Cell-Secreted Vesicles: Methods and Protocols (Methods in Molecular Biology, 2668)

Description

This detailed volume presents hands-on technological protocols used to target an array of cell-secreted extracellular vesicles (EVs) in a variety of biological systems. Beginning with methods for EV purification and analysis, the book continues with sections on the study of EV functions as well as specific systems and models allowing for the study of EVs of different origin. 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, Cell-Secreted Vesicles: Methods and Protocols serves as an ideal guide to conducting systematic assays in an effort to further our understanding of the mode of assembly, secretion, and targeting of EVs which will serve eventually as new therapeutic openings. Preface Contents Contributors Part I: Purification and Analysis of Single or Multiple EVs Chapter 1: Plasmon-Enhanced Characterization of Single Extracellular Vesicles 1 Introduction 2 Materials 2.1 Extracellular Vesicle Isolation 2.2 Reagents for nPLEX-FL Assays 2.3 Fabrication for nPLEX-FL Chips 2.4 nPLEX Measurement System 2.5 Image Analysis 3 Methods 3.1 Extracellular Vesicle Isolation 3.2 Biotinylation of Extracellular Vesicles 3.3 nPLEX-FL Chip Fabrication 3.4 nPLEX-FL Assay 3.5 Image Analysis 4 Notes References Chapter 2: Detection of Cell-Derived Exosomes Via Surface-Enhanced Raman Scattering Using Aggregated Silver Nanoparticles 1 Introduction 2 Materials 2.1 Preparation of Aggregator 2.2 Western Blotting 3 Methods 3.1 Sample Pretreatment 3.1.1 Preparation of Silver Nanoparticles 3.1.2 Cell Culture 3.1.3 Exosome Isolation and Preparation 3.2 Identification of Exosomes 3.3 Detection of Exosomes Using the SERS Method (see Fig. 2) 4 Notes References Chapter 3: In Vivo Analysis of Heterogeneous Extracellular Vesicles Using a Red-Shifted Bioluminescence Resonance Energy Trans... 1 Introduction 2 Materials 2.1 Cell Culture and Animals 2.2 Plasmids (see Fig. 1) 2.3 EV Isolation and Characterization 2.4 Ex Vivo Bioluminescence Measurements 2.5 Fluorescence Microscopy 2.6 In Vivo Bioluminescence Imaging (BLI) 3 Methods 3.1 Preparation of PalmReNL-EV Producer Cells and Conditioned Media 3.2 Enrichment of PalmReNL-EVs from Conditioned Media 3.3 Characterization of PalmReNL-EVs by Bioluminescence Measurement and Fluorescence Microscopy 3.4 Retro-Orbital (RO) or Intraperitoneal (IP) Injection of PalmReNL-EVs in Mice 3.5 Analysis of PalmReNL-EVs Circulating in the Blood 4 Notes References Chapter 4: Characterization of Extracellular Vesicles by Transmission Electron Microscopy and Immunolabeling Electron Microsco... 1 Introduction 2 Materials 2.1 EV Deposition 2.2 Fixation 2.3 Quenching/Blocking 2.4 Contrasting 2.5 Immunolabeling 3 Method 3.1 Conventional Transmission Electron Microscopy (TEM) 3.1.1 Immunolabeling 4 Notes References Chapter 5: Extracellular Vesicle Isolation by a Tangential-Flow Filtration-Based Large-Scale Purification Method 1 Introduction 2 Materials 2.1 Medium Conditioning and Initial Clarification 2.2 Tangential Flow Filtration 2.3 PEG Precipitation and PBS Resuspension 2.4 Capto Core 700 Chromatography 3 Methods 3.1 Medium Conditioning and Initial Clarification 3.2 Tangential Flow Filtration 3.3 PEG Precipitation and