1Department of Pediatrics, Emory University School of Medicine, 2Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 3Aflac Cancer Center and Blood Disorders Service of Children's Healthcare of Atlanta, 4Winship Cancer Institute of Emory University
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Myers, D. R., Sakurai, Y., Tran, R., Ahn, B., Hardy, E. T., Mannino, R., et al. Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases. J. Vis. Exp. (64), e3958, doi:10.3791/3958 (2012).
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.
1. Fabrication of the Endothelial Microdevice
2. PDMS (Polydimethylsiloxane) Preparation
3. Seeding the Microfluidic Device with Endothelial Cells
4. Representative Results
Using this protocol, standard lithographic microfabrication techniques are used to create the mold needed to produce the microfluidic channels that physiologically mimic the sizescale of the microvasculature (Figure 1A). Using an optimized perfusion technique, endothelial cells then seed and confluently culture the entire inner surface of the microfluidic system within 24-48 hours of cell seeding (Figure 1B). As the microfluidic system is transparent, the entire microdevice can be placed on a brightfield/fluorescence microscope stage for imaging and data collection.
Our system can then be applied to study hematologic diseases that involve altered biophysical properties, such as sickle cell disease, in which the increased rigidity of sickled red cells and aberrant leukocyte and endothelial adhesion contribute to microvascular obstruction. A clinically approved medication, hydroxyurea, ameliorates symptoms but its direct effect on microvascular flow is unknown. Our assay takes into account both cell rigidity and adhesion, and demonstrates that hydroxyurea significantly ameliorates flow in sickle cell disease (Figure 2).
Sickle cell disease is only one example of an application for the microvasculature-on-a-chip, as this system is ideally suited to study any hematologic process in which blood cells interact with each other and endothelial cells in the microvasculature. Other clinically relevant applications include inflammatory disorders, sepsis/lung injury, thrombotic microangiopathies, malaria, and cancer metastasis while more basic applications include leukocyte biology and hematopoietic stem cell biology, among many others.
Figure 1. A) the initial PDMS microfluidic device before endothelialization. Here, the microdevice is injected with food coloring to illustrate sizescale and overall design of the system. B) Brightfield microscopy shows the microfluidic system is completely endothelialized within 48 hours of cell seeding using the protocol described here.
Figure 2. A) The microvasculature-on-a-chip with microchannels approximates the size of post-capillary venules (30 μm), the site of most sickle cell microvascular obstructive events. Whole blood from sickle cell patients receiving hydroxyurea and from patients not receiving hydroxyurea is flowed through two different microvasculature-on-a-chip devices. B) Whole blood from sickle cell patients receiving hydroxyurea flows with relative ease within the endothelialized microchannels. C) Under the same hemodynamic conditions, whole blood from sickle cell patients not receiving hydroxyurea, however, exhibits much more sluggish flow with microchannel obstruction. The bottom channel is completely obstructed with no flow and the flow velocities in the other endothelialized microchannels are significantly lower than in the hydroxyurea condition. The scale bar in all three images is 30 μm.
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 fabrication procedure to allow other researchers to readily apply this system to their own work. In addition, the presence of the cultured endothelial cells naturally "rounds out" the effective lumen, enabling the system to be more physiologic. Furthermore, our fluid dynamic modeling reveal that the flow conditions in our system are comparable to that in the in vivo microvasculature. Finally, recent work characterizing blood flow in square and rectangular microchannels has shown that those geometries are suitable for blood rheology experiments15.
Finally, our assay is not intended to measure, in isolation, distinct cellular biophysical properties that lead to microvascular occlusion. Techniques such as atomic force microscopy, micropipette aspiration, and optical trapping have been well characterized for those types of experiments. Instead, the value of our microsystem is its capability to recapitulate, simultaneously and within a single in vitro system, an ensemble of physiological processes and biophysical properties, including adhesion molecule expression, aberrant blood cell-endothelial cell interactions, blood cell aggregation (e.g. thrombosis), cell deformability, cell size/shape, microvascular geometry, and hemodynamics, all of which contribute to pathologic microvascular cellular interactions in different disease states.
No conflicts of interest declared.
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.
|blunt point needle||OK International||920050-TE||Precision TE needle 20 Gauge x 1/2", pink|
|Hole puncher (pin vise)||Technical Innovations|
|Human umbilical cord endothelial cells (HUVECs)||Lonza||CC-2519|
|Polydimethylsiloxane (PDMS)||Fisher Scientific||NC9285739||Sylgard 184 Silicone Elastomer KIT|
|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|