Thomas Gervais

Microfluidic quadrupole and floating concentration gradient

The concept of fluidic multipoles, in analogy to electrostatics, has long been known as a particular class of solutions of the Navier-Stokes equation in potential flows, however, experimental observations of fluidic multipoles and of their characteristics have not been reported yet. Here we present a two-dimensional microfluidic quadrupole and a theoretical analysis consistent with the experimental observations. The microfluidic quadrupole was formed by simultaneously injecting and aspirating fluids from two pairs of opposing apertures in a narrow gap formed between a microfluidic probe and a substrate. A stagnation point was formed at the center of the microfluidic quadrupole, and its position could be rapidly adjusted hydrodynamically. Following the injection of a solute through one of the poles, a stationary, tunable, and movable – i.e. “floating” – concentration gradient was formed at the stagnation point. Our results lay the foundation for future combined experimental and theoretical exploration of microfluidic planar multipoles including convective-diffusive phenomena.

Empirical chemosensitivity testing in a spheroid model of ovarian cancer using a microfluidics-based multiplex platform.

The use of biomarkers to infer drug response in patients is being actively pursued, yet significant challenges with this approach, including the complicated interconnection of pathways, have limited its application. Direct empirical testing of tumor sensitivity would arguably provide a more reliable predictive value, although it has garnered little attention largely due to the technical difficulties associated with this approach. We hypothesize that the application of recently developed microtechnologies, coupled to more complex 3-dimensional cell cultures, could provide a model to address some of these issues. As a proof of concept, we developed a microfluidic device where spheroids of the serous epithelial ovarian cancer cell line TOV112D are entrapped and assayed for their chemoresponse to carboplatin and paclitaxel, two therapeutic agents routinely used for the treatment of ovarian cancer. In order to index the chemoresponse, we analyzed the spatiotemporal evolution of the mortality fraction, as judged by vital dyes and confocal microscopy, within spheroids subjected to different drug concentrations and treatment durations inside the microfluidic device. To reflect microenvironment effects, we tested the effect of exogenous extracellular matrix and serum supplementation during spheroid formation on their chemotherapeutic response. Spheroids displayed augmented chemoresistance in comparison to monolayer culturing. This resistance was further increased by the simultaneous presence of both extracellular matrix and high serum concentration during spheroid formation. Following exposure to chemotherapeutics, cell death profiles were not uniform throughout the spheroid. The highest cell death fraction was found at the center of the spheroid and the lowest at the periphery. Collectively, the results demonstrate the validity of the approach, and provide the basis for further investigation of chemotherapeutic responses in ovarian cancer using microfluidics technology. In the future, such microdevices could provide the framework to assay drug sensitivity in a timeframe suitable for clinical decision making.

Two-Aperture Microfluidic Probes as Flow Dipole: Theory and Applications

A microfluidic probe (MFP) is a mobile channel-less microfluidic system under which a fluid is injected from an aperture into an open space, hydrodynamically confined by a surrounding fluid, and entirely re-aspirated into a second aperture. Various MFPs have been developed, and have been used for applications ranging from surface patterning of photoresists to local perfusion of organotypic tissue slices. However, the hydrodynamic and mass transfer properties of the flow under the MFP have not been analyzed, and the flow parameters are adjusted empirically. Here, we present an analytical model describing the key transport properties in MFP operation, including the dimensions of the hydrodynamic flow confinement (HFC) area, diffusion broadening, and shear stress as a function of: (i) probe geometry (ii) aspiration-to-injection flow rate ratio (iii) gap between MFP and substrate and (iv) reagent diffusivity. Analytical results and scaling laws were validated against numerical simulations and experimental results from published data. These results will be useful to guide future MFP design and operation, notably to control the MFP “brush stroke” while preserving shear-sensitive cells and tissues.

Reconfigurable Microfluidic Magnetic Valve Arrays: Towards a Radiotherapy-Compatible Spheroid Culture Platform for the Combinatorial Screening of Cancer Therapies

We introduce here a microfluidic cell culture platform or spheroid culture chamber array (SCCA) that can synthesize, culture, and enable fluorescence imaging of 3D cell aggregates (typically spheroids) directly on-chip while specifying the flow of reagents in each chamber via the use of an array of passive magnetic valves. The SCCA valves demonstrated sufficient resistance to burst (above 100 mBar), including after receiving radiotherapy (RT) doses of up to 8 Gy combined with standard 37 °C incubation for up to 7 days, enabling the simultaneous synthesis of multiple spheroids from different cell lines on the same array. Our results suggest that SCCA would be an asset in drug discovery processes, seeking to identify combinatorial treatments.

