Preface
Contents
Contributors
Chapter 1: 50 Years of Myofibroblasts: How the Myofibroblast Concept Evolved
References
Part I: Fundamental Methods to Study Myofibroblast Biology
Chapter 2: Myofibroblast Functions in Tissue Repair and Fibrosis: An Introduction
1 Myofibroblasts in Repair and Fibrosis
2 Myofibroblast Plasticity
3 Myofibroblast Apoptosis
4 Reversibility of Fibrosis
5 Teleology Informs Biology and Therapeutic Opportunity
References
Chapter 3: Myofibroblast Markers and Microscopy Detection Methods in Cell Culture and Histology
1 Introduction
2 Materials
2.1 Cell Culture and Preparation
2.2 IF Staining of Cultured Cells
2.3 Tissue Preparation
2.3.1 Tissue Fixation
2.3.2 Tissue Embedding
2.4 IF Staining of Fixed Tissues
2.5 Image Analysis
3 Methods
3.1 Cell Culture
3.2 IF Staining of Fixed Cells
3.3 Tissue Preparation
3.3.1 Fixing and Paraffin-Embedding
3.3.2 Sectioning Tissues
3.4 Immunostaining of Fixed Tissues
3.5 Image Analysis
3.5.1 Quantification of Ξ±-SMA Expression in Cultured Cells Using Fiji
3.5.2 Quantification of Myofibroblasts in Tissue
4 Notes
References
Chapter 4: Fibroblast and Myofibroblast Subtypes: Single Cell Sequencing
1 Introduction
2 Materials
2.1 Digestion of Heart to Purify Myo(Fibroblasts)
2.2 Fluorescence-Activated Cell Sorting (FACS) of (Myo)Fibroblasts for scRNAseq
2.3 Preparation of Tissue Sections for Spatial Transcriptomic Profiling
2.4 Computational Analysis of scRNAseq and Spatial Transcriptomics to Identify Fibroblast Subsets
3 Methods
3.1 Digestion of Heart to Purify Myo(Fibroblasts)
3.2 Fluorescence-Activated Cell Sorting (FACS) of (Myo)Fibroblasts for scRNAseq
3.3 Preparation of Tissue Sections for Spatial Transcriptomic Profiling
3.4 Computational Analysis of scRNAseq to Identify Fibroblast Subsets
3.5 Spatial Transcriptomic Analysis with Visium
3.6 Integration of scRNAseq and Spatial Transcriptomics Datasets
4 Notes
References
Chapter 5: Myofibroblast Adhesome Analysis by Mass Spectrometry
1 Introduction
2 Materials
2.1 Coated Bead Preparations
2.2 Protein Preparation and Elution from Beads
2.3 Preparation for Mass Spectrometry
3 Methods
3.1 Bead Preparations
3.2 Protein Preparation and Elution from Beads
3.3 Protein Analysis by Mass Spectrometry
4 Notes
References
Chapter 6: Myofibroblast TGF-Ξ² Activation Measurement In Vitro
1 Introduction
2 Materials
2.1 Materials for Transformed Mink Lung Cell (TMLC) Assay
2.2 Materials for Phosphorylated Smad2 Western Blotting
3 Methods
3.1 Method for Transformed Mink Lung Cell (TMLC) Assay
3.1.1 Measurement of Global TGFΞ² Activity in Conditioned Medium
3.1.2 Measurement of Integrin-Mediated TGFΞ² Activation by TMLC Coculture Assay
3.2 Method for Measurement of pSMAD2 by Western Blotting
4 Notes
References
Chapter 7: Contraction Measurements Using Three-Dimensional Fibrillar Collagen Gel Lattices
1 Introduction
2 Materials
2.1 Preparation of Reagents for Collagen Lattices
2.2 Cell Culture
2.3 Imaging
3 Methods
3.1 Preparation of Collagen Lattice Solution
3.2 Preparation of Cells for Collagen Lattices
3.3 Seeding the Lattices
3.4 Release and Measurement
4 Notes
References
Chapter 8: Techniques to Assess Collagen Synthesis, Deposition, and Cross-Linking In Vitro
1 Introduction
2 Materials
2.1 Assessment of Collagen Synthesis and Deposition in Cultured Fibroblasts
2.2 Analysis of Lysyl Oxidase (LOX) Activity in Cell Supernatants
3 Methods
