A microfluidic biochip for the nanoporation of living cells

International audienceThis paper deals with the development of a microfluidic biochip for the exposure of living cells to nanosecond pulsed electric fields (nsPEF). When exposed to ultra short electric pulses (typical duration of 3-10ns), disturbances on the plasma membrane and on the intra cellular components occur, modifying the behavioral response of cells exposed to drugs or transgene vectors. This phenomenon permits to envision promising therapies. The presented biochip is composed of thick gold electrodes that are designed to deliver a maximum of energy to the biological medium containing cells. The temporal and spectral distributions of the nsPEF are considered for the design of the chip. In order to validate the fabricated biochip ability to orient the pulse towards the cells flowing within the exposition channels, a frequency analysis is provided. High voltage measurements in the time domain are performed to characterize the amplitude and the shape of the nsPEF within the exposition channels and compared to numerical simulations achieved with a 3D Finite-Difference Time-Domain code. We demonstrate that the biochip is adapted for 3 ns and 10 ns pulses and that the nsPEF are homogenously applied to the biological cells regardless their position along the microfluidic channel. Furthermore, biological tests performed on the developed microfluidic biochip permit to prove its capability to permeabilize living cells with nanopulses. To the best of our knowledge, we report here the first successful use of a microfluidic device optimized for the achievement and real time observation of the nanoporation of living cells

Increasing dietary oat fibre decreases the permeability of intestinal mucus

This study investigates the influence of the dietary fibre β-glucan on nutrient composition and mucus permeability. Pigs were fed a standard diet or a diet containing twice the β-glucan content for 3 days (n = 5 per group), followed by the collection of small intestinal mucus and tissue samples. Samples of the consumed diets were subjected to in vitro digestion to determine β-glucan release, nutrient profile and assessment of mucus permeability. In vitro digestion of the diets indicated that 90% of the β-glucan was released in the proximal small intestine. Measurements of intestinal mucus showed a reduction in permeability to 100 nm latex beads and also lipid from the digested enhanced β-glucan diet. The data from this study show for the first time that reducing mass transfer of bile and lipid through the intestinal mucus layer may be one way in which this decrease in bile reabsorption by soluble fibre is enabled

Food: More than the sum of its parts

The food we eat and enjoy is not just a collection of macro and micro nutrients, it has structure and texture that affect our response to it. This short review of recent research touches on many of the issues linking different types of food structure to the digestion and absorption of macronutrients. The behavior of both protein and lipid in the gastric compartment can be key to what gets emptied when and thus the kinetics of nutrient absorption, which can in turn influence risk factors for disease. Because of this there is increasing emphasis on understanding the role of gastric processing in digestion kinetics. The use of MRI has significantly improved our understanding of gastric behavior and offers new possibilities for controlling digestion kinetics. In addition, the role of fiber in the upper GI tract is also starting to be better understood. This will also lead to further opportunities to improve the health of the Western diet

Aqueous Two‐Phase System Patterning of Microbubbles: Localized Induction of Apoptosis in Sonoporated Cells

Ultrasound‐driven microbubbles produce mechanical forces that can disrupt cell membranes (sonoporation). However, it is difficult to control microbubble location with respect to cells. This lack of control leads to low sonoporation efficiencies and variable outcomes. In this study, aqueous two‐phase system (ATPS) droplets are used to localize microbubbles in select micro‐regions at the surface of living cells. This is achieved by stably partitioning microbubbles in dextran (DEX) droplets, deposited on living adherent cells in medium containing polyethylene glycol (PEG). The interfacial energy at the PEG‐DEX interface overcomes microbubble buoyancy and prevents microbubbles from floating away from the cells. Spreading of the small DEX droplets retains microbubbles at the cell surface in defined lateral positions without the need for antibody or cell‐binding ligand conjugation. The patterned microbubbles are activated on a cell monolayer exposed to a broadly applied ultrasound field (center frequency 1.25 MHz, active element diameter 0.6 cm, pulse duration 8 μs or 30 s). This system enables efficient testing of different ultrasound conditions for their effects on sonoporation‐mediated membrane disruption and cell viability. Regions of cells without patterned microbubbles show no injury or membrane disruption. In microbubble patterned regions, 8 μs ultrasound pulses (0.2‐0.6 MPa) produce cell death that is primarily apoptotic. Ultrasound‐induced apoptosis increases with higher extracellular calcium concentrations, with cells displaying all of the hallmarks of apoptosis including annexinV labeling, loss of mitochondrial membrane potential, caspase activation and changes in nuclear morphology. A new method is described for patterning microbubbles on cell monolayers to target ultrasound treatment to cells. This novel platform provides a controlled system for high throughput testing of the effects of ultrasound‐mediated cell membrane disruption on cell physiology. Using this patterning method, it is possible to induce apoptosis in select populations of cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/99004/1/3420_ftp.pd

