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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Micropatterning and Assembly of 3D Microvessels

Published: September 9th, 2016

DOI:

10.3791/54457

1Department of Bioengineering, University of Washington, 2Center for Cardiovascular Biology, Institute for Stem Cell and Regenerative Medicine, University of Washington
* These authors contributed equally

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 µm. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

The microvasculature in each organ helps define the tissue microenvironment, maintain tissue homeostasis and regulate inflammation, permeability, thrombosis, and fibrinolysis 1,2. Microvascular endothelium, in particular, is the interface between blood flow and the surrounding tissue and therefore plays a critical role in modulating vascular and organ function in response to stimuli such as hydrodynamic forces and circulating cytokines and hormones 3-5. Understanding the detailed interactions between the endothelium, blood, and the surrounding tissue microenvironment is important for the study of vascular biology and disease....

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1. Microfabrication of Patterned Polydimethylsiloxane (PDMS) with Network Design

  1. Wafer Fabrication to Create a Negative Template of the Network Design
    1. Create a network pattern using any computer-aided design (CAD) software. Ensure that the diagonal dimension between the inlet and outlet match the distance between the inlet and outlet reservoirs on the housing devices in future steps (see 2.1.1).
      Note: The design of the pattern itself is custom depending on the specific res.......

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The engineered vessel platform creates functional microvasculature embedded within a natural collagen type I matrix and allows for tight control of the cellular, biophysical and biochemical environment in vitro. To fabricate engineered microvessels, human umbilical vein endothelial cells (HUVECs) are perfused through the collagen-embedded microfluidic network where they attach to form a patent lumen and confluent endothelium. As illustrated in Figure 1A-C, the ve.......

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Engineered microvessels are an in vitro model where physiological characteristics such as luminal geometry, hydrodynamic forces, and multi-cellular interactions are present and tunable. This type of platform is powerful in that it offers the ability to model and study endothelial behavior in a variety of contexts where the in vitro culture conditions can be matched to that of the microenvironment in question. For example, the mechanisms driving endothelial processes, such as angiogenesis, are known to o.......

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The authors would like to acknowledge the Lynn and Mike Garvey Imaging Laboratory at the Institute for Stem Cell and Regenerative Medicine as well as the Washington Nanofabrication Facility at the University of Washington. They also acknowledge the financial support of National Institute of Health grants DP2DK102258 (to YZ), and training grants T32EB001650 (to SSK and MAR) and T32HL007312 (to MAR).

....

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Name Company Catalog Number Comments
Wafer Fabrication
AutoGlow Plasma System AutoGlow
Headway Spin Coater Headway Research, Inc  PWM32 Spin Coater 
ABM Contact Aligner AB-M
Alpha Step Profilometer Tencor Alpha Step 200
SU-8 Developer Microchem Y020100
SU-8 Resist Microchem SU-8 2000
8" silicon wafer Wafer World Inc.
Tabletop Micro Pattern Generator Heidelberg Instruments μPG 101 For generation of photomask
Hot plate VWR 97042-646
Ispropyl alcohol Avantor Performance Materials 9088
Petri dishes (120 x 120 mm, square) Sigma-Aldrich Z617679
Trichloro(3,3,3-trifluoropropyl)silane Sigma-Aldrich MKBG3805V
Polydimethylsiloxane (PDMS) elastomer base and curing agent Dow Corning Sylgard 184 Mixed at 10:1 (w/w)
Vacuum desiccator Sigma-Aldrich Z119024-1EA
Oven VWR 9120976
Device Fabrication and Culture
poly(methyl methacrylate) (PMMA) Plexiglas
Corona Treater Electro-Technic Products, Inc. BD-20 Handheld device for plasma treatment of PMMA devices and PDMS molds
Soldering Iron Weller  WTCPS
Stainless Steel Truss Head Slotted Machine Screw McMaster-Carr  91785A096
Stainless steel dowel pins McMaster-Carr  93600A060
Tweezers  Miltex 24-572 Any similar tweezers may be used
Spatula (Micro Spoon) Electron Microscopy Services 62410-01
Screw driver Any flat head screwdriver may be used, autoclaved
Glass coverslips (22 x 22 mm) Fisher Scientific 12-542B
Bleach Clorox 4460030966
Petri dishes (150 X 25mm) Corning 430599
Petri dishes (100 X 20 mm) Corning 2909
Cotton, cut into 1 cm x 3 cm pieces Autoclaved
Polyethyleneimine (PEI) Sigma-Aldrich P3143 Dilute to 1% in cell culture grade water
Glutaraldehyde Sigma-Aldrich G6257 Dilute to 0.1% in cell culture grade water
Sterile H2O Autoclaved DI H2O
Type I collagen, dissolved in 0.1% acetic acid Isolated from rat tails as described in Rajan et. al. 2006 (ref #37)
1 mL syringe BD 309659
10 mL syringe BD 309604
15 mL conical tubes Corning 352097
30 mL conical tubes Corning 352098
M199 10X Media  Life Technologies 11825-015
1N NaOH (sterile) Sigma-Aldrich 415413 Dilute to 1N in cell culture grade water
HUVECs  Lonza
Endothelial growth media Lonza CC-3124
Trypsin Corning 25-052-CI
Fetal bovine serum (FBS) Thermofisher Scientific 10082147
Dextran from Leuconostoc spp. (70kDa) Sigma-Aldrich 31390
Phosphate Buffered Saline (PBS) Corning 21-031-CV
Hemocytometer Hausser Scientific Co. 3200
Gel loading tips VWR 37001-152
18G Blunt Fill Needle BD  305180
20G Stainless Steel Dispensing Needle McMaster-Carr 75165A123
Tygon 1/32” ID, 3/32" OD Silicon Tubing Cole-Parmer EW-95702-00
1/16" Tube-to-tube Coupling McMaster-Carr 5116K165
90° Elbow Connectors, Tube-to-Tube McMaster-Carr 5121K901
Luer Lock Coupling (Female, 1/16" ID) McMaster-Carr 51525K211
Plastic Forceps, with Jaw Grips Electron Microscopy Services 72971
Dual Syringe Pump Harvard Apparatus 70-4505
5 mL Polystyrene Round-bottom tube Fisher Scientific 14-959-2A
Device Analysis
Formaldehyde Sigma-Aldrich F8775
Bovine Serum Albumin Sigma-Aldrich A8806-5G
Triton X-100 Sigma-Aldrich T-9284
Rabbit anti-hCD31 Abcam ab32457 1:25 working dilution
FITC conjugated anti-von Willebrand Factor antibody Abcam ab8822 1:100 working dilution
Goat anti-rabbit 568 secondary antibody Thermofisher Scientific A-11011 1:100 working dilution
Hoescht Thermofisher Scientific H1399 Resuspended in DMSO
Sodium cacodylate  Sigma-Aldrich C0250 To make 0.2M cacodylate buffer
Ethanol VWR International BDH1164-4LP
40kDa FITC-conjugated Dextran Sigma-Aldrich FD40S 
Additional Culture Reagents 
CHIR-99021 Selleck Chem S2924 Small molecule GSK-3 inhibitor
Human recombinant VEGF Peprotech 100-20
Human recombinant bFGF Peprotech AF-100-18B

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