Name: establishing villous and decidual organ cultures as ex vivo models of human maternal-fetal interface
Uploaded: 2023-10-31T11:45:33.000+00:00
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Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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

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

Using sterile technique in a tissue culture hood, thaw extracellular matrix files on ice, then, dilute the warmed extracellular matrix at a 1 to 1 ratio with ice cold collection medium. Mix the suspension with pipetting, taking care not to induce bubbles.
Next, place transwell inserts with 0.4-micron pores into individual wells of a 6-well tissue culture plate and coat each insert with 100 microliters of the extracellular matrix suspension. Place the plate on ice, and rinse the specimens 2 times in collection medium, transferring the tissue to a sterile petri dish under a dissecting microscope after the second wash.
The specimens contain 2 distinct types of decidual tissues; decidua parietalis and decidua capsularis. Using sterile springer dissecting scissors and forceps, microdissect the decidua parietalis into 3-cubic-millimeter pieces, using the forceps to tease away any clotted maternal blood and use forceps to transfer 3 or 4 pieces of decidua into each transwell.
Identify the well-vascularized villi with prominent extravillous trophoblast cell columns and use forceps to transfer 3 or 4 villous trees into each transwell. Adjust the branches, so that the trees are positioned flat atop the extracellular matrix and separate any clumped branches. Then, add 1 milliliter of collection medium to the bottom of each well and incubate the cultures overnight at 37 degrees Celsius in 5% carbon dioxide. 12 to 16 hours later, add 1 milliliter of the appropriate culture medium to the top of each transwell.

