Publications

Many patients develop coagulation abnormalities due to chronic and hereditary disorders, infectious disease, blood loss, extracorporeal circulation, and oral anticoagulant misuse. These abnormalities lead to bleeding or thrombotic complications, the risk of which is assessed by coagulation analysis. Current coagulation tests pose safety concerns for neonates and small children due to large sample volume requirement and may be unreliable for patients with coagulopathy. This study introduces a containerless drop-of-blood method for coagulation analysis, termed "integrated quasi-static acoustic tweezing thromboelastometry" (i-QATT™), that addresses these needs. In i-QATT™, a single drop of blood is forced to levitate and deform by the acoustic radiation force. Coagulation-induced changes in drop turbidity and firmness are measured simultaneously at different instants. The parameters describing early, intermediate, and late stages of the coagulation process are evaluated from the resulting graphical outputs. i-QATT™ rapidly (<10 min) detected hyper- and hypo-coagulable states and identified single deficiency in coagulation factors VII, VIII, IX, X, and XIII. The linear relationship (r² > 0.9) was established between fibrinogen concentration and two i-QATT™ parameters: maximum clot firmness and maximum fibrin level. Factor XIII activity was uniquely measured by the fibrin network formation time (r² = 0.9). Reaction time, fibrin formation rate, and time to firm clot formation were linearly correlated with heparin concentration (r² > 0.7). tPA-induced hyperfibrinolysis was detected in the clot firmness output at 10 min. i-QATT™ provides comprehensive coagulation analysis in point-of-care or laboratory settings, well suited to the needs of neonatal and pediatric patients and adult patients with anemia or blood collection issues.

Although vertebrates are indispensable to biomedical research, studies are often limited by factors such as cost, lengthy internal review, and ethical considerations. We present the earthworm as an alternative, low-cost, invertebrate applicable to certain preliminary vasculature studies. Due to the surgical availability of the earthworm's dorsal vessels, ventral vessels, and five pairs of pseudo hearts, earthworms are readily accessible, offer low-cost maintenance, and require administration of only small doses of a given compound. The earthworm model provides a simple closed vascular circulatory system with a hemoglobin structure similar to human blood. A protocol is provided for anaesthetizing the earthworms and performing surgical incisions to expose relevant blood vessels. Micropipettes for compound administration are formed by heating and pulling glass with a pipette puller and using a beveling system to create a micron-scale fine needle tip. The tips are then used with a micropositioner and microinjector to inject arbitrary compounds into the vascular system of an earthworm, repeatably, with the availability of large sample sizes and small compound volumes. Details on the intricacies of injection procedure are provided. The small vessel size of the earthworm is challenging, particularly in the case of the ventral vessel; however, mastery of the techniques presented offers high repeatability as a low-cost solution, making studies of very large sample size practical.

One of the promising approaches for high-throughput screening of cell mechanotype is microfluidic deformability cytometry (mDC), in which the apparent deformation index (DI) of the cells stretched by extensional flow at the stagnation point of a cross-slot microchannel is measured. The DI is subject to substantial measurement errors due to cell offset from the flow centerline and velocity fluctuations in inlet channels, leading to artificial widening of DI versus cell size plots. Here, we simulated an mDC experiment using a custom computational algorithm for viscoelastic cell migration. Cell motion and deformation in a cross-slot channel was modeled for fixed or randomized values of cellular mechanical properties (diameter, shear elasticity, cortical tension) and initial cell placement, with or without sinusoidal fluctuations between the inlet velocities. Our numerical simulation indicates that mDC loses sensitivity to changes in shear elasticity when the offset distance exceeds 5 μm, and just 1% velocity fluctuation causes an 11.7% drop in the DI. The obtained relationships between the cell diameter, shear elasticity, and offset distance were used to establish a new measure of cell deformation, referred to as the "elongation index" (EI). In the randomized study, the EI scatter plots were visibly separated for the low- and high-elasticity populations of cells, with a mean of 300 and 3500 Pa, whereas the standard DI output was unable to distinguish between these two groups of cells. The successful suppression of the offset artifacts with a narrower data distribution was shown for the EI output of MCF-7 cells.

