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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 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....

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........

Representative Results

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.......

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 sprouting22,32,40,46,54, but the mechanical microenvironment is still hard to maintain constantly. It remains a difficult subject to mimic the initia.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
0.25% Trypsin-EDTAGenviewGP3108
Collagen I, rat tailCorning354236
DAPISigma-AldrichD9542
Electromagnetic pinch valveWokun TechnologyWK02-308-1/3
Endothelial cell medium (ECM)Sciencell1001
Fetal bovine serum (FBS)Every GreenNA
FibronectinCorning354008
FITC-dextranMiragen60842-46-8
Graphical programming environmentLab VIEWNA
Image editing softwarePhotoShopNA
Image processing programImageJNA
IsopropanolSigma-Aldrich91237
Lithography equipmentInstitute of optics and electronics, Chinese academy of sciencesURE-2000/35
MethanolSigma-Aldrich82762
Micro-peristaltic pumpLead FluidBT101L
Micro-syringe pumpLead FluidTYD01
Oxygen plasmaMING HENGPDC-MG
ParaformaldehydeSigma-AldrichP6148
PBS (10x)BeyotimeST448
Permanent epoxy negative photoresistMicrochemSU-8 2075
Phenol Red sodium saltSigma-AldrichP5530
Polydimethylsiloxane (PDMS)Dow CorningSylgard 184
Poly-D-lysine hydrobromide (PDL)Sigma-AldrichP7886
PolytetrafluoroethyleneTeflonNA
Program softwareMATLABNA
Recombinant Human VEGF-165StemImmune LLCHVG-VF5
Sodium hydroxide (NaOH)Sigma-Aldrich1.06498
Stage top incubatorTokai HitNA
SU-8 developerMicrochemNA
Trichloro(1H,1H,2H,2H-perfluorooctyl)silaneSigma-Aldrich448931
TRITC PhalloidinSigma-AldrichP5285

References

  1. Potente, M., Gerhardt, H., Carmeliet, P. Basic and therapeutic aspects of angiogenesis. Cell. 146 (6), 873-887 (2011).
  2. Barger, A. C., Beeuwkes, R. D., Lainey, L. L., Silverman, K. J.

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Microfluidic ModelNeovascularizationAngiogenesisEndothelial CellsVascular Endothelial Growth FactorHydrogelShear StressCell CultureFibronectinMicrofluidic Control SystemPerfusionSprouting

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