3.1 Assessment of Collagen Synthesis and Deposition in Cultured Fibroblasts
3.2 Analysis of Lysyl Oxidase (LOX) Activity in Cell Supernatants
4 Notes
References
Chapter 9: Methods for Studying Myofibroblast Apoptotic Pathways
1 Introduction
1.1 Control of Mitochondrial Apoptosis by the BCL-2 Family of Proteins
1.2 Priming States
1.3 Overview of BH3 Profiling Assay
1.4 Measuring Overall Mitochondrial Apoptotic Priming and Antiapoptotic Dependencies
1.5 Extrinsic Pathway of Apoptosis
2 Materials
2.1 Assessment of Intrinsic Apoptotic Pathways via BH3 Profiling Assay
2.1.1 Buffers and Reagents
2.1.2 Staining
2.1.3 Equipment
2.2 Measuring Extrinsic Apoptotic Pathway via Annexin V Staining
2.2.1 Buffers and Reagents
2.2.2 Staining
2.2.3 Equipment
2.3 Human Primary Dermal Fibroblasts
3 Methods
3.1 Measuring Myofibroblast Intrinsic Apoptotic Pathways via BH3 Profiling Assay
3.1.1 Viability Staining
3.1.2 Preparation of 96-Well Plate with Permeabilization Buffer and BH3 Peptides and Drugs
3.1.3 BH3 Profiling Assay
3.1.4 Cytochrome c Staining
3.1.5 Flow Cytometry Analysis
3.2 Assessment of Myofibroblast Extrinsic Pathway of Apoptosis via Annexin V Staining
3.2.1 Fas Ligand Treatment
3.2.2 Sample Collection and Staining Procedure
3.2.3 Flow Cytometry Analysis
4 Notes
References
Chapter 10: Determination of Senescent Myofibroblasts in Precision-Cut Lung Slices
1 Introduction
2 Materials
2.1 Preparation of PCLS
2.2 Ξ²-Galactosidase Activity: X-Gal Staining
2.3 OCT Blocks and Sectioning
2.4 Immunofluorescence Staining
2.5 Imaging and Quantification
3 Methods
3.1 Ξ²-Galactosidase Staining
3.2 OCT Block Generation and Cryostat Sectioning
3.3 Immunofluorescence Staining
3.4 Imaging and Quantification
4 Notes
References
Chapter 11: The Scar-in-a-Jar: In Vitro Fibrosis Model for Anti-Fibrotic Drug Testing
1 Introduction
2 Materials
2.1 Cell Culture and Scar-in-a-Jar Procedure
2.2 Immunocytochemistry
2.3 Optical High-Content Screening and Analysis of Data
3 Methods
3.1 Cell Culture and Scar-in-a-Jar Procedure
3.2 Immunocytochemistry
3.3 Optical High-Content Screening and Analysis of Data
4 Notes
References
Part II: Myofibroblast Mechanobiology
Chapter 12: Why Stress Matters: An Introduction
1 Introduction
2 Extracellular Matrix as Scaffold, Signal, and Stress-Bearing Structure
3 ECM and Mechanosensing
4 Stress Generation by Myofibroblasts and Its Consequences
5 Emerging Methods to Study Myofibroblast Mechanobiology
6 Conclusion
References
Chapter 13: Soft Substrate Culture to Mechanically Control Cardiac Myofibroblast Activation
1 Introduction
2 Materials
2.1 Elastic Cell Culture Surfaces
2.2 Culture Surface Coating
2.3 Primary Rat Cardiac Fibroblast Isolation and Maintenance
2.4 Primary Mouse Cardiac Fibroblast Isolation
3 Methods
3.1 Coating Elastic Tissue Culture Surfaces
3.2 Primary Rat Cardiac Fibroblast Isolation and Culture
3.3 Primary Mouse Cardiac Fibroblast Isolation and Culture
4 Notes
References
Chapter 14: Quantitative Analysis of Myofibroblast Contraction by Traction Force Microscopy
1 Introduction
2 Materials
2.1 Activation of Coverslips
2.2 Preparation of Fibronectin-Coated TFM Substrates
2.3 Differentiation and Culturing of Myofibroblasts
2.4 Immunofluorescence Staining of Differentiation Markers
2.5 Live-Cell Imaging
2.6 Quantification of TFM Substrate Deformation and Calculation of Traction Force
3 Methods