INFOGEST static in vitro simulation of gastrointestinal food digestion

peer-reviewedSupplementary information is available at http://dx.doi.org/10.1038/s41596-018-0119-1 or https://www.nature.com/articles/s41596-018-0119-1#Sec45.Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.COST action FA1005 INFOGEST (http://www.cost-infogest.eu/ ) is acknowledged for providing funding for travel, meetings and conferences (2011-2015). The French National Institute for Agricultural Research (INRA, www.inra.fr) is acknowledged for their continuous support of the INFOGEST network by organising and co-funding the International Conference on Food Digestion and workgroup meeting

Impact of in vitro gastrointestinal digestion on peptide profile and bioactivity of cooked and non-cooked oat protein concentrates

Oat (Avena sativa) is one of the most cultivated and consumed cereals worldwide. Recognized among cereals for its high protein content (12% to 24%), it makes it an excellent source of bioactive peptides, which could be modified during processes such as heating and gastrointestinal digestion (GID). This work aims to evaluate the impact of heat treatment on the proteolysis of oat proteins and on the evolution of antioxidant peptide release during in vitro static GID, in terms of comparative analysis between cooked oat protein concentrate (COPC) and non-heated oat protein concentrate (OPC) samples. The protein extraction method and cooking procedure used showed no detrimental effects on protein quality. After GID, the proportion of free amino acids/dipeptides (40% for both samples (OPC and COPC), thus producing peptides with low molecular weight and enhanced bioactivity. Furthermore, during GID, the amino acid profile showed an increase in essential, positively-charged, hydrophobic and aromatic amino acids. At the end of GID, the reducing power of OPC and COPC increased >0.3 and 8-fold, respectively, in comparison to the non-digested samples; while ABTS•+ and DPPH• showed a >20-fold increase. Fe2+ chelating capacity of OPC and COPC was enhanced >4 times; similarly, Cu2+ chelation showed a >19-fold enhancement for OPC and >10 for COPC. β-carotene bleaching activity was improved 0.8 times in OPC and >9 times in COPC; the oxygen radical antioxidant capacity assay increased 2 times in OPC and >4.7 times in COPC, respectively. This study suggests that OPC after cooking and GID positively influenced the nutritional and bioactive properties of oat peptides. Thus, COPC could be used as a functional food ingredient with health-promoting effects, as hydrothermal treatment is frequently used for this type of cereals

Étude des effets d'impulsions électriques ultra-courtes sur des cellules vivantes : mise au point de nouveaux outils, développement théorique et premières applications in vivo