EV Resuspension 3.4 Capto Core 700 Chromatography 4 Notes References Chapter 6: Metabolomics Analysis of Urinary Extracellular Vesicles by Nuclear Magnetic Resonance and Liquid Chromatography-Mas... 1 Introduction 2 Materials 2.1 Urine Collection 2.2 Urinary EVs Isolation 2.3 EVs Characterization by Electron Microscopy 2.4 Metabolites Extraction 2.5 Untargeted Analysis by Nuclear Magnetic Resonance (NMR) 2.6 Targeted Analysis by Liquid Chromatography and Mass Spectrometry in Tandem (LC-MS/MS) 3 Methods 3.1 Urine Collection for EVs Analysis 3.2 EVs Isolation by Ultracentrifugation 3.3 EVs Characterization by Electron Microscopy 3.4 Metabolites Extraction from EVs 3.5 Metabolomic Analysis by NMR 3.5.1 Metabolomics Analysis by 1H NMR 3.5.2 Metabolomic Analysis by HR-MAS 3.6 Metabolomic Analysis by Targeted Mass Spectrometry 4 Notes References Chapter 7: Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium 1 Introduction 2 Materials 2.1 EV Production 2.1.1 MCB (P2) Thawing 2.1.2 CPC Expansion 2.1.3 Production of Conditioned Medium 2.1.4 CPC End of Production Cell (EPC) Freezing 2.1.5 Tangential Flow Filtration (TFF) and EV Enrichment 2.2 CPC-EV Quality Control Tests 2.2.1 Identity and Potency 2.2.2 Purity 2.2.3 Safety 3 Methods 3.1 EV Production 3.1.1 MCB (P2) Thawing 3.1.2 CPC Expansion 3.1.3 Production of Conditioned Medium 3.1.4 End of Production Cell (EPC) Freezing 3.1.5 Tangential Flow Filtration (TFF) and EV Enrichment 3.2 EV Quality Control 3.2.1 Identity and Potency 3.2.2 Purity 3.2.3 Safety 3.2.4 EV Stability 4 Notes References Chapter 8: Automated On-Line Isolation and Fractionation Method for Subpopulations of Extracellular Vesicles 1 Introduction 2 Materials 3 Methods 3.1 Immobilization of Antibodies on 1,1´-Carbonyldiimidazole Disks 3.2 Isolation of Subpopulations of Extracellular Vesicles by Immunoaffinity Chromatography 3.3 Fractionation of Subpopulations of Extracellular Vesicles by Asymmetrical Flow Field-Flow Fractionation On-Line Coupled to... 4 Notes References Part II: Targeting Cell Behavior Functions of EVs Chapter 9: A Novel Assay for Investigating the Role of Exosomes in Tumor Cell-Endothelial Cell Crosstalk 1 Introduction 2 Materials 2.1 Differential Centrifugation 2.2 Conditional Medium Transfer Assay 2.3 Co-Culture (HUVECs/HCC) Assay 3 Methods 3.1 Exosomes Isolation by Differential Centrifugation 3.2 Conditional Medium Transfer During HUVECs and HCC Cells´ Crosstalk 3.3 Role of HUVECs-Derived Exosomes on Tumor Cells´ Tubulogenesis by Co-Culture Assay 4 Notes References Chapter 10: Nanoparticle (NP) Loading by Direct Incubation with Extracellular Vesicles-Secretor Cells: NP Encapsulation and Ex... 1 Introduction 2 Materials 2.1 Cytotoxicity Test 2.2 Internalization Time Evaluation 2.3 Recovery of Loaded Exosomes 2.4 Characterization of the Recovered Exosomes 2.4.1 Protein Quantification 2.4.2 Exosomes Count by Nanoparticle Tracking Analysis (NTA) 2.4.3 Exosome Markers Evaluation by Immunoblot Detection 2.4.4 Evaluation of NP Loading 3 Methods 3.1 Cytotoxicity Test 3.2 Internalization Time Evaluation 3.3 Recovery of Loaded Exosomes 3.4 Characterization of the Recovered Exosomes 3.4.1 Protein Quantification 3.4.2 Exosome Count by Nanoparticle Tracking Analysis (NTA) 3.4.3 Exosome