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Technical Program TRACK 3

duced in the context of an engineering challenge will be discussed. Our re- Abhishek Jain, Andries D. van der Meer, Anne-Laure Papa, Ric-
cent work on digital immunoassays with single molecule detection and noise cardo Barrile, Wyss Institute for Biologically Inspired Engineering
rejection by spatial exclusion will be introduced, and results for ultrasensitive at Harvard University, Boston, MA, United States, Angela Lai, Car-
assays presented. Strategies for multiplexing will be presented, along with negie Mellon University, Pittsburgh, PA, United States, Benjamin
preliminary results. Next, we will present capillaric circuits that are capillary Schlechter, Beth Israel Deaconess Medical Center at Harvard
powered microfluidics for autonomous fluidic operations that are ideally suit-
ed for point-of-care applications. Newly 3D printed capillaric circuits made Medical School, Boston, MA, United States, Monicah A. Otieno,
by rapid prototyping will be introduced, and the design rules discussed. Calvert S. Louden, Janssen Pharmaceutical Research and De-
The application of rapidly prototyped capillarics to bacterial detection and velopment, Pre-Clinical Development and Safety,, Spring House,
immunoassays by taking advantage of sequential flow operations and large PA, United States, Geraldine A. Hamilton, Emulate Bio Inc., Cam-
volume flow for rapid and high sensitivity results will be shown. bridge, MA, United States, Alan D. Michelson, Andrew L. Frelinger
III, Boston Children’s Hospital at Harvard Medical School, Boston,
10:00am Self-Contained Magnetic Levitation Platform for Sickle MA, United States, Donald E. Ingber, Wyss Institute for Biologically
Cell Disease Diagnostics Inspired Engineering at Harvard University, Boston, MA, United
States
Technical Presentation. NEMB2016-5990
Mutual signaling between inflamed endothelium and activated platelets is
Bekir Yenilmez, University of Connecticut, Storrs, CT, United States, widely recognized as critical for regulation of hemostasis, initiation of plate-
Stephanie Knowlton, University of Connecticut, Mech. Eng., Storrs, let aggregation and thrombotic disorders associated with various diseases,
CT, United States, Savas Tasoglu, UConn, Storrs, CT, United States including atherosclerosis, sepsis, and diabetes. Yet no reliable and practical
diagnostic assays exist that can measure the effects of cross-talk between
Low-cost and portable point of care technology is critical for enabling rapid, platelets and inflamed vessel walls in the presence of physiological shear.
Over the last decade or so, multiple flow chambers and microfluidic devices
on-site disease diagnostics. A particular need exists for sickle cell disease
diagnostics, as the majority of those carrying the disease live in Sub-Saharan that contain microchannels with a physiologically-relevant size have been
Africa, where ubiquitous testing procedures have yet to be implemented. lined by living endothelium and exposed to flowing blood to study the basic
A serious complication of sickle cell disease is stroke and many of these science, but they have not been used in clinical diagnostic settings due to
strokes are “silent” strokes which do not present the typical noticeable the difficulty in maintaining living endothelial cells in devices. Specifically,
because it is extremely difficult to maintain the stability, sterility and storage
symptoms and are generally only detectable through imaging, such as with
an MRI. 17% of children under the age of 14 and 23% under the age of 18 with of living cell cultures for extended times in non-controlled settings, it would
sickle cell disease are estimated to suffer from silent strokes. be virtually impossible to maintain the robustness, sensitivity and standard-
Here, we present a simple yet powerful platform to perform density-based ization of these assays.
