Microfluidic Systems for Cancer Diagnosis

Description

This detailed volume explores recent developments in microfluidics technologies for cancer diagnosis and monitoring. The book is divided into two sections that delve into techniques for liquid biopsy for cancer diagnosis and platforms for precision oncology or personalized medicine in order to create effective patient avatars for testing anti-cancer drugs. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.  Authoritative and practical, Microfluidic Systems for Cancer Diagnosis serves as an ideal guide that will be helpful to either replicate the construction of microfluidic devices specifically developed for cancer diagnosis or to catalyze development of new and better cancer diagnostic devices. Preface Contents Contributors Chapter 1: Lateral Filter Array Microfluidic Devices for Detecting Circulating Tumor Cells 1 Introduction 2 Materials 2.1 Silicon Master Fabrication 2.1.1 Instruments 2.1.2 Master Fabrication 2.2 Device Fabrication 2.3 Device Functionalization 2.4 Processing Clinical Samples 3 Methods 3.1 Silicon Master Fabrication 3.2 LFAM Device Fabrication 3.3 LFAM Device Functionalization 3.4 Processing Clinical Samples Using LFAM Devices 4 Notes References Chapter 2: Circulating Tumor Cell Cluster Sorting by Size and Asymmetry 1 Introduction 2 Materials 2.1 Master Mold Fabrication 2.1.1 Equipment 2.1.2 Consumables 2.2 Soft Lithography 2.2.1 Equipment 2.2.2 Consumables 2.3 Device Preparation and Operation 2.3.1 Equipment 2.3.2 Consumables 3 Methods 3.1 Wafer Fabrication 3.2 Soft Lithography 3.3 Device Preparation 3.4 Device Operation 4 Notes References Chapter 3: Digital Microfluidics with an On-Chip Drug Dispenser for Single or Combinational Drug Screening 1 Introduction 2 Materials 2.1 MCF-10A Cell Culture 2.2 MDA-MB-231 Cell Culture 2.3 DMF System Setup and Chip Fabrication 2.4 On-Chip and Off-Chip Drug Screening Tests 3 Methods 3.1 Cell Culture and Passage 3.2 DMF Chip Design 3.3 Dielectric Layer (10 μm SU-83010 Photoresist) Coating on the Bottom Plate 3.4 Hydrophobic Layer (Teflon) Coating 3.5 DMF System Setup 3.6 Drug Concentration Quantification 3.7 On-Chip Single Drug Screening 3.8 On-Chip Multidrug Screening 3.9 Off-Chip Drug Screening 4 Notes References Chapter 4: Affinity-Based Microfluidics Combined with Atomic Force Microscopy for Isolation and Nanomechanical Characterizatio... 1 Introduction 2 Materials 2.1 Materials Common to all Protocols 2.1.1 Reagents 2.1.2 Equipment 2.2 Materials Specific for AFM-Chip 2.2.1 Reagents 2.2.2 Equipment 2.3 Materials Specific for HB-MFP 2.3.1 Reagents 2.3.2 Equipment 3 Methods 3.1 Methods for AFM-Chip 3.1.1 Device Design 3.1.2 Device Fabrication Using Soft Lithography in PDMS 3.1.3 PDMS Replica Molding 3.1.4 Glass Cleaning and Silanization 3.1.5 Reversible AFM-Chip Assembly 3.1.6 Antibody Immobilization on Glass 3.1.7 Antibody Coverage Characterization 3.1.8 Blood Processing Using AFM-Chip 3.2 Methods for HB-MFP 3.2.1 Device Design 3.2.2 Device Fabrication Using Stereolithography 3D Printing 3.2.3 Microfluidic Chip for Antibody Patterning 3.2.4 Glass Cleaning and Activation 3.2.5 Blood Processing Using HB-MFP 3.3 Methods Common for AFM-Chip and HB-MFP 3.3.1 CTC Capture and Purity Evaluation 3.3.2 Further CTC Verification 3.3.3 AFM Force Measurements and Analysis 3.3.4 Waste and Material