Cultivation and Imaging of S. latissima Embryo Monolayered Cell Sheets Inside Microfluidic Devices.

The culturing and investigation of individual marine specimens in lab environments is crucial to further our understanding of this highly complex ecosystem. However, the obtained results and their relevance are often limited by a lack of suitable experimental setups enabling controlled specimen growth in a natural environment while allowing for precise monitoring and in-depth observations. In this work, we explore the viability of a microfluidic device for the investigation of the growth of the alga Saccharina latissima to enable high-resolution imaging by confining the samples, which usually grow in 3D, to a single 2D plane. We evaluate the specimen's health based on various factors such as its growth rate, cell shape, and major developmental steps with regard to the device's operating parameters and flow conditions before demonstrating its compatibility with state-of-the-art microscopy imaging technologies such as the skeletonisation of the specimen through calcofluor white-based vital staining of its cell contours as well as the immunolocalisation of the specimen's cell wall. Furthermore, by making use of the on-chip characterisation capabilities, we investigate the influence of altered environmental illuminations on the embryonic development using blue and red light. Finally, live tracking of fluorescent microspheres deposited on the surface of the embryo permits the quantitative characterisation of growth at various locations of the organism.

Artificially decreasing cortical tension generates aneuploidy in mouse oocytes.

Human and mouse oocytes' developmental potential can be predicted by their mechanical properties. Their development into blastocysts requires a specific stiffness window. In this study, we combine live-cell and computational imaging, laser ablation, and biophysical measurements to investigate how deregulation of cortex tension in the oocyte contributes to early developmental failure. We focus on extra-soft cells, the most common defect in a natural population. Using two independent tools to artificially decrease cortical tension, we show that chromosome alignment is impaired in extra-soft mouse oocytes, despite normal spindle morphogenesis and dynamics, inducing aneuploidy. The main cause is a cytoplasmic increase in myosin-II activity that could sterically hinder chromosome capture. We describe here an original mode of generation of aneuploidies that could be very common in oocytes and could contribute to the high aneuploidy rate observed during female meiosis, a leading cause of infertility and congenital disorders.

Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration.

The migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function.

Myosin II Activity Is Selectively Needed for Migration in Highly Confined Microenvironments in Mature Dendritic Cells.

Upon infection, mature dendritic cells (mDCs) migrate from peripheral tissue to lymph nodes (LNs) to activate T lymphocytes and initiate the adaptive immune response. This fast and tightly regulated process is tuned by different microenvironmental factors, such as the physical properties of the tissue. Mechanistically, mDCs migration mostly relies on acto-myosin flow and contractility that depend on non-muscular Myosin IIA (MyoII) activity. However, the specific contribution of this molecular motor for mDCs navigation in complex microenvironments has yet to be fully established. Here, we identified a specific role of MyoII activity in the regulation of mDCs migration in highly confined microenvironments. Using microfluidic systems, we observed that during mDCs chemotaxis in 3D collagen gels under defined CCL21 gradients, MyoII activity was required to sustain their fast speed but not to orientate them toward the chemokine. Indeed, despite the fact that mDCs speed declined, these cells still migrated through the 3D gels, indicating that this molecular motor has a discrete function during their motility in this irregular microenvironment. Consistently, using microchannels of different sizes, we found that MyoII activity was essential to maintain fast cell speed specifically under strong confinement. Analysis of cell motility through micrometric holes further demonstrated that cell contractility facilitated mDCs passage only over very small gaps. Altogether, this work highlights that high contractility acts as an adaptation mechanism exhibited by mDCs to optimize their motility in restricted landscapes. Hence, MyoII activity ultimately facilitates their navigation in highly confined areas of structurally irregular tissues, contributing to the fine-tuning of their homing to LNs to initiate adaptive immune responses.

Size control in mammalian cells involves modulation of both growth rate and cell cycle duration.

Despite decades of research, how mammalian cell size is controlled remains unclear because of the difficulty of directly measuring growth at the single-cell level. Here we report direct measurements of single-cell volumes over entire cell cycles on various mammalian cell lines and primary human cells. We find that, in a majority of cell types, the volume added across the cell cycle shows little or no correlation to cell birth size, a homeostatic behavior called "adder". This behavior involves modulation of G1 or S-G2 duration and modulation of growth rate. The precise combination of these mechanisms depends on the cell type and the growth condition. We have developed a mathematical framework to compare size homeostasis in datasets ranging from bacteria to mammalian cells. This reveals that a near-adder behavior is the most common type of size control and highlights the importance of growth rate modulation to size control in mammalian cells.

Leukocyte Migration and Deformation in Collagen Gels and Microfabricated Constrictions.

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.

Monodisperse colloids synthesized with nanofluidic technology.

Limitations in the methods employed to generate micrometric colloidal droplets hinder the emergence of key applications in the fields of material science and drug delivery. Through the use of dedicated nanofluidic devices and by taking advantage of an original physical effect called capillary focusing, we could circumvent some of these limitations. The nanofluidic (i.e., submicrometric) devices introduced herein are made of soft materials, and their fabrication relies upon rapid technologies. The objects that we have generated are simple droplets, multiple droplets, particles, and Janus particles whose sizes lie between 900 nm and 3 microm (i.e., within the colloidal range). Colloidal droplets have been assembled on-chip into clusters and crystals, yielding discrete diffraction patterns. We illustrate potential applications in the field of drug delivery by demonstrating the ability of multiple droplets to be phagocytosed by murine macrophage-type cells.

Soft microflow sensors.

We present a rapid prototyping method for integrating functional components in conventional PDMS microfluidic devices. We take advantage of stop-flow lithography (D. Dendukuri, S. S. Gu, D. C. Pregibon, T. A. Hatton and P. S. Doyle, Lab Chip, 2007, 7, 818)(1) to achieve the in situ fabrication of mobile and deformable elements with a controlled mechanical response. This strategy is applied to the fabrication of microflow sensors based on a deformable spring-like structure. We show that these sensors have a large dynamic range, typically 3 to 4 orders of magnitude, and that they can be scaled down to measure flows in the nl per min range. We prepared sensors with different geometries, and their flow-elongation characteristics were modeled with a simple hydrodynamic model, with good agreement between model and experiments.

Surface treatment and characterization: perspectives to electrophoresis and lab-on-chips.

The control and modification of surface state is a major challenge in bioanalytical sciences, and in particular in electrokinetic separation methods, due to the importance of electroosmosis. This topic has gained recently a renewed interest, associated with the development of "lab-on-chips" systems that extend the range of materials in which separation channels are fabricated. The surface science community has developed through the years a large toolbox of characterization tools and surface modification protocols, which is not yet fully exploited in the bioanalytical world. In this paper, we try and present an overview of these tools, in order to stimulate new ideas for improved and more controlled surface treatment strategies for separations in capillaries and microchannels. We briefly describe some physical and chemical aspects of electroosmosis (global and spatially resolved), streaming current, and streaming potential. We also review the main strategies for surface coating, and compare the advantages of physisorption, well-organized thin self-assembled monolayers, or conversely thick polymer "brushes". Examples of existing applications to electrophoresis in microchannel are also given.