3.1 Activation of Coverslips
3.2 Preparation of Fibronectin-Coated TFM Substrates
3.3 Differentiation and Culturing of Myofibroblasts
3.4 Immunofluorescence Staining of Differentiation Markers
3.5 Acquisition of TFM Images
3.6 Quantification of TFM Substrate Deformation and Calculation of Traction Force
4 Notes
References
Chapter 15: Nucleocytoplasmic Shuttling of the Mechanosensitive Transcription Factors MRTF and YAP/TAZ
1 Introduction
1.1 Nucleocytoplasmic Traffic of Transcriptional Regulators: Key Role in Fibrosis
1.2 Quantitation of Nuclear Traffic of MRTF and YAP/TAZ: Importance and Challenges
1.3 The Nuclear Shuttling of MRTF: Overview and Open Questions
1.4 The Nuclear Shuttling of YAP and TAZ: Overview and Open Questions
1.5 New Tools and Strategies for Studying Nucleocytoplasmic Traffic
2 Materials
2.1 Cell Culture
2.2 Cell Transfection
2.3 Cell Stimulation
2.4 Immunofluorescence
2.5 Imaging Equipment and Software
3 Methods
3.1 Automated Acquisition and Analysis Using Fixed Cells in 96-Well Plates
3.1.1 Cell Culturing and Stimulation
3.1.2 Cell fixation and staining
3.1.3 Image Acquisition and Analysis Using the ImageXpress Micro Platform with Integrated MetaXpress Imaging and Analysis Soft...
3.1.4 Data Analysis
3.2 Live Cell Imaging: Kinetics of Nuclear Import Using the Rapamycin Sequestration System
3.2.1 Cell Culturing and Cell Transfection
3.2.2 Cell Stimulation and Live-Cell Imaging
3.2.3 Data Analysis
4 Notes
References
Chapter 16: Atomic Force Microscopy for Live-Cell and Hydrogel Measurement
1 Introduction
2 Materials
2.1 Hardware
2.2 Disposables for General AFM
2.3 Disposables for AFM on Gels
2.4 Disposables for AFM with Live Cells
2.5 Software
3 Methods
3.1 AFM General Setup
3.2 Hydrogel Analysis Using AFM
3.3 Live Cell Analysis Using AFM
4 Notes
References
Chapter 17: Method for Investigating Fibroblast Durotaxis
1 Introduction
2 Materials
2.1 Reagents
2.2 Deactivating the Surface of Coverslip (25 mm)
2.3 Preparation of 1:10 (v/v) Acetic Acid Solution
2.4 Preparation of Methacrylate Solution
2.5 Preparation of 5% IrgaCure Solution
2.6 Preparation of High-Resolution Photomask
2.7 Preparation of Sulfo-SANPAH Solution
2.8 Fibronectin/Collagen Coating Solution
2.9 Equipment
3 Methods
3.1 Design and Fabrication of Mechanically Patterned Soft-Stiff Hydrogel Onto a Coverslip
3.1.1 Activation of Glass Coverslips (18 mm)
3.1.2 Fabrication of the Soft Layer
3.1.3 Fabrication of the Stiff Layer on Top of the Soft Layer
3.2 Seeding of Lung Fibroblasts in Mechanically Patterned Hydrogel
3.2.1 Surface Activation and Coating of the Soft-Stiff Patterned Hydrogel Surface
3.2.2 Seeding of Lung Fibroblasts Onto the Patterned Hydrogel
3.3 Assessment of Durotactic Index
3.4 Mechanistic Studies Investigating Molecular Control of Durotaxis
4 Notes
References
Chapter 18: Decellularized Extracellular Matrix (ECM) as a Model to Study Fibrotic ECM Mechanobiology
1 Introduction
2 Materials
2.1 Human Lung Decellularization
2.2 Mouse Lung Decellularization After Bleomycin Treatment
2.3 Decellularizing In Vitro Cell Generated ECM
2.4 Fabrication of Hydrogels with Varying Stiffness
2.5 In-Solution Digestion of ECM Proteins to Peptides for Mass Spectrometry
2.6 In-Gel Digestion of Proteins for Mass Spectrometry Analysis
2.7 AFM
3 Methods
3.1 Human Lung Decellularization
3.2 Decellularizing Mouse Lung After Bleomycin Treatment