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis worldwide. Currently, no curative treatment is available for early-to-intermediate stage HCC, characterized by large and/or multifocal tumors. If left untreated, HCC rapidly progresses to a lethal stage due to favorable conditions for metastatic spread. Mechanochemical disruption of cellular structures can potentially induce phenotypic alterations in surviving tumor cells that prevent HCC progression. In this paper, HCC response to mechanical vibration via high-intensity focused ultrasound and a chemical disruptive agent (ethanol) was examined in vitro and in vivo. Our analysis revealed that mechanochemical disruption caused a significant overproduction of reactive oxygen species (ROS) in multiple HCC cell lines (HepG2, PLC/PRF/5, and Hep3B). This led to a decrease in cell viability and long-term proliferation due to increased expression and activity of death receptors TNFR1 and Fas. The cells that survived mechanochemical disruption had a reduced expression of cancer stem cell markers (CD133, CD90, CD49f) and a diminished colony-forming ability. Mechanochemical disruption also impeded HCC migration and their adhesion to vascular endothelium, two critical processes in hematogenous metastasis. The HCC transformation to a non-tumorigenic phenotype post mechanochemical disruption was confirmed by a lack of tumor spheroid formation in vitro and complete tumor regression in vivo. These results show that mechanochemical disruption inhibits uncontrolled proliferation and reduces tumorigenicity and aggressiveness of HCC cells through ROS overproduction and associated activation of TNF- and Fas-mediated cell death signaling. Our study identifies a novel curative therapeutic approach that can prevent the development of aggressive HCC phenotypes.

Background: Thromboelastography is widely used as a tool to assess the coagulation status of critical-care patients. It allows observation of changes in the material properties of whole blood brought about by clot formation and clot lysis. However, contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks, while also requiring large sample volumes.

Objectives: To develop a non-contact drop oscillation rheometry (DOR) method to measure the viscoelastic properties of blood clots and to compare the results with current laboratory standard measurements.

Methods: Drops of human blood and plasma (5-10 μL) were acoustically levitated. Acoustic field modulation induced drop shape oscillations, and the viscoelastic properties of the sample were calculated by measuring the resonance frequency and damping ratio.

Results: DOR showed sensitivity to coagulation parameters. An increase in platelet count resulted in an increase in the maximum clot stiffness. An increase in the calcium ion level enhanced the coagulation rate prior to saturation. An increase in hematocrit resulted in a higher rate of clot formation and increased clot stiffness. Comparison of the results with those obtained with thromboelastography showed that coagulation started sooner with DOR, but with a lower rate and lower maximum stiffness.

Conclusions: DOR can be used as a monitoring tool to assess blood coagulation status. The advantages of small sample size, the lack of contact and small strain (linear viscoelasticity) makes this technique unique for real-time monitoring of blood coagulation.

Keywords: acoustics; blood coagulation tests; rheology; thromboelastography; viscoelastic drop.

The majority of microfluidic technologies for cell sorting and isolation involve bifurcating (e.g., Y- or T-shaped junction) microchannels to trap the cells of a specific type. However, the microfluidic trapping efficiency remains low, independently of whether the cells are separated by a passive or an active sorting method. Using a custom computational algorithm, we studied the migration of separated deformable cells in a Y-junction microchannel, with a bifurcation angle ranging from 30° to 180°. Single or two cells of initially spherical shape were considered under flow conditions corresponding to inertial microfluidics. Through the numerical simulation, we identified the effects of cell size, cytoplasmic viscoelasticity, cortical tension, flow rate, and bifurcation angle on the critical separation distance for cell trapping. The results of this study show that the trapping and isolation of blood cells, and circulating tumor cells in a Y-junction microchannel was most efficient and least dependent on the flow rate at the bifurcation angle of 120°. At this angle, the trapping efficiency for white blood cells and circulating tumor cells increased, respectively, by 46% and 43%, in comparison with the trapping efficiency at 60°. The efficiency to isolate invasive tumor cells from noninvasive ones increased by 32%. This numerical study provides important design criteria to optimize microfluidic technology for deformability-based cell sorting and isolation.

Chemical-based medicine that targets specific oncogenes or proteins often leads to cancer recurrence due to tumor heterogeneity and development of chemoresistance. This challenge can be overcome by mechanochemical disruption of cancer cells via focused ultrasound (FUS) and sensitizing chemical agents such as ethanol. We demonstrate that this disruptive therapy decreases the viability, proliferation rate, tumorigenicity, endothelial adhesion, and migratory ability of prostate cancer cells in vitro. It sensitized the cells to TNFR1-- and Fas--mediated apoptosis and reduced the expression of metastatic markers CD44 and CD29. Using a prostate cancer xenograft model, we observed that the mechanochemical disruption led to complete tumor regression in vivo. This switch to a nonaggressive cell phenotype was caused by ROS and Hsp70 overproduction and subsequent impairment of NFκB signaling. FUS induces mechanical perturbations of diverse cancer cell populations, and its combination with agents that amplify and guide remedial cellular responses can stop lethal cancer progression. IMPLICATIONS: Mechanochemical disruption therapy in which FUS is combined with ethanol can be curative for locally aggressive and castration-resistant prostate cancer.

