Micropatterning and Assembly of 3D Microvessels

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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 research goals of the user (see Figure 1 for example patterns).
   2. Generate a mask of the network pattern using high resolution pr....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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 25 mm)	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
1 N NaOH (sterile)	Sigma-Aldrich	415413	Dilute to 1 N 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. (70 kDa)	Sigma-Aldrich	31390
Phosphate Buffered Saline (PBS)	Corning	21-031-CV
Hemocytometer	Hausser Scientific Co.	3200
Gel loading tips	VWR	37001-152
18 G Blunt Fill Needle	BD	305180
20 G 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 0.2 M cacodylate buffer
Ethanol	VWR International	BDH1164-4LP
40 kDa 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