Name: isolation of primary human decidual cells from the fetal membranes of term placentae
Uploaded: 2018-04-30T14:45:00
Description: Watch this Scientific Journal Video about Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae at JoVE.com

The authors would like to thank the donors, the RCWIH BioBank, and the Mount Sinai Hospital/UHN Department of Obstetrics and Gynaecology for the human specimens used in this study. We would like to thank the members of the Lye lab, particularly Dr. Caroline Dunk for her help with the method development. This work is supported by the Burroughs Welcome Fund (grant #1013759).

Placentae are collected from healthy term, not in labor women undergoing elective cesarean sections. The Collection, processing, and disposable of human samples adhere to the guidelines of the Mount Sinai Hospital Ethics board. A written consent is obtained from each patient. This study is approved by the Research Ethics Board at Mount Sinai Hospital.
1. Preparations
NOTE: All steps must be conducted under a fume hood and all surgical equipment must be sterilized via ...

<ol>
	<li>Brosens, N., Hayashi, N., White, J. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+receptor+regulates+decidual+prolactin+expression+in+differentiating+human+endometrial+stromal+cells.">Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.</a> Endocrinology. 140, 4809-4820 (1999).</li><li>Bell, S. C., D'Arcangues, C., Frase, I. S., Newton, J. R., Odlind, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Decidualization and relevance to menstruation. , 187-212 (1990).</li><li>Kariya, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-1+inhibits+in+vitro+decidualization+of+human+endometrial+stromal+cells.">Interleukin-1 inhibits in vitro decidualization of human endometrial stromal cells.</a> Journal of Clinical Endocrinology and Metabolism. 73, 1170-1174 (1991).</li><li>Dimitriadis, E., Robb, L., Salamonsen, L. 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The protocol described here demonstrates a cost and time effective method for isolating primary decidual cells collected from the fetal membranes of whole human term placentae that is highly accessible and straightforward. The success of this protocol is dependent on two critical factors, (1) efficiency of decidual scraping from the chorion layer of the fetal membranes and (2) the care with which the decidual cells are handled throughout the protocol. It is important that chorion tissue contamination is controlled by ens...

The endometrium, one of the most active adult female tissues, undergoes dramatic remodeling each menstrual cycle in response to stimulation by ovarian hormones, estrogen (E2) and progesterone (P4). The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue that is formed by the end of the postovulatory phase as a result of P4-driven differentiation following the E2-dominant proliferative phase. Decidual cells are responsible for the secretion of hormonal factors for successful blastocyst implantation and for development of the utero-placental interface for maintaining maternal tolerance to the fetal allograft.
Decidualization is required for implantation and subsequent remodeling of the decidual spiral arteries. Endometrial stromal cells undergo decidualization, under the control of P4 and cAMP, during the late luteal phase of the menstrual cycle1. This process is initiated around the blood vessels and spreads throughout the stroma, suggesting its role in vasculature remodeling and leukocyte trafficking regulation. This cellular transformation is characterized by a circular morphology, increased nuclear size, and expansion of the rough endoplasmic reticulum and Golgi apparatus2. Decidualized stromal cells are capable of producing paracrine factors supporting blastocyst implantation and characterized by the secretion of numerous hormones (i.e. prolactin), angiogenic growth factors, insulin growth factor binding protein-1 (IGFBP-1), prostaglandin (PG) E (stimulator of intracellular cAMP), cytokines, extracellular matrix components and nutrients essential for placental implantation and development3,4,5,6.
The decidual cell population is not solely comprised of decidualized stromal cells but also contains large, pregnancy-specific decidual leukocyte populations. Decidualization involves transient localized oedema and influx of Natural killer (NK) cells, T-cells, dendritic cells, and macrophages. The largest leukocyte subpopulation is the uterine NK cells, comprising approximately 50-70% of all maternal leukocytes infiltrating the decidua which are a source of cytokines and angiogenic factors which may aid in the decidualization process and increase in number throughout pregnancy7. Macrophages, being the second largest subpopulation of immune cells, are found around the implantation site and increase during pregnancy8. They are a source of cytokines and growth factors such as colony stimulating factor (CSF-1)9, tumor necrosis factor &#945; (TNF&#945;)10 and prostaglandin (PG) E11.
Throughout pregnancy, and before term labor, the decidua is a major source of cytokines and chemokines responsible for maternal peripheral leukocyte activation and subsequent migration into the uterine tissues to initiate labor. Animal studies showed that numerous pro-inflammatory cytokines are up-regulated in the mouse decidua during labor, such as TNF-a, IL-6, IL-12, and IL-1b12. In the human decidua, pro-inflammatory cytokines IL-1b, IL-6 and IL-8 (major neutrophil chemoattractant) exhibit higher expression during labor compared to not in labor13. These secreted cytokines result in an activation and influx of leukocytes into the decidual tissues14; an increase in decidual macrophage and neutrophil infiltration in both the human and rat is seen during term labor, with decidual infiltration preceding myometrial 4-fold greater, indicating a cascade of activation between this two-adjacent uterine tissues15. These infiltrating leukocytes produce PGs capable of activating synchronous contractions of the myometrium16, matrix metalloproteinases (MMPs) to initiate membrane rupture17,18, as well as pro-inflammatory cytokines to amplify the uterine activation process (&#39;cytokine storm&#39;).
Due to many important functions of decidual cells, such as playing a critical role in the implantation process, maintaining maternal-fetal tolerance in early gestation and participating in the activation of labor at term, different pathologies can arise during pregnancy. For instance, (1) infertility due to recurrent implantation failure and recurrent pregnancy loss can result from a failure of decidual maturation; (2) intrauterine growth restriction (IUGR) and preeclampsia due to improper development and dysfunction of the decidua/placenta or compromised vascular transformation at the decidual-myometrial junction; as well as (3) preterm birth which can result from premature decidual activation.
In light of these major disorders, coupled with the ethical and practical limitations of human in vivo studies, establishing primary human decidual cell lines is essential for in vitro analysis with the purpose of better understanding and improving clinical management of pregnancy complications. Therefore, the objective of our research was to develop a protocol which allows for the isolation of human primary decidual cells with high cell yield and viability collected from the fetal membranes of term placentae. This current protocol clearly describes a time- and cost-effective method for isolating of specific subtypes of decidual cells which be used for a variety of in vitro analyses. Characterization of the abundance and phenotype of decidual sub-populations at term and comparison to first or second trimester is crucial to defining their roles throughout human gestation.

