Séverine Le Gac

Tissue deformation spatially modulates VEGF signaling and angiogenesis

Physical forces play a major role in the organization of developing tissues. During vascular development, physical forces originating from a fluid phase or from cells pulling on their environment can alter cellular signaling and the behavior of cells. Here, we observe how tissue deformation spatially modulates angiogenic signals and angiogenesis. Using soft lithographic templates, we assemble three-dimensional, geometric tissues. The tissues contract autonomously, change shape stereotypically and form patterns of vascular structures in regions of high deformations. We show that this emergence correlates with the formation of a long-range gradient of Vascular Endothelial Growth Factor (VEGF) in interstitial cells, the local overexpression of the corresponding receptor VEGF receptor 2 (VEGFR-2) and local differences in endothelial cells proliferation. We suggest that tissue contractility and deformation can induce the formation of gradients of angiogenic microenvironments which could contribute to the long-range patterning of the vascular system.

ART culture conditions change the probability of mouse embryo gestation through defined cellular and molecular responses

STUDY QUESTION Do different human ART culture protocols prepare embryos differently for post-implantation development? SUMMARY ANSWER The type of ART culture protocol results in distinct cellular and molecular phenotypes in vitro at the blastocyst stage as well as subsequently during in vivo development. WHAT IS KNOWN ALREADY It has been reported that ART culture medium affects human development as measured by gestation rates and birthweights. However, due to individual variation across ART patients, it is not possible as yet to pinpoint a cause-effect relationship between choice of culture medium and developmental outcome. STUDY DESIGN, SIZE, DURATION In a prospective study, 13 human ART culture protocols were compared two at a time against in vivo and in vitro controls. Superovulated mouse oocytes were fertilized in vivo using outbred and inbred mating schemes. Zygotes were cultured in medium or in the oviduct and scored for developmental parameters 96 h later. Blastocysts were either analyzed or transferred into fosters to measure implantation rates and fetal development. In total, 5735 fertilized mouse oocytes, 1732 blastocysts, 605 fetuses and 178 newborns were examined during the course of the study (December 2010-December 2011). PARTICIPANTS/MATERIALS, SETTING, METHODS Mice of the B6C3F1, C57Bl/6 and CD1 strains were used as oocyte donors, sperm donors and recipients for embryo transfer, respectively. In vivo fertilized B6C3F1 oocytes were allowed to cleave in 13 human ART culture protocols compared with mouse oviduct and optimized mouse medium (KSOM(aa)). Cell lineage composition of resultant blastocysts was analyzed by immunostaining and confocal microscopy (trophectoderm, Cdx2; primitive ectoderm, Nanog; primitive endoderm, Sox17), global gene expression by microarray analysis, and rates of development to midgestation and to term. MAIN RESULTS AND THE ROLE OF CHANCE Mouse zygotes show profound variation in blastocyst (49.9-91.9%) and fetal (15.7-62.0%) development rates across the 13 ART culture protocols tested (R(2)= 0.337). Two opposite protocols, human tubal fluid/multiblast (high fetal rate) and ISM1/ISM2 (low fetal rate), were analyzed in depth using outbred and inbred fertilization schemes. Resultant blastocysts show imbalances of cell lineage composition; culture medium-specific deviation of gene expression (38 genes, ≥ 4-fold) compared with the in vivo pattern; and produce different litter sizes (P ≤ 0.0076) after transfer into fosters. Confounding effects of subfertility, life style and genetic heterogeneity are reduced to a minimum in the mouse model compared with ART patients. LIMITATIONS, REASONS FOR CAUTION This is an animal model study. Mouse embryo responses to human ART media are not transferable 1-to-1 to human development due to structural and physiologic differences between oocytes of the two species. WIDER IMPLICATIONS OF THE FINDINGS Our data promote awareness that human ART culture media affect embryo development. Effects reported here in the mouse may apply also in human, because no ART medium presently available on the market has been optimized for human embryo development. The mouse embryo assay (MEA), which requires ART media to support at least 80% blastocyst formation, is in need of reform and should be extended to include post-implantation development.

