Organ Regeneration: Methods and Protocols (Methods in Molecular Biology, 1001)

✍ Scribed by joydeep basu (editor), John W Ludlow (editor)

• Publisher: Humana
• Year: 2013
• Tongue: English
• Leaves: 380
• Category: Library

✦ Synopsis

Tissue engineering and regenerative medicine represents a wide array of cell, biomaterial and cell/biomaterial based approaches focusing on the repair, augmentation, and regeneration of diseases tissues and organs. Organ Regeneration: Methods and Protocols has been assembled in response to the growing interest in organ and tissue regeneration as a means to treat disease. Topics cover methods such as isolation and characterization of cells from selected soft tissues and solid organs, preparation and evaluation of natural and synthetic biomaterial scaffolding, implantation of regenerative constructs within experimental animals, and evaluation of regenerative outcomes by molecular and histological methodologies. Written in the successful Methods in Molecular Biology^TMseries format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Authoritative and easily accessible, Organ Regeneration: Methods and Protocols serves as a detailed guide to aid newcomers and seasoned veterans in their developmental and experimental work in tissue engineering and regenerative medicine.

✦ Table of Contents

Organ Regeneration
Preface
Contents
Contributors
Chapter 1: Isolation and Characterization of Human Adipose-Derived Stem Cells for Use in Tissue Engineering
1 Introduction
2 Materials
2.1 Equipment
2.2 Adipose Tissue Digestion
2.3 Growth, Maintenance, and Cryopreservation of ASCs
2.4 Analysis of Stem Cell Surface Markers
2.5 Determining Stem Cell Potential
3 Methods
3.1 Preparation of Lipoaspirate
3.2 Collagenase Digestion
3.3 Isolation of the Stromal Vascular Fraction
3.4 Plating and Expansion of ASCs
3.5 Harvest and Cryopreservation of ASCs
3.6 Analysis of Stem Cell Surface Markers
3.7 Adipogenesis
3.8 Osteogenesis
3.9 Chondrogenesis
4 Notes
References
Chapter 2: Isolation of Smooth Muscle Cells from Bladder for Generation of Engineered Urologic Organs
1 Introduction
2 Materials
2.1 Explantation Method
2.2 Enzymatic Digestion Method
3 Methods
3.1 Explantation Method
3.1.1 Biopsy/Tissue Handling and Preparation
3.1.2 Tissue Dissection
3.1.3 Smooth Muscle Cell Subculturing
3.2 Enzymatic Isolation Method
3.2.1 Enzyme Preparation
3.2.2 Biopsy / Tissue Handling and Preparation
3.2.3 Tissue Dissection
3.2.4 Smooth Muscle Cell Subculturing
4 Notes
References
Chapter 3: Isolation of Urothelial Cells from Bladder Tissue
1 Introduction
2 Materials
2.1 Tissue Culture
2.2 Reagents
2.3 Other Equipment
3 Methods
3.1 UC Cell Extraction
3.2 UC Feeding
3.3 UC Passaging
3.4 UC Cryopreservation
4 Notes
References
Chapter 4: Isolation of Pulsatile Cell Bodies from Esophageal Tissue
1 Introduction
2 Materials
2.1 Adult Rat Esophagus
2.2 Culture Plasticware
2.3 Stainless Steel Instruments and Surgical Material
2.4 Equipment
2.5 Culture Media Components and Supplements
2.6 Formulations
3 Methods
3.1 Preparation of Matrigel Plates
3.2 Esophageal Organoid Isolation and Pulsatile Body Culturing
4 Notes
References
Chapter 5: Cell Isolation Through Whole-Liver Perfusion and Preparation of Hepatocytes for Cytochrome P450 Analysis
1 Introduction
2 Materials
2.1 Chemicals
2.2 Culture Media and Supplements
2.3 Buffers and Solutions
2.4 Tissue Culture Materials
2.5 Hardware
2.6 Surgical Instruments
2.7 Equipment
3 Methods
3.1 Preparation ( See Fig.  2)
3.2 Whole-Liver Perfusion
3.3 Percoll Gradient
3.4 ECOD Assay Preparation (Suspension)
4 Notes
References
Chapter 6: Ex Vivo Culture and Separation of Functional Renal Cells
1 Introduction
