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

Summary

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 model for studying diabetic endothelial dysfunction. This protocol enables quantitative measurement of oxidative stress and endothelial cell responses.

Abstract

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.

Introduction

Endothelial dysfunction is a critical early event in the development of diabetic vascular complications, driven by both metabolic disturbances and abnormal hemodynamic forces^1,2. 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 homeostasis^3,4,5. Concurrently, hemodynamic shear stress is a major determinant of endothelial phenotype, with PSS under physiological....

Protocol

Ethics statement:

NOTE: All reagents, devices, and software used in this protocol are listed in the Table of Materials.

1. Microfluidic chip fabrication

1. 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.
2. 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.
3. Place the container in a vacuum chamber under -0.....

Results

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.......

Discussion

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 signaling^4,17<.......

Materials

List of materials used in this article

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