Vascular Tissue Engineering: Methods and Protocols (Methods in Molecular Biology, 2375)

✍ Scribed by Feng Zhao (editor), Kam W. Leong (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 264
• Category: Library

✦ Synopsis

This volume explores the latest techniques used to study the field of tissue engineered vascular grafts (TEVGs). The chapters in this book cover a wide array of topics such as deriving vascular cells from monocytes and induced pluripotent stem cells; engineering vascular grafts using various biomaterials and stem cells, stem cell-derived, or primary vascular cells; biomaterial modification by anticoagulation molecules; vascular bioengineering technologies such as 3D bioprinting; and fabrication of TEVGs with different geometry and multiphase structures. This book also features protocols for grafting and evaluation of vascular grafts in animal models, vascular imaging in animals, and the quantification of blood vessel permeability. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tipson troubleshooting and avoiding known pitfalls.

Cutting-edge and practical, Vascular Tissue Engineering: Methods and Protocols is a valuable resource for biomedical engineers, cell biologists, vascular surgeons, doctors, and nurses.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Differentiating Human Pluripotent Stem Cells to Vascular Endothelial Cells for Regenerative Medicine, Tissue Engine...
1 Introduction
2 Materials
2.1 Matrigel Coating
2.2 Thawing hPSCs
2.3 Maintenance of hPSCs
2.4 Single-Cell Passaging of hPSCs
2.5 Differentiation Media
2.6 Purification of hPSC-ECs
2.7 Maintenance and Passaging of hPSC-ECs
3 Methods
3.1 Preparation of Matrigel-Coated Tissue Culture Plastic Dishes (See Note 2)
3.2 Thawing of hPSCs
3.3 Routine Clonal Passaging of hPSCs
3.4 Single-Cell Passaging of hPSCs for EC Differentiation (Day 2 to Day 0)
3.5 Endothelial Cell Differentiation (Day 0 to Day 6)
3.6 Purification of hPSC-ECs Via Magnetic-Activated Cell Sorting (Day 6)
3.7 Culture of hPSC-Derived VECAD+ Endothelial Cells
4 Notes
References
Chapter 2: Generating Monocyte-Derived Endothelial-like Cells for Vascular Regeneration
1 Introduction
2 Materials
2.1 Cell Media
2.2 Culture Surface Preparation
2.3 Other Materials
2.4 Shear Plate Preparation
3 Methods
3.1 Monocyte Isolation
3.2 Monocyte Culture and Differentiation Under Static Conditions: See Fig. 1 for Schematic of Time Course
3.3 MC-EC Culture Under Shear Conditions
4 Notes
References
Chapter 3: Methods for Differentiating hiPSCs into Vascular Smooth Muscle Cells
1 Introduction
2 Materials
2.1 In Vitro Differentiation of VSMCs Via EB Formation
2.2 In Vitro Differentiation of Lineage-Specific VSMCs Via a Chemically Defined Method
2.3 Derivation of VSMCs on Monolayer Cultures of ECM Proteins with a Pulsatile Flow
3 Methods
3.1 In Vitro Differentiation of VSMCs Via EB Formation
3.2 In Vitro Differentiation of Lineage-Specific VSMCs Via a Chemically Defined Method
3.3 Derivation of VSMCs on Monolayer Cultures of ECM Proteins with a Pulsatile Flow
4 Notes
References
Chapter 4: On-Site Differentiation of Human Mesenchymal Stem Cells into Vascular Cells on Extracellular Matrix Scaffold Under ...