Electropermeabilization is a safe and routinely used method to transfer genes or drugs in cells or tissues. Recent studies on intra-cellular effects of nanosecond pulsed electric fields opened new ways for cell manipulation. But before thinking to clinical applications, the development of new tools is necessary. In this context, we developed new exposition devices to ultra-short pulses and we showed potential therapeutic applications, especially for cancer treatment. A new concept based on insulated electrodes was proposed to avoid any electrochemical contamination or burn of cells or tissues. The implementation of such method for in vitro and in vivo experiments permitted to increase the efficiency of luciferase production after conventionnal electrogenetransfer. Finally, the combination of bleomycin with nanosecond pulses gave complete tumor regressions. A biochip for the real-time observation of exposed cells under a microscope was also realized and included as the end element of the pulse transmission line. All the results presented in this thesis showed that it is possible to consider the use of such ultra-short pulsed electric fields as a new therapeutic tool for cancer treatment and gene therapy.La perméabilisation cellulaire par l'intermédiaire de champs électriques est une des méthodes les plus sures pour le transfert de gènes et de molécules d'intérêt. La découverte des effets intra-cellulaires d'un nouveau type d'impulsions électriques - les nanopulses - a ouvert de nouvelles perspectives à l'application des impulsions électriques au vivant. La mise au point de nouveaux outils est indispensable avant de pouvoir appliquer ces techniques à l'homme. Aussi, notre but a été de mettre au point des systèmes d'exposition de cellules à ces impulsions et de montrer que de nouvelles applications thérapeutiques étaient possibles, en particulier en cancérologie. Une nouvelle méthode d'exposition de cellules ou de tissus basée sur l'utilisation d'électrodes isolées a été proposée permettant d'éviter tout risque de contamination ou brûlure électrochimique. A l'aide de cette nouvelle méthode, des expériences in vitro puis in vivo ont pu être menées et confirment la possibilité d'augmenter d'un facteur 3 à 6 la production de luciférase après un électrotransfert de ce gène. Enfin, l'exposition de tumeurs à ces impulsions en présence de bléomycine a permis d'obtenir des régressions tumorales complètes. Nous avons aussi conçu un biomicrosystème permettant d'étudier les effets de telles impulsions à l'échelle cellulaire sous microscope s'intégrant comme terminaison adaptée dans la chaîne d'exposition. L'ensemble des résultats présentés dans cette thèse montre qu'il est possible d'envisager l'utilisation de ces impulsions comme nouvel outil thérapeutique dans le traitement de cancers ou en thérapie génique

Rational formulation of cookies with satiety benefits : physicochemical, nutritional and sensory approaches

