Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Summary

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging and can be applied for microvascular research.

Abstract

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

Introduction

Microvessels, as a part of the circulation system, mediate the interactions between blood and tissues, support metabolic activities, define tissue microenvironment, and play a critical role in many health and pathological conditions. Recapitulation of functional microvessels in vitro could provide a platform for the study of complex vascular phenomena. However, conventional in vitro microvessel assays, such as endothelial cell migration assays, endothelial tube formation assays, and rat and mouse aortic ring assays, are unable to recreate the in vivo microvessels with respect to three-dimensional (3D) geometry and continuous flow control

Protocol

1. Photolithography Fabrication of Photoresist Master Mold

The following protocol shows the process to fabricate the microchannels with diameters between 30-60 μm. To get a microchannel with a smaller diameter (less than 30 μm), a single spin-coating of photoresist is needed.

1. Transfer the reflow photoresist from the refrigerator at 4 °C to the cleanroom 24 hr prior to use and allow it to warm to room temperature.
2. Clean a silicon wafer and bake it for one hr at 150 °C to allow it to dehydrate. The dehydration will assist the photoresist adhesion to the silicon substrate.
3. Spin-coat the first....

Results

Our approach to fabricate the multi-depth microchannel network mimics the complex 3D geometries of in vivo microvessels, in which the microchannels have rounded cross sections¹⁵. Additionally, the diameters of parent branching channels and the daughter channels approximately obey Murray's law for maintaining the fluid flow at a required level so that the overall channel resistance is low and flow velocities are more uniform throughout the network^16-18. The processes and results for the fabr.......

Discussion

1. Master mold fabrication

One of the designing and guiding principles for vascular morphometry is known as Murray's law¹⁶, which states that the distribution of vessel diameters throughout the network is governed by minimum energy consideration. It also states that the cube of the diameters of a parent vessel at a bifurcation equals the sum of the cubes of the diameters of the daughter vessels (

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagent/Material
Reflow Photoresist	AZ Electronic Materials	AZP4620
Developer	AZ Electronic Materials	AZ 400K
PDMS	Dow Corning Corporation	Sylgard 184
MCDB 131 Culture Medium	Invitrogen	10372-019
NacBlue Nuclei Staining	Invitrogen	H1399
PKH Red Stain	Sigma	MINI26 and PKH26GL
Fibronectin	Gibco	PHE0023
L-Glutamine	Sigma	G7513
Phosphate Buffered Saline	Invitrogen	14040-133
HEPES Buffered Saline Solution	Lonza	CC-5024
Trypsin/EDTA	Invitrogen	25300-062
Trypsin Neutralizing Solution	Lonza	CC-5002
PDMS Curing Agent	Dow Corning Corporation	Sylgard 184
Primary Human Umbilical Vein Endothelial Cells	Lonza	CC-2517
Fetal Bovine Serum	Lonza	14-501F
Diluent C	Sigma	CGLDIL
Hoechst33342	Invitrogen, Molecular Probes	R37605
Dextran	Sigma	95771
3.5% Paraformaldehyde	Electron Microscopy Science	15710-S
Equipment
Spinner	Laurell Technologies Corporation	WS-400BZ-6NPP/LITE
Desiccator	BelArt Products	999320237
Inverted Microscope	Nikon	Eclipse Ti
Syringe Pump System	Harvard Apparatus	PHD Ultra
Laminar Biosafety Hood	Thermo Scientific	1300 Series A2
Planetary Centrifugal Mixer	Thinky	ARE-310
Isotemp Oven	Fisher Scientific	13-246-516GAQ
Optical Microscope	Zeiss	Invertoskop 40C
Plasma Cleaner	Harrick Plasma	PDC-32G
Hotplate	Barnstead/Thermolyne Cimarec	SP131635
Laser Scanning Confocal Microscope	Zeiss	LSM 510