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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>Prepare an adequate volume (100 ml per specimen) of 0.22 &#181;M sterile-filtered Collection media (refer to Table 1 for the recipe).</li>
	<li>Aliquot Extracellular Matrix (ECM, refer to Table of Reagents for details) for long-term storage.</li>
	<li>Store ECM in solid form at -20 &#176;C. For best performance, avoid repeat freeze-thaw. Thaw bottle to a viscous solution at 4 &#176;C, and prepare 300 &#181;l aliquots in the cold room with chilled pipette tips. Store aliquots at -20 &#176;C.</li>
</ol>
2. Collection of Fresh Tissue
CAUTION: When working with fresh human tissue, researchers must follow Universal Precautions (OSHA) for preventing transmission of Bloodborne Pathogens, and must have received proper institutional training. Proper personal protective equipment, including gloves, eye protection, and lab coats are necessary.
<ol>
	<li>Transport autoclaved strainers (one per specimen), autoclaved forceps, Wash buffer (carboy), Collection media (25 ml per specimen in a 50 ml conical tube), spray bottle, 10% bleach, 70% ethanol, tissue collection tray, sharpie, and ice bucket/cooler to hospital/clinic. 
	NOTE: Specimens are received in a research-designated space located in a room close to the operating suite. A sterile tissue culture hood is not required, but the collection should occur expeditiously and the researcher should appropriately sanitize surfaces, as described below.</li>
	<li>Place carboy of Wash buffer adjacent to the sink, and spray the spigot with 10% bleach and 70% ethanol. Place a glass tissue collection tray on the light source (light pad or lightbox), and sanitize with 10% bleach and 70% ethanol. Allow to air dry.</li>
	<li>Have the clinician carry specimens from the operating suite to the specimen preparation room. At the sink, pour the specimen atop a sterile hand-held strainer, rinse the tissue several times in Wash buffer, and transfer the entire specimen in the buffer to a sanitized glass tray atop the light source. 
	NOTE: Assessment of tissue by the clinician is of the highest priority, thus the following steps are only performed after the clinician has verbally indicated that he/she has finished the evaluation.</li>
	<li>Use sterile forceps to select placental villi and decidua (Figures 2A and 3A) for collection, and transfer to separate 50 ml conical tubes. Label the tubes with gestational age. 
	NOTE: Preferred gestational ages are less than 9 weeks and less than 16 weeks for placenta and decidua, respectively. At the institution, only a fraction of each specimen is procured for research purposes, as adequate tissue must remain for clinical pathologic diagnosis.</li>
	<li>Store and transport the specimen on ice to the research lab.</li>
	<li>To collect more than one specimen, sanitize the glass tray as described above with 10% bleach and 70% ethanol between the collection of each specimen.</li>
</ol>
3. Preparation of Organ Culture Plates
NOTE: The following steps should be performed using sterile technique in a tissue culture hood. It is ideal for the dissecting microscope to be located in a sterile field. However, this is not absolutely necessary as long as micro-dissection is performed expeditiously, and instruments are dipped frequently in 70% ethanol.
<ol>
	<li>Thaw ECM vial(s) on ice. Dilute the ECM 1:1 with ice-cold Collection media, and mix by pipetting. Pipette slowly to avoid introducing bubbles. Each well of a 6-well tissue culture plate requires 100 &#181;l of this suspension and will accommodate 3-4 organ cultures.</li>
	<li>Place transwell inserts (pore size 0.4 &#181;M) into each well of the 6-well tissue culture plate.</li>
	<li>Coat each transwell insert with a thin layer (100 &#181;l) of the ECM/media suspension.</li>
	<li>Place the plate on ice and immediately proceed to the establishment of organ cultures. Alternatively, prepare the plate prior to tissue collection, in which case wrap it in para-film and store it at 4 &#176;C for later use. Storage longer than 6 hr is not recommended as the ECM will dry out.</li>
</ol>
4. Establishment of Organ Cultures
NOTE: This step describes how to place decidual organ cultures and villous organ cultures in separate transwells. The tissues are not in contact.
<ol>
	<li>Rinse specimens twice in Collection media, and centrifuge at 1,000 x g between each rinse. Transfer tissue to a sterile petri dish and place it on the stage of a dissecting microscope. Keep a 6-well plate on ice adjacent to the microscope.</li>
	<li>Micro-dissect tissue with sterile springer dissecting scissors and forceps, to establish 2-3 mm villous organ cultures (Figure 1A). 
	NOTE: Villous organ cultures are small, well-vascularized &#34;trees&#34; with 2 or 3 branches, and with prominent extravillous trophoblast (EVT) cell columns11. It is important to utilize only villous tissue from &#60; 9-week first trimester specimens, as the invasion of extravillous trophoblasts into ECM is most pronounced at these young ages.</li>
	<li>Select well-vascularized villi with prominent EVT cell columns. EVT cell columns are visualized as bulbous, &#34;fuzzy&#34;, &#34;fluffy&#34; ends (refer to arrowheads in Figure 1A).</li>
	<li>Use sterile forceps to transfer 3 or 4 villous trees into each transwell. Adjust branches so the tree is positioned flat atop ECM and separate clumped branches.</li>
	<li>Micro-dissect decidual tissue with sterile springer dissecting scissors and forceps, to establish 3 mm3 decidual organ cultures (Figure 2A). 
	NOTE: Specimens contain two distinct types of decidual tissues, decidua parietalis and decidua capsularis (left and right tissue fragments, respectively, Figure 2A).</li>
	<li>Trim with scissors to create 3 mm3 pieces of decidua parietalis. Decidua capsularis is not used for these cultures. Use forceps to tease away any clotted maternal blood.</li>
	<li>Use sterile forceps to transfer 3-4 pieces of decidua into each transwell.</li>
	<li>Add 1 ml of Collection media to the bottom of the well. Incubate plate overnight at 37 &#176;C in 5% CO2</li>
	<li>For experiments where it is important to mimic normal first-trimester placental oxygen tension, maintain villous organ cultures in relative hypoxia (3% O2). 
	NOTE: Laboratory options include small hypoxia incubator chambers that fit inside existing incubators or a tri-gas incubator that introduces external N2 gas to lower oxygen concentration.</li>