analysis of micro-particles, such as cells, using magnetic levitation. Under Here, we describe a simple microfluidic device lined by a chemically
deoxygenated conditions, red blood cells from patients with sickle cell dis-
ease have been shown to attain higher densities than those from healthy preserved human endothelium that retains its ability to support thrombus
patients. This is a portable, self-contained device to levitate particles of inter- formation and platelet adhesion as blood flows through its channels at an
est, image them using an embedded low-cost optical system and a camera arterial shear rate. We demonstrate the potential clinical value of this chemo-
module, and process the captured images in order to assess the distribu- preserved endothelialized device by showing that thrombus formation and
platelet function can be measured within minutes using a small volume (0.5
tion of the particles. All components are contained in a compact, portable
3D-printed casing. The required sample preparation is simple, requiring only mL) of whole blood taken from subjects receiving antiplatelet medications,
a drop of blood from the patient via finger stick. The blood sample is mixed and demonstrating potentially greater reliability than standard platelet func-
with a paramagnetic gadolinium medium and sodium metabisulfite (which tion tests and collagen-coated perfusion chambers.
are both low-cost reagents) then loaded into a glass microcapillary which is We first coated the inner surface of a rectangular microfluidic device with
inserted between two permanent magnets separated 1 mm apart with like
poles facing each other. The magnets form a magnetic field which exerts type I collagen and then cultured human umbilical vein endothelial cells (HU-
a force on the cells in the direction of the centerline between the magnets VECs) on all four walls of the channel to create a rectangular tube lined by a
with magnitude greatest at the surface of the magnets and approaching continuous, confluent endothelial cell monolayer. When we activated the en-
zero at the centerline. A second force, the buoyancy force, relies on the dothelium in the device by adding increasing doses of TNF-α (0, 5, and 100
ng/ml) and fixed the cells with 4% paraformaldehyde, we found that the fixed
density of the particle relative to that of the medium. Particles levitate at
a height at which the magnetic force is in equilibrium with the buoyancy endothelium continued to exhibit a dose-dependent increase in intercellular
force. Levitating blood cells are then imaged and their levitation heights are adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1), as
used to calculate the particle distribution. This value serves as an indicator well as von Willebrand factor (VWF) and tissue factor (TF). These results con-
to diagnose sickle cell disease, as demonstrated by a pilot study of blood firm that the fixed endothelium retains expression of multiple molecules that
mediate adhesion of blood cells and platelets after activation with TNF-α.
samples from patients with and without the disease. Sample preparation,
magnetic levitation, and analysis are completed within only 10 minutes. When blood from a healthy donor was flowed over an endothelium that
This device will enable rapid, on-site testing for sickle cell disease in order was fixed without prior treatment with TNF-α, there was virtually no platelet
to allow proper care to be administered and prevent complications early on adhesion on the surface, as would be expected for a healthy endothelium.
for children with the disease. The device is user-friendly, label-free, and por- In contrast, when devices were used with endothelium that was treated with
increasing doses of TNF-α prior to fixation, a dose-dependent increase in
table, making it ideas for point-of-care disease diagnostics. The platform has
been designed with easily accessible and inexpensive components, allow- platelet surface adhesion to the endothelial layer was observed. Importantly,
ing widespread use of this method by researchers, clinicians, and patients we did not observe any significant difference in platelet adhesion when we
alike. Future applications to other diseases, including cancer, malaria, and compared living versus chemopreserved endothelium and the morphology
Babessia, can have a tremendous impact on global health, particularly in de- of thrombi appeared similar to that of thrombi formed on living endothelium
in vivo. Thus, these findings confirm that the fixed surface of the endothelium
veloping countries where more advanced testing is inaccessible as well as
in-home testing and disease monitoring. retains its pro-thrombotic function after fixation and it is capable of reproduc-
ing physiological thrombosis in our microfluidic device.
10:20am Assessing Hemostasis and Platelet Function using a To establish clinical utility, we also perfused whole blood of patients who
Microfluidic Device Lined with Fixed Human Endothelium were regular users of antiplatelet drugs, including aspirin or both aspirin and
46
clopidogrel. We found that these subjects showed a significant reduction in
Technical Presentation. NEMB2016-6037 platelet aggregation when tested using our devices lined by a fixed endo-