Markers Evaluation by Immunoblot Detection 3.4.4 Evaluation of NP Loading 4 Notes References Chapter 11: Two Complementary Strategies to Quantitate Extracellular Vesicle Uptake Using Bioluminescence and Non-Lipidic Dyes 1 Introduction 2 Materials 2.1 Buffers and Solutions 2.2 Cell Line Culture and Media 2.3 Consumable Materials and Instruments 2.4 EV Uptake Reporters and Staining Reagents 3 Methods 3.1 EV Uptake Luciferase Assay 3.1.1 Cell Culture and Previous Considerations 3.1.2 Performance of the Assay 3.1.3 Quantification 3.2 EV Uptake Flow Cytometry Assay 3.2.1 Cell Culture and Previous Considerations 3.2.2 EV Staining 3.2.3 Performance of the Assay 3.2.4 Quantification 4 Notes References Chapter 12: Integrin-Mediated Exosomal Homing to Organs 1 Introduction 2 Materials 2.1 Cell Culture and Exosome Isolation 2.2 Nanoparticle Tracking Analysis (NTA) 2.3 Flow Cytometry 2.4 Analysis of Integrin-Mediated Exosomal Binding to Ligand 2.5 In Vivo Exosome Homing Assay in a Competitive Context 3 Methods 3.1 Exosome Isolation 3.2 Characterization of Exosome Size by Nanoparticle Tracking Analysis (NTA) 3.3 Analysis of Exosomal Expressions of Integrins and Tetraspanins Using Flow Cytometry 3.3.1 Immobilization of Exosomes to Beads 3.3.2 Determination of Exosomal Marker Tetraspanin (CD9, CD63, and CD81) Expressions 3.3.3 Examination of Integrin Expression on Exosomes 3.4 Analysis of Integrin-Mediated Exosomal Binding to Ligand 3.4.1 Immobilization of MAdCAM-1 to Beads 3.4.2 Fluorescent Labeling of Exosome 3.4.3 Analysis of Exosomal Binding to Integrin Ligand 3.5 Competitive In Vivo Exosomal Homing Assay 3.5.1 Fluorescent Labeling of Exosomes 3.5.2 Competitive In Vivo Exosomal Homing Assay (See Fig. 4) 4 Notes References Chapter 13: Methods and Protocols for Using Extracellular Vesicles as Delivery Vehicles in Neuronal Research 1 Introduction 2 Materials 2.1 Loading Nucleic Acids and Proteins to Exosomes 2.1.1 Loading Nucleic Acids onto Exosomes Using Transfection Reagents 2.1.2 Targeted Loading of Proteins to Exosomes Using the Intrinsic Method: Addition of Exosome-Specific Tags (XPack/XStamp) Commercially Available Plasmids and Cell Line Cultures Cloning the Gene of Interest (e.g., NGB) into a Vector to Target Its Protein Product to Exosome Immunocytochemistry (ICC) of Transfected HEK293 to Detect NGB Protein Dot Blot of Transfected HEK293 Exosomes to Detect NGB Protein 2.1.3 Targeted Loading of Proteins to Exosomes Using Extrinsic Method: Exosome Surface Engineering via Click Chemistry 2.2 Enhanced and Targeted Localization of EVs/Exosomes to Specific Cells or Organs 2.2.1 Adding Peptide Tags BHP1 (Brain Homing Peptide 1) and NCAM (Neural Adhesion Molecule) to Target EV/Exosome Localization 2.3 Visualization of EV/Exosome Uptake and Internalization 2.3.1 Staining the Cells Using Fluorescent Lipophilic Dyes 2.3.2 Coculture, Conditioned Media Exchange, or a Direct EV/Exosome Treatment 2.3.3 Confocal Microscopy for Confirmation of EV/Exosome Uptake and Internalization 3 Methods 3.1 Loading of Nucleic Acids and Proteins to Exosomes 3.1.1 Loading of Nucleic Acids to Exosomes (Fig. 2) 3.1.2 Intrinsic Loading Proteins of Interest, e.g., RFP, NGB, to Exosomes Cloning NGB into XPack MSCV-XP-MCS-EF1α-Puro Cloning Lentivector (Fig. 3) Transfection of HEK293 Cell Line with NGB-XPack Clone Immunocytochemistry (ICC) of Transfected HEK293 to Detect NGB Protein Expressed in Cytoplasm Dot Blot for the Detection of NGB Protein in HEK293 Exosomes 3.1.3 Extrinsic Loading of Proteins to Exosomes via Exosome Surface Engineering (Click Chemistry) (Fig. 5) Click Chemistry Dot Blot to Confirm Presence of Antibody on Exosomal Surface. 