Disposal and Risk Assessment 3.4 Advantages, Limitations, and Data Analysis 3.4.1 Advantages 3.4.2 Limitations 3.4.3 Data Analysis 3.5 Future Directions 4 Notes References Chapter 5: Capture and Selective Release of Viable Circulating Tumor Cells 1 Introduction 2 Materials 2.1 Solution Preparation 2.2 Microfluidic Fabrication 2.3 Device Functionalization 2.4 Selective CTC Capturing 2.5 Release of Viable CTCs 3 Methods 3.1 Microfluidic Fabrication 3.1.1 Two-Layer Herringbone Master Mold Fabrication 3.1.2 Poly(Dimethylsiloxane) (PDMS) Preparation 3.1.3 Device Bonding 3.2 Device Functionalization 3.2.1 LbL Nanocoating 3.2.2 Antibody Functionalization 3.3 Selective CTC Capturing 3.3.1 Device and Sample Preparation 3.3.2 Whole Blood Processing 3.4 Release of Viable CTCs 3.5 Results 3.5.1 Selective CTC Capturing 3.5.2 Release of Viable CTCs 4 Notes References Chapter 6: Single-Response Electronic Tongue and Machine Learning Enable the Multidetermination of Extracellular Vesicle Bioma... 1 Introduction 2 Materials 2.1 Device Microfabrication 2.2 EV Isolation from Blood Samples 2.3 EV Electrochemical Analysis 2.4 Machine Learning Analyses 3 Methods 3.1 Device Microfabrication 3.2 EV Isolation from Blood Samples 3.3 EV Electrochemical Analysis 3.4 Machine Learning Analyses 4 Notes References Chapter 7: Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and M... 1 Introduction 2 Materials 2.1 Molecular Biology 2.2 Cancer and Normal (Control) Cell Lines 2.3 Two-Dimensional (2D) and Three-Dimensional (3D) Biocompatible Hydrogels 2.3.1 2D 2.3.2 3D 2.4 Confocal Microscopy System 2.5 Inoculation of Cancer Cells into Nude Mice 2.6 Tumor Tissue Slicing 3 Methods 3.1 Production of Retrovirus and Lentivirus Encoding CaViar Components (GCaMP5G and QuasAr2) and Transduction of Human Cancer ... 3.1.1 Plasmid Isolation 3.1.2 Polymerase Chain Reaction (PCR) 3.1.3 Running Gel 3.1.4 Gibson Assembly 3.1.5 Retroviral Transduction 3.1.6 Lentiviral Transduction 3.2 Culture and Maintenance of Human Cancer Cells 3.2.1 Subculture of Adherent Cells (for T-75 Flask) 3.2.2 Cryopreservation of Cells 3.2.3 Thawing of Frozen Cells 3.2.4 Cell Viability and Counting 3.3 Preparation of 2D and 3D Biomaterials 3.3.1 PAA Gel Preparation 3.3.2 Photo-Encapsulation of HCT-8 Cells in a 3D Hydrogel 3.4 Ca2+ Imaging In Vitro 3.4.1 Hardware and Software of Microscopy 3.4.2 Live Cell Ca2+ Imaging 3.5 HCT-8 Cell Xenograft Model Generation 3.6 Ca2+ Imaging Ex Vivo 3.6.1 HCT-8 Tumor Tissue Slicing 3.6.2 Ca2+ Imaging of Tumor Slices (Fig. 2) 4 Notes References Chapter 8: Microfluidic Protocols for the Assessment of Anticancer Therapies in 3D Tumor-Stromal Cocultures 1 Introduction 2 Materials and Equipment 2.1 Device Fabrication 2.2 Preparation of Devices 2.3 Cell Seeding and 3D Cocultures in Microfluidic Devices 2.4 Labeling Cells with Membrane Dyes 2.5 Microfluidic Viability Assays 2.6 Immunotherapy and Combination Therapy Microfluidic Assays 2.7 Immunofluorescence in Microfluidic Devices 2.8 Solutions Prepared 2.8.1 Synperonic F108 Solution 2.8.2 Phosphate Buffered Saline 2.8.3 Cell Culture Media 2.8.4 Membrane Dye Solutions 2.8.5 Viability Staining Solution 2.8.6 Solutions for Immunofluorescence in Microfluidic Devices 3 Methods 3.1 Device Fabrication 3.2 Preparation of Devices 3.3 Cell Seeding and 3D Cocultures in Microfluidic Devices 3.4 Labeling Cells with Membrane Dyes 3.5 Microfluidic Viability Assays 3.6 Immunotherapy and Combination Therapy Microfluidic Assays 3.7 Immunofluorescence in Microfluidic Devices 4 Notes References Chapter 9: A Microfluidic Approach for Enrichment and Single-Cell Characterization of Circulating Tumor Cells from Peripheral ... 