2 Materials, Reagents, and Equipment
2.1 Materials
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Renal Cell Isolation
3.2 Renal Cell Cryopreservation
3.3 Renal Cell Culture and Harvest Procedure
3.4 Renal Cell Fractionation
4 Notes
References
Chapter 7: Isolation and Myogenic Differentiation of Mesenchymal Stem Cells for Urologic Tissue Engineering
1 Introduction
2 Materials
2.1 Cells Isolation and Culture
2.2 Myogenic Differentiation of Human BMSC
2.3 Flow Cytometry
2.4 Immunofluore-scence Staining
2.5 PCR
2.6 Western Blotting
3 Methods
3.1 Isolation and Culture of Bone Marrow-Derived MSC
3.2 Flow Cytometry
3.3 Differentiation of Human BMSC In Vitro: Myogenic Growth Factors
3.4 Differentiation of Human BMSC In Vitro: Cocultured with Bladder SMC or SMC Conditioned Medium
3.5 BMSC Proliferation on Bladder ECM
3.5.1 Preparation of the Bladder Tissue-Specific ECM
3.5.2 BMSC Proliferate and Myogenic Differentiation on Bladder ECM
3.6 In Vivo Myogenic Differentiation
3.7 Immunofluor-escence Staining of SMC Markers
3.8 PCR for SMC-Specific Gene Expression
3.9 Western Blot for SMC Markers
3.9.1 BMSC Cell Detachment and Cell Lysate Preparation
3.9.2 Western Blot Analysis of SMC-Specific Markers
4 Notes
References
Chapter 8: Xeno-Free Adaptation and Culture of Human Pluripotent Stem Cells
1 Introduction
2 Materials
2.1 Culture Components and Media
2.2 Cultureware
2.3 Equipment
3 Methods
3.1 Culture Dish preparation
3.2 Negative Selection
3.3 Passaging hPSCs: Enzymatic
3.4 Passaging hPSCs: Manual
3.5 Cryopreservation
3.6 Thawing
4 Notes
References
Chapter 9: Reverse Engineering Life: Physical and Chemical Mimetics for Controlled Stem Cell Differentiation into Cardiomyo...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Mold Fabrication
2.3 Hydrogel Patterning and Cell Culture
3 Methods
3.1 Stem Cell Maintenance Cultures (Fig.  1)
3.2 Preparation of Geltrex-Coated Plates (Adapted from Manufacturer’s Instructions, See Note 6)
3.3 Preparation of MEF Conditioned Medium
3.4 Growth of Human Embryonic Stem Cells
3.5 Forced Aggregation of Stem Cells
3.6 Cardiac Specification (After ~5 Days in RPMI-PVA)
3.7 Cardiomyocyte Development
3.8 Physical Signals to Enhance Cardiomyocyte Differentiation
3.8.1 Cleaning Silicon Wafers to Remove Organic Contaminants
3.8.2 Photoresist Coating (Fig.  2a)
3.8.3 Photomask Creation and Alignment (Fig.  2b)
3.8.4 Exposure, Baking, and Development of the Photoresist Layer (Fig.  2c, d)
3.8.5 Preparing Negative Polydimethylsiloxane Template (Fig.  3)
3.8.6 Cast Tissue Molds
3.8.7 Prepare PDMS for Cell-Hydrogel Molding
3.8.8 Molding the Cell-Hydrogel Mixture
4 Notes
References
Chapter 10: Phenotypic Analysis of Bioactive Cells for Application in Regenerative Medicine
1 Introduction
2 Materials
2.1 Reagents
2.2 Solutions
2.3 Equipment
3 Methods
3.1 Initial Identification of Target Cell Types from Primary Tissue Source
3.2.1 Characterization of Surface/Membrane Antigens
3.2.2 Characterization of Intracellular/Cytoplasmic Antigens
3.3 FACS Characterization of Primary Cultured Cells Using Single-Color Staining
3.3.1 Characterization of Surface/Membrane Antigens
3.3.2 Characterization of Intracellular/Cytoplasmic Antigens
3.3.3 Flow Cytometer Plot Setup for Basic Single-Color Analysis ( see Note 10)
3.4 Multicolor Immunophenotyping
3.4.1 Multicolor Indirect Immunophenotyping of Surface Antigens Utilizing Different Fluorescent Spectra on a Flow Cytometer
3.4.2 Flow Cytometer Plot Setup and Basic Two-Color Analysis of Culture Cells
3.5 Three-Dimensional Phenotypic Analysis of Cell/Biomaterial Combinations Using Confocal Microscopy
4 Notes
References
