Das Forum 2013 mit dem Thema "Automation von zellulären Applikationen / Phenotypic Screening and Cellular Models in Drug Discovery" fand am 7. März im Darmstadtium in Darmstadt statt.
While in the past cell based assays using recombinant cell lines have been the key pathway to discover new therapeutic drugs, these approaches often fail with more complex disease processes. Cell signaling is an integrated process and cross-talk between pathways is a common phenomenon. A recombinant cell line that expresses the target of interest often lacks the physiological background of the real cell interactions within the patient.
As a consequence, many positive leads that show a high activity in a recombinant cell based assay fail to show similar activity in clinical stages. Therefore, the development of more relevant systemic cell based assays which can be processed in an automated high throughput mode are an important aim for future drug discovery.
This year’s ELRIG.de Forum will focus on two aspects in achieving this goal.
- Differentiated cells derived from induced pluripotent stem cells (iPS) are physiologically relevant cells which can be produced in quantities required for HTS campaigns.
- Phenotypic screening integrates the idea of not only analyzing the drug, the target interaction and its biological activity, with the application of a more holistic viewpoint of looking for general phenotypic changes within the cell. Experiments can be performed using cells expressing endogenous levels of receptors and without the artificial impact of highly overexpressed receptors.
Cellular Screening in 2D and 3D Identification of cancer specific modulators
The talk will focus on the development of different toxicity screening platforms. As part of an FP7 funded project the NCI60 cancer cell line panel is used to assess cellular toxicity. A comparison between 2D and 3D cell culture models as well as label-free technology approaches will be presented.
Bernhard Ellinger, European ScreeningPort GmbH, Schnackenburgallee 114, 22525 Hamburg, Germany
Cell-based label-free dynamic mass redistribution assay to study pleiotropic seven transmembrane receptor signaling
Seven transmembrane receptors (7TMR) also referred to as G protein-coupled receptors (GPCR) make up the largest transmembrane protein family in the human genome. Due to the versatile and important role of these integral proteins for physiological and pathophysiological events, they represent key targets for therapeutic interventions and about 30% of marketed drugs modulate the activity of GPCRs. The majority of techniques currently employed to examine signaling behavior of GPCRs requires artificial labels on either receptor or ligand. In contrast to these classical methods, a number of novel label-free technology platforms have emerged recently that are competent to resolve receptor activity as integrated cellular response. Such platforms should be ideally suited to observe phenomena such as pleiotropic 7TMR signaling and ligand bias. The present talk will introduce a label-free method based on detection of dynamic mass redistribution (DMR) and attempt to show the strengths of holistic label-free detection as compared with classical, single component functional assays but also highlight the challenges such as the need for signal deconvolution to interpret the complex cellular response profiles.
Stephanie Hennen, University of Bonn, Molecular-, Cellular-, and Pharmacobiology Section, Nussallee 6, 53115 Bonn, Germany
Potential of impedance technology to study G-protein-coupled receptor biology
G-protein-coupled receptors (GPCRs) are the target of more drugs than any other class of proteins due to their drugability and due to their specific involvement in many physiological and pathophysiological processes. In pharmaceutical drug discovery it is of great interest to study GPCRs in a therapeutically relevant cell setting. However, this requires demanding cell culture and difficult assay development. Recently, the non-invasive and label-free impedance technology for GPCRs became available. This method measures changes in electrical impedance of cell monolayers, which result from changes in cellular morphology after GPCR activation.
At Actelion Pharmaceuticals, we integrated this technology into our drug discovery programs to characterize GPCR responses in secondary screenings, with emphasis on non-recombinant cells. In this presentation we give examples how impedance can be used to decipher the mechanism of action of compounds including kinetics of GPCR activation, receptor desensitization or GPCR receptor subtype activation. Furthermore, we show how compound testing can be performed in disease-relevant cells expressing the native receptors. We also use impedance technology to measure long-term cellular phenotypic changes and their modulation. We conclude that impedance is a very sensitive and unbiased method, which provides complementary and novel insights in drug discovery.
Katrin Sobel, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, 4123 Allschwil, Switzerland
High Content Live Cell Imaging with Chromobodies
Usually, the detection of endogenous intracellular proteins is limited to immunofluorescent stainings with conventional antibodies in fixed and permeabilized cells. However, live cell information of spatial and temporal distribution of proteins dramatically increases the quality and relevance of any assay, also phenotypic high content assays. Until now, such important analysis can only be accomplished through artificial introduction of fluorescently labeled counterparts of the proteins of interest.
Now, the discovery of Chromobodies® and advancements in high content imaging systems with improved optical properties and live cell capabilities has opened the door to visualize endogenous components of the cell in real-time in living cells. Chromobodies® are a new class of fluorescent antibodies derived from Camelidae, which are characterized by a much smaller size and simpler organization than conventional antibodies. The antigen-binding domains (VHHs) of these antibodies are formed by only one polypeptide chain allowing their straightforward expression in heterologous systems. By fusing a fluorescent protein to these antigen-binding domain we create Chromobodies®, the fluorescent biosensors working in living cells.
