Extracellular Vesicles : Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Extracellular Vesicles: A Brief Overview and Its Role in Precision Medicine
	1 Tissue Biopsy versus Liquid Biopsy
	2 What are Extracellular Vesicles?
	3 Biogenesis of EVs
	4 Diagnostic Potential of EVs
	5 Therapeutic Potential of EVs
	6 Conclusion
	References
Chapter 2: Red Blood Cells: A Source of Extracellular Vesicles
	1 Introduction
	2 The Mechanisms of EV Generation During RBC Storage
	3 Functional Consequences of EV Formation During Storage
	4 EVs Generated by RBC in Circulation by Complement
	References
Part I: Isolation of Extracellular Vesicles
	Chapter 3: Isolation of Extracellular Vesicles by Ultracentrifugation
		1 Introduction
		2 Materials
		3 Methods
			3.1 Differential Centrifugation/Ultracentrifugation Protocols
			3.2 Sucrose Gradient Separation Protocol
		4 Notes
		References
	Chapter 4: Sequential Filtration: A Gentle Method for the Isolation of Functional Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Prefiltration
			2.2 Tangential Flow Filtration (TFF) System
			2.3 Track Etch Filtration
		3 Methods
			3.1 Prefiltration of Biofluid
			3.2 Tangential Flow Filtration
			3.3 Track-Etch Filtration
		4 Notes
		References
	Chapter 5: Paper-Based for Isolation of Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Collection of Serum Samples
			2.2 Fabrication of Paper Devices
			2.3 Immunopurification
			2.4 Scanning Electromicrographs (SEM)
			2.5 RNA Isolation
			2.6 Paper-Based ELISA
		3 Methods
			3.1 Collection of Serum Samples
			3.2 Fabrication of Paper Devices
			3.3 Immunopurification
			3.4 SEM Analysis
			3.5 RNA Isolation
			3.6 Paper-Based ELISA
		4 Notes
		References
	Chapter 6: Filter-Based Extracellular Vesicle mRNA Isolation and High-Throughput Gene Expression Analysis
		1 Introduction
		2 Materials
			2.1 Urine Collection and Processing
			2.2 Ascites Collection
			2.3 Plasma Collection
			2.4 Extracellular Vesicle Capture
			2.5 mRNA Hybridization and Isolation
			2.6 cDNA Synthesis
			2.7 Real-Time PCR Analysis
		3 Methods
			3.1 Urine Collection and Processing
			3.2 Ascites Collection and Processing
			3.3 Plasma Collection and Processing
			3.4 Collection Tube Extracellular Vesicle Capture and mRNA Isolation
			3.5 Filterplate Extracellular Vesicle Capture and mRNA Isolation
			3.6 cDNA Synthesis
			3.7 Real-Time PCR Quantitation and Analysis
		4 Notes
		References
	Chapter 7: Specific and Generic Isolation of Extracellular Vesicles with Magnetic Beads
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Immunomagnetic Isolation
			2.3 Flow Cytometry Analysis
			2.4 Electrophoresis
			2.5 Western Blot
			2.6 Generic Capture and Release Buffers
			2.7 Transmission Electron Microscopy
		3 Methods
			3.1 Sample Preparation
			3.2 Exosome Enrichment from Urine
			3.3 Exosome Enrichment from Plasma and Serum
			3.4 Isolation of Exosomes with Magnetic Beads
				3.4.1 Specific and Direct Isolation of Exosomes
				3.4.2 Semi Generic and Direct Isolation of Exosomes
				3.4.3 Generic and Direct Isolation of Exosomes
			3.5 Release of Magnetic Bead-Bound Exosomes
				3.5.1 Release of Exosomes After Generic and Direct Isolation
			3.6 Analysis of Exosomes
				3.6.1 Flow Analysis of Magnetic Bead-Bound Exosomes
				3.6.2 Western Blot Analysis of Magnetic Bead-Bound Exosomes
				3.6.3 Electron Microscopy Analysis of Magnetic Bead-Bound Exosomes
			3.7 Function
				3.7.1 Analysis of Released Exosomes by Flow Cytometry
				3.7.2 Analysis of Released Exosomes by Electron Microscopy
			3.8 Complex Starting Materials
