Spatial and temporal characterization of cytoskeletal reorganizations in adherent platelets.

The functional role of platelets is intricately linked to the dynamic organization of two main components of the cytoskeleton, microtubules and actin fibers. Throughout the phases of platelet activation, spreading, and retraction, both of these essential polymers undergo continuous and orchestrated reorganization. Our investigation of the dynamic cytoskeletal changes during these phases highlights a sequential remodeling of the actin cytoskeleton in adherent platelets from the formation of initial actin nodules through the development of stress fibers and a subsequent return to nodular structures.

Microfluidic system for in vitro epithelial folding and calcium waves induction.

Epithelial folding is a fundamental process where initially flat monolayers transform into functional 3D structures. This protocol details fabrication steps for a polycarbonate microfluidic platform which enables triggering epithelial folds that recapitulate stereotypical cell shape changes and folding-associated mechanical stresses. We describe the steps for cell seeding to form a monolayer on the chip, and subsequent approach to trigger calcium waves in the epithelial monolayer through local epithelial deformation.

Direction of epithelial folding defines impact of mechanical forces on epithelial state.

Cell shape dynamics during development is tightly regulated and coordinated with cell fate determination. Triggered by an interplay between biochemical and mechanical signals, epithelia form complex tissues by undergoing coordinated cell shape changes, but how such spatiotemporal coordination is controlled remains an open question. To dissect biochemical signaling from purely mechanical cues, we developed a microfluidic system that experimentally triggers epithelial folding to recapitulate stereotypic deformations observed in vivo.

Cell fluorescence photoactivation as a method to select and study cellular subpopulations grown in mechanically heterogeneous environments.

A central challenge to the biology of development and disease is deciphering how individual cells process and respond to numerous biochemical and mechanical signals originating from the environment. Recent advances in genomic studies enabled the acquisition of information about population heterogeneity; however, these so far are poorly linked with the spatial heterogeneity of biochemical and mechanical cues. Whereas in vitro models offer superior control over spatiotemporal distribution of numerous mechanical parameters, researchers are limited by the lack of methods to select subpopulations of cells in order to understand how environmental heterogeneity directs the functional collective response.

Extracellular matrix in multicellular aggregates acts as a pressure sensor controlling cell proliferation and motility.

Imposed deformations play an important role in morphogenesis and tissue homeostasis, both in normal and pathological conditions. To perceive mechanical perturbations of different types and magnitudes, tissues need appropriate detectors, with a compliance that matches the perturbation amplitude. By comparing results of selective osmotic compressions of CT26 mouse cells within multicellular aggregates and global aggregate compressions, we show that global compressions have a strong impact on the aggregates growth and internal cell motility, while selective compressions of same magnitude have almost no effect.

Measuring cell displacements in opaque tissues: dynamic light scattering in the multiple scattering regime.

Coherent light scattered by tissues brings structural and dynamic information, at depth, that standard imaging techniques cannot reach. Dynamics of cells or sub-cellular elements can be measured thanks to dynamic light scattering in thin samples (single scattering regime) or thanks to diffusive wave spectroscopy in thick samples (diffusion regime). Here, we address the intermediate regime and provide an analytical relationship between scattered light fluctuations and the distribution of cell displacements as a function of time.

Deciphering Cell Intrinsic Properties: A Key Issue for Robust Organoid Production.

We highlight the disposition of various cell types to self-organize into complex organ-like structures without necessarily the support of any stromal cells, provided they are placed into permissive 3D culture conditions. The goal of generating organoids reproducibly and efficiently has been hampered by poor understanding of the exact nature of the intrinsic cell properties at the origin of organoid generation, and of the signaling pathways governing their differentiation. Using microtechnologies like microfluidics to engineer organoids would create opportunities for single-cell genomics and high-throughput functional genomics to exhaustively characterize cell intrinsic properties.

Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity.

Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis.

A 3D Toolbox to Enhance Physiological Relevance of Human Tissue Models.

We discuss the current challenges and future prospects of flow-based organoid models and 3D self-assembling scaffolds. The existing paradigm of 3D culture suffers from a lack of control over organoid size and shape; can be an obstacle for cell harvesting and extended cellular and molecular analysis; and does not provide access to the function of exocrine glands. Moreover, existing organ-on-chip models are mostly composed of 2D extracellular matrix (ECM)-coated elastomeric membranes that do not mimic real organ architectures.

Effect of an osmotic stress on multicellular aggregates.

There is increasing evidence that multicellular structures respond to mechanical cues, such as the confinement and compression exerted by the surrounding environment. In order to understand the response of tissues to stress, we investigate the effect of an isotropic stress on different biological systems. The stress is generated using the osmotic pressure induced by a biocompatible polymer.

Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development.

3D culture systems are a valuable tool for modeling morphogenesis and carcinogenesis of epithelial tissue in a structurally appropriate context. We present a novel approach for 3D cell culture based on a flow-focusing microfluidic system that encapsulates epithelial cells in Matrigel beads. As a model we use prostatic and breast cells and assay for development of acini, polarized cellular spheres enclosing lumen.

Facile bench-top fabrication of enclosed circular microchannels provides 3D confined structure for growth of prostate epithelial cells.

We present a simple bench-top method to fabricate enclosed circular channels for biological experiments. Fabricating the channels takes less than 2 hours by using glass capillaries of various diameters (from 100 µm up to 400 µm) as a mould in PDMS. The inner surface of microchannels prepared in this way was coated with a thin membrane of either Matrigel or a layer-by-layer polyelectrolyte to control cellular adhesion.

The modulation of attachment, growth and morphology of cancerous prostate cells by polyelectrolyte nanofilms.

The behaviour of cancerous epithelial prostatic cells (PC3) growing on polyelectrolytes (PE) coatings was compared to the behaviour of immortalized normal prostatic cells (PNT-2). The cell behaviour was evaluated and quantified in terms of initial cell attachment, growth, metabolic activity, morphometry, adhesion, apoptosis and stress related gene expression. Both the anionic PSS (poly(sodium 4-styrenesulphonate))-terminated surface and cationic PAH (poly(allylamine hydrochloride))-terminated surfaces were not cytotoxic.

Label-free analysis of prostate acini-like 3D structures by lensfree imaging.

We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view.

Iterative operations on microdroplets and continuous monitoring of processes within them; determination of solubility diagrams of proteins.

We demonstrate a technique for controlling the content of multiple microdroplets in time. We use this system to rapidly and quantiatively determine the solubility diagrams of two model proteins (lysozyme and ribonuclease A).