	<li>Add 1 ml of Villous or Decidual medium (Table 1) to the top of the transwell after 12-16 hr of culture. 
	NOTE: The absence of media during the first overnight in culture allows the extravillous trophoblast cell columns to invade (villous culture), and for the tissue (both villous and decidual) to become securely embedded in ECM. In our experience, decidual organ cultures remain viable for 72 hr and villous organ cultures for 96 hr. We recommend experimental endpoints that fall within this timeframe.&#160; See Table 1 for the composition of Villous and Decidual medium.&#160; The villous medium has a high percentage of FBS, while the Decidual medium is supplemented with pregnancy hormones (progesterone, 17&#946;-estradiol) that maintain the decidualized state (Table 1).</li>
</ol>
Table 1: Media recipes. Components and concentrations for preparation of Wash buffer, Collection medium, Villous medium, and Decidual medium.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Wash buffer</td>
			<td>Collection medium</td>
			<td>Villous medium</td>
			<td>Decidual medium</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>DMEM/F-12 with GlutaMAX</td>
			<td>DMEM/F-12 with GlutaMAX</td>
			<td>DMEM/F-12 with GlutaMAX</td>
		</tr>
		<tr>
			<td>Penicillin 100 IU/ml</td>
			<td>Fetal Bovine Serum 2.5%</td>
			<td>Fetal Bovine Serum 20%</td>
			<td>Fetal Bovine Serum 2.5%</td>
		</tr>
		<tr>
			<td>Streptomycin 100 &#956;g/ml</td>
			<td>Penicillin 100 IU/ml</td>
			<td>Penicillin 100 IU/ml</td>
			<td>17&#946;-estradiol 300 pg/ml</td>
		</tr>
		<tr>
			<td>Gentamicin 50 &#956;g/ml</td>
			<td>Streptomycin 100 &#956;g/ml</td>
			<td>Streptomycin 100 &#956;g/ml</td>
			<td>Progesterone 20 ng/ml</td>
		</tr>
		<tr>
			<td>Amphotericin B 1.25 &#956;g/ml</td>
			<td>Gentamicin 50 &#956;g/ml</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Amphotericin B 1.25 &#956;g/ml</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sterilization pouches</td>
			<td>Fisher Scientific</td>
			<td>01-812-54</td>
			<td>For autoclaving individual dissecting tools</td>
		</tr>
		<tr>
			<td>Fine mesh strainer</td>
			<td>Cuisinart (Amazon.com)</td>
			<td>NA</td>
			<td>Wrap completely in aluminum foil and autoclave prior to tissue collection.</td>
		</tr>
		<tr>
			<td>Carboy with spigot</td>
			<td>Fisher Scientific</td>
			<td>03-007-647</td>
			<td>For large volume preparation of Wash buffer.</td>
		</tr>
		<tr>
			<td>Ice packs</td>
			<td>Nortech labs</td>
			<td>GB8818</td>
			<td>These do not have to be purchased, rather they can be recycled/reused from any routine laboratory shipment that includes them in the packaging.</td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>VWR</td>
			<td>V1001</td>
			<td>70% solution made by adding dH20 to 190 or 200 proof research grade alcohol</td>
		</tr>
		<tr>
			<td>Micro dissecting forceps</td>
			<td>Stoelting</td>
			<td>52102-43</td>
			<td>4 inches, 1 x 2 x 0.5 mm3 , Slight Curve</td>
		</tr>
		<tr>
			<td>Micro dissecting forceps</td>
			<td>Stoelting</td>
			<td>52102-06</td>
			<td>4 inches, Straight Fine, Sharp</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica Microsystems</td>
			<td>MZ16 or M60</td>
			<td>We have had success with the listed models. External gooseneck flexible light sources are helpful but not necessary.</td>
		</tr>
		<tr>
			<td>50 ml conical tubes</td>
			<td>Sigma-Aldrich (Corning)</td>
			<td>CLS4558</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco (ThermoFisher Scientific)</td>
			<td>10010023</td>
			<td>We purchase from our university Tissue Culture Core facility, alternate options such as this are available.</td>
		</tr>
		<tr>
			<td>10x Phosphate Buffered Saline</td>
			<td>Teknova</td>
			<td>P0195</td>
			<td>For preparation of Wash buffer we use 10x PBS</td>
		</tr>
		<tr>
			<td>DMEM/F-12 nutrient mixture (Ham&#39;s) with GlutaMAX</td>
			<td>Gibco (Life Technologies)</td>
			<td>10565-018</td>
			<td>We purchase this specific media formulation, containing 2.438 g/ L sodium bicarbonate, 55 mg/L sodium pyruvate, and 4.5 g/L 
			glucose</td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>BD Falcon</td>
			<td>353224</td>
			<td>Polystyrene, Tissue culture treated</td>
		</tr>
		<tr>
			<td>6-well transwells</td>
			<td>Millipore</td>
			<td>PICM03050</td>
			<td>Insert - 30 mm diameter, 0.4 &#956;m pore size hydrophilic PTFE membrane</td>
		</tr>
		<tr>
			<td>Extracellular Matrix (for example, Matrigel Matrix)</td>
			<td>BD Biosciences</td>
			<td>354234</td>
			<td>We have utilized Matrigel Matrix in our studies. It is a solid at room temperature and at -20 &#176;C. Avoid repeat freeze/thawing. Thaw bottle to viscous solution at 4 &#176;C, and prepare ~ 300 &#956;L aliquots in the cold room with chilled pipette tips. Store aliquots at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% w/v aqueous solution</td>
			<td>Alfa Aesar</td>
			<td>30525-89-4</td>
			<td>For tissue fixation, a fresh preparation of 4% paraformaldehyde is made by diluting this stock in PBS.</td>
		</tr>
		<tr>
			<td>Tissue culture incubator, maintained at 37 &#176;C, 5% CO2, 3% oxygen (optional for villous organ cultures)</td>
			<td></td>
			<td></td>
			<td>For some experiments, hypoxia may be preferred. This can be established multiple ways, including addition of exogenous nitrogen via gas cylinder, Tygon tubing, and a regulator.</td>
		</tr>
	</tbody>
</table>

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...

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.

40 Aufrufe. Etablierung von Zotten- und Dezidualorgankulturen als ex vivo Modelle der menschlichen Maternal-Fetal-Schnittstelle

Serie: Etablierung von Zotten- und Dezidualorgankulturen als ex vivo Modelle der menschlichen Maternal-Fetal-Schnittstelle - Experiment

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.

siRNA-Transfektion und EMSA-Analysen auf frisch isolierten menschlichen Villöse Cytotrophoblasten

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

Forschung

JoVE Journal

Genetik

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.

Dreidimensionale Darstellung und Analyse der Immunolabeled, geklärt menschlichen Plazenta Zotten vaskulärer Netzwerke

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

Entwicklungsbiologie

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.

Isolation von primären menschlichen Deziduale Zellen aus der fetalen Membranen der Begriff Plazentas

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

Transkript

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