Des biscuits enrichis en protéines et/ou en fibres, contenant peu de matières grasses et de sucres ont été formulés, en optimisant à la fois les paramètres de composition et notamment la teneur en eau de la pâte ; mais également les paramètres du procédé, comme ceux du pétrissage, du façonnage par mouleuse rotative et de la cuisson. Des outils d'aide à la formulation ont été développés afin d'optimiser cette phase. Il s'agit d'un outil sensoriel prédictif de la machinabilité des pâtes moulées et d'un outil mathématique de prédiction de l'hydratation des pâtes biscuitières en fonction de leur composition en protéines et/ou en fibres. La première étape de prototypage, réalisée à l'échelle du laboratoire, a aboutit à la formulation d'environ 250-300 recettes. Les prototypes ont ensuite subit trois sélections multicritères successives (250-300 -> 40 -> 14 -> 4 recettes), avec une fabrication des produits réalisée à l'échelle pilote. Le transfert d'échelle, laboratoire vers pilote, a pu être optimisé en identifiant de façon précise et détaillée les variables de contrôle et les variables d'état du diagramme de production des biscuits. L'étape de prototypage a permis de sélectionner les candidats (protéines et fibres) pertinents pour l'enrichissement. Parmi les 250-300 recettes initiales, 40 recettes ont été sélectionnées sur des critères de composition et de machinabilité et ont été caractérisées d'un point de vue sensoriel (réalisé par Kraft). Parmi ces 40 recettes, 14 recettes ont été sélectionnées pour être représentatives de l'espace sensoriel engendré par les 40 recettes. Sur ces 14 recettes, des études de rassasiement et de satiété, ont été réalisées par d'autres acteurs du projet (tâche 2 et tâche3), pour aboutir à la sélection finale de 4 recettes avec une recette contrôle, une recette enrichie en protéines, une recette enrichie en fibres et une recette enrichie en protéines + fibres. Des digestions in vitro ont été réalisées sur ces 4 maquettes, à l'aide d'un outil sophistiqué et dynamique le TIM-1, pour suivre l'évolution de la viscosité, et de l'hydrolyse des protéines et de l'amidon, le long du tractus gastro-intestinal. Les caractérisations instrumentales réalisées sur les produits sélectionnés aux différentes étapes de la sélection ont permis de montrer l'importance de l'effet de l'hydratation de la pâte et/ou de l'enrichissement en protéines et/ou en fibres sur i) le dimensionnel des pâtons et des biscuits, ii) sur les propriétés rhéologiques à la rupture des biscuits et iii) sur l'état de l'amidon après cuisson et iv) sur l'apport de viscosité et la dégradation des composants durant la digestion. Ces caractérisations ont également permis de s'assurer de la bonne reproductibilité lors des différentes phases de production industrielle. Les résultats obtenus lors des digestions in vitro seront comparés avec ceux de l'étude in vivo.Cookies enriched with protein and/or fiber, and containing little fat and sugar, were formulated by optimizing both compositional parameters, in particular the water content of the dough, and process parameters such as kneading, shaping by rotary molding machine and baking. Formulation tools have been developed to optimize this phase. These include a sensory tool for predicting the machinability of molded doughs, and a mathematical tool for predicting the hydration of cookie doughs as a function of their protein and/or fiber composition. The first prototyping stage, carried out on a laboratory scale, resulted in the formulation of around 250-300 recipes. The prototypes then underwent three successive multi-criteria selections (250-300 -> 40 -> 14 -> 4 recipes), with product manufacture carried out at pilot scale. The transfer of scale from laboratory to pilot was optimized by precisely identifying the control and state variables in the cookie production diagram. The prototyping stage enabled the selection of relevant candidates (proteins and fibers) for enrichment. From the initial 250-300 recipes, 40 were selected on the basis of composition and machinability criteria, and were characterized from a sensory point of view (carried out by Kraft). From these 40 recipes, 14 were selected to be representative of the sensory space generated by the 40 recipes. On these 14 recipes, satiation and satiety studies were carried out by other project participants (task 2 and task 3), leading to the final selection of 4 recipes: a control recipe, a protein-enriched recipe, a fiber-enriched recipe and a protein + fiber-enriched recipe. In vitro digestions were carried out on these 4 models, using a sophisticated and dynamic tool, TIM-1, to follow the evolution of viscosity and hydrolysis of proteins and starch along the gastrointestinal tract. The instrumental characterizations carried out on selected products at different stages of the selection process demonstrated the importance of the effect of dough hydration and/or protein and/or fiber enrichment on i) the dimensional properties of dough pieces and cookies, ii) the rheological properties at cookie breakage, iii) the state of starch after baking, and iv) the contribution to viscosity and component degradation during digestion. These characterizations also ensured good reproducibility during the different phases of industrial production. The results of the in vitro digestions will be compared with those of the in vivo study

勘驗協助義務

International audienceProTIS (Procédés de Traitement de l'Information et du Signal) est un module d'enseignement en auto-apprentissage, par le biais de tutoriels en ligne, à l'attention d'étudiant·e·s de L3 et M1 en école d'ingénieurs. Les objectifs principaux de ce module sont l'appropriation d'une démarche d'ingénierie et l'acquisition de compétences techniques dans les domaines de l'électronique pour le traitement de l'information et de l'informatique embarquée. Les élèves ont pour objectif technique de concevoir et de réaliser un dispositif électronique de leur choix dans son intégralité, par équipe de 4. Chaque équipe sélectionne les fonctionnalités dont elle a besoin dans une liste préétablie et organise la collaboration entre ces membres (collaboration technique mais également liée au bon déroulement du projet). Chaque élève établit ensuite son plan de formation à la mise en œuvre d'une partie de ces fonctionnalités.Les modalités particulières de cet enseignement permettent à chaque élève de progresser à son rythme à l'aide d'un support pédagogique original. Chaque étape est guidée par l'équipe enseignante et une évaluation détaillée des compétences acquises est mise en place. Cette approche du travail par projet a pour ambition, outre l'amélioration de l'expérience pédagogique pour les apprenant·e·s et pour l'équipe enseignante, la construction de compétences solides en vue de la mise en œuvre de projets plus complets dans des domaines connexes