Name: Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface
Uploaded: 2023-10-31T11:45:33
Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Preparation, Prior to Collection Day
<ol>
	<li>Autoclave strainers/forceps for collection, and scissors/forceps for micro-dissection.</li>
	<li>Prepare an adequate volume (500 ml per specimen) of Wash buffer (refer to Table 1 for the recipe).</li>
	<li>...

establishing villous and decidual organ cultures as ex vivo models of human maternal-fetal interface

Source: Rizzuto, G. A., et al. <a href="https://app.jove.com/v/54237">Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface.</a> J. Vis. Exp. (2016).
This video demonstrates the establishment of primary human placental villous and decidual organ cultures in ex vivo conditions. These models are well-suited for studying pathogenesis at the human maternal-fetal interface.

<img alt="Figure 1" src="/files/ftp_upload/21715/21715_Figure1.jpg" /> 
Figure 1: Villous organ cultures &#8211; Representative gross and microscopic images. (A) Two terminal villous trees with a gestational age of 6 weeks, as viewed under a dissecting microscope. Note the &#34;fluffy&#34; ends (arrowheads) and prominent fetal vasculature coursing through the branches of the tree on the left that make this piece suitable for organ cu...

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

Begin with placental villous and decidual parietalis specimens.
Dissect the villous &#8212; a well-vascularized tissue containing extravillous trophoblast,&#160; or EVT, cell columns.
Transfer the villous over an extracellular matrix, ECM-coated transwell placed in a tissue culture well.
Take the next specimen; microdissect the decidua &#8212; a uterine tissue surrounding the developing fetus.
Place the decidua over another ECM-coated transwell.
Add a suitable medium to the tissue culture wells to maintain humidity, while keeping the transwells devoid of medium.
Without a medium, the EVT cell columns secrete proteinases that cleave ECM proteins.
This initiates ECM remodeling, creating space for the EVT cells to invade and firmly adhere to the matrix.
Meanwhile, the other cells of the villous and decidua attach to the ECM by activating integrin receptors, which cluster to bind cooperatively to the matrix.
These processes strengthen the cell-to-ECM adhesion, securely embedding the tissues.
Finally, add tissue-specific media to the transwells for culture growth.

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Research

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

In Vitro Models

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

JoVE Journal

Genetics

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Developmental Biology

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Establishing Villous and Decidual Organ Cultures as Ex Vivo Models of Human Maternal-Fetal Interface

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