Micro-Electrical Impedance Spectroscopy and Identification of Patient-Derived, Dissociated Tumor Cells.

Fine needle aspirate sampling of tumors requires acquisition of sufficient cells to complete a diagnosis. Aspirates through such fine needles are typically composed of small cell clusters in suspension, making them readily amenable to microfluidic analysis. Here we show a microfluidic device with integrated electrodes capable of interrogating and identifying cellular components in a patient-derived sample of dissociated tumor cells using micro-electrical impedance spectroscopy ( µ EIS). We show that the µ EIS system can distinguish dissociated tumor cells in a sample consisting of red blood cell (RBCs) and peripheral blood mononucleated cells (PBMCs). Our µ EIS system can also distinguish dissociated tumor cells from normal cells and we show results for five major cancer types, specifically, lung, thyroid, breast, ovarian, and kidney cancer. Moreover, our µ EIS system can make these distinctions in a label-free manner, thereby opening the possibility of integration into standard clinical workflows at the point of care.

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.

Inertio-elastic focusing of bioparticles in microchannels at high throughput.

Controlled manipulation of particles from very large volumes of fluid at high throughput is critical for many biomedical, environmental and industrial applications. One promising approach is to use microfluidic technologies that rely on fluid inertia or elasticity to drive lateral migration of particles to stable equilibrium positions in a microchannel. Here, we report on a hydrodynamic approach that enables deterministic focusing of beads, mammalian cells and anisotropic hydrogel particles in a microchannel at extremely high flow rates. We show that on addition of micromolar concentrations of hyaluronic acid, the resulting fluid viscoelasticity can be used to control the focal position of particles at Reynolds numbers up to Re≈10,000 with corresponding flow rates and particle velocities up to 50 ml min(-1) and 130 m s(-1). This study explores a previously unattained regime of inertio-elastic fluid flow and demonstrates bioparticle focusing at flow rates that are the highest yet achieved.

Cellular bias on the microscale: probing the effects of digital microfluidic actuation on mammalian cell health, fitness and phenotype.

The potential benefits of using new technologies such as microfluidics for life science applications are exciting, but it is critical to understand and document potential biases imposed by these technologies on the observed results. Here, we report the first study of genome-level effects on cells manipulated by digital microfluidics. These effects were evaluated using a broad suite of tools: cell-based stress sensors for heat shock activation, single-cell COMET assays to probe changes in DNA integrity, and DNA microarrays and qPCR to evaluate changes in genetic expression. The results lead to two key observations. First, most DMF operating conditions tested, including those that are commonly used in the literature, result in negligible cell-stress or genome-level effects. Second, for DMF devices operated at high driving frequency (18 kHz) and with large driving electrodes (10 mm × 10 mm), there are significant damage to DNA integrity and differential genomic regulation. We hypothesize that these effects are caused by droplet heating. We recommend that for DMF applications involving mammalian cells that driving frequencies be kept low (≤ 10 kHz) and electrode sizes be kept small (≤ 5 mm) to avoid detrimental effects.

Mitochondrial localization and the persistent migration of epithelial cancer cells.

During cancer cell invasion, faster moving cancer cells play a dominant role by invading further and metastasizing earlier. Despite the importance of these outlier cells, the source of heterogeneity in their migratory behavior remains poorly understood. Here, we show that anterior localization of mitochondria, in between the nucleus and the leading edge of migrating epithelial cancer cells, correlates with faster migration velocities and increased directional persistence. The asymmetry of mitochondrial localization along the axis of migration is absent during spontaneous cell migration on two-dimensional surfaces and only occurs in the presence of chemical attractant cues or in conditions of mechanical confinement. Moreover, perturbing the asymmetric distribution of mitochondria within migrating cells by interfering with mitochondrial fusion (opa-1) or fission (drp-1) proteins, significantly reduces the number of cells with anterior localization of mitochondria and significantly decreases the velocity and directional persistence of the fastest moving cells. We also observed similar changes after perturbing the linkage between mitochondria and microtubules by the knockdown of mitochondrial rhoGTPase-1 (miro-1). Taken together, the changes in migration velocity and directional persistence in cells with anterior-localized mitochondria could account for an order of magnitude differences in invasive abilities between cells from otherwise homogenous cell populations.

Cell-based sensors for quantifying the physiological impact of microsystems.

