Endothelialized على microfluidics لدراسة التفاعلات الاوعية الدموية الدقيقة في الأمراض الدموية
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 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.
Protocol
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 fabricated10,11, we opted to use a more simplified and standard fabricatio…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Name of the reagent | Company | Catalogue 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 |
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