Chondrocytes: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Isolation of Chondrocytes from Human Cartilage and Cultures in Monolayer and 3D
1 Introduction
2 Materials
3 Methods
3.1 Chondrocyte Isolation
3.2 Isolation of Chondrocytes with Sequential Digestion (Recommended for Articular Cartilage)
3.3 Propagation and Routine Maintenance of Chondrocytes in 2D
3.4 Trypsinization of Monolayer-Cultured Chondrocytes
3.5 Chondrocyte Culture in 3D Aggregate Culture
4 Notes
References
Chapter 2: Protocol for the Isolation of Intact Chondrons from Healthy and Osteoarthritic Human Articular Cartilage
1 Introduction
1.1 Cartilage Structure
1.2 Chondrocytes
1.3 Chondrons as a Niche for Chondrocytes
2 Materials
2.1 Reagents and Kits
2.2 Incubation Medium
2.3 Chondrogenic Medium
3 Methods
3.1 Preparation of Human Articular Cartilage Samples
3.2 Digesting Cartilage Samples for Chondron Isolation
3.3 Analysis of Chondrocyte Viability in Chondrons
4 Notes
References
Chapter 3: Cartilage-Specific Cre Recombinase Transgenes/Alleles in the Mouse
1 The Cre-loxP and Tet-ON/Tet-OFF Technologies
2 Chondrocyte-Specific Promoters
2.1 Col2a1
2.2 Col11a2
2.3 Col10a1
2.4 Acan
2.5 Matn1
2.6 Gdf5
2.7 Prg4
2.8 Sox9
3 Recommendation for the Use of the Cre-lox System to Target Chondrocytes
4 Concluding Remarks
5 Notes
References
Chapter 4: Isolation of Mouse Growth Plate and Articular Chondrocytes for Primary Cultures
1 Introduction
2 Materials
3 Methods
3.1 Isolation of Chondrocytes from Growth Plate Cartilage
3.1.1 Mouse and Tissue Source
3.1.2 Solutions
3.1.3 Dissection of the Thoracic Cage
3.1.4 Growth Plate Chondrocyte Release from Tissue and Plating
3.2 Isolation of Chondrocytes from Articular Cartilage
3.2.1 Mouse and Tissue Source
3.2.2 Solutions
3.2.3 Dissection of Articular Cartilage
3.2.4 Articular Chondrocyte Release from Tissue and Plating
4 Notes
References
Chapter 5: Fetal Growth Plate Cartilage: Histological and Immunohistochemical Techniques
1 Introduction
2 Materials
2.1 Isolating Murine Embryos for Paraffin Sections
2.2 Isolating Murine Embryos for Fresh Frozen Sections
2.3 Fetal Growth Plate Hematoxylin and Eosin Staining
2.4 Safranin O Staining for the Evaluation of GAGs in the Murine Growth Plate
2.5 In Situ Cell Death Detection (TUNEL Assay)
2.6 Immunohistochemistry on Formalin-Fixed, Paraffin-Embedded Tissues
2.7 EdU Assay for Evaluating Cellular Proliferation
2.8 EF5 Staining to Detect Hypoxia in the Developing Growth Plate
2.9 Fluorescent Image Quantification Using ImageJ
3 Methods
3.1 Isolating Murine Embryos for Paraffin Sections
3.2 Isolating Murine Embryos for Fresh Frozen Sections
3.3 Fetal Growth Plate Hematoxylin and Eosin Staining
3.4 Safranin O Staining for the Evaluation of GAGs in the Murine Growth Plate
3.5 In Situ Cell Death Detection (TUNEL Assay)
3.6 Immunohistochemistry on Formalin-Fixed, Paraffin-Embedded Tissues
