Trophoblasts: Methods and Protocols (Methods in Molecular Biology, 2728)

✍ Scribed by Sandeep Raha (editor)

• Publisher: Humana
• Year: 2023
• Tongue: English
• Leaves: 249
• Category: Library

✦ Synopsis

This volume explores the latest approaches used to assess trophoblast angiogenesis, transport function, cellular respirations, migration, and invasion. The chapters in this book cover topics such as various methods to study and manipulate primary trophoblast cells; strategies using liposomes to deliver biomolecules to trophoblasts; in vitro models of the placenta that emulate the cellular interactions in the 3D uterine environment; and models exploring heterogenous cell types and 3D-organoid structures that represent cell-to-cell interactions in vivo. 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 tips on troubleshooting and avoiding known pitfalls.

Cutting-edge and thorough, Trophoblasts: Methods and Protocols is a valuable resource that will help researchers better understand the function of the placenta and its complex contribution to maternal health and fetal development, and also provide greater insights to the area of study known as the developmental origins of health and diseases (DoHaD).

✦ Table of Contents

Preface
Contents
Contributors
Part I: Isolation and Culture Methods
Chapter 1: Isolation and Maintenance in Culture of Primary Human Trophoblast from Term Placentae
1 Introduction
2 Materials
2.1 Equipment and Materials
2.2 Chemical Reagents and Media
3 Methodology
3.1 Isolation Protocol
3.2 Plating Protocol
4 Notes
References
Chapter 2: Isolation of Human Villous Cytotrophoblastic Cells from Term Placenta for Transfection
1 Introduction
2 Materials
2.1 Dissection of Placental Tissue
2.2 Tissue Digestion
2.3 5-70% Discontinuous Percoll Gradient
2.4 Cell Culture Medium
2.5 Microporation of vCTB
3 Methods
3.1 Placental Tissue Dissection
3.2 Percoll Gradient Preparation and Loading of Cells
3.3 Harvesting of vCTB from Gradient and Washing
3.4 Transfection of Villous Cytotrophoblastic Cells with siRNA by Microporation
4 Notes
References
Chapter 3: Enzymatic Digestion and Single Cell Isolation of Peri-implantation Stage Human Trophoblast Cells
1 Introduction
2 Materials
2.1 Extended Culture During the Peri-implantation Period
2.1.1 Human Embryo Warming
2.1.2 Removal of the Zona Pellucida
2.1.3 Fibronectin-Coated Chambered Coverslips (See Note 2)
2.1.4 IVC1/IVC2
2.2 Single-Cell Digestion
2.3 Collection for Downstream Assays
3 Methods
3.1 Extended Culture During the Peri-implantation Period
3.2 Enzymatic Digestion into Single Cells
3.3 Collection of Single Cells for Downstream Assays
4 Notes
References
Chapter 4: Precision-Cut Slice Culture Method for Rat Placenta
1 Introduction
2 Materials
2.1 Culture Medium and Containers
2.2 Chemicals and Accessories
2.3 Instruments
2.4 Animals
3 Methods
3.1 Animal Breeding
3.2 Preparation
3.3 Tissue Harvest
3.4 Preparing Organotypic Slice Cultures
3.5 Slice Culture Maintenance
3.6 Harvesting the Precision-Cut Placental Tissue Slices
4 Notes
References
Part II: Analysis of Fusion
Chapter 5: Evaluation of Molecular Interactions and Cellular Dynamics at the Maternal-Fetal Interface During Placental Morphog...
1 Introduction
1.1 Structural Organization of Maternal-Fetal Interface in Mice, Rats, and Human
1.2 Strategies to Study Maternal-Fetal Interface
2 Materials
2.1 Cell Culture Reagents
2.2 Flow Cytometry
2.3 In Situ Hybridization
2.4 Immunohistochemistry
2.5 Immunofluorescence
2.6 qRT-PCR
2.7 Western Blot
2.8 Co-culture Without Physical Contact
2.9 Co-culture with Physical Contact
2.10 Spheroid Assay
2.11 Elisa
2.12 MS
2.13 Invasion Assay
2.14 Scratch Wound Assay
3 Methods
3.1 Harvesting Mouse/Rat Utero-placental Tissues
3.2 Mouse Trophoblast Stem Cell Culture and Induction of Differentiation
3.3 Harvesting Human Placental Tissue
3.4 Mouse Endothelial Cell Line, MS1 (MILE SEVEN1)
