Integrated platform for culture, observation, and parallelized electroporation of spheroids.

Reversible electroporation is a method to introduce molecules into cells by increasing the permeability of their membranes, thanks to the application of pulsed electric fields. One of its main biomedical applications is electro-chemotherapy, where electroporation is used to deliver anticancer drugs into tumor tissues. To improve our understanding of the electroporation effect on tissues and select efficient treatments, in vitro tumor models are needed. Cell spheroids are relevant models as they can reproduce tumor microenvironment and cell-cell interactions better than 2D cell cultures. Various methods offering a relatively simple workflow are now available for their production. However, electroporation protocols usually require handling steps that may damage spheroids and result in random spacing, inducing variations in electric field distribution around spheroids and non-reproducible electroporation conditions. In addition, only a few microsystems allow the production and electroporation of spheroids, and the spheroids produced lack reproducibility in size and location. To overcome these issues, we developed a unique device enabling culture, monitoring, and electroporation of hundreds of regular spheroids in parallel, with a design ensuring that all spheroids are submitted to the same electric field conditions. It is comprised of a microfluidic chamber encompassing a micro-structured agarose gel, allowing easy medium exchange while avoiding spheroid handling. It also enables optical imaging of spheroids in situ, thanks to transparent electrodes. In this paper, we describe the fabrication and characterization of the developed microsystem and demonstrate its applicability to electroporation of a network of spheroids. We present a first successful application as an anticancer drug testing platform, by evaluating the bleomycin effect on HT29 colorectal cancer cell spheroids. This work opens new perspectives in the development of in vitro assays for the preclinical evaluation of electroporation-based treatment.

Eukaryotic Cell Capture by Amplified Magnetic in situ Hybridization Using Yeast as a Model.

A non-destructive approach based on magnetic in situ hybridization (MISH) and hybridization chain reaction (HCR) for the specific capture of eukaryotic cells has been developed. As a prerequisite, a HCR-MISH procedure initially used for tracking bacterial cells was here adapted for the first time to target eukaryotic cells using a universal eukaryotic probe, Euk-516R. Following labeling with superparamagnetic nanoparticles, cells from the model eukaryotic microorganism Saccharomyces cerevisiae were hybridized and isolated on a micro-magnet array. In addition, the eukaryotic cells were successfully targeted in an artificial mixture comprising bacterial cells, thus providing evidence that HCR-MISH is a promising technology to use for specific microeukaryote capture in complex microbial communities allowing their further morphological characterization. This new study opens great opportunities in ecological sciences, thus allowing the detection of specific cells in more complex cellular mixtures in the near future.

Dielectrophoretic cell trapping for improved surface plasmon resonance imaging sensing.

The performance of conventional surface plasmon resonance (SPR) biosensors can be limited by the diffusion of the target analyte to the sensor surface. This work presents an SPR biosensor that incorporates an active mass-transport mechanism based on dielectrophoresis and electroosmotic flow to enhance analyte transport to the sensor surface and reduce the time required for detection. Both these phenomena rely on the generation of AC electric fields that can be tailored by shaping the electrodes that also serve as the SPR sensing areas. Numerical simulations of electric field distribution and microparticle trajectories were performed to choose an optimal electrode design. The proposed design improves on previous work combining SPR with DEP by using face-to-face electrodes, rather than a planar interdigitated design. Two different top-bottom electrode designs were experimentally tested to concentrate firstly latex beads and secondly biological cells onto the SPR sensing area. SPR measurements were then performed by varying the target concentrations. The electrohydrodynamic flow enabled efficient concentration of small objects (3 µm beads, yeasts) onto the SPR sensing area, which resulted in an order of magnitude increased SPR response. Negative dielectrophoresis was also used to concentrate HEK293 cells onto the metal electrodes surrounded by insulating areas, where the SPR response was improved by one order of magnitude.

Performance improvement of plasmonic sensors using a combination of AC electrokinetic effects for (bio)target capture.

Analytes concentration techniques are being developed with the appealing expectation to boost the performance of biosensors. One promising method lies in the use of electrokinetic forces. We present hereafter a new design for a microstructured plasmonic sensor which is obtained by conventional microfabrication techniques, and which can easily be adapted on a classical surface plasmon resonance imaging (SPRI) system without further significant modification. Dielectrophoretic trapping and electro-osmotic displacement of the targets in the scanned fluid are performed through interdigitated 200 µm wide gold electrodes that also act as the SPR-sensing substrate. We demonstrate the efficiency of our device's collection capabilities for objects of different sizes (200 nm and 1 µm PS beads, as well as 5-10 µm yeast cells). SPRI is relevant for the spatial analysis of the mass accumulation at the electrode surface. We demonstrate that our device overcomes the diffusion limit encountered in classical SPR sensors thanks to rapid collection capabilities (<1 min) and we show a consequent improvement of the detection limit, by a factor >300. This study of an original device combining SPRI and electrokinetic forces paves the way to the development of fully integrated active plasmonic sensors with direct applications in life sciences, electrochemistry, environmental monitoring and agri-food industry.