1 Introduction 2 Materials 2.1 Sample Collection and RBC Lysis 2.2 CTC Enrichment Using Trapezoidal Spiral Microfluidics 2.3 Static Droplet Microfluidic Device Loading 2.4 On-Chip Staining, Enumeration, and Live Cell Imaging 2.5 Retrieval and Molecular Studies 3 Methods 3.1 Sample Collection and RBC Lysis 3.2 CTC Enrichment Using Trapezoidal Spiral Microfluidics 3.3 Static Droplet Microfluidic Device Loading 3.4 On-Chip Staining, Enumeration, and Live Cell Imaging 3.5 Retrieval and Molecular Studies 4 Notes References Chapter 10: Rapid On-Site Evaluation (ROSE): A Microfluidic Approach 1 Introduction 2 Materials 2.1 Instruments for ROSE Preparation Device Fabrication 2.2 Smearing Tool 2.3 PVA Film with Giemsa Stain 2.4 Blotting Unit 2.5 Microfluidic Chamber Fabrication 2.6 PANC-1 Cell Culture 2.7 1 cP and 57 cP FNA Model Samples 2.8 Data Acquisition 3 Methods 3.1 Smearing Tool Fabrication 3.2 PVA Film with Giemsa Stain 3.3 Blotting Unit 3.4 Microfluidic Chamber Fabrication 3.5 Instructions for Use 3.6 PANC-1 Cell Culture 3.7 1 cP and 57 cP FNA Model Samples 3.8 Cell Counting Area Frame 3.9 Smearing Speed Assessment with a Motorized Stage 3.10 Linearity Characterization 3.11 Liver, Lymph Node, and Thyroid FNA Model 3.12 5x and 40x Imaging of PANC-1, Liver, Lymph Node, and Thyroid Cells 3.13 Hemispherical Holder 3.14 Small Intestine Tissue Preparation 3.15 Small Intestine Tissue Imaging 3.16 Data Treatment 4 Notes References Chapter 11: Microfluidic Acoustic Method for High Yield Extraction of Cell-Free DNA in Low-Volume Plasma Samples 1 Introduction 2 Materials 2.1 Microfluidic Chip and Associated Components 2.1.1 Description of Fabrication Equipment 2.1.2 Description of Materials 2.1.3 Description of Components 2.2 Actuation Setup 2.3 cfDNA Extraction and Quantification 2.3.1 Reagents 2.3.2 Lab Equipment and Consumables 2.3.3 Other 3 Method 3.1 Preparation of the 3D-Printed Mold 3.2 Preparation of the Silicone Slab 3.3 Preparation of the Microfluidic Chip 3.4 Micromixer Assembly 3.5 On-Chip cfDNA Extraction (Plasma Sample 100 μL) 3.6 Cell-Free DNA Quantification and Fragment Analysis 4 Notes References Chapter 12: Isolation of Extracellular Vesicles by a Microfluidic Platform to Diagnose and Monitor Pancreatic Cancer 1 Introduction 2 Materials 2.1 Synthesis and Characterization of Fe3O4-EDC-NHS Nanoparticles 2.2 Exosomal Antibody Coupling 2.3 Microdevice Assembly 2.4 Exosomal Source (Serum and/or Blood) and Exosomal Capture 3 Methods 3.1 Synthesis and Characterization of the Fe3O4-EDC-NHS Nanoparticles Covered with the Exosomal Antibody 3.1.1 Fe3O4 Nanoparticle Synthesis 3.1.2 EDC/NHS Functionalization 3.1.3 Nanoparticle Characterization 3.1.4 Anti-CD9 Coupling 3.1.5 Nanoparticle-CD9 Complex Characterization 3.2 Fabrication of the Microdevice 3.3 Isolation of the Exosomes with the Microdevice 3.4 Characterization of the Isolated Exosomes 4 Notes References Chapter 13: High-Throughput Separation and Enrichment of Rare Malignant Tumor Cells from Large-Volume Effusions by Inertial