3.2 Enhanced and Targeted Localization of EVs/Exosomes to Specific Cells or Organs (Fig. 6) 3.2.1 Adding Peptide Tags BHP1 (Brain Homing Peptide 1) and NCAM (Neural Adhesion Molecule) to Target EV/Exosome Localization 3.3 Visualization of EV/Exosome Uptake and Internalization (Fig. 7) 3.3.1 Staining the Cells Using Fluorescent Lipophilic Dyes 3.3.2 Coculture, Conditioned Media Exchange, or a Direct EV/Exosome Treatment 3.3.3 Confocal Microscopy for Confirmation of EV/Exosome Uptake and Internalization 4 Notes References Chapter 14: Quantitative Analysis of Extracellular Vesicle Release Using Artificial MicroRNAs Abbreviations 1 Introduction 2 Materials 2.1 Design of an Artificially Barcoded Exosomal microRNAs (bEXOmiRs) 2.2 Cloning of bEXOmiRs in a Mammalian Expression Vector 2.3 Analysis of bEXOmiR Expression and Abundance in Cells and Isolated EVs 2.4 EV Isolation Materials 2.5 Cell Culture 3 Methods 3.1 Design of an Artificially Barcoded Exosomal microRNAs (bEXOmiRs) 3.2 Cloning of bEXOmiRs in a Mammalian Expression Vector 3.3 Analysis of bEXOmiR Expression in HEK293T Cells 3.4 Detection and Quantification of bEXOmiRs in Isolated EVs 4 Notes References Part III: Systems and Models to Study EVs Chapter 15: Purification of Bacterial-Enriched Extracellular Vesicle Samples from Feces by Density Gradient Ultracentrifugation 1 Introduction 2 Materials 2.1 Materials and Reagents Required 2.2 Buffers 2.3 Density Gradient Preparation 2.4 NanoSight Measurements 2.5 Immuno-Electron Microscopy 2.6 Protein Isolation and Western Blotting 2.7 Analysis of EVs Using ExoView 3 Methods 3.1 Stool Sample Filtration 3.2 Concentration of the Fecal Filtrate 3.3 Isolation of Extracellular Vesicles by Size-Exclusion Chromatography 3.4 BEV Enrichment by Density Gradient Ultracentrifugation 3.5 Methods Used for EV Characterization 3.5.1 NanoSight Measurements 3.5.2 Immuno-Electron Microscopy 3.5.3 Protein Isolation and Western Blotting 3.5.4 Analysis of EVs Using ExoView 4 Notes References Chapter 16: Isolation and Characterization of Extracellular Vesicles from Lymphocytes 1 Introduction 2 Materials 2.1 Cell Culture 2.2 Activation of Cells 2.3 Isolation of EVs 2.4 Nanoparticle Tracking Analysis (NTA) 2.5 Transmission Electron Microscopy (TEM) 2.6 ExoView 2.7 Equipment 3 Methods 3.1 Cell Line and Culture Conditions 3.2 In Vitro Activation of Jurkat T Cells 3.3 Isolation of T Cell-Derived EVs: Sequential Filtration 3.4 Characterization of EVs Isolated from T Cells 3.4.1 Extracellular Vesicle Characterization with NTA System 3.4.2 TEM Imaging of EVs 3.4.3 Extracellular Vesicles Analyses with ExoView 4 Notes References Chapter 17: Biogenesis of Mesoporous Silica Nanoparticles Enclosed in Extracellular Vesicles by Mouse Renal Adenocarcinoma Cel... 1 Introduction 2 Materials 2.1 Fluorescence Mesoporous Silica Nanoparticle 25 nm TA 2.2 Producing