				3.8.1 Semi Generic and Direct Isolation of Exosomes from Plasma and Serum
				3.8.2 Generic and Direct Isolation of Exosomes from Plasma and Serum
				3.8.3 Semi Generic and Direct Isolation of Exosomes from Urine
			3.9 Analysis of Exosomes from Urine
				3.9.1 Flow Analysis of Magnetic Bead-Bound Exosomes
		4 Notes
		References
Part II: Purification of Extracellular Vesicles
	Chapter 8: Polymer-Based Purification of Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Exosome Production
			2.2 Exosome Purification
			2.3 Immunolabeling for Electron Microscopy
		3 Methods
			3.1 Exosome Generation
			3.2 Exosome Purification
			3.3 EM Immunolabeling
		4 Notes
		References
	Chapter 9: Size Exclusion Chromatography: A Simple and Reliable Method for Exosome Purification
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparing Columns and Centricons
			3.2 Concentrating Conditioned Media
			3.3 Purifying Exosomes
		4 Notes
		References
	Chapter 10: Purification Protocols for Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Ultracentrifugation
			2.2 Ultrafiltration
			2.3 Proprietary Polymer-Based Reagents
			2.4 Size Exclusion Chromatography (SEC)
			2.5 Density Gradient Centrifugation
			2.6 Immunoaffinity Isolation
		3 Methods
			3.1 Ultracentrifugation
			3.2 Ultrafiltration
			3.3 Proprietary Polymer-Based Reagents
			3.4 Size Exclusion Columns
			3.5 Density Gradient Centrifugation
			3.6 Immunoaffinity Isolation
		4 Notes
		References
Part III: Characterization of Extracellular Vesicles
	Chapter 11: Characterization of Extracellular Vesicles by Surface Plasmon Resonance
		1 Introduction
		2 Materials
			2.1 Extracellular Vesicle Isolation from Whole Blood
			2.2 Reagents for nPLEX Assays
			2.3 Fabrication of nPLEX Chip and Microfludic Flow Cell
			2.4 nPLEX Measurement System
		3 Methods
			3.1 Extracellular Vesicle Isolation from Whole Blood
			3.2 Antibody Biotinylation for nPLEX
			3.3 nPLEX Chip Fabrication
			3.4 Microfluidic Flow Cell Fabrication
			3.5 nPLEX Assay
		4 Notes
		References
	Chapter 12: Extracellular Vesicle Isolation and Analysis by Western Blotting
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 EV Isolation
			2.3 Western Blot
		3 Methods
			3.1 EV Isolation
			3.2 Western Blot
		4 Notes
		References
	Chapter 13: Analysis of Extracellular Vesicles Using Fluorescence Nanoparticle Tracking Analysis
		1 Introduction
		2 Materials
			2.1 NanoSight Instrumentation
			2.2 NanoSight Instrument Performance Check
			2.3 Preparation of CellMask Orange (CMO) Labeled EVs
			2.4 Preparation of EV Standards for Immunobead Depletion and Antibody Q-Dot Labeling
			2.5 Preparation of Qdot-Conjugated Antibodies
		3 Methods
			3.1 NTA: Instrument Performance Check
			3.2 Labeling of EVs Using CMO
			3.3 fl-NTA of Qdot-Conjugated-Antibody-Labeled EVs
				3.3.1 Preparation of EV Standards: Immunobead Depletion
				3.3.2 Antibody Qdot Conjugation
				3.3.3 Labeling the EV Standard with Qdot-Conjugated Antibodies
				3.3.4 NTA of the EV Standard Labeled with Qdot-Conjugated Antibodies
				3.3.5 Titration of Qdot-Conjugated Antibodies Using the EV Standard
		4 Notes
		References
	Chapter 14: Characterization of Extracellular Vesicles by Flow Cytometry
		1 Introduction
		2 Materials
			2.1 Dragon Green Polystyrene Beads: Bangs Laboratories, Inc. 9025 Technology Drive Fishers, Indiana 46038-2886 USA
			2.2 The ApogeeMix Product (Cat #1493): Apogee Flow Systems Unit 7, Grovelands, Boundary Way, Hemel Hempstead, HP2 7TE, UK
			2.3 Photon Correlation Spectroscopy Latex Beads 6602336: Beckman Coulter, Inc. Life Science Division Headquarters 5350 Lakevie...
