VASCULAR MORPHOGENESIS : methods and protocols.

Preface
Contents
Contributors
Chapter 1: Historical Overview of In Vivo and In Vitro Angiogenesis Assays
	1 The Past Will Always Govern the Present!
	2 Modern Era of Angiogenesis Assays
		2.1 Corneal Neovascularization Assay
		2.2 In Vitro Vasculogenesis Assays
		2.3 Experiments ``Designed´´ by Nature
	References
Chapter 2: The Fundamental Contribution of Judah Folkman in the Setting of Angiogenesis Assays
	1 Development of Assays to Study Angiogenesis
	2 Tumor Growth in Isolated Perfused Organs
	3 The Rabbit Cornea
	4 The Chorioallantoic Membrane
	5 Culture of Endothelial Cells
	References
Chapter 3: Identification of Endothelial Cells and Their Progenitors
	1 Introduction
	2 Materials
		2.1 Reagents
		2.2 Equipment
	3 Methods
		3.1 ECFC Isolation and Expansion
		3.2 ECFC Cryopreservation
		3.3 Thawing ECFC
		3.4 ECFC Culture and Expansion
		3.5 Single-Cell Preparation
		3.6 Single-Cell Clonogenic Assay
	4 Notes
	References
Chapter 4: In Vitro Coculture Assays of Angiogenesis
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Reagents for Cell Staining
	3 Methods
		3.1 Cell Culture
		3.2 The Standard Coculture Assay
		3.3 Staining Cocultures for Quantification
		3.4 Preparation of Cocultures for Immunofluorescence Microscopy
		3.5 Preparation of Human Macrophages and Incorporation into Assay
		3.6 Preparation of Tumoroids and Incorporation into Assay
	4 Notes
	References
Chapter 5: Endothelial Cells: Co-culture Spheroids
	1 Introduction
	2 Materials
		2.1 Tissue
		2.2 Reagents for Isolation and Culture of HUVECs
		2.3 Reagents for HBMSC Isolation and Culture
		2.4 Reagents for Monoculture and Co-culture Spheroids
	3 Methods
		3.1 Isolation and Culture of HUVEC
		3.2 Isolation and Culture of HBMSC
		3.3 Generation of Co-culture 3D Spheroids
	4 Notes
	References
Chapter 6: Microfluidic Device Setting by Coculturing Endothelial Cells and Mesenchymal Stem Cells
	1 Introduction
	2 Materials
		2.1 Fabrication of Master Molds (Photolithography)
		2.2 Fabrication of Microfluidic Devices (Soft Lithography)
		2.3 Formation of Microchannels and Surface Treatment
		2.4 Gel Formation in the Gel Region
		2.5 HUVEC-MSC Coculture in a Microfluidic Device
	3 Methods
		3.1 Fabrication of Master Molds (Photolithography)
		3.2 Fabrication of Microfluidic Devices (Soft Lithography)
		3.3 Formation of Microchannels and Surface Treatment
		3.4 Gel Formation in the Gel Region
		3.5 HUVEC-MSC Coculture in a Microfluidic Device
	4 Notes
	References
Chapter 7: Spatial Statistics-Based Image Analysis Methods for the Study of Vascular Morphogenesis
	1 Introduction
	2 Materials
		2.1 Software Tools
		2.2 Analysis of Vessel Tree Growth and Distribution
		2.3 Analysis of Microvessel and Tissue Cell Codistribution
	3 Methods
		3.1 Imaging Chick Embryo Area Vasculosa
		3.2 Imaging Mast Cells and Microvessels in Tissue Sections
		3.3 Morphometric Characterization of Vascular Branching
		3.4 Morphometric Characterization of the Vascular Tree Spatial Distribution
		3.5 Morphometric Characterization of the Spatial Relationship Between Cells and Vessels
	4 Notes
	References
Chapter 8: Studying Angiogenesis in the Rabbit Corneal Pocket Assay
