Name: microfluidic model to mimic initial event of neovascularization
Uploaded: 2021-04-10T13:00:00
Description: Watch this Scientific Journal Video about Microfluidic Model to Mimic Initial Event of Neovascularization at JoVE.com

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).

1. Wafer preparation
NOTE: This protocol is specific for the SU-8 2075 negative photoresist used during this research.
<ol>
	<li>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.</li>
	<li>Transfer the silicon wafer to a hotplate, which is preheated to 180 &#176;C and bake the wafer for 10 min.</li>
	<li>Remove the silicon wafer from the hotplate and cool it to room temperature....

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...

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 different parts of the vasculature, resulting in important effects on vascular cells8,9,10,11,12. Previous studies have shown that shear stress may affect various aspects of ECs, including cell phenotypic changes, signal transduction, gene expression, and the communication with mural cells13,14,15,16,17,18,19,20; hence, regulate neovascularization21,22,23,24.
Therefore, to better understand neovascularization, it is important to reconstruct the process in natural cellular microenvironment in vitro. Recently, many models have been established to create micro-vessels and provide precise control of microenvironment25,26,27, taking advantage of advances in microfabrication and microfluidic technology. In these models, micro-vessels can be generated by hydrogel28,29, polydimethylsiloxane (PDMS) microfluidic chips30,31,32 or 3D bioprinting33,34. Some aspects of the microenvironment, such as luminal shear stress22,23,35,36, transendothelial flow37,38,39,40, biochemical gradient of angiogenic factors41,42, strain/stretch43,44,45, and co-cultured with other types of cells32,46 have been mimicked and controlled. Usually, a large reservoir or syringe pump was used to provide perfused medium. Transendothelial flow in these models was created by pressure drop between the reservoir and micro-tube22,23,38,40. However, the mechanical microenvironment was hard to maintain constantly in this way. Transendothelial flow would increase and then exceed the physiological level if a high flow rate with high shear stress was used for perfusion. Previous study showed that at the initial period of neovascularization, the velocity of transendothelial flow is very low due to the intact ECs and basement membrane, usually under 0.05 &#181;m/s8. Meanwhile, though luminal shear stress in vascular system varies greatly, it is relatively high with mean values of 5-20 dyn/cm2,11,47. For now, the velocity of transendothelial flow in previous works have been generally kept between 0.5-15 &#181;m/s22,38,39,40, and the luminal shear stress was usually under 10 dyn/cm2&#160;23. It remains a difficult subject to constantly expose ECs to high luminal shear stress and physiological level of transendothelial flow simultaneously.
In the present study, we describe an in vitro 3D model to mimic the initial event of neovascularization (MIEN). We developed a microfluidic chip and an automatic control, highly efficient circulation system to form perfusion micro-tubes and simulate the process of sprouting48. With the MIEN model, the microenvironment of ECs stimulated at the initial period of neovascularization are firstly recapitulated. ECs can be stimulated by high luminal shear stress, physiological level of transendothelial flow and various VEGF distribution simultaneously. We describe the steps of establishing the MIEN model in detail and the key points to be paid attention to, hoping to provide a reference for other researchers.

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

microfluidic model to mimic initial event of neovascularization

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.

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...

Watch this Scientific Journal Video about Microfluidic Model to Mimic Initial Event of Neovascularization at JoVE.com

Microfluidic Model to Mimic Initial Event of Neovascularization

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 ...

Research

JoVE Journal

Bioengineering

Tri-layered Electrospinning to Mimic Native Arterial Architecture using Polycaprolactone, Elastin, and Collagen: A Preliminary Study

Throughout native artery, collagen and elastin play an important role, providing a mechanical backbone, preventing vessel rupture, and promoting recovery ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Initial Evaluation of Antibody-conjugates Modified with Viral-derived Peptides for Increasing Cellular Accumulation and Improving Tumor Targeting

Antibody-conjugates (ACs) modified with virus-derived peptides are a potentially powerful class of tumor cell delivery agents for molecular payloads used ...