Chapter 11: Preparation and Evaluation of Natural Scaffold Materials for Kidney Regenerative Applications
1 Introduction
2 Materials
2.1 Biomaterial Preparation
2.1.1 Microsphere Cross-Linking
2.1.2 Microsphere Purification
2.2 Biomaterial Evaluation
2.2.2 Microsphere Sizing
2.2.3 Biocompatibility
3 Methods
3.1 Gelatin Microspheres Preparation
3.1.2 Bead Cross-Linking
3.1.3 Optional: Bead Sizing ( see Notes 5 and 6)
3.2 Gelatin Microsphere Characterization
3.2.2 In Vitro Enzymatic Degradation Assay ( see Notes 12 and 13)
3.2.3 Quantification of Primary Amines ( see Note 16): Option 1 ( see Note 17)
3.2.4 Quantification of Primary Amines: Option 2 ( see Note 19)
3.3 Gelatin Microsphere Cytocompatibility Evaluation ( see Note 20)
3.4 Gelatin Microsphere Biocompatibility
3.4.2 Construct Microinjection (Fig.  3)
4 Notes
References
Chapter 12: Rapid Production of Autologous Fibrin Hydrogels for Cellular Encapsulation in Organ Regeneration
1 Introduction
2 Materials
3 Methods
3.1 Whole Blood Plasma Separation
3.2 Fibrinogen Concentration
3.3 Cell Encapsulation and Gelation
4 Notes
References
Chapter 13: Electrospinning Tubular Scaffolds with Tissue-Like Mechanical Properties and Biomimetic Surface Features
1 Introduction
1.1 What This Technique Accomplishes
2 Materials
2.1 Selection of Primary Component (Elastic)
2.2 Creation of Electrospinning Working Solution of Elastic Component
2.3 Selection of Secondary Component
2.4 Electrospinning Target
2.5 High-Voltage Power Source
2.6 Syringe Pump/Stand
2.7 Delivery Needle/Syringe Selection
3 Methods
3.1 Preparation: Setup
3.1.1 Equipment Positioning
3.2 Electrospinning Inner Elastic Tube
3.3 Preparation for the Addition of Outer/Second Layer
3.4 Electrospinning of Outer/Second Layer
3.5 Final Processing of Scaffold
4 Notes
Sec001319
Sec001320
Sec001321
Sec001322
Sec001323
Sec001324
Sec001325
Sec001326
References
Chapter 14: Design, Fabrication, and Preparation of Synthetic Scaffolds for Urologic Tissue Engineering
1 Introduction
1.1 Scaffolds
1.2 Urologic Organ Regeneration
1.3 Scaffold Design Considerations
2 Materials
2.1 Scaffold Fabrication
2.2 Prewetting (Preparation for Cell Seeding)
3 Methods
3.1 Scaffold Fabrication
3.2 Scaffold Preparation and Sterilization
3.3 Prewetting (Preparation for Cell Seeding)
3.3.1 Method 1: Vacuum Prewetting
3.3.2 Method 2: Ethanol Prewetting
4 Notes
References
Chapter 15: Characterization of a PGA-Based Scaffold for Use in a Tissue-Engineered Neo-Urinary Conduit
1 Introduction
2 Materials
2.1 Scaffold Fabrication
2.2 Degradation/Mass Loss Profile of PGA-Based Scaffold
2.3 Cell Culture on PGA-Based Scaffold
3 Methods
3.1 Scaffold Fabrication
3.2 Tensile Testing of PGA-Based Scaffold
3.3 Mass Loss of PGA-Based Scaffold
3.4 Cell Viability
3.5 Cell Proliferation
4 Notes
References
Chapter 16: Migration Assay to Evaluate Cellular Interactions with Biomaterials for Tissue Engineering/Regenerative Medicin...
1 Introduction
2 Materials
2.1 Migration Assay Components
2.2 Components for Characterizing Migration
2.2.1 Microscopy-Related Characterization
2.2.2 Metabolic or Cell Proliferation-Based Assays for Cell Quantitation
2.2.3 RNA/DNA Characterization of Cells
3 Procedure
3.1 Migration Assay
3.1.1 Preparation of Biomaterials to Be Tested
3.1.2 Preparation of Donor Tissue
3.1.3 Preparation of Cell-Seeded Inserts
3.1.4 Establishing Migration Assay
4 Characterization of Migration
4.1 How Much Migration Occurred?
4.1.1 Live/Dead Stain
4.1.2 PrestoBlue Quantitation
5 What Types of Cells Migrated?
5.1 Immuno-fluorescent Labeling
5.2 PCR Analysis of Migrated Cells
6 Notes
References
Chapter 17: Care of Rodent Models Used for Preclinical Evaluation of Tissue-Engineered/Regenerative Medicine Product Candid...