Dielectrophoresis-assisted creation of cell aggregates under flow conditions using planar electrodes.

We present a microfluidic platform allowing dielectrophoresis-assisted formation of cell aggregates of controlled size and composition under flow conditions. When specific experimental conditions are met, negative dielectrophoresis allows efficient concentration of cells towards electric field minima and subsequent aggregation. This bottom-up assembly strategy offers several advantages with respect to the targeted application: first, dielectrophoresis offers precise control of spatial cell organization, which can be adjusted by optimizing electrode design. Then, it could contribute to accelerate the establishment of cell-cell interactions by favoring close contact between neighboring cells. The trapping geometry of our chip is composed of eight electrodes arranged in a circle. Several parameters have been tested in simulations to find the best configurations for trapping in flow. Those configurations have been tested experimentally with both polystyrene beads and human embryonic kidney cells. The final design and experimental setup have been optimized to trap cells and release the created aggregates on demand.

MyDEP: A New Computational Tool for Dielectric Modeling of Particles and Cells.

Dielectrophoresis (DEP) and electrorotation (ROT) are two electrokinetic phenomena exploiting nonuniform electric fields to exert a force or torque on biological particles suspended in liquid media. They are widely used in lab-on-chip devices for the manipulation, trapping, separation, and characterization of cells, microorganisms, and other particles. The DEP force and ROT torque depend on the respective polarizabilities of the particle and medium, which in turn depend on their dielectric properties and on the field frequency. In this work, we present a new software, MyDEP, which implements several particle models based on concentric shells with adjustable dielectric properties. This tool enables the study of the variation in DEP and ROT spectra according to different parameters, such as the field frequency and medium conductivity. Such predictions of particle behavior are very useful for choosing appropriate parameters in DEP experiments. The software also enables the study of the homogenized properties of spherical or ellipsoidal multishell particles and provides a database containing published cell properties. Equivalent electrical conductivity and relative permittivity of the cell alone and in suspension can be calculated. The software also offers the ability to create graphs of the evolution of the crossover frequencies with the electric field frequency. These graphs can be directly exported from the software.

Magnetic nanoparticle DNA labeling for individual bacterial cell detection and recovery.

A culture independent approach was developed for recovering individual bacterial cells out of communities from complex environments including soils and sediments where autofluorescent contaminants hinder the use of fluorescence based techniques. For that purpose fifty nanometer sized streptavidin-coated superparamagnetic nanoparticles were used to chemically bond biotin-functionalized plasmid DNA molecules. We show that micromagnets can efficiently trap magnetically labeled transformed Escherichia coli cells after these bacteria were subjected to electro-transformation by these nanoparticle-labeled plasmids. Among other applications, this method could extend the range of approaches developed to study DNA dissemination among environmental bacteria without requiring cultivability of recombinant strains or expression of heterologous genes in the new hosts.

From bipolar to quadrupolar electrode structures: an application of bond-detach lithography for dielectrophoretic particle assembly.

We describe a new, simple process for fabricating transparent quadrupolar electrode arrays enabling large-scale particle assembly by means of dielectrophoresis. In the first step, interdigitated electrode arrays are made by chemical wet etching of indium tin oxide (ITO). Then, the transition from a bipolar to a quadrupolar electrode arrangement is obtained by covering the electrode surface with a thin poly(dimethylsiloxane) (PDMS) film acting as an electrical insulation layer in which selective openings are formed using bond-detach lithography. The PDMS insulating layer thickness was optimized and controlled by adjusting experimental parameters such as the PDMS viscosity (modulated by the addition of heptane) and the PDMS spin-coating velocity. The insulating character of the PDMS membrane was successfully demonstrated by performing a dielectrophoretic assembly of polystyrene particles using interdigitated electrodes with and without a PDMS layer. The results show that the patterned PDMS film functions properly as an electrical insulation layer and allows the reconfiguration of the electric field cartography. Electric field simulations were performed in both configurations to predict the dielectrophoretic behavior of the particles. The simulation results are in perfect agreement with experiments, in which we demonstrated the formation of concentrated clusters of polystyrene particles and living cells of regular size and shape.

Improvements in the extraction of cell electric properties from their electrorotation spectrum.

In this paper, we propose a new approach to perform cell dielectric characterization from their electrorotation spectrum. At first, a variance analysis is carried out to quantify the dispersion in electrorotation spectra due to the different parameters involved. On this basis, the impact of each parameter is emphasized by weighing the spectrum with an appropriate frequency-dependent coefficient: this technique enables to minimize the coupling effects which deteriorate the accuracy of parameter extraction. In addition, the Nelder-Mead simplex algorithm used in the identification procedure is modified to account for bounded intervals in which the unknown parameters are expected to vary. Both these techniques have proven to give increased confidence levels compared to previous work reported in the literature.