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TRACK 3 TRACK 3 Technical Program

on critical parameters such as enzyme kinetics, dosage, and probe stability. 12:40pm Point-of-Care Monitoring of Red Blood Cell Adhesion
According to this model, synthetic biomarkers that circulate in stealth but and Deformability as a Marker of Disease Severity and Treat-
then activate at sites of disease have the theoretical capacity to discriminate ment Response in Sickle Cell Disease
tumors as small as 5 mm in diameter–a threshold sensitivity that is otherwise
challenging for medical imaging and blood biomarkers to achieve. This Technical Presentation. NEMB2016-5994
model may be adapted to describe the behavior of additional activity-based
approaches to allow cross-platform comparisons, and to predict allometric
scaling across species. Yunus Alapan, Anima Adhikari, Ceonne Kim, Evren Gurkan-Ca-
vusoglu, Jane Little, Umut Gurkan, Case Western Reserve Univer-
12:20pm Detecting and Trapping of a Single C. elegans Worm sity, Cleveland, OH, United States
in a Microfluidic Chip for Automated Microplate Dispensing
Abnormal adhesion of blood cells to endothelium are pathophysiologically
Technical Presentation. NEMB2016-5966 central to Sickle Cell Disease (SCD). Despite recent advances in identifying
and targeting cellular adhesion in SCD, knowledge of abnormal cellular ad-
hesion has not been integrated into routine clinical care or trial design, due
Israel Desta, Abdelrazak Al-Sharif, Nour AlGharibeh, Nahal to a requirement for complicated custom-designed systems, highly trained
Mustafa, Nikolas Giakoumidis, New York University Abu Dhabi, personnel, and extensive sample manipulation. The SCD Biochip is a novel
Abu Dhabi, Abu Dhabi, United Arab Emir., Kris Gunsalus, New York point-of-care (POC) microfluidic assay that allows rapid, preprocessing-free,
University, New York, NY, United States, Yong-Ak Song, New York and standardized interrogation of red blood cell (RBC) adhesion to endothe-
University Abu Dhabi, Abu Dhabi, Abu Dhabi, United Arab Emir. lium components (fibronectin, FN, or laminin, LN) in whole blood.
After a complete blood count as a standard of care, 15 µL of surplus whole
Caenorhabditis elegans worm is one of the small invertebrate animals, about blood was injected, without processing, on to FN- or LN-functionalized Bio-
1 mm in length and 80 um in diameter, that is most studied in the genetic, chips at or above the physiological shear stresses of post-capillary venules
2
developmental, and biochemical researches. It is transparent at all stages of (1-5 dyne/cm ). Serial quantitative and qualitative evaluations of RBC adhe-
life and reproduces asexually making the population genetically invariant. sion and deformability, using standardized protocols, were performed on
Furthermore, the possibility of observing important signaling pathways that >>100 SCD adult subjects with correlative clinical data.
are quite similar to human pathways makes C. elegans an important animal
model in high-throughput drug screening. To dispense C. elegans worms Greater RBC adherence per unit area to FN or LN was seen in HbSS com-
into microwell plates for drug screening, sorting system such as COPAS Bio- pared with compound heterozygous SCD. HbSS samples with a low fetal
sort is currently used that is based on the fluorescence-activated cell sorting hemoglobin (HbF) displayed greater adhesion to FN or LN than did samples
(FACS) technology. Both counting and sorting of worms require fluorescence with a high HbF (8% HbF as the cutoff). RBC adhesion was also significantly
labeling of worms prior to sorting and dispensing, making the process te- greater in samples from HbSS patients with higher LDH levels compared to
dious and time-consuming. In addition, because of the high cost of such those with lower LDH levels (500 IU/L as the cutoff). Qualitative analyses
automatic sorting systems, most of the labs still use a simple volume-based revealed deformable and non-deformable HbSS-containing RBCs. Deform-
dispensing method with a varying number of worms depending on the able RBCs adhere less to FN when exposed to higher shear stresses, and
concentration of worm suspension. To accurately quantify and compare the the most tightly adherent cells were non-deformable RBCs. At highest shear
effect of drugs between individual wells, there is a need for simple and inex- stress (50 dyne/cm2), 75±17% of adhered HbSS RBCs were non-deformable
pensive dispensing unit to count and dispense a specific number of worms, (n=9). Overall, there was a significant association between adhered non-de-
starting from a single to multiple worms into each microwell accurately and formable RBCs (%) and serum LDH levels (n=21, Pearson correlation coeffi-
rapidly. cient of 0.79, p<0.005).

Due to the comparable size scale, there has been a great deal of efforts in Longitudinal analyses performed on individual subjects ~ 1 month apart
the microfluidic community to find ways of manipulating C. elegans worms showed stable RBC adhesion to FN or LN being treated for SCD with trans-
using microfluidic systems. As evidenced in the diverse applications of fusions or hydroxyurea (stable levels of HbA or HbF). In two patients moni-
microfluidic systems ranging from laser surgery to phenotypical analysis, tored over 4 months, adhesion to FN or LN dropped >>60% from baseline
microfluidic systems have been proven useful for manipulation of C. elegans after episodes of transfusion (elevated levels of HbA) or initiation of hydroxy-
worms. However, no microfluidic system has yet been demonstrated for urea (elevated levels of HbF).
direct dispensing of C. elegans worms into microwell plates. In this paper,
we demonstrate a microfluidic system in PDMS, poly(dimethylsiloxane), for The SCD Biochip evaluates, simply and with small sample volumes, complex
label-free detecting, trapping and dispensing of a single C. elegans worm adhesion properties, which reflect clinical phenotypes, including hemoglo-
into a microplate. It consisted of two PDMS layers, a flow and a control layer bin composition, hemolysis, and treatment status. Applied serially and under
underneath. Using five microfluidic pneumatic valves, a single worm was varied clinical scenarios, this adaptable POC technology will yield a more
trapped upon an optical detection with a pair of optical fibers integrated precise characterization of abnormal adhesive events in a given individual
perpendicular to the constriction channel and then dispensed into each mi- and a more accurate assessment of response to therapy overall.
crowell using a robotic handling system. To integrate a standard optical fiber
of 125 um in diameter to a channel height of 37 um, we applied a two-step
PDMS molding process. On the launching side, the optical fiber was con- 3-4
nected to a laser light source with a wavelength of 660 nm. On the detec- TISSUE ENGINEERING
tion side, the optical fiber was connected to a photodiode and coupled to a
LabVIEW program through an Arduino board. Up on a signal received from Navarro 4:00pm - 5:40pm
the photodiode, the pneumatic valves were actuated to trap and dispense
a single worm into a microwell with a constant amount of M9 buffer solution. Session Organizer: Laxman Saggere, Univ Of Illinois/Chicago, Chi-
Several parameters were significant in improving the dispensing yield. The cago, IL, United States
most critical ones were the flow rate and the concentration of worms in the
suspension. At a concentration of 20-25 worms/10 uL and a flow rate of 10
uL/min, we could dispense a single worm into each microwell rapidly and 4:00pm Matrix Composition and Biophysical Characteristics
with high accuracy. Due to its simple design and facile fabrication, we ex- Coordinately Influence Liver Progenitor Differentiation
pect that this microfluidic detection and trapping chip can be expanded to
a multiplexed dispensation system of C. elegans worms for high-throughput Technical Presentation. NEMB2016-5999 35
drug screening purposes.