3.5 Rat Aortic Smooth Muscle Cell Culture
3.6 Human Trophoblast Cell Lines and Culture Conditions
3.7 Cell Lines and Culture Conditions of Cells Used to Study Cell-Cell Interaction
3.8 Analysis of Cell Composition at the Maternal-Fetal Interface
3.8.1 Flow Cytometry
3.8.2 Histological Analysis: In Situ Hybridization
3.8.3 Immunohistochemistry
3.8.4 Immunocytochemical Analyses: Direct and Indirect Immunofluorescence
3.8.5 Biochemical Analyses: qRT-PCR Analysis
3.8.6 Western Blotting Analysis
3.9 Characterization of the Cellular Interactions at the Maternal-Fetal Interface
3.9.1 Co-culture Without Physical Contact
3.9.2 Co-culture with Physical Contact
3.9.3 Spheroid Assay
3.10 Analysis of Trophoblast Cell Function
3.10.1 Analysis of Secretory Cytokines/Chemokines from Trophoblast Cells Using ELISA
3.10.2 Mass Spectrometry to Analyze Proteome
3.10.3 Invasion Assay
3.10.4 Scratch Wound Assay
4 Notes
References
Chapter 6: In Vivo Quantitative Assessment of Gestational Choriocarcinoma Development and Progression Using Luminescent Tropho...
1 Introduction
2 Materials
2.1 Animals and Reagents
3 Methods
3.1 Animal Use
3.2 JEG-3-Luc Cell Preparation
3.3 The Surgery
3.4 Bioluminescence Imaging
3.5 Immunohistology Characterization
4 Notes
References
Chapter 7: Analyzing Trophoblast Fusion Using Immunofluorescence and Split Protein Complementation Assays
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescence
2.3 Split GFP Complementation Assay
3 Methods
3.1 Cell Culture
3.2 Analysis of BeWo Cell Fusion Using Immunofluorescence Staining
3.3 Lentiviral Production and Transduction of Split GFP Expression Vectors
3.4 Analysis of BeWo Cell Fusion Using a Split GFP Complementation Assay
4 Notes
References
Chapter 8: Quantification of Trophoblast Syncytialization by Fluorescent Membrane Labeling
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Fluorescent Membrane Potential Dye Di-8-ANEPPS
3.2 Forskolin-Induced BeWo Cell Fusion
3.3 Membrane Labeling with Di-8-ANEPPS
3.4 Fluorescence Imaging of Trophoblast Fusion
3.5 Fusion Index Quantification
4 Notes
References
Chapter 9: Assessment of Placental Sodium-Independent Leucine Uptake and Transfer in Trophoblast Cells
1 Introduction
2 Materials
2.1 General Cell Culture
2.2 Leucine Uptake
2.3 Leucine Transfer
3 Methods
3.1 Preparation of BeWo Cells
3.2 Leucine Uptake
3.2.1 Preparation for Leucine Uptake Dose-Response Assay
3.2.2 Leucine Uptake
3.3 Leucine Transfer Across the Placental Barrier Using Transwell Technique
3.3.1 Preparation for Leucine Transport Across a Placental Barrier Model
3.3.2 Trans-epithelial Electrical Resistance (TEER)
3.3.3 Apparent Permeability Coefficient (Papp)
3.3.4 Leucine Transfer
4 Notes
References
Chapter 10: Assessing Cholesterol Efflux on Primary Human Trophoblast Cells
1 Introduction
2 Materials
3 Methods
3.1 Seeding the Cells and Labeling Cellular Cholesterol
3.2 Equilibration
3.3 Acceptor Incubation
3.4 Sample Processing
3.5 Analysis of Results and Calculations
4 Notes
References
Chapter 11: RGD-Based Fluorescence to Assess Placental Angiogenesis
1 Introduction
2 Materials
3 Methods
3.1 αvb3 Labeling with Angiostamp700
3.2 2D-Fluorescence In Vivo Imaging
3.3 3D-Fluorescence and microCT In Vivo Imaging
3.4 Ex Vivo Fluorescence Imaging
4 Notes
References
Chapter 12: Real-Time Assessment of Mitochondrial Function in Cytotrophoblast and Syncytialized Trophoblast Cells Using the Se...
1 Introduction
2 Materials
3 Methods
3.1 Washing the Cells
3.2 Preparing Stock Compounds (See Note 12)
3.3 Loading Compounds into Sensor Cartridge (See Note 15)
3.4 BCA Analysis
4 Notes
References
Chapter 13: Quantifying Trophoblast Function Using the xCELLigence System
1 Introduction
2 Materials
2.1 Equipment
2.2 Cells
2.3 Reagents
3 Methods
3.1 Setting Up the Analyzer
3.2 Proliferation
3.3 Cell Preparation
3.4 Set Up Experimental Protocol
3.5 Background Readings