Here we present novel high content screening assays to:
• Accurately plot and quantify each sub phase of DNA replication for each individual cell with the Cell Cycle Chromobody (Burgess et al., 2012, PLoS One)
• Monitor reorganization, disassembly and reassembly of actin cytoskeleton in real time using the Actin Chromobody
• Non-invasive visualization of the nuclear lamina and its fragmentation during apoptosis (Zolghadr et al., 2012, Methods Mol Biol)
Kourosh Zolghadr, ChromoTek GmbH, Am Klopferspitz 19, 82152 Martinsried, Germany
CAP and CAP-T cells: a new Protein Production Platform based on Immortalized Human Amniocytes and it's Potential Applications in Cell Based Assays
Human CAP (CEVECŒs Amniocyte Production) cells allow for stable and high yield production of recombinant proteins, with excellent biologic activity and therapeutic efficacy, as a result of authentic posttranslational modification. Based on CAP cells a transient expression system has been developed, that enables extremely high production yields of recombinant proteins within a few days. Besides the use in early preclinical development through to clinical supply of recombinant biotherapeutics the cells also show the promise to be an efficient tool for drug screening purposes.
Hartmut Tintrup, CEVEC Pharmaceuticals GmbH, Gottfried-Hagen-Str. 62, 51105 Cologne, Germany
High-content screens for drug sensitizers and biomarkers
Cenix BioScience conducts preclinical contract research and technology development, focused on high-throughput screening studies using RNAi, miRNA modulation and compound treatments, for a wide range of applications including target discovery & validation, lead compound identification & optimization, drug mechanism of action analyses, predictive toxicology and biomarker discovery. Our presentation will focus on the example of RNAi/drug modifier screens, which have proven to be valuable for identification of drug sensitizers and biomarkers. We will present our own approaches to such high-content microscopy-based screens, always aiming for a high complexity of phenotypic readouts, whilst exploiting relatively simple, inexpensive labeling and analysis strategies.
Christoph Sachse, Cenix BioScience GmbH, Tatzberg 47, 01307 Dresden, Germany
Cells in the "Q": Phenotypic Screening of Cells in Solution
High content methods that make multi-parameter measurements on single cells have sparked a resurgence of phenotypic screening. IntelliCyt's new system, iQue, rapidly screens cell and bead populations in solution. A recent phenotypic screen for modulators of leukemia using IntelliCyt technology will be discussed.
Steve Davenport, IntelliCyt Corporation, 9620 San Mateo Blvd. NE, Albuquerque, NM 87113, USA
Organotypic 3D microtissue spheroids for drug testing
Cell-based assays, being a highly versatile tool to assess cell response to biological and chemical stimuli, have become a powerful tool in the research lab. They can be tailored to evaluate many cellular and biochemical functions. On an industrial level they are used to discover and develop new drugs and test the toxicity of new and old chemicals. To further improve the biology of cells grown in vitro, cell technologies which allow a 3-diemensional structure towards a more organotypic "tissue" model are being used more intensively. These culture systems are becoming increasingly complex being composed not only of a single cell type but, similar to native tissue, incorporating several cell populations to mimick native tissue as close as possible. The increasing use of more physiological 3-dimensional cell model systems for drug and substance testing entails the development of more sophisticated analysis systems which allow parallel multiplexing of several read outs. It becomes especially intricate if a drug response has to be correlated to a specific cell population in a multi-cell type system. Here we demonstrate combinations of a novel size-reduced Luciferase and fluorescent reporter proteins to measure drug effects on specific cell populations in multi-cell type tumor spheroid models. Tumor spheroids were generated from a cancer cell lines harboring the Luciferase of fluorescent proteins and mouse fibroblasts expressing a red fluorescent protein. With a multi plate reader drug effects on the different cell populations were assessed combining high sensitive luminescence quantification, representing the impact on the cancer cell population, and fluorescence read out, signifying the impact on the stroma cell population. Non-disruptive and analysis technologies have to be adapted to gain the full benefit of more complex organotypic multi-cell type models. The combination of fluorescent reporter and secreted luciferase allows to tailor high-throughput-compatible analysis according to the spheroid system applied, to detect drug efficacy on cancer and stroma cell populations within the same model.
