Multiorgan-on-a-Chip: A Systemic Approach To Model and Decipher Inter-Organ Communication.

Multiorgan-on-a-chip (multi-OoC) platforms have great potential to redefine the way in which human health research is conducted. After briefly reviewing the need for comprehensive multiorgan models with a systemic dimension, we highlight scenarios in which multiorgan models are advantageous. We next overview existing multi-OoC platforms, including integrated body-on-a-chip devices and modular approaches involving interconnected organ-specific modules. We highlight how multi-OoC models can provide unique information that is not accessible using single-OoC models. Finally, we discuss remaining challenges for the realization of multi-OoC platforms and their worldwide adoption. We anticipate that multi-OoC technology will metamorphose research in biology and medicine by providing holistic and personalized models for understanding and treating multisystem diseases.

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

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.

Direct transfection of clonal organoids in Matrigel microbeads: a promising approach toward organoid-based genetic screens.

Organoid cultures in 3D matrices are relevant models to mimic the complex in vivo environment that supports cell physiological and pathological behaviors. For instance, 3D epithelial organoids recapitulate numerous features of glandular tissues including the development of fully differentiated acini that maintain apico-basal polarity with hollow lumen. Effective genetic engineering in organoids would bring new insights in organogenesis and carcinogenesis. However, direct 3D transfection on already formed organoids remains challenging. One limitation is that organoids are embedded in extracellular matrix and grow into compact structures that hinder transfection using traditional techniques. To address this issue, we developed an innovative approach for transgene expression in 3D organoids by combining single-cell encapsulation in Matrigel microbeads using a microfluidic device and electroporation. We demonstrate that direct electroporation of encapsulated organoids reaches up to 80% of transfection efficiency. Using this technique and a morphological read-out that recapitulate the different stages of tumor development, we further validate the role of p63 and PTEN as key genes in acinar development in breast and prostate tissues. We believe that the combination of controlled organoid generation and efficient 3D transfection developed here opens new perspectives for flow-based high-throughput genetic screening and functional genomic applications.

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. A more complete understanding of the development of organoids would enhance their relevance as models to study organ morphology, function, and disease and would open new avenues in drug development and regenerative medicine.

Comparative study of fully three-dimensional reconstruction algorithms for lens-free microscopy.

We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multiangle acquisitions on 3D cell cultures embedded in extracellular matrices. Lens-free microscopy acquisitions present some inherent issues such as the lack of phase information on the sensor plane and a limited angular coverage. We developed and compared three different algorithms based on the Fourier diffraction theorem to obtain fully 3D reconstructions. These algorithms present an increasing complexity associated with a better reconstruction quality. Two of them are based on a regularized inverse problem approach. To compare the reconstruction methods in terms of artefact reduction, signal-to-noise ratio, and computation time, we tested them on two experimental datasets: an endothelial cell culture and a prostate cell culture grown in a 3D extracellular matrix with large reconstructed volumes up to ∼5 mm3 with a resolution sufficient to resolve isolated single cells. The lens-free reconstructions compare well with standard microscopy.

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. 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.

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

Video 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.

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. Each individual bead on average acts as a single 3D cell culture compartment generating one acinus per bead. Compared to standard protocols microfluidics provides increased control over the environment leading to more a uniform acini population. The increased facility of bead manipulation allowed us to isolate single cells which are self-sufficient to fully develop into acini in presence of Matrigel. Furthermore, combination of our microfluidic approach with large particle FACS opens new avenues in high throughput screening on single acini or spheroids.

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.

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. The initial attachment of cells was better on the PAH-terminated surface compared to fibronectin. However, the proliferation rate of PC3 cells was reduced on the PAH-terminated surface and slightly increased on the PSS coatings. Only PAH prevented the clustering phenotype of PC3 and reduced the number of focal adhesion points as compared to fibronectin or PSS coatings. In contrast, none of the PE surfaces significantly affected the biological responses of PNT-2 cells. PAH-terminating films provide a tool to preferentially modulate the growth of some cancerous phenotypes, in this case as a micro-environment that reduces the growth of metastatic PC3 cells.

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. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.

A miniaturized planar patch-clamp system for transportable use.

In the last decade, planar patch-clamp (PPC) has emerged as an innovative technology allowing parallel recordings of cellular electrophysiological activity on planar substrates. If PPC is widely adopted by the pharmaceutical sector, it remains poorly extended to other areas (i.e. environment and safety organizations) probably because of the large, expensive and non-easily transportable format of those commercial equipments. The present work describes for the first time a new compact and transportable planar patch-clamp system (named Toxint'patch or TIP, for Toxin detection with integrated patch-clamp) focusing on environmental matters and meant to be used in coastal laboratories, for direct on-site monitoring of the seawater and shellfish quality. The TIP system incorporates silicon chips tailored to monitor cellular ionic currents from cultured cells stably expressing a phycotoxin molecular target. The functionality of this novel briefcase-sized PPC system is described in terms of fluidic control, electronic performances with amplifying and filtering boards and of user interface for data acquisition and control implemented on a computer.