	1 Introduction
		1.1 Rabbit Cornea Pocket Assay
	2 Materials
		2.1 Animals
		2.2 Reagents and Drugs
		2.3 Facilities, Equipment, and Materials
	3 Methods
		3.1 Sample Preparation
		3.2 Surgery
		3.3 Quantification of Neovascular Growth
		3.4 Histological Examination and Immunohistochemical Analysis
		3.5 Gene and Protein Expression
		3.6 Drug Treatments and Pharmacokinetic Studies
		3.7 Advantages and Limitations of Rabbit Cornea Assay
	4 Notes
	References
Chapter 9: Avians as a Model System of Vascular Development
	1 Introduction
	2 Materials
		2.1 Fertilized Eggs
		2.2 Incubators
		2.3 Dissection Tools (Fig. 3)
		2.4 Optical Equipment
		2.5 Electroporation/Viral Injection Equipment (Fig. 5)
		2.6 Solutions
		2.7 Preparation for Agar-Albumen Culture Dishes for Modified New culture (EC Culture, Related to Subheading 3.1.3)
		2.8 Reagents of Viral Production in Replication-Incompetent Retroviral System Using (Related to Subheading 3.3.4)
	3 Techniques for Handling the Embryo
		3.1 Culture of the Avian Embryo
			3.1.1 In Ovo Culture: Preparation, In Ovo Operation, and Incubation After the Surgery
			3.1.2 Shell-Less Culture
			3.1.3 New Culture: Modified EC Culture
				Making Culture Plates
				Making Filter Paper Rings
				Dissection of Embryos for EC Culture (Fig. 8)
		3.2 Labeling Techniques
			3.2.1 Tracing Cells by Vital Dye Staining
		3.3 Somatic Transgenesis
			3.3.1 Electroporation
				In Ovo Electroporation (Fig. 9)
				Ex Ovo Electroporation (Fig. 10)
			3.3.2 Lipofection
			3.3.3 Adenovirus
			3.3.4 Replication-Incompetent Retroviral-Mediated Gene Transfer
				Transfection
				Virus Production
		3.4 Genomics
		3.5 Chorioallantoic Membrane Assay (CAM)
		3.6 Other Approaches
		3.7 Conclusion
	4 Notes
	References
Chapter 10: Dynamic Imaging of Mouse Embryos and Cardiac Development in Static Culture
	1 Introduction
	2 Materials
		2.1 Embryo-Dissection Medium
		2.2 Rat Serum Extraction
		2.3 Embryo Dissection
		2.4 Embryo-Culture Medium
	3 Methods
		3.1 Rat Serum Extraction (See Note 1)
		3.2 Embryo Dissections for Live Imaging
		3.3 Imaging Setup
		3.4 Visualization of Embryonic Structures Using OCT
		3.5 Cardiodynamic Analysis
		3.6 Imaging the Vasculature Using Speckle Variance
		3.7 Live Imaging of Hemodynamics Using Doppler OCT
		3.8 Conclusion
	4 Notes
	References
Chapter 11: High-Resolution Confocal Imaging of Pericytes in Human Fetal Brain Microvessels
	1 Introduction
	2 Materials
	3 Methods
		3.1 Fixation and Sectioning
		3.2 Immunofluorescence Staining
		3.3 High-Resolution Confocal Imaging
		3.4 Image Processing and 3D Reconstruction
	4 Representative Results
	References
Chapter 12: Quantification of Tumor Vasculature by Analysis of Amount and Spatial Dispersion of Caliber-Classified Vessels
	1 Introduction
	2 Materials
	3 Methods
		3.1 Vessel Staining
		3.2 Image Acquisition
		3.3 Images to Stack
		3.4 Making Stack Isotropic
		3.5 Contrast Enhancement and Image Thresholding
		3.6 Vessel Fill-Up [macro: 3D_Close&Fill.txt]
		3.7 Bandpass Caliber Masks [macro: ClassifyVessels.txt]
		3.8 Classification of Original Binary Voxels in Caliber Classes [macro: OriSignalRecovery.txt]