Microsystems are increasingly used in the manipulation, patterning and sorting of cells. Critical to the widespread adoption of these new technologies is development of an understanding of their impact on cellular physiology. Here we show the integration of a cell-based sensor, a microfabricated electrical screening platform, and quantitative imaging to enable the first large-scale physiological screens of the impact of microsystems on cells. To perform physiological screening, we developed a cell-based sensor that reports on stress-mediated transcription (via Heat Shock Factor 1 induced expression of GFP). This cell-based sensor was quantitatively characterized using automated imaging. The integration of this quantitative physiological sensor with a microfabricated system enabled the execution of multiplexed screens across electric field strength, frequency, and application duration. Voltage sweeps indicate increasing physiological stress with increasing voltage due to Joule heating, while frequency sweeps indicate increased stress at lower frequencies (<500 kHz) compared with higher frequencies (>1 MHz) due to generation of reactive species at lower frequencies. Combined voltage and frequency sweeps enable the generation of complex maps of physiological state.

Electroactive hydrodynamic weirs for microparticle manipulation and patterning.

We present a platform for parallelized manipulations of individual polarizable micron-scale particles (i.e., microparticles) that combines negative dielectrophoretic forcing with the passive capture of hydrodynamic weir-based trapping. Our work enables manipulations using ejection- andor exclusion-based methods. In ejection operations, we unload targeted weirs by displacing microparticles from their capture faces via electrode activation. In exclusion-based operations, we prevent weir loading by activating selected on-chip electrodes before introducing microparticles into the system. Our work describes the device's passive loading dynamics and demonstrates enhanced functionalities by forming a variety of particle patterns.

Plastic masters-rigid templates for soft lithography.

We demonstrate a simple process for the fabrication of rigid plastic master molds for soft lithography directly from (poly)dimethysiloxane devices. Plastics masters (PMs) provide a cost-effective alternative to silicon-based masters and can be easily replicated without the need for cleanroom facilities. We have successfully demonstrated the use of plastics micromolding to generate both single and dual-layer plastic structures, and have characterized the fidelity of the molding process. Using the PM fabrication technique, world-to-chip connections can be integrated directly into the master enabling devices with robust, well-aligned fluidic ports directly after molding. PMs provide an easy technique for the fabrication of microfluidic devices and a simple route for the scaling-up of fabrication of robust masters for soft lithography.

Electrically addressable vesicles: tools for dielectrophoresis metrology.

Dielectrophoresis (DEP) has emerged as an important tool for the manipulation of bioparticles ranging from the submicron to the tens of microns in size. Here we show the use of phospholipid vesicle electroformation techniques to develop a new class of test particles with specifically engineered electrical propserties to enable identifiable dielectrophoretic responses in microfabricated systems. These electrically addressable vesicles (EAVs) enable the creation of electrically distinct populations of test particles for DEP. EAVs offer control of both their inner aqueous core and outer membrane properties; by encapsulating solutions of different electrolyte strength inside the vesicle and by incorporating functionalized phospholipids containing poly(ethylene glycol) (PEG) brushes attached to their hydrophilic headgroup in the vesicle membrane, we demonstrate control of the vesicles' electrical polarizabilities. This combined with the ability to encode information about the properties of the vesicle in its fluorescence signature forms the first steps toward the development of EAV populations as metrology tools for any DEP-based microsystem.

A photopatternable silicone for biological applications.

We show the application of a commercially available photopatternable silicone (PPS) that combines the advantageous features of both PDMS and SU-8 to address a critical bioMEMS materials deficiency. Using PPS, we demonstrate the ability to pattern free-standing mechanically isolated elastomeric structures on a silicon substrate: a feat that is challenging to accomplish using soft lithography-based fabrication. PPS readily integrates with many cell-based bioMEMS since it exhibits low autofluorescence and cells easily attach and proliferate on PPS-coated substrates. Because of its inherent photopatternable properties, PPS is compatible with standard microfabrication processes and easily aligns to complex featured substrates on a wafer scale. By leveraging PPS' unique properties, we demonstrate the design of a simple dielectrophoresis-based bioMEMS device for patterning mammalian cells. The key material properties and integration capabilities explored in this work should present new avenues for exploring silicone microstructures for the design and implementation of increasingly complex bioMEMS architectures.