Mi... 1 Introduction 2 Materials 2.1 Materials for Chip Fabrication of Microfluidic Sorter and Concentrator 2.2 Clinical Sample Preparation 2.3 Immunofluore-scence Staining 3 Methods 3.1 Fabrication of Microfluidic Sorter 3.2 Fabrication of Microfluidic Concentrator 3.3 Sample Preparation 3.4 MTC Separation and Enrichment Operation 3.5 Immunofluorescence Staining 4 Notes References Chapter 14: SAIF: Label-Free Separation of Circulating Tumor Cells Using a Self-Amplified Inertial Focusing Microfluidic Chip 1 Introduction 2 Materials 2.1 Materials for Fabrication of Silicon Masters 2.2 Materials and Equipment for Fabrication of PDMS Devices 2.3 Materials and Equipment for Blood Sample Processing 2.4 Materials and Equipment for Microfluidic Device Operation 2.5 96-Well Plate Treatment 2.6 Immunofluorescence Staining 3 Methods 3.1 Fabrication of Silicon Masters 3.2 Fabrication of PDMS Devices 3.3 Sample Processing 3.4 96-Well Plate Treatment with Polylysine 3.5 Operation of Microfluidic Device 3.6 CTC Separation 3.7 Immunofluore-scence Staining 3.8 Enumeration of CTCs 4 Notes References Chapter 15: Patient-Specific Microfluidic Cancer Spheroid Cultures for Testing Cancer Therapies 1 Introduction 2 Materials 2.1 Medium for Organoid Cultivation (Denoted as ``Organoid Media´´) 2.2 Digestion of Cancer Tissues and Biopsies 2.3 Maintenance and Propagation of Cancer Organoids 2.4 Fabrication of Microfluidic Device with Microwells 2.5 Seeding of Cancer Spheroids in Microfluidic Devices 2.6 Testing Cellular Response Against Drug in the Microfluidic Device 3 Methods 3.1 Preparation of Wnt-3A, R-Spondin, and Noggin Conditioned Medium (Denoted as ``L-WRN Conditioned Medium´´) 3.1.1 L-WRN Cell Culture 3.1.2 Collection of L-WRN Conditioned Medium 3.2 Digestion of Cancer Tissues and Biopsies and Seeding of Extracted Cells on Matrigel (Fig. 1) 3.3 Maintenance and Propagation of Cancer Organoids (Fig. 1; Matrigel culture) 3.4 Preparation of Microfluidic Device with Microwells (Fig. 2a and b) 3.5 Digestion of Organoids to Single Cells for Seeding into Microfluidic Device 3.6 Seeding and Maintenance of Cancer Spheroids in Microfluidic Device (Fig. 2c) 3.7 Testing Chemotherapies Using Microfluidic Cancer Cultures (Fig. 2e) 3.8 Assessment of Cellular Response against Drug in the Microfluidic Device (Fig. 2e) 3.8.1 Spheroid Size Growth Assessment Using ImageJ Software 3.8.2 Viability Assessment Using Live/Dead Staining Kit (Fig. 3) 4 Notes References Chapter 16: Isolation of Cancer Cells from Liquid Biopsies Using 3D-Printed Affinity Devices 1 Introduction 2 Materials 2.1 3D-Printed Microfluidic Device Fabrication 2.1.1 Instruments for 3D-Printed Microfluidic Device 2.1.2 Designs and Printing for 3D-Printed Microfluidic Device 2.2 3D-Printed Microfluidic Device Surface Modification/Antibody Conjugation 2.3 Detection of Clinical Cancer Cells 2.4 Imaging of Isolated Cancer Cells 3 Methods 3.1 3D Printing Microfluidic Device 3.2 Surface Modification 3.3 Isolation of Cancer Cells with 3D-Printed Microfluidic Device 3.4 Imaging 4 Notes References Chapter 17: A Microfluidic SERS Assay to Characterize the Phenotypic Heterogeneity in Cancer-Derived Small Extracellular Vesic... 