Progress and future of in vitro models to study translocation of nanoparticles

AbstractThe increasing use of nanoparticles in products likely results in increased exposure of both workers and consumers. Because of their small size, there are concerns that nanoparticles unintentionally cross the barriers of the human body. Several in vivo rodent studies show that, dependent on the exposure route, time, and concentration, and their characteristics, nanoparticles can cross the lung, gut, skin, and placental barrier. This review aims to evaluate the performance of in vitro models that mimic the barriers of the human body, with a focus on the lung, gut, skin, and placental barrier. For these barriers, in vitro models of varying complexity are available, ranging from single-cell-type monolayer to multi-cell (3D) models. Only a few studies are available that allow comparison of the in vitro translocation to in vivo data. This situation could change since the availability of analytical detection techniques is no longer a limiting factor for this comparison. We conclude that to further develop in vitro models to be used in risk assessment, the current strategy to improve the models to more closely mimic the human situation by using co-cultures of different cell types and microfluidic approaches to better control the tissue microenvironments are essential. At the current state of the art, the in vitro models do not yet allow prediction of absolute transfer rates but they do support the definition of relative transfer rates and can thus help to reduce animal testing by setting priorities for subsequent in vivo testing.

A planar on-chip micro-nib interface for NanoESI–MS microfluidic applications

We present a novel nanoelectrospray emitter tip based on the principle of a nib rather than a nozzle for nano-electrospray ionization–mass spectrometry (ESI–MS) applications. The fabrication of the micro-nibs relies on micromachining techniques using the epoxy-based negative photoresist SU-8. A double exposure photolithographic process has been employed to form a nib in a membrane-like structure. The nibs contained a capillary slot measuring 20 µm at the tip end. The nib sources were successfully tested on an ion trap mass spectrometer using standard peptide samples at low concentrations, down to 1 µM. High voltage (HV) supply was achieved using platinum wire inserted in a liquid reservoir. A Taylor cone was clearly seen protruding from the nib tip and was determined by the dimensions of the capillary slot.

Controlled cavitation–cell interaction: trans-membrane transport and viability studies

Cavitation bubble dynamics close to a rigid surface gives rise to a rapid and transient fluid flow. A single bubble is created with a laser pulse at different stand-off distances from the rigid surface, where the stand-off distance gamma is defined by gamma = h/R(max), with h being the initial distance and R(max) being the maximum bubble radius. When the surface is covered with adherent cells, molecular delivery and cell detachment after single cavitation activity are observed at different locations. We find a maximum of cell detachment at a normalized stand-off distance of gamma approximately 0.65. In contrast, the maximum of the molecular uptake is found when gamma approaches 0. The single cavitation event has only little effect on the viability of cells in the non-detached area. We find apoptosis of cells only very close to the area of detachment and, additionally, the metabolism of the non-detached cells shows no pronounced difference compared to control cells according to an MTS assay. Thus, although the cavitation event is responsible for the detachment of cells, only few of the remaining cells undergo a permanent change.

Superstructures of chiral nematic microspheres as all-optical switchable distributors of light

Light technology is based on generating, detecting and controlling the wavelength, polarization and direction of light. Emerging applications range from electronics and telecommunication to health, defence and security. In particular, data transmission and communication technologies are currently asking for increasingly complex and fast devices, and therefore there is a growing interest in materials that can be used to transmit light and also to control the distribution of light in space and time. Here, we design chiral nematic microspheres whose shape enables them to reflect light of different wavelengths and handedness in all directions. Assembled in organized hexagonal superstructures, these microspheres of well-defined sizes communicate optically with high selectivity for the colour and chirality of light. Importantly, when the microspheres are doped with photo-responsive molecular switches, their chiroptical communication can be tuned, both gradually in wavelength and reversibly in polarization. Since the kinetics of the “on” and “off” switching can be adjusted by molecular engineering of the dopants and because the photonic cross-communication is selective with respect to the chirality of the incoming light, these photo-responsive microspheres show potential for chiroptical all-optical distributors and switches, in which wavelength, chirality and direction of the reflected light can be controlled independently and reversibly.

Low-temperature, simple and fast integration technique of microfluidic chips by using a UV-curable adhesive

In the fields of MicroElectroMechanical Systems (MEMS) and Lab On a Chip (LOC), a device is often fabricated using diverse substrates which are processed separately and finally assembled together using a bonding process to yield the final device. Here we describe and demonstrate a novel straightforward, rapid and low-temperature bonding technique for the assembly of complete microfluidic devices, at the chip level, by employing an intermediate layer of gluing material. This technique is applicable to a great variety of materials (e.g., glass, SU-8, parylene, UV-curable adhesive) as demonstrated here when using NOA 81 as gluing material. Bonding is firstly characterized in terms of homogeneity and thickness of the gluing layer. Following this, we verified the resistance of the adhesive layer to various organic solvents, acids, bases and conventional buffers. Finally, the assembled devices are successfully utilized for fluidic experiments.

