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International audience; Electropulsation increases membrane permeability for ions and molecules, including DNA or low-permeant cytotoxic drugs. This mechanism is exploited in Electrochemotherapy (ETC), a treatment that consists in exposing cutaneous or subcutaneous tumors to electric pulses in order to enhance delivery of chemotherapeutic agents such as bleomycin or cisplatin into cancer cells. ECT is currently disseminated in more than 130 cancer centers in Europe and notably applied to the treatment of cutaneous metastases and of tumors located in complex anatomic sites such as head and neck. However, the full development of this therapeutic approach requires proper understanding of electric field impact on biological tissues. While in vitro studies performed on isolated cells provide useful information on how field pulses induce biological membrane permeabilization, more realistic 3D in vitro models are required to mimic the behavior of cells in a tumor or a tissue. 3D models can be used as a convenient approach for optimization of electropulsation protocols, offering a partial alternative to in vivo studies performed on sacrified animals.We present different microfluidic platforms allowing dielectrophoresisassisted formation of cell aggregates of regular size and shape. When specific experimental conditions are met (medium composition, electric field intensity and frequency), negative dielectrophoresis (DEP) 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, DEP offers precise control of cell spatial organization, which can be adjusted by optimizing electrode design. Then, rapid establishment of cell-cell interactions is allowed thanks to dipoledipole attraction forces between neighboring cells. Lastly, the electrodes used for DEP can also be subsequently used to perform cell aggregate electroporation or characterization by electrorotation or impedance spectroscopy.Three different electrode configurations will be presented. The first one is composed of a planar quadrupolar electrode array fabricated by selectively coating a bipolar electrode array with a thin micropatterned membrane playing the role of an insulating layer. The second aims at further exploiting 3D effects by using thick electrodes made of carbon-doped PDMS. The last one is based on an original approach developed at EPFL, known as “liquid electrodes”. This configuration exploits planar electrodes fabricated on the bottom of deadend chambers placed on each side of a microfluidic channel and acting similarly to thick electrodes embedded in the channel walls.

The design of micro and nanomaterials with controlled properties requires the development of new bottom- up assembly approaches. The work presented in this manuscript aims to control the formation of aggregates of colloidal particles and cells, with potential applications in the fields of biosensors, microelectronics, optics and tissue engineering. The proposed approach for assembling colloids into organized structures is based on the use of dielectrophoresis, a phenomenon observed when polarizable particles are placed in a non-uniform electric field. One of the drawbacks of this technique is the reversibility of the assemblies thus formed, the cancellation of the electric field causing the redispersion of the colloids. As a solution to this problem, we proposed to use a coupling agent to maintain the cohesion of the structures formed in the liquid medium. In order to better control the dielectrophoretic behavior and the chemical reactions exploited, electrical and chemical characterizations of the manipulated particles were carried out. Moreover, different types of microelectrodes have been studied for the generation of the electric field required for the assembly. A new approach has been developed for the fabrication of transparent micro patterned ITO microelectrodes and their integration in microfluidic systems, based on the exploitation of a thin micro-structured PDMS membrane used as an insulating layer. The method has been applied to the fabrication of "vertical" microelectrodes and of quadrupolar electrode arrays. The latter were used to obtain permanent assemblies of carboxylic acid functionalized, fluorescent, polystyrene particles, by combining negative dielectrophoresis with the use of a chemical coupling agent (Jeffamine). HEK 293 cell aggregates were also produced by negative dielectrophoresis. We have demonstrated that it was possible, under certain conditions, to preserve the permanent character of the cell aggregates after field removal., La réalisation de micro et nanomatériaux avec des propriétés contrôlées nécessite le développement de nouvelles voies d’élaboration dites ascendantes « ou Bottom-up ». Les travaux présentés dans ce manuscrit visent à maîtriser la formation d’agrégats de particules colloïdales et de cellules, avec des applications potentielles dans les domaines des biocapteurs, de la microélectronique, de l’optique et de l’ingénierie tissulaire. L’approche proposée pour assembler les particules en structures organisées se base sur l’emploi de la diélectrophorèse, reposant sur l’application d’un champ électrique non- uniforme. L’un des inconvénients de cette technique tient au caractère réversible des assemblages ainsi formés, l’annulation du champ électrique entrainant la redispersion des colloïdes. Afin d’apporter une solution à ce problème, il est possible de recourir à un couplage chimique pour maintenir la cohésion des structures formées dans le milieu liquide. Afin de mieux maîtriser le comportement diélectrophorétique et les réactions chimiques exploitées, des travaux de caractérisation électrique et chimique des particules manipulées ont été réalisés. D’autre part, différents types de microélectrodes ont été étudiées pour la génération du champ électrique nécessaire à l’assemblage. Une nouvelle filière technologique a été développée pour la réalisation de microélectrodes transparentes en ITO et leur intégration en système microfluidique, basée sur l’exploitation du pouvoir isolant d’une fine couche de PDMS micro-structurée. La méthode a été appliquée à la fabrication de microélectrodes « verticales » puis à la réalisation de matrices d’électrodes quadripolaires. Ces dernières ont permis d’obtenir des assemblages permanents de particules de polystyrène fluorescentes présentant des groupements carboxyliques en surface, en combinant l’emploi de la diélectrophorèse négative et l’utilisation d’un agent de couplage chimique (Jeffamine). Des agrégats de cellules HEK 293 ont également été réalisés par diélectrophorèse négative. Nous avons démontré qu’il était possible, sous certaines conditions, de préserver le caractère permanent des agrégats cellulaires après coupure du champ.