			2.4 0.1% Tween 20 in PBS
			2.5 Specialized Instrumentation
		3 Methods
			3.1 Dragon Green Beads: Evaluation of Size and Fluorescence
			3.2 Photon Correlation Spectroscopy Beads (See Note 11)
			3.3 ApogeeMix Sensitivity and Resolution Performance
			3.4 100 nm Photon Correlation Spectroscopy Bead: Lower Detection Limit (See Note 15)
			3.5 Acquisition of EVs
			3.6 Sorting of EVs
		4 Notes
		References
	Chapter 15: Characterization of Extracellular Vesicles by Size-Exclusion High-Performance Liquid Chromatography (HPLC)
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Vesicle-Depleted Fetal Bovine Serum (FBS)
			3.2 Preprocessed Cell Culture Supernatant for Isolation of EVs
			3.3 EV Isolation from FBS or Preprocessed Cell Culture Supernatant
			3.4 Size-Exclusion HPLC Analysis
		4 Notes
		References
	Chapter 16: Multi-Surface Antigen Staining of Larger Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Solutions
			2.2 Antibodies
			2.3 Software
			2.4 Hardware
		3 Methods
		4 Notes
		References
	Chapter 17: Microcapillary Chip-Based Extracellular Vesicle Profiling System
		1 Introduction
		2 Materials
			2.1 Analytical Instrumentation
			2.2 Analysis Chip
		3 Methods
			3.1 Preparation of Microfluidic Chips
			3.2 Preparation of EV Samples
			3.3 Evaluation of Size, Zeta Potential, and Concentration of EVs
			3.4 Surface Protein Analysis of Individual EVs
			3.5 Multiparametic Characterization; Evaluation of Both Diameter and Surface Protein Expression of Individual Exosomes
		4 Notes
		References
Part IV: Imaging of Extracellular Vesicles
	Chapter 18: Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy
		1 Introduction
		2 Materials
		3 Methods
			3.1 Negative Staining
			3.2 Immunogold Labeling
			3.3 Cryo-Electron Microscopy
			3.4 TEM-/TSEM-Image Processing
		4 Notes
		References
	Chapter 19: Imaging of Isolated Extracellular Vesicles Using Fluorescence Microscopy
		1 Introduction
		2 Materials
		3 Methods
			3.1 Clean Glass Coverslip Preparation
			3.2 Sample Preparation
			3.3 Sample Addition to Coverslips and Imaging
			3.4 Imaging
		4 Notes
		References
Part V: Labeling and Tracking of Extracellular Vesicles
	Chapter 20: In Vivo Tracking of Extracellular Vesicles in Mice Using Fusion Protein Comprising Lactadherin and Gaussia Lucifer...