3.6 Addition of Cells
3.7 Start Experimental Measurements
3.8 Graphing the Data
3.9 Adhesion
3.10 Migration
3.11 Invasion
3.12 Examination of Cells Post-experiment
4 Notes
References
Part III: Targeted Therapy
Chapter 14: Liposome-Encapsulated Anti-inflammatory Proteins for Targeted Delivery to the Placenta to Treat Fetal Growth Restr...
1 Introduction
2 Materials
3 Methods
3.1 Synthesis of the Liposomes and Encapsulation of C43
3.2 Determine the Effect of Liposome-Encapsulated C43 on Placental Trophoblast or Endothelial Cell Viability and Proliferation...
4 Notes
References
Chapter 15: Trophoblast-Targeted Liposomes for Placenta-Specific Drug Delivery
1 Introduction
2 Materials
3 Methods
3.1 Film Method to Synthesize Liposomes
3.2 Conjugation of Peptides to Liposomes
3.2.1 Ester Activation
3.2.2 Amine Reaction
3.3 Characterization of the plCSA-liposomes
3.3.1 Determination of the Size of plCSA-liposomes
3.3.2 Determination of the Encapsulation Efficiency and Loading Efficiency
3.3.3 Determination of the Conjugation Efficiency
4 Notes
References
Part IV: Advanced 3D Models
Chapter 16: Methods for Co-culture of Primary Human Extravillous Trophoblast Cells and Uterine Natural Killer Cells
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions and Media
3 Methods
3.1 Tissue Preparation
3.2 First Trimester Villous Explant Outgrowth
3.3 First Trimester Villous Enzymatic Digestion
3.4 Isolate EVT from Term Placenta with Enzymatic Digestion
3.5 uNK Cell Isolation Using Magnetic Sorting
3.6 uNK Isolation by Flow Cytometry
3.7 EVT and uNK Cell Co-culture
3.8 Direct Co-culture
3.9 Indirect Co-culture
4 Notes
References
Chapter 17: Trophoblast Organoids as a Novel Tool to Study Human Placental Development and Function
1 Introduction
2 Materials
2.1 Tissue Culture Plastic
3 Methods
3.1 General Instructions
3.1.1 Preparation of Solutions
3.2 Isolation of vCT
3.2.1 Tissue Preparation
3.2.2 Tissue Digestion
3.2.3 Percoll Gradient Enrichment for vCT
3.2.4 Removal of Erythrocytes
3.3 Formation of Trophoblast Organoid (TB-ORG) Domes
3.4 Splitting of TB-ORG
3.4.1 Splitting of Four TB-ORG Domes with High Density of Organoids
3.4.2 Splitting of Two Domes Containing Large TB-ORG
3.5 Freezing and Thawing of TB-ORG
3.5.1 Freezing of TB-ORG
3.5.2 Thawing of TB-ORG
3.6 TB-ORG Differentiation
3.6.1 EVT Induction Directly After Splitting
3.6.2 EVT Induction of TB-ORG
3.6.3 Isolation of Trophoblast Subtypes from TB-ORG
3.7 Molecular and Histological Analysis of TB-ORG
3.7.1 TB-ORG Lysis for RNA and Protein Preparation
3.7.2 Preparation of Paraffin-embedded TB-ORG Tissue Blocks
4 Notes
References
Chapter 18: A Three-Dimensional Trophoblast Invasion Microfluidic Platform for Toxicological Screening
1 Introduction
2 Materials
2.1 Instruments
2.2 Reagents
3 Methods
3.1 Chip Priming
3.2 Fibronectin Coating
3.3 Cell Seeding
3.4 Media Flow
3.5 Real-time Imaging
3.6 Antibody Staining and Imaging
3.7 Cell Harvesting
4 Notes
References
Chapter 19: Three-Dimensional In Vitro Human Placental Organoids from Mononuclear Villous Trophoblasts or Trophoblast Stem Cel...
1 Introduction
2 Materials
2.1 Isolation of Mononuclear Villous Trophoblasts
2.2 Culture of Side-population Trophoblasts and Okae Human TSC
2.3 Trophoblast Organoid Formation
2.4 Preparation of Organoids for Histological Analysis
2.5 Fixation and Immunohistochemical Staining of Organoids
3 Methods
3.1 Generation of First Trimester Trophoblast Organoids: Isolation of First Trimester Mononuclear Villous Trophoblasts
3.2 Side-population Trophoblast or Okae TSC Culture
3.3 Trophoblast Organoid Formation
3.4 Organoid Passaging, Freezing, or Stimulation of Extravillous Trophoblast Outgrowth
3.5 Histological Assessment of Trophoblast Organoids
3.5.1 Fixation and Embedding of Trophoblast Organoids for Immunohistochemical Analysis
3.5.2 Whole Mount Staining of Trophoblast Organoids
4 Notes
References
Index