1 Introduction
2 Materials
2.1 Decellularization
2.2 Cell Culture and Differentiation
2.3 Real-Time RT-PCR Analysis
3 Methods
3.1 Preparation of Decellularized ECM Scaffolds (See Note 5)
3.2 Cell Culture and On-Site Differentiation of MSCs on Decellularized Vascular ECM Scaffold in a Rotary Bioreactor
3.2.1 Cell Maintenance Culture
3.2.2 MSCs Differentiation into ECs
3.2.3 MSCs Differentiation into VSMCs
3.3 Real-Time RT-PCR Analysis
4 Results and Discussion
5 Notes
References
Chapter 5: End-Point Immobilization of Heparin on Electrospun Polycarbonate-Urethane Vascular Graft
1 Introduction
2 Materials
2.1 Fabrication of Electrospun PCU Vascular Graft
2.2 Surface Amination of PCU Vascular Graft by Plasma Treatment
2.3 Preparation of Aldehyde-Terminated Heparin
2.4 End-Point Heparin Immobilization of Aminated Vascular Grafts
2.5 Amine Detection and Quantification of Plasma-Treated Grafts
2.6 Heparin Quantification of Heparin-Modified Vascular Grafts
2.7 Antithrombogenic Evaluation of Heparin-Modified Vascular Grafts
3 Methods
3.1 Fabrication of Electrospun PCU Vascular Graft
3.2 Surface Amination of PCU Vascular Graft by Plasma Treatment
3.3 Preparation of Aldehyde-Terminated Heparin
3.4 End-Point Heparin Immobilization of Aminated Vascular Grafts
3.5 Amine Detection and Quantification of Plasma-Treated Grafts
3.6 Heparin Quantification of Heparin-Modified Vascular Grafts
3.7 Antithrombogenic Evaluation of Heparin-Modified Vascular Grafts
4 Notes
References
Chapter 6: Microfluidic Coaxial Bioprinting of Hollow, Standalone, and Perfusable Vascular Conduits
1 Introduction
2 Materials
2.1 Bioink
2.2 Cells
2.3 Media and Other Cell Reagents
2.4 MCCES
2.5 Bioprinting of the Vascular Tubes
2.6 Structural and Functional Properties of Bioprinted Vascular Tubes
3 Methods
3.1 Preparation of GelMA
3.2 Preparation of the Blend Bioink
3.3 Maintenance and Preparation of Cells
3.4 Fabrication of the Three-Layered MCCES
3.5 Bioprinting of Multilayered Vascular Conduits Using the MCCES
3.6 Visualization of the Bioprinted Hollow Tubes Using Fluorescent Microbeads
3.7 Viability of Cells Within Bioprinted Vascular Conduits
3.8 Functionality of Cells Within Bioprinted Vascular Conduits
4 Notes
References
Chapter 7: In Situ Fabrication and Perfusion of Tissue-Engineered Blood Vessel Microphysiological System
1 Introduction
2 Materials
2.1 Cells
2.2 Cell Culture Media
2.3 Cell Culture Supplies
2.4 Materials for Mold Assembly
2.5 Materials for TEBV Fabrication
2.6 Materials for TEBV Perfusion
2.7 Materials for Vasoactivity Testing
3 Methods
3.1 Set up Mandrels and Side Connectors
3.2 Passage Primary Cells (ECs, hNDFs, and hMSCs)
3.3 Passage viSMCs and viECs
3.4 TEBV Fabrication
3.5 Plastic Compression and Dehydration of TEBVs In Situ
3.6 Endothelial Seeding of TEBVs
3.7 TEBV Perfusion
3.8 Vasoactivity Testing
3.9 En Face Immunofluorescence Imaging of TEBV Endothelium
4 Notes
References
Chapter 8: Peritoneal Pre-conditioning Method for In Vivo Vascular Graft Maturation Utilizing a Porous Pouch
1 Introduction
2 Materials
2.1 Electrospinning
2.2 Hydrogel Pouch Fabrication
2.3 Pouch Implantation in the Peritoneal Cavity and Aortal Grafting
3 Methods
3.1 Electrospinning
3.2 Hydrogel Pouch Fabrication
3.3 Pouch Implantation in the Peritoneal Cavity and Aortal Grafting
3.4 Graft Monitoring and Removal
4 Notes
References
Chapter 9: Fabrication of a Completely Biological and Anisotropic Human Mesenchymal Stem Cell-Based Vascular Graft
1 Introduction
2 Materials
2.1 Aligned Polydimethylsiloxane (PDMS) Substrate Preparation
2.2 Cell Culture Reagents
2.3 Reagents for Sample Preparation
2.4 Other Equipment
3 Methods
3.1 Development of Aligned PDMS
3.2 hDF Sheet Culture
3.3 hDF Sheet Decellularization
3.4 hMSC-ECM Sheet Culture
3.5 TEVG Assembly
3.6 ECM Alignment Examination by SEM
3.7 H&E Staining
3.8 Differentiation Examination
3.9 Mechanical Strength Test
4 Notes
References
Chapter 10: Engineering Vascular Grafts with Multiphase Structures
1 Introduction
2 Materials
2.1 Required Reagents
2.2 Electrospinning Equipment
2.3 Mechanical Characterization Equipment
3 Methods
3.1 Synthesis of POC Pre-polymer
3.2 Intimal Layer Formation: Dipcoating
3.3 Medial Layer Formation: Electrospinning
3.4 Mechanical Characterization of the Graft
4 Notes
References
Chapter 11: Fabrication of Small-Diameter Tubular Grafts for Vascular Tissue Engineering Applications Using Mulberry and Non-m...