		3.9 Assembly of Progressively Reconstructed Angioarchitectures (PRA) [macro: BuildProgressive-Angioarchitectures.txt]
		3.10 Normalization [macro: NormalizingVolume.txt]
		3.11 Analysis of PRAs by Signal Amount and Spatial Dispersion [plugin: Spatial_Dispersion.class]
		3.12 Linear Regression and Important Descriptive Parameters
		3.13 Biological Meaning of Descriptive Parameters
		3.14 Visual Workflow Control by Volume Viewing (3D Renderings)
		3.15 Identification of Discarded Vascular Structures and Signals
		3.16 Analysis Scale-Up
		3.17 Statistical Analyses for Comparison of Angioarchitectures
	4 Notes
	5 Rationale for Plugin ``Spatial Dispersion´´ [https://github.com/nHv95/Vessel_analysis/Spatial_Dispersion.java]
	References
Chapter 13: A Xenograft Model for Venous Malformation
	1 Introduction
	2 Materials
		2.1 Cell Culture
		2.2 Subcutaneous Injection of Cells into Mouse
		2.3 Lesion Plug Measurements and Collection
		2.4 Tissue Staining
	3 Methods
		3.1 Cell Plating and Expansion
		3.2 Day Before Injection
		3.3 Endothelial Cell Preparation and Counting
		3.4 Syringe Preparation
		3.5 Subcutaneous Injection into Mouse
		3.6 Lesion Growth Monitoring
		3.7 Tissue Collection and Processing
		3.8 Staining of Lesion Sections
			3.8.1 Hematoxylin and Eosin (H&E)
			3.8.2 Ulex europaeus Agglutinin-1 (UEA-1) Immunohistochemistry (IHC)
		3.9 Vascular Channel Analysis
	4 Notes
	References
Chapter 14: In Vivo Vascular Network Forming Assay
	1 Introduction
	2 Materials
		2.1 Cell Culture of Human ECFCs and MSCs
		2.2 Collagen-Fibrin Hydrogel
		2.3 Immunofluorescent Staining
	3 Methods
		3.1 Preparation of Collagen-Fibrin Hydrogel
		3.2 Preparation of Cell-Hydrogel Mixture for Injection
		3.3 Injection into Immunodeficient Nude Mice
		3.4 Harvesting the Implants
		3.5 Evaluation of Vascular Networks in Explanted Collagen-Fibrin Plugs
		3.6 Evaluation of Human Lumens and Perivascular Coverage by Immunofluorescent Staining
		3.7 Evaluation of Cell Proliferation and Apoptosis in Vascular Networks
	4 Notes
	References
Chapter 15: Assessment of Vascular Patterning in the Zebrafish
	1 Introduction
		1.1 Morpholinos
		1.2 Genetic Mutants
		1.3 Assessing Vascular Phenotypes in Loss-of-Function Models
			1.3.1 Where Is the Gene Expressed?
			1.3.2 Assessing General Morphology and Development in Morphants or Mutants
			1.3.3 Assessing Circulation and Cardiac Function
			1.3.4 Assessing Vascular Patterning
		1.4 Summary of Considerations in Assessing Vascular Phenotypes
	2 Materials
		2.1 Microangiography
		2.2 Long-Term Time-Lapse Imaging
	3 Methods
		3.1 Microangiography
			3.1.1 Preparation of the Apparatus
			3.1.2 Experimental Procedure
		3.2 Long-Term Time-Lapse Imaging
			3.2.1 Mounting Animals for Long-Term Time-Lapse Imaging
	4 Notes
	References
Chapter 16: By the Skin of Your Teeth: A Subcutaneous Mouse Model to Study Pulp Regeneration
	1 Introduction
	2 Materials
	3 Methods
		3.1 Isolation and Culture of Dental Pulp Stem Cells (DPSC)
		3.2 Preparation of the Scaffold
		3.3 Implantation of the Scaffolds
		3.4 Tissue Isolation and Embedding into Paraffin
		3.5 Masson´s Trichrome Staining
		3.6 Hematoxylin-Eosin
	4 Notes
	References
Index