1 Introduction
2 Materials
3 Pre-surgical Preparation
4 Surgery Suite Setup
5 Analgesics, Anesthetics, and Antibiotics
6 Postsurgical Care
7 Harvesting the Construct
8 Lessons Learned
9 Notes
References
Chapter 18: Bioreactor Design Considerations for Hollow Organs
1 Introduction
2 Materials
2.1 Component List (Fig.  1)
2.2 Bioreactor Construction Components
2.3 Cleaning, Packaging, and Desiccation
2.4 Independent Testing
2.5 Details
3 Methods
3.1 Assembly and Sterilization
3.2 Seeding of Cells
3.3 Incubation and Maturation
3.4 Shipping
4 Notes
Chapter 19: Construction of a Multicoaxial Hollow Fiber Bioreactor
1 Introduction
2 Materials
3 Methods
3.1 Assembly of Manifold #1
3.2 Assembly of Manifold #2
3.3 Assembly of Manifold #3
3.4 Attachment of Tube Connectors
3.5 Quality Assurance and Sterilization
3.6 Fiber Wetting
4 Notes
References
Chapter 20: Isolation of Human Cadaveric Pancreatic Islets for Clinical Transplantation
1 Introduction
2 Materials
2.1 Specific Materials for Steps of the Isolation of Pancreatic Islets
2.1.1 Decontamination of the Pancreas
2.1.2 Making Liberase Enzyme
2.1.3 Digestion System Priming System
2.1.4 Digesting the Human Pancreas
2.1.5 Counting Islets
2.1.6 Density Gradient Separation and Harvesting of Human Islets
3 Methods
3.1 Human Pancreas Decontamination Setup
3.2 Formulating Liberase Enzyme ( See Note 2)
3.3 Trim and Decontaminate Human Cadaveric Pancreas
3.4 Cannulation and Distension of the Human Cadaveric Pancreas
3.5 Digesting the Human Pancreas
3.6 Counting Islets
3.6.1 Making Dithizone
3.6.2 Staining Islets with Dithizone
3.6.3 Performing Islet Count for Islet Equivalent Calculation
3.7 Density Gradient Separation and Harvesting of Human Islets
3.7.1 Making 1.077 g/ml Ficoll Gradient
3.7.2 Making 1.1 g/ml Ficoll Gradient
3.7.3 Transplant Media
3.8 Setting up the COBE Prior to Use
3.9 Using the COBE for Isolating Islets on a Density Gradient
3.10 Loading Islets into Bag for Transplantation
4 Notes
References
Chapter 21: Microencapsulation of Pancreatic Islets for Use in a Bioartificial Pancreas
1 Introduction
2 Materials
2.1 Chemicals
2.2 Equipment
3 Methods
4 Notes
References
Chapter 22: Bioengineered Skin Substitutes
1 Introduction
1.1 Skin Structure and Function
1.2 Skin Problems and Diseases
1.3 Different Types of Tissue-Engineered Skin Models
1.4 Tissue-Engineered Skin Substitutes as Models for Toxicological Testing
1.5 Gene-Modified Skin as Bioreactor for Therapeutic Protein Delivery
1.6 Description of Our Engineered Skin Model and Its Advantages
2 Materials
2.1 Acellular Dermis
2.2 Human Primary Keratinocyte Isolation and Maintenance
2.3 Epidermal Skin Equivalents
3 Methods
3.1 Acellular Dermis
3.2 Human Primary Keratinocyte Isolation and Maintenance
3.3 Epidermal Skin Equivalents
4 Notes
References
Chapter 23: Formulation of Selected Renal Cells for Implantation into a Kidney
1 Introduction
2 Materials
2.1 Formulation of Rat Renal Cells
2.2 Cell Culture Media and Reagents
2.3 Tissue Digestion
2.4 Cell Separation
2.5 Cell Formulation
2.6 Creating a Gel
2.7 Equipment
2.8 Plastic Supplies and Cultureware
2.9 Stainless Steel Instruments and Surgical Material
3 Method
3.1 Rat Renal Cell Isolation Method
3.2 Harvesting the Cells for Formulation
3.3 Formulation in Gelatin Hydrogel
3.3.1 After Counting the Cells a Final Wash Should Be Performed Using Gelatin
4 Notes
References
Chapter 24: Human Liver Bioengineering Using a Whole Liver Decellularized Bioscaffold
