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

Summary

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.

Abstract

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.

Introduction

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^1,2. 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....

Protocol

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 rpm at 100 rpm/sec acceleration for 10 sec, followed by 1,000 rpm at 300 rpm/sec acceleration for 30 sec. (see Supplemental Video 1)
   3. Pre-bake t....

Results

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 (Figure 1B

Discussion

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.......

Materials

List of materials used in this article

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 4.0.0.982
Images acquire and analyse for NO production	Universal Imaging	Metafluor