Jens M. Kelm, InSphero AG, Wagistrasse 27, 8952 Schlieren, Switzerland
Der neue PIPETMAX® - offene Plattform zur Automatisierung von Pipettier-Protokollen
Automatisierter Probendurchsatz beim Plattenpipettieren. PIPETMAX® zeichnet sich durch eine einfache und flexible Steuerung aus und trägt zur Kostenminimierung durch die Automatisierung von Routine Pipettieraufgaben bei. Der PIPETMAX® Pipettierautomat unterstützt mehrere Pipettierköpfe während eines Laufs. Der Computer ist nicht direkt mit der Plattform verbunden: Die Protokolle können zunächst am Arbeits-PC erstellt werden und die Methode kann anschließend über einen USB Stick in das Gerät geladen werden. PIPETMAX® ist für folgende Anwendungen geeignet: PCR/qPCR, cell-based assays, NGS prep, ELISA prep, tip-based sample prep.
Ute Pfeifle-Tiller, Gilson International B.V. Deutschland, Hoenbergstraße 6, 65555 Limburg, Germany
Viral vector technology in cell based assays
Requirements for cell based assays have induced a rapid development of novel cell models in the past years. To improve the effectiveness of target research and compound development the use of patient or stem cell-derived cell models is gaining constantly in importance. SIRION Biotech specializes in viral vector technology and provides sophisticated cell modeling. Viral vector technology enables the generation of primary cell-based models and cell models with homogenous gene expression for high content screening and phenotypic analysis. The viral vector technology can be combined with a unique RNAiONE technology for efficient target research, and inducible gene expression systems for generation of cell models for difficult-to-screen targets.
Kathrin Schmitt, SIRION Biotech GmbH, Am Klopferspitz 19, 82152 Planegg, Germany
Advancements in the Use of iPS Cell-derived model systems for In Vitro Disease Modeling and Phenotypic Screening
Induced pluripotent stem cell (iPSC) technology enables the creation of biologically relevant in vitro cell models for discovery of novel therapeutic targets and phenotypic screening approaches to drug development. Cellular Dynamics International (CDI) is the world's largest producer of fully functional human iPSC-derived cell types and has commercialized various iCell® Products – including iCell Cardiomyocytes, Neurons, Astrocytes and others. The functional properties of each of these human cell types recapitulate the expected in vivo characteristics of the relative native cells in the human body, making them ideal for use in disease modeling and drug discovery. Here, we present case studies demonstrating the use of iPSC-derived human cells for disease modeling, target validation, and drug screening. Specifically, these case studies include screening assay development for modulators of cardiac hypertrophy; high-throughput assay application for neuronal synaptic function; and assay design for compound screening and target validation for the prevention of β-amyloid induced toxicity in neurons. These examples provide evidence of the rapid implementation of human iPSC-derived cell models into drug discovery campaigns.
Giorgia Salvagiotto, Cellular Dynamics International, Inc, 525 Science Drive, Madison WI, 53711, USA
Analyzing contractility of human iPS derived cardiomyocytes using engineered heart tissue
Human cardiomyocytes can be derived in vitro from pluripotent stem cells, but techniques to study physiological contractile parameters are limited. To overcome this limitation, the technique of fibrin-based engineered heart tissue (EHT) was applied to human iPS cell derived cardiomyocytes. In this format, contractile parameters can be measured by a custom made and automated device, which uses figure recognition to detect contractions. This setup enables new possibilities, for example in the fields of drug screening and disease modeling, since the effect of various interventions on contractile parameters can be directly analyzed.
Sebastian Schaaf, University Medical Center Hamburg-Eppendorf, Institute of Experimental and Clinical Pharmacology and Toxicology, Martinistraße 52, 20246 Hamburg, Germany
Stem-cell-based phenotypic screening efforts at Roche
Pluripotent stem cells (PSCs) provide a unique source to generate cells of any tissue facilitating relevant cellular in vitro models that closely mimic human physiologic properties. To this end, we have derived human neural stem cells (hNSC) from human embryonic stem cells (hESC) to perform a neurogenesis drug discovery program aiming at identifying compounds that increase endogenous adult human neurogenesis and synaptic maturation through in vitro phenotypic screenings.
Moreover, the recent advent of induced pluripotent stem cell technology provides unparalleled access to any tissue cells from any healthy or diseased individual enabling to model a "patient in a dish". In collaboration with research teams at Massachusetts General Hospital and Harvard University we have generated induced pluripotent stem cells (iPSCs) from patients with Type 2 Diabetes associated with macro-vascular complications such as Coronary Artery Disease to investigate the underlying mechanisms of endothelial dysfunction. For this, the patient iPSC lines have been differentiated into functional endothelial cells for disease modeling and drug discovery.
We expect that the use of relevant PSCs derived cellular models in combination with disease modeling assays represent a powerful tool to recapitulate specific properties of human physiology and pathophysiology.
Christoph Patsch, Jacques Bailly, Eva Christina Thoma, Ravi Jagasia, Pascal Petitjean, Klaus Christensen, Jean-Philippe Carralot, Simona Ceccarelli, Michael Prummer, Hoa Truong, Sannah Zoffmann, Dorothee Kling, Gregor Dernick, Thilo Enderle, Roberto Iacone, Martin Graf, Hoffmann-La Roche AG, Basel, Switzerland