Microfluidic Model to Mimic Initial Event of Neovascularization

Summary

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Abstract

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Introduction

Neovascularization happens in many normal and pathological processes^1,2,3,4, which include two major processes in adults, angiogenesis and arteriogenesis⁵. Besides the best-known growth factors, such as vascular endothelial growth factor (VEGF)⁶, mechanical stimulations, in particular the blood flow induced shear stress, is important in the regulation of neovascularization⁷. As we know, the magnitude and forms of shear stress vary dramatically and dynamically in differen....

Protocol

1. Wafer preparation

NOTE: This protocol is specific for the SU-8 2075 negative photoresist used during this research.

1. Clean the silicon wafer 3 to 5 times with methanol and isopropanol on a spin coater as follows: first spin for 15 s at 500 rpm, and then spin for 60 s at 3,000 rpm.
2. Transfer the silicon wafer to a hotplate, which is preheated to 180 °C and bake the wafer for 10 min.
3. Remove the silicon wafer from the hotplate and cool it to room temperature........

Discussion

For a long time, real-time observation of neovascularization has been a problem. Several approaches have been developed recently to create perfused vessels lining with ECs and adjacent to extracellular matrix for sprouting^{22,32,40,46,54}, but the mechanical microenvironment is still hard to maintain constantly. It remains a difficult subject to mimic the initia.......

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Genview	GP3108
Collagen I, rat tail	Corning	354236
DAPI	Sigma-Aldrich	D9542
Electromagnetic pinch valve	Wokun Technology	WK02-308-1/3
Endothelial cell medium (ECM)	Sciencell	1001
Fetal bovine serum (FBS)	Every Green	NA
Fibronectin	Corning	354008
FITC-dextran	Miragen	60842-46-8
Graphical programming environment	Lab VIEW	NA
Image editing software	PhotoShop	NA
Image processing program	ImageJ	NA
Isopropanol	Sigma-Aldrich	91237
Lithography equipment	Institute of optics and electronics, Chinese academy of sciences	URE-2000/35
Methanol	Sigma-Aldrich	82762
Micro-peristaltic pump	Lead Fluid	BT101L
Micro-syringe pump	Lead Fluid	TYD01
Oxygen plasma	MING HENG	PDC-MG
Paraformaldehyde	Sigma-Aldrich	P6148
PBS (10x)	Beyotime	ST448
Permanent epoxy negative photoresist	Microchem	SU-8 2075
Phenol Red sodium salt	Sigma-Aldrich	P5530
Polydimethylsiloxane (PDMS)	Dow Corning	Sylgard 184
Poly-D-lysine hydrobromide (PDL)	Sigma-Aldrich	P7886
Polytetrafluoroethylene	Teflon	NA
Program software	MATLAB	NA
Recombinant Human VEGF-165	StemImmune LLC	HVG-VF5
Sodium hydroxide (NaOH)	Sigma-Aldrich	1.06498
Stage top incubator	Tokai Hit	NA
SU-8 developer	Microchem	NA
Trichloro(1H,1H,2H,2H-perfluorooctyl)silane	Sigma-Aldrich	448931
TRITC Phalloidin	Sigma-Aldrich	P5285