lens-free-video-microscopy-for-dynamic-quantitative-analysis-adherent

Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator ...

Université Grenoble Alpes

Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling.

Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics.

The cell is a very complex system and although molecular biology allowed spectacular progresses, biologists are currently trying to tackle complexity at the system level, to unravel cell mysteries. The inference of transcriptional regulatory networks, the characterization of their topology, their dynamic, their role in major cell functions is at the heart of this strategy. We are proposing a systemic approach, based on integration of large scale data such as expression profiling, ChIP on chip analysis and high throughput RNA interference using siRNA microarrays to infer these networks and characterize associated phenotypes. Ultimately, the characterization of the properties of these networks should impact our understanding of biology and offer potential applications in medicine and pharmacology.

[Inference of transcriptional regulatory networks].

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.

2D and 3D cell microarrays in pharmacology.

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.

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

Single cell analysis circumvents the need to average data from large populations by observing each cell individually, thus enabling the analysis of cell-to-cell variability. The ability to work on this scale presents many new opportunities for the life sciences and biomedical applications. Microfluidics has become a tool of choice for such studies and electrowetting on dielectric (EWOD) technology is well adapted for samples with reduced size and biological studies at the single cell level. In the present manuscript, for the first time, we present an integrated and automated system based on EWOD that can process the complete workflow on a single device, from the isolation of a single cell to mRNA purification and gene expression analysis.

An EWOD-based microfluidic chip for single-cell isolation, mRNA purification and subsequent multiplex qPCR.

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.

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

A long-sought milestone in the defense against bioterrorism is the development of rapid, simple, and near-patient assays for diagnostic and theranostic purposes. Here, we present a powerful test based on a host response to a biological weapon agent, namely the ricin toxin. A signature for exposure to ricin was extracted and characterized in mice and then integrated into a plastic microfluidic cartridge. This enabled early diagnosis of exposure to ricin in mice using a drop of whole blood in less than 1 h and 30 min. The cartridge stores the reagents and implements all of the steps of the analysis, including mRNA extraction from a drop of blood, followed by tens of parallel RT-qPCR reactions. The simple and low-cost microfluidic cartridge developed here may find other applications in point-of-care diagnostics.

Point-of-care diagnostics for ricin exposure.

A general radioprotective effect by fibroblast growth factor (FGF) has been extensively described since the early 1990s; however, the molecular mechanisms involved remain largely unknown. Radiation-induced DNA double-strand breaks (DSBs) lead to a complex set of responses in eukaryotic cells. One of the earliest consequences is phosphorylation of histone H2AX to form nuclear foci of the phosphorylated form of H2AX (γH2AX) in the chromatin adjacent to sites of DSBs and to initiate the recruitment of DNA-repair molecules. Upon a DSB event, a rapid signaling network is activated to coordinate DNA repair with the induction of cell-cycle checkpoints. To date, three kinases (ATM, ATR, and DNA-PK) have been shown to phosphorylate histone H2AX in response to irradiation. Here, we report a kinome-targeted small interfering RNA (siRNA) screen to characterize human kinases involved in H2AX phosphorylation. By analyzing γH2AX foci at a single-nucleus level, we identified 46 kinases involved either directly or indirectly in H2AX phosphorylation in response to irradiation in human keratinocytes. Furthermore, we demonstrate that in response to irradiation, the FGFR4 signaling cascade promotes JNK1 activation and direct H2AX phosphorylation leading, in turn, to more efficient DNA repair. This can explain, at least partially, the radioprotective effect of FGF.

A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation.

Phenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Φ-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Φ-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Φ-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Φ-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection.

Φ-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays.

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.

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

Modularity, feedback control, functional redundancy and bowtie architecture have been proposed as key factors that confer robustness to complex biological systems. MicroRNAs (miRNAs) are highly conserved but functionally dispensable. These antinomic properties suggest that miRNAs fine-tune gene expression rather than act as genetic switches. We synthesize published and unpublished data and hypothesize that miRNA pluripotentiality acts to buffer gene expression, while miRNA degeneracy tunes the expression of targets, thus providing robustness to gene expression networks. Furthermore, we propose a Lavallière-tie architecture by integrating signal transduction, miRNAs and protein expression data to model complex gene expression networks.

MicroRNA degeneracy and pluripotentiality within a Lavallière-tie architecture confers robustness to gene expression networks.

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.

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

Dielectrophoresis is widely used for cell characterization, and the exerted force on cells depends on the difference of polarizability between the latter and the surrounding medium. This physical phenomenon is translated by the real part of the Clausius-Mossotti factor. It is mostly modeled from the imaginary part, measured by electrorotation. The method described here measures experimentally the real part of the Clausius-Mossotti factor. It relies on the cell velocity when submitted to pure dielectrophoresis, and it was conducted on several human cell lines, at different times. A variety of cell lines was evaluated, from different organs or representative of different stages of cancer, with promising findings for early cancer detection.

Label-Free Electric Monitoring of Human Cancer Cells as a Potential Diagnostic Tool.

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.

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

A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.

Article	Year
Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics. Biology open\| PubMed ID: 27744291	2016
[Inference of transcriptional regulatory networks]. Medecine sciences : M/S\| PubMed ID: 18601881	2008
2D and 3D cell microarrays in pharmacology. Current opinion in pharmacology\| PubMed ID: 19520607	2009
The modulation of attachment, growth and morphology of cancerous prostate cells by polyelectrolyte nanofilms. Biomaterials\| PubMed ID: 24060421	2013
An EWOD-based microfluidic chip for single-cell isolation, mRNA purification and subsequent multiplex qPCR. Lab on a chip\| PubMed ID: 25080028	2014
Controlled 3D culture in Matrigel microbeads to analyze clonal acinar development. Biomaterials\| PubMed ID: 25818441	2015
Point-of-care diagnostics for ricin exposure. Lab on a chip\| PubMed ID: 25892365	2015
A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation. Cellular and molecular life sciences : CMLS\| PubMed ID: 25894690	2015
Φ-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays. Scientific reports\| PubMed ID: 26382112	2015
Time-lapse contact microscopy of cell cultures based on non-coherent illumination. Scientific reports\| PubMed ID: 26459014	2015
MicroRNA degeneracy and pluripotentiality within a Lavallière-tie architecture confers robustness to gene expression networks. Cellular and molecular life sciences : CMLS\| PubMed ID: 27038488	2016
A 3D Toolbox to Enhance Physiological Relevance of Human Tissue Models. Trends in biotechnology\| PubMed ID: 27497676	2016
Label-Free Electric Monitoring of Human Cancer Cells as a Potential Diagnostic Tool. Analytical chemistry\| PubMed ID: 27562118	2016
Deciphering Cell Intrinsic Properties: A Key Issue for Robust Organoid Production. Trends in biotechnology\| PubMed ID: 28927991	2017
High-Content Monitoring of Drug Effects in a 3D Spheroid Model. Frontiers in oncology\| PubMed ID: 29322028	2017

Xavier Gidrol

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