		1 Introduction
		2 Materials
			2.1 Transfection and EV Collection
			2.2 Evaluation of Serum EV Concentration Profile After Intravenous Injection
			2.3 In Vivo Imaging of Intravenously Administered EVs
		3 Methods
			3.1 Transfection and EV Collection
			3.2 Evaluation of Serum EV Concentration Profile of Intravenously Administered EVs
			3.3 In Vivo Imaging of Intravenously Administered EVs
		4 Notes
		References
	Chapter 21: Tracking Extracellular Vesicles Delivery and RNA Translation Using Multiplexed Reporters
		1 Introduction
		2 Materials
			2.1 Reporter Constructs, Extracellular Vesicle Donor, and Extracellular Vesicle Recipient Cells
			2.2 Extracellular Vesicle Isolation Equipment
			2.3 Extracellular Vesicle Fluorescence Assay
			2.4 EV Treatment
			2.5 Extracellular Vesicle Uptake Assay
			2.6 Extracellular Vesicle Bioluminescence and EV-RNA Translation Assays
		3 Methods
			3.1 Generation of Extracellular Vesicle Donor Cells
			3.2 Extracellular Vesicle Isolation
			3.3 Fluorescent Assay on Isolated Extracellular Vesicles
			3.4 Bioluminescent Assay on Isolated EVs or EV-RNA Translation
			3.5 Simultaneous Tracking of EV Delivery and EV-RNA Translation
		4 Notes
		References
Part VI: Downstream Extracellular Vesicle Applications: Genomics and Proteomics
	Chapter 22: Extraction and Analysis of Extracellular Vesicle-Associated miRNAs Following Antibody-Based Extracellular Vesicle ...
		1 Introduction
		2 Materials
			2.1 Preparation of Antibody-Coated Beads
			2.2 Plasma Preparation
			2.3 Isolation of EVs with Antibody-Coated Beads
			2.4 RNA Extraction and Purification
			2.5 RNA Quality and Quantity Control
			2.6 RT-qPCR of EV-miRNAs
		3 Methods
			3.1 Preparation of Antibody-Coated Beads
			3.2 Plasma Preparation
			3.3 Isolation of EVs with Antibody-Coated Beads
			3.4 RNA Extraction and Purification
			3.5 RNA Quality and Quantity Control
			3.6 RT-qPCR of EV-miRNAs
			3.7 Data Analysis and Limitations of the Protocol
		4 Notes
		References
	Chapter 23: Extracellular Vesicle miRNA Detection Using Molecular Beacons
		1 Introduction
		2 Materials
			2.1 Extracellular Vesicle-Free FBS
			2.2 Extracellular Vesicle Preparation in the Cell
			2.3 Extracellular Vesicle Isolation
			2.4 Extracellular Vesicle Quantification by ELISA (Enzyme-Linked Immunosorbent Assay)
			2.5 Detection of miRNA-21 (miR-21) in Extracellular Vesicles Using MB
			2.6 MB Delivery into Extracellular Vesicle Using Streptolysin O (SLO)
		3 Methods
			3.1 Extracellular Vesicle-Free FBS Preparation
			3.2 Extracellular Vesicle Production
			3.3 Extracellular Vesicle Isolation Using Total Exosome Isolation
			3.4 Extracellular Vesicle Isolation Using ExoQuick-TC
			3.5 Extracellular Vesicle Isolation by Differential Centrifugation
			3.6 Extracellular Vesicle Quantification by ELISA
			3.7 In Situ Detection of Extracellular Vesicle miRNA Using MB
			3.8 Enhanced Delivery of MBs into Extracellular Vesicles Using Streptolysin O (SLO)
		4 Notes
		References
	Chapter 24: Rapid Isolation of Extracellular Vesicles from Blood Plasma with Size-Exclusion Chromatography Followed by Mass Sp...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Preparation of Single-Use SEC Columns
			3.2 SEC Isolation of EVs
			3.3 Lipid Depletion Using Bligh-Dyer Liquid-Liquid Extraction Followed by Tryptic Digestion of the EV Isolate
			3.4 nLC-MS/MS Analysis
		References
	Chapter 25: An Adaptable Polyethylene Glycol-Based Workflow for Proteomic Analysis of Extracellar Vesicles
		1 Introduction
		2 Materials
			2.1 Polyethylene Glycol-Based Extracellular Vesicle Enrichment
			2.2 Optiprep Density Gradient Purification
			2.3 Protein Quantification
			2.4 Colloidal Coomassie Staining
			2.5 In-Gel Trypsin Digestion
		3 Methods
			3.1 PEG-Based Exosome Enrichment
			3.2 Optiprep Density Gradient Purification
			3.3 Western Blot Analysis
			3.4 Exosomal Protein Quantification
			3.5 Colloidal Coomassie Staining
			3.6 In-Gel Trypsin Digestion
			3.7 Summary
		4 Notes
		References
Part VII: Isolation of Extracellular Vesicles from Biofluids
	Chapter 26: Protocol for Exosome Isolation from Small Volume of Ovarian Follicular Fluid: Evaluation of Ultracentrifugation an...