Microfluidics and spectroscopic imaging for personalized medicine in ovarian cancer

With certain cancers, such as ovarian cancers, a significant number of patients are non-responsive to the standard chemotherapy treatment. These patients, therefore, experience the negative side effects of chemotherapy without clinical benefits. With the everincreasing number of available anti-cancer drug alternatives to chemotherapy, there is tremendous pressure on clinicians to make the right treatment choice. The biomarker approach—a statistical method that associates drug-response rates with specific patient characteristics—is effective for predicting which patients will respond best to a given treatment. However, only very specific cancer subtypes have associated biomarkers (i.e., the BRCA mutation for breast cancer). There is a crucial need, therefore, for a complementary predictive method that is applicable to virtually all types of cancers. Clusters of cells, known as spheroids, are the most popular 3D tissue model in cancer research. These samples, which have standard diameters of around 400 m, are relatively easy to culture and they represent patient tumors better than traditional 2D cell cultures.1 The spheroids are often formed and cultured in miniaturized fluidic systems—or microfluidic chips—in which biological assays are performed. However, spheroids have been of little use in personalized therapy because they are formed using generic cell lines that do not reproduce the specificities of a patient’s tumor. A promising alternative would be to directly test therapies on small amounts of cancer tissue from patients, but this approach has had limited success in the past because of challenges associated with culturing patient tissue outside the human body and with developing detection methods to measure drug response in 3D tissue. Figure 1. Personalized approach for the selection of an optimal anticancer treatment. Small amounts of tissue from a patient are sectioned into spheroid-sized samples. These individual samples are then introduced into a microsystem, in which different treatment options can be tested and their effects measured using various detection systems. Inset shows a top-view image of a sample trapped inside a well. The sample is labeled with fluorescent probes marking cells that are viable (green) and dead (red), and is imaged using confocal fluorescence microscopy. The results of the test may help medical specialists choose the most effective treatment for each patient.

Spectroscopic imaging system for high-throughput viability assessment of ovarian spheroids or microdissected tumor tissues (MDTs) in a microfluidic chip

There is a growing effort in the biomicrosystems community to develop a personalized treatment response assay for cancer patients using primary cells, patient-derived spheroids, or live tissues on-chip. Recently, our group has developed a technique to cut tumors in 350 μm diameter microtissues and keep them alive on-chip, enabling multiplexed in vitro drug assays on primary tumor tissue. Two-photon microscopy, confocal microscopy and flow cytometry are the current standard to assay tissue chemosensitivity on-chip. While these techniques provide microscopic and molecular information, they are not adapted for high-throughput analysis of microtissues. We present a spectroscopic imaging system that allows rapid quantitative measurements of multiple fluorescent viability markers simultaneously by using a liquid crystal tunable filter to record fluorescence and transmittance spectra. As a proof of concept, 24 spheroids composed of ovarian cancer cell line OV90 were formed in a microfluidic chip, stained with two live cell markers (CellTrackerTM Green and Orange), and imaged. Fluorescence images acquired were normalized to the acquisition time and gain of the camera, dark noise was removed, spectral calibration was applied, and spatial uniformity was corrected. Spectral un-mixing was applied to separate each fluorophores contribution. We have demonstrated that rapid and simultaneous viability measurements on multiple spheroids can be achieved, which will have a significant impact on the prediction of a tumor’s response to multiple treatment options. This technique may be applied as well in drug discovery to assess the potential of a drug candidate directly on human primary tissue.

Systematic analysis of microfluidic probe design and operation

Microfluidic probes are an emerging tool used in a wide range of applications including surface biopatterning, immunohistology, and cell migration studies. They control flow above a surface by simultaneously injecting and aspirating fluids from a pen-like structure positioned a few tens of microns above a surface. Rather than confining flows inside microchannels they rely on recirculating flow patterns between the probe tip and the substrate to create a hydrodynamic flow confinement (HFC) zone in which reagents can be locally delivered to the surface. In this paper, we provide a theoretical model, supported by numerical simulations and experimental data, describing the extent of the HFC as a function of the two most important probe operation parameters, the ratio of aspiration to injection flow rate, and the distance between probe apertures. Two types of probes are studied: two-aperture microfluidic probes (MFPs) and microfluidic quadrupoles (MQs). In both cases, the model yields very accurate results and suggests a simple underlying theory based on 2D potential flows to understand probe operation. We further highlight how the model can be used to precisely control the probes “brush stroke” while in surface patterning mode. The understanding of probe operation made possible through the provided analytical model should lay the bases for computer-controlled probe calibration and operation.