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^1-8. Studies of microvessels using animal models and in vivo assays, such as corneal angiogenesis assay, chick chorioallantoic membrane angiogenesis assay, and Matrigel plug assay, are more time-consuming, high in cost, challenging with respect to observation and quantifications, and raise ethical issues^{1, 9-13}.

Advances in micromanufacturing and microfluidic chip technologies have enabled a variety of insights into biomedical sciences while curtailing the high experimental costs and complexities associated with animals and in vivo studies¹⁴, such as easily and tightly controlled biological conditions and dynamic fluidic environments, which would not have been possible with conventional macroscale techniques.

Here, we present an approach to construct an endothelialized microchannels-on-a-chip which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow by using the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics.

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 layer of photoresist using the following recipe:

Step	Time (Seconds)	Speed (rpm)	Acceleration
1	2	300	highest
2	10	0
3	3	300	highest
4	60	600	highest
5	10	500	highest
6	10	600	highest

4. Then, bake the wafer on a hotplate with a temperature of 110 °C for 90 sec. After the soft bake the thickness of the photoresist will be 20-30 μm.
5. Spin coat a second layer of photoresist following the same recipe used for the first layer.
6. Soft bake the wafer again by placing it on a hotplate with a temperature of 110 °C for 90 sec. After this soft bake the thickness of the photoresist will be 40-60 μm.
7. Generate positive master patterns by exposing the photoresist to UV light with an exposure dose of 14,500 mJ/cm² through a film mask.
8. Dilute the developer with deionized (DI) water (1:2 (v/v)). Rinse the wafer repeatedly in the solution until the pattern is fully developed. Then wash the wafer with DI water and dry it using nitrogen gas.
9. Reflow: Place the wafer on a hotplate with a temperature of 120 °C for 4 min and cover with a glass Petri dish to prevent solvent evaporation. Remove the wafer from the hotplate and allow it to cool to room temperature. The photoresist thickness after reflow will be 50-60 μm.

2. Soft Lithography Fabrication of PDMS Microchannel Network

1. Prepare polydimethylsiloxane (PDMS) solution at the weight ratio of 10:1 (base:curing agent) and mix it thoroughly using a planetary centrifugal mixer.
2. Cast the PDMS solution onto the reflowed photoresist master mold. Place the casted PDMS in a desiccator for 15 min to degas. Use nitrogen gas to remove any remaining bubbles if necessary.
3. Bake the PDMS in an oven at a temperature of 60 °C for 3 hr to allow it to cure. Then remove the cured PDMS layer from the master mold.
4. Use a sharpened puncher to create inlet/outlet holes by punching holes in the channel network. Clean the surface of the PDMS using nitrogen gas.
5. Treat two PDMS layers with oxygen plasma for 30 sec inside a plasma cleaner at an operating pressure of 4.5 x 10^-2 Torr and an oxygen flow rate of 3.5 ft³/min. Then, align the surfaces of the PDMS manually under an optical microscope. Use a drop of water if necessary for a better control of the alignment.
6. Bake the device in an oven at 60 °C for 30 min to achieve permanent bonding.

3. HUVEC Culture and Seeding in the Chip

1. Culture the HUVECs with culture medium with L-glutamine supplemented with 10% fetal bovine serum (FBS). For the experiment passages between 2 and 5 were used.
2. Once the HUVECs are confluent, count the cells and harvest them by first rinsing the cells with HEPES buffered saline solution (HEPES-BSS), and then treat the cells with trypsin/EDTA and incubate for 2-6 min at 37 °C. After the trypsinization is complete, neutralize the trypsin/EDTA with trypsin neutralizing solution. Centrifuge and suspend the cells in culture medium with 8% dextran to collect them. Dextran was used to increase the medium viscosity to aid in better cell seeding and attachment.
3. Treat the device with oxygen plasma for 5 min with an operating pressure of 4.5 x 10^-2 Torr and an oxygen flow rate of 3.5 ft³/min. Then load the device with DI water and treat with UV light for 8 hr in a laminar biosafety hood for sterilization.
4. One day before the cells are ready, wash the device with 1x phosphate buffered saline (1x PBS) then coat with fibronectin (100 μg/ml, diluted with 1x PBS) and incubate in the refrigerator at 4 °C overnight.
5. After the fibronectin coating, wash the device with 1x PBS then load with culture medium. Incubate the device at a temperature of 37 °C for 15 min.
6. Load the HUVEC cells in 8% dextran culture medium with a cell concentration of 3-4 x 10⁶ cells/ml. Place a 20 μl droplet of cells at one inlet of the device and tilt it to introduce the cells into the microfluidic channel. After 15-20 min the cells will begin to attach to the side walls of the channels. Rotate the device every 15 min to create a more uniform distribution of cells. If necessary, additional loading can be performed.

4. Long-term Perfusion Setup

After 5-6 hr of static culture, the attached HUVECs will start to fully spread out. Set up perfusion using a remote controlled syringe pump system with a steady flow of 10 μl/hr. Perfusion can be adjusted for a higher flow rate, and can last for a time period between 4 days to 2 weeks.

5. Cell Staining and Microscope Characterization

1. When the cells reach confluence inside the device, firstly, wash the device with 1x PBS to thoroughly remove the medium. Then load the device with red dye diluted with diluent (4 μl-1 ml). Load the dye similar to the cell loading procedure. Incubate the device in the dark for 5 min at room temperature, and then wash the device with culture medium to stop the staining. Long incubation of the dye can cause cellular toxicity and disadhesion.
2. Load the device with blue dye diluted with 1x PBS (2 droplets per ml). Incubate in the dark for 5 min at room temperature then thoroughly wash the device with 1x PBS.
3. Examine the cell staining under an inverted optical microscope. If the staining was good, load the microchannels with fixing medium (3.5% paraformaldehyde diluted with 1x PBS), then submerge the device in fixing medium and completely cover it with aluminum foil. Store the device in the refrigerator at a temperature of 4 °C to prevent the device from drying out and photobleaching. The fixed device is now ready for confocal imaging, which can be done by a laser scanning confocal microscope.

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