Lens-free microscopy for 3D + time acquisitions of 3D cell culture

Abstract Thanks to a novel three-dimensional imaging platform based on lens-free microscopy, it is possible to perform multi-angle acquisitions and holographic reconstructions of 3D cell cultures directly into the incubator. Being able of reconstructing volumes as large as ~5 mm3 over a period of time covering several days, allows us to observe a broad range of migration strategies only present in 3D environment, whether it is single cell migration, collective migrations of cells and dispersal of cells. In addition we are able to distinguish new interesting phenomena, e.g. large-scale cell-to-matrix interactions (>1 mm), fusion of cell clusters into large aggregate (~10,000 µm2) and conversely, total dissociation of cell clusters into clumps of migrating cells. This work on a novel 3D + time lens-free microscopy technique thus expands the repertoire of phenomena that can be studied within 3D cell cultures

A 3D Toolbox to Enhance Physiological Relevance of Human Tisssue models

International audienceWe 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. A new comprehensive 3D toolbox for cell biology has emerged to address some of these issues. Advances in microfabrication and cell-culturing approaches enable the engineering of sophisticated models that mimic organ 3D architectures and physiological conditions, while supporting flow-based drug screening and secretomics-based diagnosis.TrendsMicrofluidics and microfabrication have revolutionized the way in which cells can be studied and manipulated in systems that are starting to provide 3D models and organ-on-chip devices.Individual-organ models and multiple-organ interaction models address the issue of how microengineered approaches can faithfully reproduce key elements of physiologically relevant microenvironments.The innovative technical nature of such 3D systems opens up exciting possibilities of answering several important fundamental biological questions that cannot be addressed with standard culture conditions.In the race to closely mimic the structural and physiological functions of human tissues and organs, new possibilities have emerged in the form of 3D organ-level structures that integrate dynamic mechanical cues as well as chemical signals

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 microchannels were then used as scaffolds for 3D-confined epithelial cell culture. To show that our device can be used with several epithelial cell types from exocrine glandular tissues, we performed our biological studies on adherent epithelial prostate cells (non-malignant RWPE-1 and invasive PC3) and also on breast (non-malignant MCF10A) cells We observed that in static conditions cells adhere and proliferate to form a confluent layer in channels of 150 µm in diameter and larger, whereas cellular viability decreases with decreasing diameter of the channel. Matrigel and PSS (poly (sodium 4-styrenesulphonate)) promote cell adhesion, whereas the cell proliferation rate was reduced on the PAH (poly (allylamine hydrochloride))-terminated surface. Moreover infusing channels with a continuous flow did not induce any cellular detachment. Our system is designed to simply grow cells in a microchannel structure and could be easily fabricated in any biological laboratory. It offers opportunities to grow epithelial cells that support the formation of a light. This system could be eventually used, for example, to collect cellular secretions, or study cell responses to graduated hypoxia conditions, to chemicals (drugs, siRNA, …) and/or physiological shear stress

Time-lapse contact microscopy of cell cultures based on non-coherent illumination

International audienceVideo microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell

Epithelial cellular organization and proliferation inside fabricated microchannels.

<p><b>A.</b> Schematic representation of an epithelial cell monolayer covering the inner wall of the circular channels fabricated in polydimethylo-siloxane (PDMS). <b>B.</b> Cross-section of a microchannel formed in PDMS and coated with Matrigel. Cells infused into channels adhere to 3D-confined scaffolds and form a confluent layer (Actin in red, nuclei in blue). Bar 50 µm. <b>C.</b> Organization of MCF10A cells forming a confluent monolayer after 5 days of growth inside a Matrigel-coated tube (diameter 150 µm). Phase contrast images. Note that the focus of each image is at different plane (top, middle and bottom of the tube as indicated in A.). Scale bar = 100 µm. <b>D.</b> Apotome Z-stack of PC3 cells on PSS substrate stained with Hoechst (top panel) and Phalloidin (lower panel). Separate-channel images are shown and three positions of the tubes (as indicated in A) are viewed.</p

Visualization 3: Comparative study of fully three-dimensional reconstruction algorithms for lens-free microscopy

Visualization_3_RWPE1_3D_view Originally published in Applied Optics on 01 May 2017 (ao-56-13-3939

Microchannels fabrication process.

<p>Scheme of the device (<b>a</b>); Microphotograph of the device. One Euro coin added as a scale (<b>b</b>); Phase contrast pictures of cross section of channels fabricated in PDMS and agarose (<b>c</b>); Steps of fabrication (<b>d</b>) and (<b>e</b>). More detailed information provided in Figure S1. Scale bar 50 µm.</p

PC3 cell viability in microchannels.

<p>Calcein AM/propidium iodide test on PC3 cells in tubes of 105 µm (right) and 240 µm (left) coated with polyelectrolytes (PAH or PSS) and Matrigel. Note the limited adhesion and proliferation of cells in channels coated with PAH and non-coated (CTRL). In 105 µm channels we observe increased cellular death and the same effect of the type of coating. Scale bar 50 µm.</p