Label-Free Monitoring of Cells in vitro (Bioanalytical Reviews Book 2)

This book is dedicated to label-free, non-invasive monitoring of cell-based assays and it comprises the most widely applied techniques. Each approach is described and critically evaluated by an expert in the field such that researchers get an overview on what is possible and where the limitations are. The book provides the theoretical basis for each technique as well as the most successful and exciting applications. Label-free bioanalytical techniques have been known for a long time as valuable tools to monitor adsorption processes at the solid-liquid interface in general – and biomolecular interaction analysis (BIA) in particular. The underlying concepts have been progressively transferred to the analysis of cell-based assays. The strength of these approaches is implicitly given with the name 'label-free': the readout is independent of any label, reagent or additive that contaminates the system under study and potentially affects its properties. Thus, label-free techniques provide an unbiased analytical perspective in the sense that the sample is not manipulated by additives but pure. They are commonly based on physical principles and read changes in integral physical properties of the sample like refractive index, conductivity, capacitance or elastic modulus to mention just a few. Even though it is not implied in the name, label-free approaches usually monitor the cells under study non-invasively meaning that the amplitude of the signal (e.g. electric field strength, mechanical elongation) that is used for the measurement is too low to interfere or affect. In contrast to label-based analytical techniques that are commonly restricted to a single reading at a predefined time point, label-free approaches allow for a continuous observation so that the dynamics of the biological system or reaction become accessible. Preface 7 Contents 11 Impedance-Based Assays Along the Life Span of Adherent Mammalian Cells In Vitro: From Initial Adhesion to Cell Death 12 1 Introduction 13 2 Physical Background of Impedance-Based Assays 16 2.1 Behavior of Cells in Electric Fields 16 2.2 Physical Basics of Impedance Sensing 19 2.3 Experimental Setup for Impedance Measurements of Adherent Cells In Vitro 21 2.4 Popular Electrode Designs and Commercial Systems 25 2.5 The Measurement Principle 27 2.6 The ECIS Model: Correlating Impedance Readings with Cell Morphology 29 3 Impedance-Based Assays to Study Cell Phenotypes and Behavior In Vitro 31 3.1 Cell-Matrix Adhesion 31 3.2 Cell Proliferation 36 3.3 Cell Layer Maturation: Barrier Function as a Prominent Example 40 3.4 Micromotion: An Indicator for Viability and Motility 42 3.5 Cell Migration: The Electrical Wound-Healing Assay and 3D Approaches 46 3.6 Morphology Changes in Response to Biological, Chemical, or Physical Stimuli 49 3.6.1 Receptor Activation and Signal Transduction 49 Impedance-Based Assays in Receptor-Mediated Signaling Research 52 Impedance-Based Assays in Drug Discovery Targeting Cell Surface Receptors 53 3.6.2 Cell Differentiation 54 3.6.3 Cell-Cell and Cell-Pathogen Interactions 56 3.6.4 Response to Physical Challenges: Invasive Electric Fields, Fluid Shear, and More 57 3.7 Monitoring Cell Death and Cytotoxicity 59 4 Concluding Remarks 65 References 67 Transistor-Based Impedimetric Monitoring of Single Cells 87 1 Introduction 88 2 Materials and Methods 92 2.1 Field-Effect Transistors 92 2.2 Measurement Setups 93 2.3 Chip Encapsulation 95 2.4 Preparation of Devices and Cells 96 3 Results and Discussion 98 3.1 Low Density Cultures 98 3.2 Single Cell Impedance 99 3.3 TTF Spectra and Modeling 101 3.4 Optimization of Devices for Single Cell FETCIS 105 3.5 Recordings from Primary Neuronal Cultures 107 3.6 Human T-Cell Experiments 108 3.7 Adjustment of Medium Conductivity for Improved FETCIS 113 4 Conclusion and Outlook 117 References 118 Label-Free Monitoring of 3D Tissue Models via Electrical Impedance Spectroscopy 121 1 Introduction 122 1.1 Microelectrode Design and Spatial Resolution 123 1.2 Optimization of Measurement Parameters 124 1.3 Literature Review on the Optimization of Electrode Design 125 2 Real-Time Monitoring of 3D Tissue Models 125 2.1 Scaffold-Based 3D Cultures 126 2.2 EIS in Scaffold-Based 3D Cultures 127 2.3 Scaffold-Free 3D Cultures 130 2.4 EIS in Scaffold-Free 3D Cultures 131 3 A Linear Electrode Array Integrated in a Microfluidic Channel for Spheroid Measurements 133 4 Modeling of 3D Microelectrodes 137 5 The Future of EIS-Based Monitoring of 3D Tissue Models 139 References 140 On the Use of the Quartz Crystal Microbalance for Whole-Cell-Based Biosensing 145 1 Introduction 146 2 Instrumental Aspects 148 3 Background: The Non-gravimetric QCM 149 3.1 Full-Fledged Viscoelastic Modeling Is Difficult for Biological Cells 151 4 Simple Models and Situations: Where These Can Be Applied 153 4.1 Cell Layers as Semi-infinite Viscoelastic Media 153 4.2 Torsional Resonators Can Measure the Thickness of Biofilms 155 4.3 Coupled Resonances 157 5 Other Dimensions of Sensing 160 5.1 Variation of Amplitude 160 5.2 Piezoelectric Stiffening 162 5.3 Temporal Variations 165 5.4 Temperature Sweeps 165 6 Combined Instruments 166 7 Conclusions 167 References 168 Microphysiometry 173 1 Introduction 174 1.1 What Is Microphysiometry? 