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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.
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.
Neovascularization happens in many normal and pathological processes1,2,3,4, 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....
1. Wafer preparation
NOTE: This protocol is specific for the SU-8 2075 negative photoresist used during this research.
The in vitro 3D model to mimic the initial event of neovascularization (MIEN) presented here consisted of a microfluidic sprouting chip and a microfluidic control system. The microfluidic sprouting chip was optimized from previous publications22,23,37,40,51,52,53. Briefly, it contai.......
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 sprouting22,32,40,46,54, but the mechanical microenvironment is still hard to maintain constantly. It remains a difficult subject to mimic the initia.......
This work was supported by the National Natural Science Research Foundation of China Grants-in-Aid (grant nos. 11827803, 31971244, 31570947, 11772036, 61533016, U20A20390 and 32071311), National key research and development program of China (grant nos. 2016YFC1101101 and 2016YFC1102202), the 111 Project (B13003), and the Beijing Natural Science Foundation (4194079).
....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 |
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