1 Introduction
2 Materials
2.1 Bioreactor System
2.2 Decellularization
2.3 Human Tissue Processing and Cell Isolation
3 Methods
3.1 Harvesting Cadaveric Animal Livers
3.2 Liver Decellularization
3.3 Isolation of Human Fetal Liver Progenitor Cells
3.4 hUVEC Isolation and Expansion
3.5 Scaffold Preparation and Sterilization for Bioreactor
3.6 Bioreactor Assembly and Recellularization
3.7 hUVECs Preparation for Bioreactor Seeding
3.8 Human Liver Progenitor Cell Preparation for Bioreactor Seeding
3.9 Bioscaffold Seeding of Human Liver Progenitor Cells and hUVECs
4 Notes
References
Chapter 25: A “Living Bioreactor” for the Production of Tissue-Engineered Small Intestine
1 Introduction
2 Materials
2.1 Experimental Animals
2.2 Organoid Units
2.3 Digestion Enzyme
2.4 Operating Instruments and Medications
3 Methods
3.1 Recovery of Small Intestine from Donor Mice
3.2 Removal of Fecal Material from Intestine
3.3 Mincing of Intestine
3.4 Enzyme Digestion
3.5 Isolation of Organoid Units
3.6 Implantation of Loaded Scaffold
3.7 Recovery of TESI
4 Notes
References
Chapter 26: Tissue Engineering of Esophagus and Small Intestine in Rodent Injury Models
1 Introduction
1.1 Esophagus
1.2 Small Intestine
2 Materials
2.1 Ad-SMC Isolation and Culture
2.2 Biomaterials
2.3 Animal Model
3 Methods
3.1 Isolation of Ad-SMC
3.2 Preparation of the Biomaterials Scaffolds
3.3 Preparation of the Construct
3.4 Animal Surgery
4 Notes
References
Chapter 27: Scanning Electron Microscopy Evaluation of Endothelialized Tissue-Engineered Constructs
1 Introduction
2 Materials
3 Methods
3.1 Primary Fixation
3.2 Osmium Tetroxide Post-fixation
3.3 Ethanol Dehydration
3.4 Critical Point Drying
3.5 Sputter Coating
4 Notes
References
Chapter 28: Genotypic and Phenotypic Analysis of In Vivo Tissue Regeneration in an Animal Model
1 Introduction
2 Materials
2.1 Buffers and Reagents
2.2 Equipment
2.3 Commercial Kits
3 Methods
3.1 RNA Isolation and cDNA Synthesis
3.2 qRT-PCR
3.3 Protein Extraction
3.4 SDS-PAGE and Western Blot
3.5 Regenerative Response Induction Calculation
References
Chapter 29: Histological Evaluation of Tissue Regeneration Using Biodegradable Scaffold Seeded by Autologous Cells for Tubu...
1 Introduction
2 Materials and Methods
2.1 Study Design
2.2 Tissue Collection
2.3 Tissue Fixation
2.4 Tissue Sampling
2.5 Histology Processing
2.6 Immunohisto-chemistry
2.7 Reagents and Solutions
2.7.1 Solutions
3 Immunohisto-chemistry Staining Protocols
3.1 Cytokeratin AE1/AE3 Protocol
3.1.1 Procedure
3.1.2 Manual Staining Procedure for Cytokeratin AE1/AE3
3.1.3 Acceptance Criteria ( see Fig.  2a)
3.2 Cytokeratin 7 Protocol
3.2.1 Solutions
3.2.2 Procedure
3.2.3 Manual Staining Procedure for Cytokeratin 7
3.2.4 Acceptance Criteria ( see Fig.  2b)
3.3 Calponin Protocol
3.3.1 Solutions
3.3.2 Procedure
3.3.3 Manual Staining Procedure for Calponin
3.3.4 Acceptance Criteria ( see Fig.  2c)
4 Statistical Analysis
5 Tissue Evaluation
6 Histological Assessment
6.1 Grading Scheme
6.2 Immunohisto-chemistry Grading
7 Functional Parameters
7.1 Epithelialization
7.2 Luminal Surface Fibrovascular Response
7.3 Muscular Layer Fibrovascular Response
7.4 Myofibroblastic Reaction
7.5 Peritoneal Surface Integration
8 Safety Parameters
8.1 Tissue Necrosis
8.2 Luminal Surface Fibrous Tissue
8.3 Muscular Layer Fibrous Tissue
8.4 Inflammatory Response
8.5 Scaffold Degradation
8.6 Calcification and/or Heterotopic Bone Formation
9 Histo-morphometry
10 Notes
References
Index