		1 Introduction
			1.1 General Notes
		2 Materials
			2.1 Follicular Fluid Collection
			2.2 Ultracentrifugation
			2.3 Commercial Kits
			2.4 Controls
		3 Methods
			3.1 Human Follicles Sample Collection
			3.2 Equine and Bovine Follicles Sample Collection
			3.3 Clearing Protocol (Sample Processing)
			3.4 Ultracentrifugation Procedure
			3.5 Commercial Isolation Techniques (Summarized in Table 1)
			3.6 Positive and Negative Controls
		4 Support Protocols
			4.1 Granulosa Cells (GCs) Collection
			4.2 Cumulus Cells (CCs) Collection
			4.3 Exosome-Free Fetal Calf Serum (Exo-FBS) Preparation
		5 Notes
		References
	Chapter 27: Isolation of Extracellular Vesicles in Saliva Using Density Gradient Ultracentrifugation
		1 Introduction
		2 Materials
			2.1 Chemicals
			2.2 Plastic Tubes
			2.3 Centrifuges and Rotors
			2.4 Other Instruments
		3 Methods
			3.1 Saliva Collection
			3.2 Pretreatment (See Note 11)
			3.3 Preparation of Crude EVs
			3.4 Fractionation of Crude EVs Based on Densities (See Note 13)
			3.5 NTA Analysis [17] (See Note 8)
		4 Notes
		References
	Chapter 28: Isolation of Extracellular Vesicles from Breast Milk
		1 Introduction
		2 Materials
		3 Methods
			3.1 Breast Milk Collection
			3.2 Sample Preparation
			3.3 EV Isolation
		4 Notes
		References
	Chapter 29: An Integrated Double-Filtration Microfluidic Device for Detection of Extracellular Vesicles from Urine for Bladder...
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Urine Samples
			2.3 Devices
			2.4 ELISA
		3 Methods
			3.1 Device Fabrication
			3.2 Cell Culture
			3.3 EV Isolation Using Ultracentrifugation
				3.3.1 EVs Isolation from Cell Medium
				3.3.2 EVs Isolation from Urine
			3.4 EV Isolation Using Double Filtration
			3.5 Microplate ELISA
			3.6 Microchip ELISA
		4 Notes
		References
Part VIII: Extracellular Vesicles Isolated from Cell Culture, Parasites and Stem Cells
	Chapter 30: Electric Field-Induced Disruption and Releasing Viable Content from Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Ultracentrifugation-Based Extracellular Vesicle Extraction
			2.2 Radioimmunoprecipitation Assay (RIPA) Buffer-Based Extracellular Vesicle Cargo Unloading
			2.3 Magnetic Bead-Based Extracellular Vesicle Extraction
			2.4 Electric Field Induced Release and Measurement of Exosomal Content (EFIRM)
		3 Methods
			3.1 Ultracentrifugation-Based Extracellular Vesicle Extraction from Cell Culture
			3.2 Magnetic Bead-Based Extracellular Vesicle Extraction
			3.3 Radioimmunoprecipitation Assay (RIPA) Buffer-Based Extracellular Vesicle Cargo Unloading
			3.4 Electric Field Induced Release and Measurement of Extracellular Vesicle Content (EFIRM)
				3.4.1 Precoating of Electrode with a DNA Probe
				3.4.2 Extracellular Vesicle Cargo Unloading
				3.4.3 Reporter Antibody and Readout
		4 Notes
		References
	Chapter 31: Production and Characterization of Extracellular Vesicles in Malaria
		1 Introduction