3.2.1 Bleomycin Intratracheal Administration in Mice
3.2.2 Mouse Lung Harvest
3.3 Decellularizing In Vitro Cell Generated ECM
3.3.1 Surface Treatment of PDMS Sheets
3.3.2 Decellularized Cell-Based ECM
3.4 Fabrication of Polyacrylamide Hydrogels with Varying Stiffness
3.4.1 Coverslip Preparation
3.4.2 Solubilized dECM Preparation
3.4.3 Hydrogel Preparation
3.5 In-Solution Digestion of dECM Proteins to Peptides for Mass Spectrometry Analysis
3.6 In-Gel Digestion of Proteins for Mass Spectrometry Analysis
3.7 AFM Calibration and Experiment Preparation
3.7.1 Laser Alignment
3.7.2 Cantilever Calibration
3.7.3 Tip Preparation
3.8 dECM Sample Preparation
3.8.1 Tissue Thickness
3.8.2 Mounting dECM
3.9 AFM Settings/Parameters for Obtaining Force Curves
3.9.1 Ramping Rate
3.9.2 Loading Rate and Velocity
3.9.3 Probing Area
3.9.4 Tip Indentation Depth
3.10 Force Curve Interpretation and Data Analysis
3.10.1 Basic Principles of FD Curve
3.10.2 FD Analysis
4 Notes
References
Chapter 19: Fibrosis on a Chip for Screening of Anti-Fibrosis Drugs
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Micropillar Array Device and Collagen Prepolymer
2.3 Pharmacological Treatment
2.4 Immunofluorescence
2.5 Microscopy and Image Analysis
2.6 Centrifuges and Vacuum Chamber
3 Methods
3.1 Microtissue Device Fabrication
3.2 Microtissue Seeding and Pharmacological Treatment
3.3 Force Measurement
3.4 Stiffness Measurement
3.5 Immunostaining and Microscopy
4 Notes
References
Part III: Animal and Human Models to Study Myofibroblast Biology
Chapter 20: Animal and Human Models of Tissue Repair and Fibrosis: An Introduction
1 Introduction: Different Philosophies to Study Myofibroblasts
2 Advanced Human Culture Models
3 Mouse Models of Tissue Repair and Fibrosis
4 Genetic Strategies to Study Myofibroblast Functions In Vivo
References
Chapter 21: Mouse Models of Lung Fibrosis
1 Introduction
1.1 Selection of Experimental Rodent Models That Express The Target(s) of Interest
1.2 Selection of Route of Administration, Dose, and Frequency
1.3 Selection of Control Groups and Control Treatments, Statistical and Species Sex Considerations
1.4 Analyzing a Discovery Cohort and Selection of Primary and Secondary Outcomes
1.5 Design and Execution of Validation Experiments
2 Materials
2.1 Intratracheal Administration of Drugs
2.2 Endpoint Measures: Respiratory Mechanics
2.3 Endpoint Measures: Blood Collection for Plasma Analysis
2.4 Endpoint Measures: BALF Collection for Immune Cell Differentials
2.5 Endpoint Measures: Lung Processing for Histopathology, Flow Cytometry, and Single-Cell RNA Sequencing
2.6 Analysis of Homogenized Tissue Samples
2.7 Quantification Using Digitalized TMAs
3 Methods
3.1 Intratracheal Administration of Drugs
3.2 Endpoint Measures: Respiratory Mechanics
3.3 Endpoint Measures: Blood Collection for Plasma Analysis
3.4 Endpoint Measures: BALF Collection for Immune Cell Differentials
3.5 Lung Processing for Histopathology, Flow Cytometry, and Single-Cell RNA Sequencing
3.5.1 Processing of the Right Superior and Inferior Lobes for Flow Cytometry or scRNAseq Assessment
3.5.2 Processing of the Right Middle and Post-Caval Lung Lobes for RNA Analysis
3.5.3 Processing of the Left Lung Lobe for Histology
3.5.4 Crushing of Snap-Frozen Lung and Homogenization for RNA and Protein Assessment
3.6 Analysis of Homogenized Tissue Samples
3.6.1 RNA Extraction
3.6.2 Collagen Assessment