Advances in methods that determine cell mechanical phenotype, or mechanotype, have demonstrated the utility of biophysical markers in clinical and research applications ranging from cancer diagnosis to stem cell enrichment. Here, we introduce quantitative deformability cytometry (q-DC), a method for rapid, calibrated, single-cell mechanotyping. We track changes in cell shape as cells deform into microfluidic constrictions, and we calibrate the mechanical stresses using gel beads. We observe that time-dependent strain follows power-law rheology, enabling single-cell measurements of apparent elastic modulus, E_a, and power-law exponent, β. To validate our method, we mechanotype human promyelocytic leukemia (HL-60) cells and thereby confirm q-DC measurements of E_a = 0.53 ± 0.04 kPa. We also demonstrate that q-DC is sensitive to pharmacological perturbations of the cytoskeleton as well as differences in the mechanotype of human breast cancer cell lines (E_a = 2.1 ± 0.1 and 0.80 ± 0.19 kPa for MCF-7 and MDA-MB-231 cells). To establish an operational framework for q-DC, we investigate the effects of applied stress and cell/pore-size ratio on mechanotype measurements. We show that E_a increases with applied stress, which is consistent with stress stiffening behavior of cells. We also find that E_a increases for larger cell/pore-size ratios, even when the same applied stress is maintained; these results indicate strain stiffening and/or dependence of mechanotype on deformation depth. Taken together, the calibrated measurements enabled by q-DC should advance applications of cell mechanotype in basic research and clinical settings.

This study reports, for the first time, development of tyrosine kinase inhibitor-loaded, thermosensitive liposomes (TKI/TSLs) and their efficacy for treatment of renal cell carcinoma when triggered by focused ultrasound (FUS). Uptake of these nanoparticles into renal cancer cells was visualized with confocal and fluorescent imaging of rhodamine B-loaded liposomes. The combination of TKI/TSLs and focused ultrasound was tested in an in vitro tumor model of renal cell carcinoma. According to MTT cytotoxic assay and flow cytometric analysis the combined treatment led to the least viability (23.4 ± 2.49%, p<0.001), significantly lower than that observed from treatment with FUS (97.6 ± 0.67%, n.s.) or TKI/TSL (71.0 ± 3.65%, p < 0.001) at 96 hours compared to control. The importance of this unique, synergistic combination was demonstrated in viability experiments with non-thermosensitive liposomes (TKI/NTSL+FUS: 58.8 ± 1.5% vs TKI/TSL+FUS: 36.2 ± 1.4%, p<0.001) and heated water immersion (TKI/TSL+WB43°: 59.3 ± 2.91% vs TKI/TSL+FUS: 36.4 ± 1.55%, p < 0.001). Our findings coupled with the existing use of focused ultrasound in clinical practice make the proposed combination of targeted chemotherapy, nanotechnology, and focused ultrasound a promising platform for enhanced drug delivery and cancer treatment.

Background: Thromboelastography is widely used as a tool to assess the coagulation status of surgical patients. It allows observation of changes in material properties of whole blood, beginning with early stages of clot formation and ending with clot lysis. However, the contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks.
Objectives: To develop acoustic tweezing thromboelastometry as a noncontact method for perioperative assessment of whole blood coagulation.
Methods: Acoustic tweezing is used to levitate microliter drops of biopolymer and whole blood samples. By quasi-statically changing the acoustic pressure we control the sample drop location and deformation. Sample size, deformation and location are determined by digital imaging at each pressure.
Results: Simple Newtonian liquid solutions maintain a constant, reversible location vs deformation curve. In contrast, the deformation/location curves for gelatin, alginate and whole blood uniquely change as the samples solidify. Increasing elasticity causes the sample to deform less, leading to steeper stress/strain curves. By extracting a linear regime slope, we show that whole blood exhibits a unique slope profile as it begins to clot. By exposing blood samples to pro- or anti-coagulants, the slope profile changes, allowing detection of hyper- or hypo-coagulable states.