Mouse Renal Adenocarcinoma Cell-Derived EV-FMSN 2.3 Western Blot 2.4 Negative Staining TEM and Immuno-TEM 3 Methods 3.1 Preparing Fluorescence Mesoporous Silica Nanoparticle 25 nm TA 3.2 Producing Mouse Renal Adenocarcinoma Cell-Derived EV-FMSN 3.3 Characterization of Renca EV-FMSN 3.3.1 Nanotracking Analysis (Time 1-2 h) 3.3.2 Zeta Potential Measurement (Time 1-1.5 h) 3.3.3 Western Blot (Time 13 h) 3.3.4 Negative Staining TEM and Immuno-TEM 3.4 Anticipated Results 4 Notes References Chapter 18: Magnetic Separation of Cell-Secreted Vesicles with Tailored Magnetic Particles and Downstream Applications 1 Introduction 2 Materials 2.1 Covalent Immobilization of Antibodies on Tosylactivated Magnetic Particles 2.2 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by ELISA 2.3 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by Bradford 2.4 Immunomagnetic Separation of the Exosomes on Tailored Magnetic Particles 2.5 Covalent Immobilization of Exosomes on Tosylactivated Magnetic Particles 2.6 Characterization of the Exosomes by Flow Cytometry 2.7 Characterization of the Exosomes by Confocal Microscopy 2.8 Quantification of the Exosomes by Magneto-Actuated Immunoassay 3 Methods 3.1 Covalent Immobilization of Antibodies on Tosylactivated Magnetic Particles 3.2 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by ELISA 3.3 Determination of the Amount of Antibody Immobilized on Tailored Magnetic Particles by Bradford 3.4 Immunomagnetic Separation of the Exosomes on Tailored Magnetic Particles 3.5 Covalent Immobilization of Exosomes on Tosylactivated Magnetic Microparticles 3.6 Characterization of the Exosomes by Flow Cytometry 3.7 Characterization of the Exosomes by Confocal Microscopy 3.8 Quantification of the Exosomes by Magneto-Actuated Immunoassay 4 Notes References Chapter 19: Cilia-Derived Extracellular Vesicles in Caenorhabditis Elegans: In Vivo Imaging and Quantification of Extracellula... 1 Introduction 2 Materials 3 Methods 3.1 Preparation of NGM Plates 3.2 OP50 Bacterial Stock, OP50 Liquid Culture, and Seeding Plates 3.2.1 OP50 Bacterial Stock 3.2.2 OP50 Liquid Culture 3.2.3 Seeding Plates 3.3 Synchronizing Worm Populations by Egg-Laying Window 3.4 Imaging of EV Release from Ciliated Neurons in Living Animals 3.4.1 Sample Preparation 3.4.2 Time-Lapse Imaging Using Confocal Microscopy 3.4.3 Quantification of the Number of Released EVs/Time 3.5 Imaging and Quantification of Ciliary-Derived EVs in Their Capturing Tissue 3.5.1 Sample Preparation 3.5.2 Imaging (Z-Stacks) Using Confocal Microscopy 3.5.3 Quantification of EV Fluorescence in a ROI 3.5.4 Quantification of EV Number in a ROI Using ComDet 3.6 Markers for the Study of Ciliary EVs in C. Elegans 4 Notes References Chapter 20: Exosome-Based COVID-19 Vaccine 1 Introduction 2 Materials 2.1 Cell Culture and Lentiviral Transduction 2.2 Ultracentrifugation 2.3 Immunoblotting 2.4 Transmission Electron Microscopy (TEM) 3 Methods 3.1 Cell Culture and Lentiviral Transduction 3.2 Ultracentrifugation-Based Exosome Isolation 3.3 Characterization of Exosomes 3.3.1 Immunoblotting 3.3.2 Immunoblotting: Characterization of S, M, E Exosomal Vaccines 3.3.3 Immuno-Transmission Electron Microscopy (TEM) 3.3.4 Transmission Electron Microscopy 4 Notes References Index