1 Introduction
2 Materials
2.1 Silk Fibroin (SF) Source
2.2 Silk Fibroin (SF) Extraction
2.3 Micro-patterned Polydimethylsiloxane (PDMS) Molds and Silk Film Fabrication
2.4 Isolation of Vascular Cells from Porcine Aorta
2.5 Multilayered Silk Film-Based Tubular Graft Fabrication and Maturation
2.6 Mold Parts (for Bi-layered Silk Scaffold-Based Vascular Graft)
2.7 Bi-layered Tubular Graft Fabrication
3 Methods
3.1 GRAFT I (Multilayered Silk Films-Based Vascular Graft)
3.1.1 Fabrication of Silk Films (See Note 7)
3.1.2 Isolation and Culture of Vascular Cells (See Note 11)
3.1.3 Culture of Vascular Cells onto Patterned Silk Films
3.1.4 Fabrication and Maturation of Multilayered Vascular Graft
3.2 GRAFT II (Bi-layered Silk Scaffold-Based Vascular Graft)
4 Notes
References
Chapter 12: Fabrication and Evaluation of Tissue-Engineered Vascular Grafts with Hybrid Fibrous Structure
1 Introduction
2 Materials
2.1 Electrospinning
2.2 Surgical Preparation
2.3 Histological Analysis
3 Methods
3.1 Co-electrospinning of Vascular Grafts
3.1.1 Fabrication of PCL/PDS Vascular Grafts
3.1.2 Fabrication of PCL/COL-7A Vascular Grafts
3.2 Rat Abdominal Aorta Replacement Model
3.3 Histological Analysis
3.3.1 H&E Staining
3.3.2 Immunofluorescence Staining
4 Notes
References
Chapter 13: Controlling Pore Size of Electrospun Vascular Grafts by Electrospraying of Poly(Ethylene Oxide) Microparticles
1 Introduction
2 Materials
2.1 Preparation of Polymer Solution
2.2 Removal of PEO Microparticles
3 Methods
3.1 Fabrication of Electrospraying PEO Microparticles
3.2 Characterization of PEO Microparticles
3.3 Fabrication of PCL/PEO Composite Vascular Grafts
3.4 Removal of PEO Microparticles from PCL/PEO Composite Vascular Grafts
3.5 Characterization of Porogenic PCL Grafts
3.5.1 SEM Analysis
3.5.2 Mechanical Testing
4 Notes
References
Chapter 14: Injectable Hydrogels for Vascular Tissue Engineering
1 Introduction
2 Materials
2.1 Reagents
2.2 Instruments
2.3 Cell Lines
2.4 Animals
3 Methods
3.1 Synthesis of Biomaterials
3.1.1 Synthesis of PPC
3.1.2 Synthesis of PPC-ET
3.1.3 Synthesis of CRGDS-Functionalized 8-Arm PEG Derivatives
3.1.4 Hydrogel Fabrication
3.2 Characterization of Biomaterials
3.2.1 Dynamic Rheology Measurement
3.2.2 Swelling and Porosity Measurement
3.2.3 Morphology of Hydrogels
3.2.4 In Vitro Degradation of Hydrogels
3.3 Release Kinetics Study
3.4 In Vitro Studies
3.4.1 Cytotoxicity Study
3.4.2 Cell Adhesion Study
3.4.3 Effects of Mydgf Composite Hydrogels on the Vascularization of HUVECs
3.5 Rat Acute Myocardial Infarction (AMI) Model and Evaluation of Heart Functions After Hydrogel Injection
3.5.1 AMI Model
3.5.2 Hydrogel Injection
3.5.3 Cardiac Echocardiography and Micro-PET Measurements
3.6 Morphometric and Histological Studies for Heart Structure Evaluation
3.7 Immunohistochemistry in the Evaluation of Neovascularization
3.8 Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling (TUNEL) Staining to Measure Apoptosis
4 Notes
References
Chapter 15: Rabbit Surgery Protocol for End-to-End and End-to-Side Vascular Graft Anastomosis
1 Introduction
