Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Summary

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (<30 μm) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Abstract

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales ^1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions^3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions^6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.

Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

Protocol

1. Fabrication of the Endothelial Microdevice

1. Create a photomask by submitting a computer assisted design (CAD) drawing of the microfluidic device to an outside mask vendor. The mask used was composed of a chrome layer on soda lime glass. In this case the microfluidic channel width was 30 μm.
2. Clean a bare silicon wafer with piranha (10:1 ratio of sulfuric acid and hydrogen peroxide) for 15 minutes and dip in hydrofluoric acid for 30 seconds. Rinse with deionized (DI) water for approximately 10 seconds.
3. Using a spin coater, spin Microchem SU-8 2025 photoresist onto the wafer to a height of 30 μm. For SU-8 2025, a spin speed....

Discussion

Our endothelialized microdevice system is best suited when used in conjunction with in vivo experiments, and its reductionist approach may help elucidate the biophysical mechanisms of hematologic processes that are observed in humans and animal models. Furthermore, our system is not without limitations. For instance, our microfluidic channels are square in cross-section. Although technically circular microchannels can be fabricated^10,11, we opted to use a more simplified and standard fabricatio.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
blunt point needle	OK International	920050-TE	Precision TE needle 20 Gauge x 1/2", pink
dextran	Sigma-Aldrich	31392
Fibronectin	Sigma-Aldrich	F0895
Hole puncher (pin vise)	Technical Innovations
Human umbilical cord endothelial cells (HUVECs)	Lonza	CC-2519
Plasma cleaner	Plasma	PDC-326
Polydimethylsiloxane (PDMS)	Fisher Scientific	NC9285739	Sylgard 184 Silicone Elastomer KIT
Sigmacote	Sigma-Aldrich	SL2
SU-8 2025	Microchem	Y111069
SU-8 Developer	Microchem	Y020100
Syringe pump	Harvard Apparatus	70-3008	PHD-ULTRA
tubing(larger)	Cole-Parmer Instrument Company	06418-02	Tygonreg microbore tubing, 0.020" ID x 0.060" OD
tubing(smaller)	Cole-Parmer Instrument Company	06417-11	PTFE microbore tubing, 0.012" ID x 0.030" OD