		2 Materials
			2.1 Preparation of EV-Depleted Serum
			2.2 Cell Culture and Production of EVs
			2.3 Purification of EVs
			2.4 Size Fractionation of Cell Extracts Using a Fast Protein Liquid Chromatography System
		3 Methods
			3.1 Preparation of EV-Depleted Serum
			3.2 Cell Culture of iRBCs and Production of EVs
			3.3 Purification of EVs
			3.4 Size Fractionation of Purified EVs Using a Fast Protein Liquid Chromatography System
		4 Notes
		References
	Chapter 32: Isolation of Extracellular Vesicles from Stem Cells
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Reagent Setup
		3 Methods
			3.1 Cell Culture Using EV Depleted Culture Media
			3.2 Purification of EV by Precipitation Using PEG (See Note 2)
			3.3 Imaging IPSC-EV Using Transmission Electron Microscopy (TEM)
			3.4 Measuring the Size Distribution Using Nanoparticle Tracking Analysis (NTA)
		4 Notes
		References
Part IX: Extracellular Vesicles in Mouse Models
	Chapter 33: The Use of Peripheral Extracellular Vesicles for Identification of Molecular Biomarkers in a Solid Tumor Mouse Mod...
		1 Introduction
		2 Materials
			2.1 Cancer Stem Cell (CSCs) Isolation from Solid Tumor and Culture
			2.2 Xenograft Model
			2.3 Peripheral Blood Isolation
			2.4 Extracellular Vesicle Isolation
		3 Methods
			3.1 CSCs Isolation from Solid Tumor and Cell Culture
			3.2 Xenograft Model
				3.2.1 Orthotopic Model (Brain Location)
				3.2.2 Heterotopic Model (Flanks Location)
			3.3 Peripheral Blood Collection
			3.4 Isolation of Extracellular Vesicles
			3.5 gDNA Biomarkers
		4 Notes
		References
Part X: Therapeutic Applications of Extracellular Vesicles
	Chapter 34: Therapeutic Applications of Extracellular Vesicles: Perspectives from Newborn Medicine
		1 Introduction
		2 Stem Cell-Based Therapies in Newborn Medicine
		3 MSC Based Therapies for BPD
		4 Cell-Free Based Therapies: Extracellular Vesicles
		5 Therapeutic Application of MSC-EVs
		6 Application and Regulation of EV Therapies
		7 Stem Cell Origin of EVs
		8 EV Isolation
		9 Dose Evaluation
		10 Developing an EV Potency Assay
		11 Manufacturing, Scale-Up, Storage, and Reproducibility
		12 Toxicology and Safety
		13 Summary
		References
	Chapter 35: Therapeutic Use of Tumor Cell-Derived Extracellular Vesicles
		1 Introduction
		2 Materials
			2.1 Drug Loaded EVs
			2.2 Oncolytic Adenovirus Loaded EVs
			2.3 Mice Model
		3 Methods
			3.1 Preparation of Drug Loaded EVs
			3.2 Preparation of EVs Loaded Oncolytic Adenovirus
			3.3 Preparation of Oncolytic Adenovirus-Infected Cells EVs
			3.4 Preparation of Mouse Tumor Model
			3.5 Delivery of Drug Loaded EV to Mice
			3.6 Delivery of Drug Loaded EV to Tumor Patients
			3.7 T-EV-Loaded DCs act as Therapeutic Vaccines
				3.7.1 Isolation of Tumor Cell Derived EVs
				3.7.2 Preparation of T-EVs-Loaded DCs
				3.7.3 T-EV-Loaded DCs Exhibit Therapeutic Effects in Various Tumor Models
		4 Notes
		References
Index