We thank T. Hunt, M. Rosenbluth, and the Lam Lab for their advice and useful discussions. We acknowledge the support from G. Spinner and the Institute for Electronics and Nanotechnology at the Georgia Institute of Technology. Financial support for this work was provided by an NIH grant K08-HL093360, UCSF REAC award, an NIH Nanomedicine Development Center Award PN2EY018244, and funding from the Center for Endothelial Cell Biology of Children's Healthcare of Atlanta.

1. Fabrication of the Endothelial Microdevice
<ol>
 <li>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 &mu;m.</li>
 <li>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 approxima...

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No conflicts of interest declared.

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 fabricated10,11, we opted to use a more simplified and standard fabricatio...

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

endothelialized microfluidics for studying microvascular interactions in hematologic diseases

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 conditions3-6. 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 interactions6-13.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.

Watch this Scientific Journal Video about Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases at JoVE.com

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

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

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

endothelialized-microfluidics-for-studying-microvascular-interactions

Research

JoVE Journal

Bioengineering

15.8K Görüntüleme.  Emory University School of Medicine. Mikrovasküler ölçekli kanallar (