Maternal Placental Interface: Methods and Protocols (Methods in Molecular Biology, 2781)

Dedication
Preface
Contents
Contributors
Chapter 1: Histological Analysis of Trophoblast Cells in the Mouse Placental Labyrinth Zone
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Specific Solutions and Reagents
	3 Methods
		3.1 Tissue Collection
		3.2 Processing for Paraffin Sectioning
		3.3 Sectioning Paraffin Blocks
		3.4 Fluorescent Immunohistochemistry
		3.5 Image Analysis
	4 Notes
	References
Chapter 2: Isolation and Immunophenotyping of Leukocytes from the Human Maternal-Fetal Interface
	1 Introduction
	2 Materials
		2.1 Biological Samples
		2.2 Specific Solutions and Reagents
		2.3 Tissue Digestion
		2.4 Cell Sorting
		2.5 Density Gradient Separation
		2.6 Immunophenotyping
	3 Methods
		3.1 Isolation of Choriodecidual Leukocytes
			3.1.1 Tissue Digestion
		3.2 Cell Sorting
		3.3 Isolation of Placental Blood Leukocytes
			3.3.1 Placental Blood Collection
			3.3.2 Cell Isolation by Density Gradient
		3.4 Immunophenotyping by Flow Cytometry
		3.5 Preparing Leukocytes for Nucleic Acid Isolation
	4 Notes
	References
Chapter 3: Evaluation of Leukocyte Chemotaxis Induced by Human Fetal Membranes in an In Vitro Model
	1 Introduction
	2 Materials
		2.1 Culture and Stimulation of Fetal Membranes
		2.2 Isolation of Umbilical Cord Blood Mononuclear Cells (UCBMC)
		2.3 Chemotaxis Assay
		2.4 Migrant Leukocyte Counting
		2.5 Leukocyte Immunophenotyping
	3 Methods
		3.1 Culture and Stimulation of Fetal Membranes (Cultivation Time: 3 Days)
		3.2 Isolation of Umbilical Cord Blood Mononuclear Cells (UCBMC)
		3.3 Chemotaxis Assay
		3.4 Migrant Leukocyte Counting
		3.5 Leukocyte Immunophenotyping
	4 Notes
	References
Chapter 4: In Vitro Culturing of Human Term Placental Explants
	1 Introduction
	2 Materials
		2.1 Disposable Reagents
		2.2 Non-disposable Sterilized Materials
		2.3 Equipment
	3 Methods
		3.1 Placenta Collection
		3.2 Dissection of Placental Explants
	4 Notes
	References
Chapter 5: In Vitro Culturing of Human Trophoblasts from Term Placenta
	1 Introduction
	2 Materials
		2.1 Disposable Reagents
		2.2 Non-disposable Sterilized Materials
		2.3 Equipment
	3 Methods
		3.1 Percoll Gradient Preparation
		3.2 Placenta Collection
		3.3 Isolation of Villous Trophoblasts
		3.4 Trophoblast Culture
	4 Notes
	References
Chapter 6: Culture of Human Fetal Membranes in a Two Independent Compartment Model: An Ex Vivo Approach
	1 Introduction
	2 Material
		2.1 Fetal Membrane Dissection and Washing
		2.2 Two Independent Compartment Culture Model
		2.3 Differential Stimulation: An Ex Vivo Approach
	3 Methods
		3.1 Fetal Membrane Dissection and Washing
		3.2 Two Independent-Compartment Culture Model
		3.3 Differential Stimulation: An Ex Vivo Approach
	4 Notes
	References
Chapter 7: Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae
	1 Introduction
	2 Materials (See Note 1)
		2.1 Equipment
		2.2 Solutions and Reagents
	3 Methods
		3.1 Biological Samples
		3.2 Scraping and Collection of Decidua
		3.3 Enzymatic Digestion
		3.4 Discontinuous Percoll Gradient (See Note 6)
		3.5 Cell Counting
		3.6 Morphological Characterization
		3.7 Vimentin Expression
	4 Notes
	References
Chapter 8: Generation of Knockout Mouse Trophoblast Stem Cells by CRISPR/Cas9
	1 Introduction
	2 Material
		2.1 Cells
		2.2 Cell Media Composition
		2.3 Harvesting, Transfecting, and Passaging Cells
		2.4 CRISPR/Cas9 Plasmid Cloning
	3 Methods
		3.1 Selection of the Target Exon and sgRNA Design
		3.2 CRISPR Plasmid Cloning
		3.3 CRISPR Plasmids Delivery into mTSC
		3.4 Single-Cell Sorting and Expansion
		3.5 Clonal KO mTSC Verification
	4 Notes
	References
Chapter 9: CRISPR Activation in Mouse Trophoblast Stem Cells
	1 Introduction
	2 Material
		2.1 Cell Lines