1 Introduction 2 Materials 2.1 Cell Culture, Cell Passage, and sEV Generation 2.2 sEV Isolation Via Size Exclusion Chromatography 2.3 Fabrication of ESCP 2.4 Gold Nanoparticle Synthesis and SERS Nanotag Preparation 2.5 ESCP Assay 3 Methods 3.1 Cell Culture 3.2 Cell Passage 3.3 sEV Generation 3.4 Column Preparation for Size Exclusion Chromatography 3.5 sEV Isolation 3.6 ESCP Device Fabrication 3.7 ESCP Device Functionalization 3.8 Gold Nanoparticle Synthesis 3.9 SERS Nanotag Preparation 3.10 ESCP Assay 3.11 Raman Scanning and Data Processing 4 Notes References Chapter 18: Cluster-Wells: A Technology for Routine and Rapid Isolation of Extremely Rare Circulating Tumor Cell Clusters from... 1 Introduction 2 Materials 2.1 Micromachining of Silicon Wafers 2.1.1 Instruments 2.1.2 Materials for Wafer Fabrication 2.2 Fabrication of PDMS Molds 2.3 Fabrication of Devices 2.4 Device Preparation and Sample Processing 2.5 Immunofluorescence Staining 2.6 Preparation for Imaging 3 Methods 3.1 Micromachining of Silicon Wafers 3.1.1 Processing the First Mask Layer 3.1.2 Processing the Second Mask Layer 3.1.3 Processing the Third Mask Layer 3.2 Fabrication of PDMS Molds 3.3 Fabrication of Devices 3.4 Device Preparation and Sample Processing 3.5 Immunofluorescence Staining 3.6 Preparation for Imaging 4 Notes References Chapter 19: Secretion Function Analysis of Ex Vivo Immune Cells in an Integrated Microfluidic Device 1 Introduction 2 Materials 2.1 Master Mold Fabrication 2.2 Device Fabrication and Assembly 2.2.1 PDMS Replica 2.2.2 Glass Slide Modification 2.2.3 Chip Assembly 2.2.4 Microfluidic Control 2.2.5 Chip Setup 2.3 Immune Cell Isolation and Culture 2.4 On-Chip Immunoassay 2.5 Data Acquisition and Analysis 3 Methods 3.1 Master Mold Fabrication 3.1.1 Flow Layer Mold Fabrication 3.1.2 Control Layer Mold Fabrication 3.2 Device Fabrication 3.3 Glass Slide Modification 3.4 Microfluidic Device Assembly 3.5 Microfluidic Device Setup 3.6 Monocyte Isolation from Peripheral Blood and Culture (See Note 17) 3.7 Monocyte Seeding to Microfluidic Device 3.8 On-Chip Cell Secretory Immunophenotyping 3.9 Image Acquisition and Analysis 4 Notes References Chapter 20: Dynamic Tumor Perfusion and Real-Time Monitoring in a Multiplexed 3D Printed Microdevice 1 Introduction 2 Materials 2.1 Cells 2.2 Reagents 2.3 Equipment 3 Methods 3.1 Preparation for 3D Printing 3.2 3D Printing 3.3 3D Printed Device Post Processing 3.3.1 Removal and Cleaning 3.3.2 Polishing Procedure 3.3.3 Curing 3.3.4 Micromold Creation 3.3.5 Spheroid Formation Protocol 3.3.6 Platform Setup and Spheroid Loading/Staining 3.3.7 Imaging Setup 4 Notes References Chapter 21: Capture and Release of Cancer Cells Through Smart Bioelectronics 1 Introduction 2 Materials 2.1 Polymer Preparation 2.2 Microfabrication of Gold Electrodes 2.2.1 Instruments 2.2.2 Gold Electrode Microfabrication 2.2.3 PDMS Gasket Fabrication 2.3 Collection of Cells Using the Bioelectronic Device 2.3.1 Media Preparation 2.3.2 Cell Culture 2.3.3 Cell Capture and Release 3 Methods 3.1 Polymer Preparation 3.2 Microfabrication of Bioelectronic Devices 3.3 Adhesion of the PDMS Gasket 3.4 Preparation of Cell Suspension 3.5 Collection of Cells Using the Bioelectronic Device 4 Notes References Chapter 22: Fabrication of Multilayer Microfluidic Arrays for Passive, Efficient DNA Trapping and Profiling 1 Introduction 2 Materials 2.1 PDMS 2.2 Partitioning Oil 2.3 Tubing 3 Methods 3.1 Mold Fabrication 3.2 Device Fabrication 3.3 Device Loading 4 Notes References Index