174 1.2 A Brief Review of Microphysiometry 176 2 Parameters and Sensors 177 2.1 Extracellular Acidification and pH Sensors 177 2.2 Cellular Oxygen Uptake and Sensors for Dissolved Oxygen 179 2.3 Sensors for CO2 180 2.4 Sensors for Glucose and Lactate 181 2.5 Sensing Metabolic Heat 181 3 Experimental Conditions and Automation 182 3.1 Climate Control and Fluidic Systems 182 3.2 Microreaction Volumes 183 3.3 Automation 184 4 Cell Culture 185 4.1 Cell Models 185 4.2 Culture Systems 185 5 Data Processing and Numerical Models 187 5.1 Dynamic Data Analysis 187 5.2 Numerical Models for Reaction and Diffusion 188 6 Selected Applications 189 6.1 Toxicology 189 6.2 Clinical Cancer Research 191 References 193 Optical Waveguide-Based Cellular Assays 199 1 Introduction 200 2 RWG Biosensors 200 3 RWG Readers 201 3.1 Angular Interrogation-Based Imaging Systems 202 3.2 Wavelength Interrogation-Based Scanning Systems 203 3.3 Whole-Plate Imaging Systems 204 3.4 High-Resolution Imaging Systems 205 3.5 High-Frequency Intensity-Based Imaging System 205 3.6 Microfluidic Biosensor Systems 206 4 Optical Waveguide-Based Cell Assays 207 4.1 Cell Phenotype profiling 208 4.2 Assay Formats 209 5 Applications in Drug Discovery 211 5.1 Screening 212 5.2 Lead Optimization 213 5.3 Lead Selection 215 5.4 Target Identification 216 5.5 Discovery of Novel Receptor Biology 218 5.6 Single-Cell Analysis 219 6 Summary and Perspective 220 References 221 Label-Free Quantitative In Vitro Live Cell Imaging with Digital Holographic Microscopy 228 1 Introduction 230 2 Digital Holographic Microscopy for Live Cell Imaging 231 2.1 Principles of Off-Axis Digital Holographic Microscopy 232 2.2 Evaluation of Digital Off-Axis Holograms 235 2.3 Spatial Phase Shifting-Based Reconstruction of Off-Axis Holograms 239 2.4 Hologram Evaluation by Spatial Filtering 241 2.5 Automated Holographic Refocusing 242 2.6 Evaluation of Quantitative DHM Phase Images by Image Segmentation 244 2.7 Automated Single Cell Tracking and Quantification of Cell Motility 246 2.8 Refractive Index, Volume, and Dry Mass Determination of Suspended Cells 248 3 Selected Applications in Cell-Based Assays 250 3.1 Characterization of Suspended Single Cells and Subcellular Organelles 251 3.2 Monitoring of the Intracellular Water Content 253 3.3 Monitoring the Influence of Cytokines 256 3.4 Analysis of Cell Volume and Refractive Index Changes After Transfection with MicroRNA 258 3.5 Quantification of Cell Motility as an Indicator for Invasiveness in Endometriosis Research 258 3.6 Imaging of Cytokinesis After Downregulation of Survivin in Ewing ́s Sarcoma Cells 260 3.7 Label-Free Phenotyping by Cell Thickness 262 3.8 Multimodal Growth and Morphology Analysis of Mixed Cell Cultures 262 3.9 Multiparameter Monitoring of In Vitro Wound Healing Assays 266 3.10 Label-Free In Vitro Toxicity Testing of Pathogens 268 3.11 Analysis of Cell-Nanomaterial Interactions 269 3.12 Challenges and Future Prospects of Quantitative DHM Phase Imaging 271 4 Conclusions 274 References 274 Index 282 Front Matter ....Pages i-xi Impedance-Based Assays Along the Life Span of Adherent Mammalian Cells In Vitro: From Initial Adhesion to Cell Death (Judith A. Stolwijk, Joachim Wegener)....Pages 1-75 Transistor-Based Impedimetric Monitoring of Single Cells (F. Hempel, J. K. Y. Law, S. Ingebrandt)....Pages 77-110 Label-Free Monitoring of 3D Tissue Models via Electrical Impedance Spectroscopy (Frank Alexander Jr., Sebastian Eggert, Dorielle Price)....Pages 111-134 On the Use of the Quartz Crystal Microbalance for Whole-Cell-Based Biosensing (D. Johannsmann)....Pages 135-162 Microphysiometry (Martin Brischwein, Joachim Wiest)....Pages 163-188 Optical Waveguide-Based Cellular Assays (Y. Fang)....Pages 189-217 Label-Free Quantitative In Vitro Live Cell Imaging with Digital Holographic Microscopy (B. Kemper, A. Bauwens, D. Bettenworth, M. Götte, B. Greve, L. Kastl et al.)....Pages 219-272 Back Matter ....Pages 273-277