The development of high quality seals for silicon patch-clamp chips.

Planar patch-clamp is a two-dimensional variation of traditional patch-clamp. By contrast to classical glass micropipette, the seal quality of silicon patch-clamp chips (i.e. seal resistance and seal success rate) have remained poor due to the planar geometry and the nature of the substrate and thus partially obliterate the advantages related to planar patch-clamp. The characterization of physical parameters involved in seal formation is thus of major interest. In this paper, we demonstrate that the physical characterization of surfaces by a set of techniques (Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM), X-ray Photoelectron Spectroscopy (XPS), surface energy (polar and dispersive contributions), drop angles, impedance spectroscopy, combined with a statistical design of experiments (DOE)) allowed us discriminating chips that provide relevant performances for planar patch-clamp analysis. Analyses of seal quality demonstrate that dispersive interactions and micropore size are the most crucial physical parameters of chip surfaces, by contrast to surface roughness and dielectric membrane thickness. This multi-scale study combined with electrophysiological validation of chips on a diverse set of cell-types expressing various ion channels (IRK1, hERG and hNa(v)1.5 channels) unveiled a suitable patch-clamp chip candidate. This original approach may inspire novel strategies for selecting appropriate surface parameters dedicated to biochips.

A simple method for the reconstitution of membrane proteins into giant unilamellar vesicles.

A simple method for the reconstitution of membrane protein from submicron proteoliposomes into giant unilamellar vesicles (GUVs) is presented here: This method does not require detergents, fusion peptides or a dehydration step of the membrane protein solution. In a first step, GUVs of lipids were formed by electroformation, purified and concentrated; and in a second step, the concentrated GUV solution was added to a small volume of vesicles or proteoliposomes. Material transfer from submicron vesicles and proteoliposomes to GUVs occurred spontaneously and was characterized with fluorescent microscopy and patch-clamp recordings. As a functional test, the voltage-dependent, anion-selective channel protein was reconstituted into GUVs, and its electrophysiological activity was monitored with the patch clamp. This method is versatile since it is independent of the presence of the protein, as demonstrated by the fusion of fluorescently labeled submicron vesicles and proteoliposomes with GUVs.

2D and 3D cell microarrays in pharmacology.

To analyze the phenotypic consequences of perturbing mammalian cells with drugs, there is an increasing need for systematic cell-based assays in an HTS format. Cell microarrays provide an attractive solution as they offer more than a simple miniaturization and mechanization of conventional microtiter plates. While standard monolayer two-dimensional culture conditions are poor mimics of the cellular environment in situ, microfabricated systems enable three-dimensional organotypic cell cultures and have the potential to provide biological insight not achievable before. This article compares different cell microarray formats and evaluates their potential use in the drug discovery process.

Influence of glass and polymer coatings on CHO cell morphology and adhesion.

Successful development of cell-on-chip microsystems where living cells are deposited and grown in microfabricated structures is highly dependent on the control of cell/substrate interactions. In this study, several materials of interest were tested for CHO cell growth and morphology: (i) glass, fibronectin-, poly-L-lysine- and 3-aminopropyltriethoxysilane (APTES)--treated glass and UV/O(3)-modified PDMS coating on glass as well as (ii) silicon, poly-L-lysine-, APTES-, O(2) plasma-treated and oxide-coated silicon. In addition, we quantitatively characterized cell adhesion to these substrates using a radial flow detachment assay. Lack of correlation between cell adhesion and cell morphology was systematically observed for all substrates. In particular, we show that PDMS coatings on glass can be finely tuned by UV/O(3) treatment to enhance cell adhesion and induce elongated morphology. Moreover, we observed a low shear stress cell detachment mechanism on silicon oxide coatings on silicon wafers. It is therefore possible with these coatings to selectively influence either cell adhesion or morphology.

Hourglass SiO2 coating increases the performance of planar patch-clamp.

Obtaining high-throughput electrophysiological recordings is an ongoing challenge in ion channel biophysics and drug discovery. One particular area of development is the replacement of glass pipettes with planar devices in order to increase throughput. However, successful patch-clamp recordings depend on a surface coating which ideally should promote and stabilize giga-seal formation. Here, we present data supporting the use of a structured SiO(2) coating to improve the ability of cells to form a "seal" with a planar patch-clamp substrate. The method is based on a correlation study taking into account structure and size of the pores, surface roughness and chip capacitance. The influence of these parameters on the quality of the seal was assessed. Plasma-enhanced chemical vapour deposition (PECVD) of SiO(2) led to an hourglass structure of the pore and a tighter seal than that offered by a flat, thermal SiO(2) surface. The performance of PECVD chips was validated by recording recombinant potassium channels, BK(Ca), expressed in stable HEK-293 cell lines and in inducible CHO cell lines and low conductance IRK1, and endogenous cationic currents from CHO cells. This multiparametric investigation led to the production of improved chips for planar patch-clamp applications which allow electrophysiological recordings from a wide range of cell lines.