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Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Published: May 19th, 2016



1Department of Cellular and Molecular Physiology, College of Medicine, Penn State University, 2Lane Department of Computer Science and Electrical Engineering, West Virginia University
* These authors contributed equally

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype1,2. The microfluidics technology enables a precisely controlled fluid through a geometrically constrained microscale (sub-millimeter) channels3, which provides the opportunity for cultured cells, especially for vascular ECs, to grow under desired flow conditions. These features make the cell culture conditions closer to in vivo than the conventional, static 2D cell cultures. They are extremely important when the microfluidic devices are use....

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1. Microfluidic Device Fabrication

  1. Standard Photolithography Fabrication of a SU-8 50 Master Mold
    1. Clean the silicon wafer before spin-coating. Rinse a bare 2 inch silicon wafer with acetone for 15 min followed by isopropyl alcohol (IPA) for 15 min. Dehydrate the wafer by placing it on a hotplate at 150 °C for 1 hr. After dehydration, cool the wafer at room temperature.
    2. Spin-coat the silicon wafer with SU-8 photoresist. Add 2 ml SU-8 photoresist onto the wafer. Ramp the wafer to 500.......

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This section shows some of the results obtained with the cultured microvessel network developed with this protocol. The microchannel pattern is a three level branching network (Figure 1A). In this design, a 159 µm wide mother channel branches into two 126 µm wide channels, and branches again into four 100 µm wide daughter channels. A 3D numerical simulation was performed to estimate the shear stress distributions under the flow rate of 0.35 µl/min (

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In this article, we present detailed protocols for the development of cultured microvessel network, the characterization of EC junctions and cytoskeleton F-actin distribution, and the quantitative measurements of EC [Ca2+]i and NO production using a microfluidic device. The perfused microfluidic device provides an in vitro model that allows a close simulation of the in vivo microvascular geometries and shear flow conditions. Since the cultured microvessel network can be formed by p.......

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This work was supported by National Heart, Lung, and Blood Institute grants HL56237, National Institute of Diabetes and Digestive and Kidney Diseases Institute DK97391-03, National Science Foundation (NSF-1227359 and EPS-1003907).


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Name Company Catalog Number Comments
ATP Sigma-Aldrich A2383
Acetone Fisher Scientific A929
Biopsy punch Miltex 33-31 AA
Bovine Albumin MP Biomedicals 810014
Bovine Brain Extract (BBE) Lonza CC-4098
Cover-slip Fisher Scientific 12-542C
DAF-2 DA Calbiochem 251505
Dextran Sigma-Aldrich 31390
Donkey anti-Goat IgG (H+L) Secondary Antibody Life technologies A-11055
DPBS, no calcium, no magnesium Gibco 14190-250
DRAQ5 (nuclei staining)  Cell Signaling Technology 4084
Endothelial Cell Growth Supplement (ECGS) Sigma-Aldrich E2759-15MG
Fetal Bovine Serum Gibco 16000-044
Fibronectin Gibco PHE0023
Fluo-4 AM Life technologies F-14201
Gelatin from porcine skin Sigma-Aldrich G1890-100G
Gentamicin (50 mg/mL) Gibco 15750-078
Glass coverslip Fisher Scientific 12-548B
Glass Pasteur pippette VWR 14672
Heparin sodium salt from porcine intestinal mucosa Sigma-Aldrich H3393-10KU
HEPES Buffered Saline Solution Lonza CC-5024
Human umbilical vein endothelial cells (HUVECs) Lonza CC-2517
Isopropyl alcohol (IPA) VWR 89125
L-Glutamine (200 mM) Gibco 25030-081
Mammalian Ringer Solution Ingredient
NaCl (132 mM) Fisher Scientific S671-3
KCL (4.6 mM) Fisher Scientific P217-500
CaCl2 · 2H2O (2.0 mM) Fisher Scientific C79-500
MgSO4 ·7H2O (1.2 mM) Fisher Scientific M63-500
Glucose(5.5 mM) Fisher Scientific BP350-1
NaHCO3 (5.0 mM) Fisher Scientific S233-500
Hepes Salt (9.1 mM) Research Organics 6007H
Hepes Acid (10.9 mM) Research Organics 6003H
MCDB 131 Culture Medium Life technologies 10372-019
Paraformaldehyde Electron Microscopy Sciences 15710
Phalloidin (F-actin staining) Sigma-Aldrich P1951
Phosphate Buffered Saline  Life technologies 14040-133
Polydimethylsiloxane (PDMS) Dow Corning Corporation Sylgard 184
Scalpel Exel Int 29552
Scotch tape 3M 34-8711-3070-3
Silicon wafer VWR 14672
SU-8 photoresist MicroChem SU-8 2050 Y111072
SU-8 developer MicroChem Y020100
tissue culture flasks Sigma-Aldrich Z707503-100EA
Triton X-100 Chemical Book T6878
Trypsin Neutralizer solution Gibco R-002-100
Trypsin/EDTA Solution (TE) Gibco R-001-100
Tubing Cole-Parmer PTFE microbore tubing, 0.012" ID x 0.030" OD
VE-cadherin Santa Cruz Biotechnology SC-6458
Name of Equipment Company Catalog Number Comments/Description
Biosafety Laminar hood NuAire NU-425 Class II, Type A2
CCD camera Hamamatsu ORCA
Confocal microscope Leica TCS SL
Desiccator Bel-Art F42022
Hotplate Wenesco HP-1212
Incubator Forma Scientific 3110
Isotemp oven Barnstead 3608-5
Lithography bench Karl Suss MA6 Contact Lithography
Optical microscope Nikon L200 ND & Diaphod 300
Shutter for the CCD camera Sutter Instrument Lambda 10-2
Plasma cleaner PVA TePla/Harrick plasma M4L/PDC-32G
Spin coater Brewer Science Cee 200X
Syringe pump system Harvard Apparatus 703005
Name of Software Company Catalog Number Comments/Description
CAD software Autodesk AutoCAD 2015
CFD simulation software COMSOL COMSOL Multiphysics
Images acquire and analyse for NO production  Universal Imaging Metafluor

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