3.7 EdU Assay for Evaluating Cellular Proliferation
3.8 EF5 Staining to Detect Hypoxia in the Developing Growth Plate
3.9 Fluorescent Image Quantification Using ImageJ
4 Notes
References
Chapter 6: Preparation of Adult Mouse Skeletal Tissue Sections for RNA In Situ Hybridization
1 Introduction
2 Materials
2.1 Reagents and Solutions
2.2 Histology and Other Supplies
2.3 Equipment
2.4 RNAscope Reagents (ACD)
3 Methods
3.1 Preparation of Tissue Sections
3.2 RNA In Situ Hybridization
3.3 Image Acquisition and Data Analysis
4 Notes
References
Chapter 7: MicroRNA In Situ Hybridization in Paraffin-Embedded Human Articular Cartilage and Mouse Knee Joints
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Deparaffinization
2.3 In Situ Hybridization
3 Methods
3.1 Sample Preparation
3.2 Deparaffinization
3.3 In-Situ Hybridization
4 Notes
References
Chapter 8: Laser Capture Microdissection of Mouse Growth Plate Cartilage
1 Introduction
2 Materials
2.1 Slide Pretreatment
2.2 Tissue Sectioning
2.3 Staining
2.4 LCM
2.5 RNA Isolation and Analysis
3 Methods
3.1 Poly-l-Lysine Slide Coating
3.2 CryoStat Sectioning
3.3 Frozen Section Staining Methods
3.3.1 Frozen Section H&E Staining
3.3.2 Frozen Section Cresyl Violet Staining
3.4 Laser Capture Microdissection
3.4.1 Preparation of Lysis Buffer Solution
3.4.2 Computer Set-Up and Software Settings
3.4.3 Loading Caps and Slide Samples
3.4.4 Cap Placement and Marking Region of Interest
3.4.5 Capture and Cut
3.5 RNA Extraction
4 Notes
References
Chapter 9: RNA Isolation from Articular Cartilage Tissue
1 Introduction
2 Materials
2.1 Tools and Equipment
2.2 Reagents
3 Methods
3.1 Harvest and Freeze Cartilage from Murine Femoral Heads
3.2 Harvest and Freeze Articular Cartilage from Human Osteoarthritic Knee Joint Specimens
3.3 RNA Purification Using a TRIzol Reagent and Spin Column Chromatography
4 Notes
References
Chapter 10: Small Nucleolar RNA Expression Profiling in Cartilage
1 Introduction
2 Materials
2.1 Collection of Cartilage Biopsies for RNA Extraction and Histological Grading (See Note 1)
2.2 Micro-dismembrator Tissue Homogenization for RNA Extraction (See Notes 2 and 3)
2.3 RNA Isolation from Cartilage and Subsequent Production of Poly(a) RNA
2.4 Poly(A) RNA Isolation
2.5 Poly(A) cDNA Synthesis
2.6 SnoRNA Primer Design
2.7 qRT-PCR
2.8 CAP-CLIP Pyrophosphatase Pretreatment Prior to snoRNA-seq Library Preparation
3 Methods
3.1 Collection of Cartilage Explants for RNA Extraction and Histological Grading
3.2 Micro-dismembrator Tissue Homogenization for RNA Extraction
3.3 RNA Isolation from Cartilage for Subsequent Poly-A RNA
3.4 Poly(A) RNA Isolation
3.5 Poly(A) cDNA Synthesis
3.6 SnoRNA Primer Design
3.7 qRT-PCR
3.8 CAP-CLIP Pyrophosphatase Pretreatment Prior to snoRNA-Seq Library Preparation for Hydrolysis of the Pyrophosphate Bonds of...