The influence of different membrane components on the electrical stability of bilayer lipid membranes

A good understanding of cell membrane properties is crucial for better controlled and reproducible experiments, particularly for cell electroporation where the mechanism of pore formation is not fully elucidated. In this article we study the influence on that process of several constituents found in natural membranes using bilayer lipid membranes. This is achieved by measuring the electroporation threshold (V(th)) defined as the potential at which pores appear in the membrane. We start from highly stable 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) membranes (V(th) approximately 200 mV), and subsequently add therein other phospholipids, cholesterol and a channel protein. While the phospholipid composition has a slight effect (100 mV<or=V(th)<or=290 mV), cholesterol gives a concentration-dependent effect: a slight stabilization until 5% weight (V(th) approximately 250 mV) followed by a noticeable destabilization (V(th) approximately 100 mV at 20%). Interestingly, the presence of a model protein, alpha-hemolysin, dramatically disfavours membrane poration and V(th) shows a 4-fold increase ( approximately 800 mV) from a protein density in the membrane of 24x10(-3) proteins/microm(2). In general, we find that pore formation is affected by the molecular organization (packing and ordering) in the membrane and by its thickness. We correlate the resulting changes in molecular interactions to theories on pore formation.

Parallel single-cell analysis microfluidic platform.

We report a PDMS microfluidic platform for parallel single‐cell analysis (PaSCAl) as a powerful tool to decipher the heterogeneity found in cell populations. Cells are trapped individually in dedicated pockets, and thereafter, a number of invasive or non‐invasive analysis schemes are performed. First, we report single‐cell trapping in a fast (2–5 min) and reproducible manner with a single‐cell capture yield of 85% using two cell lines (P3x63Ag8 and MCF‐7), employing a protocol which is scalable and easily amenable to automation. Following this, a mixed population of P3x63Ag8 and MCF‐7cells is stained in situ using the nucleic acid probe (Hoechst) and a phycoerythrin‐labeled monoclonal antibody directed at EpCAM present on the surface of the breast cancer cells MCF‐7 and absent on the myeloma cells P3x63Ag8 to illustrate the potential of the device to analyze cell population heterogeneity. Next, cells are porated in situ using chemicals in a reversible (digitonin) or irreversible way (lithium dodecyl sulfate). This is visualized by the transportation of fluorescent dyes through the membrane (propidium iodide and calcein). Finally, an electrical protocol is developed for combined cell permeabilization and electroosmotic flow (EOF)‐based extraction of the cell content. It is validated here using calcein‐loaded cells and visualized through the progressive recovery of calcein in the side channels, indicating successful retrieval of individual cell content.

Microstamped Petri dishes for scanning electrochemical microscopy analysis of arrays of microtissues.

While scanning electrochemical microscopy (SECM) is a powerful technique for non-invasive analysis of cells, SECM-based assays remain scarce and have been mainly limited so far to single cells, which is mostly due to the absence of suitable platform for experimentation on 3D cellular aggregates or microtissues. Here, we report stamping of a Petri dish with a microwell array for large-scale production of microtissues followed by their in situ analysis using SECM. The platform is realized by hot embossing arrays of microwells (200 μm depth; 400 μm diameter) in commercially available Petri dishes, using a PDMS stamp. Microtissues form spontaneously in the microwells, which is demonstrated here using various cell lines (e.g., HeLa, C2C12, HepG2 and MCF-7). Next, the respiratory activity of live HeLa microtissues is assessed by monitoring the oxygen reduction current in constant height mode and at various distances above the platform surface. Typically, at a 40 μm distance from the microtissue, a 30% decrease in the oxygen reduction current is measured, while above 250 μm, no influence of the presence of the microtissues is detected. After exposure to a model drug (50% ethanol), no such changes in oxygen concentration are found at any height in solution, which reflects that microtissues are not viable anymore. This is furthermore confirmed using conventional live/dead fluorescent stains. This live/dead assay demonstrates the capability of the proposed approach combining SECM and microtissue arrays formed in a stamped Petri dish for conducting cellular assays in a non-invasive way on 3D cellular models.