		2.2 Cell Media Composition
		2.3 Harvesting, Transfecting, and Passaging Cells
		2.4 CRISPR/dCas9 Plasmid Cloning
	3 Methods
		3.1 Lentiviral Production
			3.1.1 sgRNA Designing and Golden-Gate sgRNA Plasmid Cloning
			3.1.2 Lentiviral Packaging
		3.2 Generation of SAM mTSCs
		3.3 Generation of the sgRNA SAM mTSCs
	4 Notes
	References
Chapter 10: Feto-Maternal Interface Organ-on-Chip: A New Technology to Study Ascending Infection
	1 Introduction
	2 Materials
	3 Methods
		3.1 Master Mold Fabrication-Photo-Lithography and Machine Milling
		3.2 Fabrication of the Device
		3.3 Preparing the Device Before Loading
		3.4 Loading Cells into the Devices
		3.5 Propagation Experiments
	4 Notes
	References
Chapter 11: Culture and Maintenance of Immune Cells to Model Innate Immune Status at the Feto-maternal Interface
	1 Introduction
	2 Materials
		2.1 HL-60 Cell Culture and Differentiation
		2.2 THP-1 Cell Culture and Differentiation
		2.3 NK-92 Cell Culture and Differentiation
		2.4 BeWo Cell Culture
	3 Methods
		3.1 Maintenance of HL-60 Cell Line
		3.2 Differentiation of HL-60 into Neutrophil-Like Cells
		3.3 Maintenance of THP-1 Cell Line
		3.4 Differentiation of THP-1 into Macrophage-Like Cells
		3.5 Maintenance of NK-92 Cell Line
		3.6 Maintenance of BeWo Cell Line
		3.7 Differentiation of NK-92 into Decidual NK-Like Cells
		3.8 Cryopreservation of Cells
	4 Notes
	References
Chapter 12: Placental Trophoblast Cell Isolation from the Term Placenta
	1 Introduction
	2 Materials
		2.1 Reagents Required
		2.2 Solutions Required
		2.3 Specialist Equipment Required
	3 Methods
		3.1 Placenta Tissue Preparation and Enzymatic Digestion
		3.2 Modified Percoll Gradient Density Separation
		3.3 Immunopurification
		3.4 Maintenance of Trophoblast Culture
	4 Notes
	References
Chapter 13: Dissociation of Placental Tissues for Single-Cell Techniques
	1 Introduction
	2 Materials
		2.1 Human Placental Samples
		2.2 Reagents for Processing of Placental Samples
		2.3 Equipment for Processing of Placental Samples
	3 Methods
		3.1 Preparation of Single-Cell Suspension from the Basal Plate with Placental Villi (BP + PV)
		3.2 Preparation of Single-Cell Suspension from the Chorioamniotic Membranes (CAM)
		3.3 Cell Counting and Cryopreservation
		3.4 Thawing of Cryopreserved Single-Cell Suspensions
		3.5 10x Genomics Library Preparation, Sequencing, and Analysis
	4 Notes
	References
Chapter 14: Immunophenotyping of Leukocytes in Amniotic Fluid
	1 Introduction
	2 Materials
		2.1 Amniotic Fluid Cell Counting and Processing
		2.2 Immunophenotyping of Amniotic Fluid (See Note 2)
	3 Methods
		3.1 Amniotic Fluid Cell Counting and Processing
		3.2 Immunophenotyping of Amniotic Fluid (See Note 2)
	4 Notes
	References
Chapter 15: Obtaining Tissues of Human Amniotic Membrane and Identification of Pluripotent Markers
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Spesific Solutions and Reagents
	3 Methods
		3.1 Tissue Colletion
		3.2 Frozen Tissue Sections
		3.3 Sectioning Tissue-Tek Blocks
		3.4 Fluorescent Immunohistochemistry
		3.5 Image Analysis
	4 Notes
	References
Chapter 16: Isolation of total DNA from Placenta Samples, both Fresh and following Formalin and Paraffin Treatment
	1 Introduction
	2 Material
	3 Methods
		3.1 Paraffin Removal from Placenta
		3.2 Treatment of Formalin-Embedded and Fresh Tissue
		3.3 DNA Isolation
	4 Notes
	References
Chapter 17: Infection of Fetal Membranes with Mycobacterium tuberculosis and Tissue Processing to Isolate RNA for Expression A...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Culture and Cryopreservation of M. tuberculosis
		3.2 Determination of CFU/mL of the Cryopreserved Bacterial Culture
		3.3 Chorioamniotic Membrane Explants and Culture
		3.4 Chorioamniotic Membrane Infection
	4 Notes
	References
Index