as shear stress patterns, glucose oscillation frequencies, and channel geometries can be adjusted. This method serves as a versatile tool for mechanistic studies of mechanobiological pathways and drug screening for therapeutic interventions in diabetic vascular disease.

Micro-PIV validation of flow field within the microfluidic channel 
Figure 1D provides an overview of the microfluidic platform used to investigate endothelial responses to coupled metabolic and mechanical stimulation. The system integrates a straight-channel PDMS microfluidic chip with a pressure-driven flow control module, enabling the application of OG condition together with physiological PSS. A programmable valve-switching strategy was employed to generate periodic a...

Here, we present a protocol to fabricate and operate a microfluidic system that exposes endothelial cells to synchronized oscillatory hyperglycemia and pulsatile shear stress. This approach provides a physiologically relevant in vitro&#160;model for studying diabetic endothelial dysfunction. This protocol enables quantitative measurement of oxidative stress and endothelial cell responses.

Endothelial dysfunction in diabetic vascular complications involves complex interactions between metabolic disturbances, specifically oscillatory glucose ...

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.

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Research

JoVE Journal

Bioengineering

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On-Chip Endothelial Inflammatory Phenotyping

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Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

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Micropatterning and Assembly of 3D Microvessels 

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In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

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Microfluidic Model to Mimic Initial Event of Neovascularization

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Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

Explore a programmable microfluidic platform modeling endothelial dysfunction under synchronized oscillatory hyperglycemia and pulsatile shear stress. This method enables mechanistic studies and drug screening for diabetic vascular disease.

A Microfluidic System for Modeling Endothelial Dysfunction under Combined Physiological Pulsatile Shear Stress and Oscillatory Hyperglycemia (Video) | JoVE Journal

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

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