2 Materials
2.1 Supplies for Pre-surgical Procedures
2.2 Supplies for the Tubular Graft Implantation Surgery
2.3 Supplies for Post-operation Animal Care
3 Methods
3.1 Pre-surgical Procedures
3.1.1 Two to Seven Days Before Surgery
3.1.2 One Day Before Surgery
3.1.3 Surgery Day Pre-surgery Preparation
3.2 End-to-Side Tubular Graft Implantation Surgical Procedure
3.3 End-to-End Tubular Graft Implantation Surgical Procedure
3.4 Postoperative Animal Care
4 Notes
References
Chapter 16: Vascular Imaging in Small Animals Using Clinical Ultrasound Scanners
1 Introduction
2 Materials
2.1 Supplies for Imaging
2.2 Equipment for Imaging
3 Methods
3.1 Pre-imaging Preparation
3.2 Preoperative Ultrasound Imaging
3.3 Postoperative Ultrasound Imaging Without Anesthesia
3.3.1 Checklist for Suitability of Imaging Without Anesthesia
3.3.2 Imaging Steps for Suitable Animals
3.4 Ultrasound Imaging at Endpoint with Contrast Agent
4 Notes
References
Chapter 17: Vascular Graft Implantation Using a Bilateral End-to-Side Aortoiliac Preclinical Model
1 Introduction
2 Materials
2.1 Graft Implant
2.2 Graft Explant
3 Methods
3.1 Implantation of Bilateral Aortoiliac Arterial Bypass Grafts
3.2 Humane Euthanasia and Explantation of Arterial Bypass Grafts
4 Notes
References
Chapter 18: Quantification of In Vitro Blood-Brain Barrier Permeability
1 Introduction
2 Materials
2.1 Mammalian Ringer´s Solution
2.2 1% BSA Solution
2.3 Fluorescently Labeled Solute Solutions
2.4 Cells Needed to Generate the In Vitro BBB
3 Methods
3.1 Generation of In Vitro BBB
3.2 Calibration Curve for Solute Permeability Measurement
3.3 Determination of Solute Permeability of In Vitro BBB (PBBB)
3.4 Determination of Hydraulic Conductivity of In Vitro BBB (Lp BBB)
3.5 Determination of Trans-BBB Electric Resistance of In Vitro BBB (TERBBB)
3.6 Representative Results for LpBBB, PBBB, and TERBBB
4 Notes
References
Chapter 19: Subcellular Force Quantification of Endothelial Cells Using Silicone Pillar Arrays
1 Introduction
2 Materials
2.1 Microfabrication of the Micropillar Arrays
2.2 Soft Lithography of PDMS Micropillars
2.3 Micropatterning Micropillar Arrays with Stamp-Off Technique
2.4 Cell Seeding
2.5 Imaging
2.6 Traction Force Analysis
3 Methods
3.1 Microfabrication of the Micropillar Arrays
3.2 Soft Lithography of PDMS Micropillars
3.2.1 Methods for Stamp Fabrication
3.2.2 Method for Negative Molds for Micropillar Arrays
3.2.3 Method for Micropillar Arrays
3.3 Micropatterning Micropillar Arrays with Stamp-Off Technique
3.4 Cell Seeding
3.5 Imaging
3.6 Traction Force Analysis
4 Notes on Applications of Micropillars
References
Chapter 20: Computational Assessment of Hemodynamics Vortices Within the Cerebral Vasculature Using Informational Entropy
1 Introduction
2 Materials
2.1 Computational Workstation
2.2 Computational Software and Programing Languages
3 Methods
3.1 Creation of 3D Patient-Specific Vasculature Computational Structure from 3D Digital Subtraction Angiography Images in DICO...
3.2 Computational Fluid Dynamic Simulation and Flow Vortex Identification
4 Notes
References
Index