3.7 Histology Tissue Microarray (TMA) Production and Digitization
3.7.1 Creation of a TMA
3.7.2 Tissue Array Digitization
3.8 Quantification Using Digitized TMAs
3.8.1 Protein and mRNA Quantification from Digitized TMAs
3.8.2 Picrosirius Red Quantification
4 Notes
References
Chapter 22: Mouse Models of Kidney Fibrosis
1 Introduction
1.1 UUO Model
1.1.1 UUO Advantages
1.1.2 UUO Disadvantages
1.1.3 UUO Variations
1.2 IRI Model
1.2.1 IRI Advantages
1.2.2 IRI Disadvantages
1.2.3 IRI Variations
2 Materials
2.1 Materials for UUO Model (See Notes 1-6)
2.2 Materials for IRI Model (See Notes 1-6)
3 Methods
3.1 UUO Model
3.2 IRI Model
3.3 Time Points
3.4 Downstream Techniques Suitable to Investigate These Processes
4 Notes
References
Chapter 23: Mouse Models of Liver Fibrosis
1 Introduction
1.1 General Considerations
1.2 Hepatotoxin-Induced Fibrosis Models
1.2.1 Carbon Tetrachloride (CCl4)
1.2.2 Thioacetamide (TAA)
1.3 Biliary Fibrosis Models
1.3.1 Bile Duct Ligation (BDL)
1.3.2 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine (DDC) Diet
1.3.3 Mdr2-/- Mice
1.4 NASH-Associated Fibrosis
1.4.1 High-Fat High-Cholesterol Diet (HFHCD) Models
1.4.2 Choline-Deficient High-Fat Diet Models
2 Materials
2.1 CCl4 Injection
2.2 BDL Model
2.3 HFHCD Diet
2.4 Choline-Deficient High-Fat Diet Models
2.5 Harvesting Tissue and Analysis
3 Methods
3.1 CCl4 Model
3.2 BDL Model
3.3 HFHCD Diet
3.4 Choline-Deficient High-Fat Diet Models
3.5 Harvesting Tissue and Analysis
4 Notes
References
Chapter 24: Mouse Models of Muscle Fibrosis
1 Introduction
1.1 Cellular Mechanisms in Skeletal Muscle Fibrosis
1.2 Muscle Fibrosis in Mice: Muscular Dystrophy Models
1.3 Increasing Naturally Occurring Fibrosis in mdx Mice by Genetic Strategies
1.4 Accelerating and Expanding Fibrosis Development in mdx Mice
2 Materials
2.1 Materials for Traumatic Skeletal Muscle Fibrosis in mdx Mice
2.2 Morphological Assessment of Fibrosis by Picro-Sirius Red Stain
2.3 Biochemical Determination of Collagen on Muscle Sections
3 Methods
3.1 Traumatic Induction of Skeletal Muscle Fibrosis in mdx Mice
3.2 Morphological Assessment of Fibrosis by Picro-Sirius Red Stain
3.3 Biochemical Determination of Collagen on Muscle Sections
3.4 Other Downstream Techniques Suitable to Investigate Muscle Fibrosis
4 Notes
References
Chapter 25: Mouse Models of Skin Fibrosis
1 Introduction
1.1 Bleomycin-Induced Skin Fibrosis
1.2 Sclerodermatous Chronic Graft-Versus-Host Disease (SclGvHD) Model
1.3 Fibrosis Induced by Recombinant DNA Topoisomerase I and Complete FreundΒ΄s Adjuvant
1.4 Tight Skin Mice 1 (Tsk1) Mice
1.5 Fra2-Transgenic Mice
1.6 Other Genetic Models
2 Materials
2.1 Bleomycin-Induced Skin Fibrosis
3 Methods
3.1 Bleomycin-Induced Skin Fibrosis
3.2 Assessment of Skin Fibrosis
4 Notes
References
Chapter 26: Mouse Models of Intestinal Fibrosis
1 Introduction
2 Materials
2.1 DSS Colitis Model
2.2 TNBS Colitis Model
2.3 AIEC Colitis Model
2.4 IL-10-Deficiency Colitis Model
2.5 SAMP1/YitFc Ileocolitis Model
3 Methods
3.1 DSS Colitis Model
3.1.1 DSS Titration (5-8 d)
3.1.2 DSS Induction of Chronic DSS Colitis (30-42 d)
3.2 TNBS Colitis Model
3.2.1 TNBS Titration (5-8 d)
3.2.2 Chronic TNBS Colitis (56 d)
3.3 AIEC Colitis Model
3.3.1 Induction of AIEC Colitis (7-63 d)
3.4 IL-10-Deficiency Colitis Model
3.4.1 Spontaneous Colitis in IL-10 Knockout Mice (~20 Weeks)
3.5 SAMP1/YitFc Ileocolitis Model
