On-Chip Endothelial Inflammatory Phenotyping

Summary

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Abstract

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.¹ These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,² lipid-^{3, 4} and RAGE-induced⁵ inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-α. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Protocol

1. Cell Culture and Substrate Preparation

1. Cut 3-inch circular substrates from a 100 x 20 mm tissue culture dish (BD Falcon) using a lathe. Sterilize substrates by submersion in 70% ethanol. Place in a Petri dish and coat with 4 ml type-I collagen (100 μg/ml) for 1 hr at room temperature, then rinse with 4 ml 1 x PBS.
2. Suspend Human Aortic Endothelial Cells (HAEC, passage 4-6) at 6.5x10⁵ cells/ml and seed by applying 1 ml directly to substrate. Place in a 37 °C, 5% CO₂ incubator and allow cells to adhere to the substrate for 1 hr.
3. Add 9 ml of Endothelial Growth Medium-2 (EGM-2) supplemented....

Discussion

We describe the use of microfluidic PDMS devices for the on-chip assessment of endothelial inflammatory phenotype through the real-time imaging of CAM expression and monocyte adhesion. A major advantage of our approach lies in the ability to quantify outcomes associated with endothelial dysfunction in cells exposed to inflammatory mediators such as dietary lipids and cytokines under defined hydrodynamic conditions that mirror shear stress in atherogenic vessels. The VMMC facilitate the use of small amounts of reagents or.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 x 20mm Petri Dishes	BD Falcon	353003
Ethanol 95%	EMD Chemicals	EX0290-1
DPBS	Cellgro	21-031-CV
Type I Rat Tail Derived Collagen	Gibco	A10483-01
Human Aortic Endothelial Cells	Genlantis	PH30405A
Antibiotic-Antimycotic Solution	Invitrogen	15240-062
Endothelial BulletKit	Lonza	CC-4176
Endothelial Basal Media-2	Lonza	CC-3156
10 ml Syringes	BD Falcon	309604
Polyurethane tubing	Tygon	ABW0001
Leibovitz-15 Media	Gibco	11415-069
Sylgard 184 Silicone Elastomer Base	Dow Corning	184
Sylgard 184 Silicone Elastomer Curing Agent	Dow Corning	184
SU8 Photoresist Master Wafer	UC Davis Pan Lab	N/A
Eclipse TE200 Inverted Microscope	Nikon	Eclipse TE200
Syringe Pump	Harvard Apparatus	PHD2000
19 gauge hypodermic needle	Kendall	8881
THP-1 Monocytic Cell Line	ATCC	TIB-202
HBSS (Hanks Buffered Saline Solution) with Ca2+/Mg2+	Gibco	14025-092
Tumor Necrosis Factor Alpha (TNF-α)	R&D Systems	210-TA-010
Stromal Derived Factor - 1 (SDF-1)	R&D Systems	350-NS-010
RPMI 1640	Cellgro	10-040-CV
Human Serum Albumin (HSA)	ZLB Behring	NDC 0053-7680-32
Table 2. Specific reagents and equipment.