4 Notes
References
Chapter 11: MicroRNA Expression Profiling, Target Identification, and Validation in Chondrocytes
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents and Kits
2.2.1 miRNA Sequence by NGS
2.2.2 miRNA Profiling by TaqMan Array System
2.2.3 miRNA Target screening by Cell-Based Reporter Library Assay
2.2.4 miRNA and Target Interaction by CLIP
3 Methods
3.1 miRNA Expression Profiling
3.1.1 miRNA Sequence by NGS
3.1.2 miRNA Profiling by TaqMan Array System
3.2 miRNA Target Prediction by TargetScan
3.3 miRNA Target screening Using a Cell-Based Reporter Library Assay
3.4 miRNA and Target Interaction by CLIP
3.4.1 Preparation of Antibody-Coupled Beads
3.4.2 HITS-CLIP
UV Cross-Linking and Immunoprecipitation
End Labeling and Reverse Transcription
Library Enrichment and Sequencing
4 Notes
References
Chapter 12: ChIP-Seq Assays from Mammalian Cartilage and Chondrocytes
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents for Isolation of Primary Rib Chondrocytes from Newborn Mice and Cross-Link
2.3 Reagents for ChIP
2.4 Reagents for ChIP-Seq Library Construction
3 Methods
3.1 Isolation of Primary Rib Chondrocytes from Newborn Mice and Cross-Link
3.2 ChIP
3.3 ChIP-Seq Library Construction
3.4 Sequencing by NGS
3.5 ChIP-Seq Data Analysis
4 Notes
References
Chapter 13: MicroRNAs and Regulation of Autophagy in Chondrocytes
1 Introduction
1.1 MicroRNA: Biogenesis and Role in Chondrogenesis
1.2 Autophagy in Chondrocytes
1.3 Interactions of miRNA with Genes Regulating Autophagy: The Autophagy Interactome
2 Materials
2.1 Culture Media
2.2 Chemicals and Kits
3 Methods
3.1 Chondrocyte Culture
3.2 Transfection of miRNA
3.3 Quantifying miRNA Expression
4 Notes
References
Chapter 14: Mitochondrial Biogenesis, Activity, and DNA Isolation in Chondrocytes
1 Introduction
2 Materials
2.1 Cell Culture Medium and Reagents
2.2 Mitochondrial Mass Determination
2.3 Mitochondrial DNA Measurement
2.4 Mitochondrial DNA Mutation Detection
2.5 SDS/Western Blot Reagents
2.6 Assessment of Acetylation Levels of PGC-1a
2.7 siRNA Transfection Reagent
2.8 SIRT3 Deacetylation Activity Assay
2.9 Oxygen Consumption Rate (OCR) Measurement
2.10 Intracellular ATP Measurement
3 Methods
3.1 Primary Human Knee Chondrocyte Cell Preparation and Culture as Reported
3.2 Protein Expression of PGC-1α, TFAM, NRF1, NRF2, SIRT3, SOD2, OGG1, Acetylated SOD2, Acetylated OGG1, and OXPHOS
3.2.1 Cell Lysate Preparation
3.2.2 Protein Concentration Determined by BCA
3.2.3 Protein Separation and Membrane Transfer
3.2.4 Western Blotting
3.3 Assessment of Acetylation Levels of PGC-1α
3.3.1 Immunoprecipitation with PGC-1α Antibody Using Pierce Classic IP Kit (See Note 11)
3.3.2 Western Blot with Anti-Acetyl Lysine Antibody
3.4 Mitochondrial Mass by MitoTracker Green FM Staining
3.5 mtDNA Content by Real-Time PCR
3.5.1 Total DNA Is Isolated from Chondrocytes Using DNeasy Blood & Tissue Kit
3.5.2 DNA Concentration Measured by NanoDrop
3.5.3 Real-Time PCR
3.6 OCR Measurement
3.7 ATP Measurement
3.8 Knockdown of TFAM in Human Knee Articular Chondrocytes
3.8.1 Cell Preparation Before Transfection
3.8.2 Transfection (Single Well of 6-Well Plate as an Example)
3.9 mtDNA Mutation Detection by Real-Time PCR
3.10 SIRT3 Deacetylase Activity Assay (See Note 23)
4 Notes
References
Chapter 15: Assessing Chondrocyte Status by Immunofluorescence-Mediated Localization of Parkin Relative to Mitochondria
1 Introduction
2 Materials
2.1 Chondrocyte Culture
2.2 Mitochondrial Dysfunction Inducers
2.3 Staining of Mitochondria
2.4 Cell Fixation and Permeabilization
2.5 Antibodies, Blocking Agent, and Primary and Secondary Antibody Diluent
2.6 Nuclear Counterstaining and Antifade Mounting media Agent
2.7 Confocal Microscope
2.8 Buffers and Reagents
3 Methods
3.1 Chondrocyte Culture and Mitochondrial Staining with MitoTracker Deep Red
3.2 Cell Fixation and Permeabilization
3.3 Blocking
3.4 Primary Antibody Staining
3.5 Secondary Antibody Staining
3.6 DAPI Staining and Mounting
3.7 Imaging
4 Notes
References
Index