3.5.1 Spontaneous Colitis in SAMP1/YitFc Mice (~40 Weeks)
4 Notes
References
Chapter 27: A Rodent Model of Hypertrophic Scarring: Splinting of Rat Wounds
1 Introduction
2 Materials
2.1 Experimental Animals: Rats
2.2 Anesthesia, Operation Preparation, and Personal Protective Equipment (See Note 1)
2.3 Surgical Instruments
2.4 Post-Operation Treatment
2.5 Sampling
3 Methods
3.1 Animal Order and Preparation
3.2 Anesthesia and Operative Preparation
3.3 Generation of Splinted Wounds
3.4 Post-Operative Management
3.5 Sample Collection and Analysis
4 Notes
References
Chapter 28: Three-Dimensional Model of Hypertrophic Scar Using a Tissue-Engineering Approach
1 Introduction
2 Materials
2.1 Culture Media
2.1.1 Tissue Transport Medium
2.1.2 Human Fibroblast Culture Medium (Fb Medium)
2.1.3 Human Keratinocyte Culture Medium (K Medium)
2.1.4 Air Liquid Epidermis Culture Medium (A/L Medium)
2.1.5 Freezing Medium
2.2 Monolayer Culture of Human Fibroblasts
2.2.1 Extraction and Culture of Human Fibroblasts
2.2.2 Subculture of Human Fibroblasts (Passage)
2.2.3 Cryopreservation of Human Fibroblasts
2.2.4 Thawing of Human Fibroblasts
2.3 Monolayer Culture of Human Keratinocytes
2.3.1 Extraction and Culture of Human Keratinocytes
2.3.2 Subculture of Human Keratinocytes (Passaging)
2.3.3 Cryopreservation of Human Keratinocytes
2.3.4 Thawing of Human Keratinocytes
2.4 Formation of Dermal Sheets
2.5 Keratinocyte Addition on the Dermis
2.6 Skin Assembly
3 Methods
3.1 Monolayer Culture of Human Fibroblasts
3.1.1 Extraction and Culture of Human Fibroblasts
3.1.2 Subculture of Human Fibroblasts (Passaging)
3.1.3 Cryopreservation of Human Fibroblasts
3.1.4 Thawing of Human Fibroblasts
3.2 Monolayer Culture of Human Keratinocytes
3.2.1 Extraction and Culture of Human Keratinocytes
3.2.2 Subculture of Human Keratinocytes (Passage)
3.2.3 Cryopreservation of Human Keratinocytes
3.2.4 Thawing of Human Keratinocytes
3.3 Human Skin Reconstruction by the Self-Assembly Approach
3.3.1 Formation of Dermal Sheets
3.3.2 Keratinocyte Addition
3.3.3 Skin Assembly
4 Notes
References
Chapter 29: Methods for the Study of Renal Fibrosis in Human Pluripotent Stem Cell-Derived Kidney Organoids
1 Introduction
2 Materials
2.1 Plating hPSCs for Differentiation
2.2 Differentiation of hPSCs into Kidney Organoids
2.3 Injury of Kidney Organoids
2.4 Organoid Fixation and Cryopreservation
2.5 Organoid Analysis by Confocal Microscopy
2.6 Organoid Analysis by Real-Time PCR (qPCR)
3 Methods
3.1 Differentiation of hPSCs into Metanephric Mesenchyme (Fig. 1)
3.2 Induction of Organoid Nephron Damage and Interstitial Fibrosis (Fig. 2)
3.3 Assessment of Organoid Nephron Damage and Fibrosis Using Immunofluorescence Microscopy (Fig. 3)
4 Notes
References
Chapter 30: Decellularized Human Lung Scaffolds as Complex Three-Dimensional Tissue Culture Models to Study Functional Behavio...
1 Introduction
2 Materials
2.1 Generation of Human Precision-Cut Lung Slices (PCLS)
2.2 Decellularization of PCLS
2.3 Recellularization of PCLS with Primary Human Fibroblasts
3 Methods
3.1 Generation of Human Precision-Cut Lung Slices (PCLS)
3.1.1 Human Lung Tissue Inflation by Agarose Filling
3.1.2 Precision-Cut Lung Slicing of Agarose-Filled Human Lung Tissue
3.2 Decellularization of PCLS
3.3 Recellularization of PCLS with Primary Human Lung Fibroblasts
3.4 Functional Studies
4 Notes
References
Index