<table>
	<tbody>
		<tr>
			<td>Hank&#8217;s balanced salt solution with calcium and magnesium</td>
			<td>Prepared in facility (LTRI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s balanced salt solution without calcium and magnesium</td>
			<td>Prepared in facility (LTRI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diaper pads</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical scissors</td>
			<td>AL Medical</td>
			<td>2018-12-20.</td>
			<td></td>
		</tr>
		<tr>
			<td>Large surgical forceps</td>
			<td>Fine Science Tools</td>
			<td>11000-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic disposable cell scraper (25 cm)</td>
			<td>Sarstedt</td>
			<td>83.183</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mm (size 60 mesh) metal sieve</td>
			<td>Sigma-Aldrich</td>
			<td>S1020-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable scalpel with plastic handle (#21)</td>
			<td>Fisher Scientific</td>
			<td>08-927-5D</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile plastic petri dish (diameter 10 cm)</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile specimen container (urine cup, 4.5 oz)</td>
			<td>VWR</td>
			<td>25384-146</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon filter (70 mm)</td>
			<td>VWR/Corning</td>
			<td>21008-952</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythrocyte lysis buffer</td>
			<td>Qiagen</td>
			<td>79217</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue, 0.4% solution</td>
			<td>Lonza</td>
			<td>17-942E</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Fisher Scientific</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>Reichert</td>
			<td>1490</td>
			<td></td>
		</tr>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640 culture media</td>
			<td>Invitrogen</td>
			<td>11835-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Wisent</td>
			<td>080-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Normocin (50mg/ mL)</td>
			<td>Invivogen</td>
			<td>ant-nr-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic top filtration unit (0.22 mm membrane, 500 mL)</td>
			<td>Millipore</td>
			<td>SCGPT05RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2, lyophilized powder</td>
			<td>Sigma-Aldrich</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Soy bean trypsin inhibitor, powder</td>
			<td>Sigma-Aldrich</td>
			<td>T9003-250mg</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase powder</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA powder)</td>
			<td>Fisher Scientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc confocal microscope - Leica DMI 6000B</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Imaging Flow cytometer - Image Stream MK2</td>
			<td>Amnis</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IDEA software</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>APC-conjugated Vimentin antibody</td>
			<td>R&#38;D Systems</td>
			<td>IC2105A</td>
			<td></td>
		</tr>
		<tr>
			<td>APC H7-conjugated CD45 antibody</td>
			<td>BD</td>
			<td>641399</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-conjugated Cytokeratin antibody</td>
			<td>MACs Miltenyi Biotec</td>
			<td>130-080-101</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCP -conjugated Cytokeratin 7 antibody</td>
			<td>Novus</td>
			<td>NBP2-47941PCP</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor450 Fixable Viability dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>65-0863-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vimentin primary antibody</td>
			<td>Santa Cruz</td>
			<td>sc-7558</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45 primary antibody</td>
			<td>Dako</td>
			<td>M0701</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin primary antibody</td>
			<td>Dako</td>
			<td>M0821</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytokeratin 7 primary antibody</td>
			<td>Dako</td>
			<td>M7018</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Santa Cruz</td>
			<td>sc-2025</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat IgG</td>
			<td>Santa Cruz</td>
			<td>sc-2028</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A10036</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 secondary antibody</td>
			<td>Fisher Scientific</td>
			<td>A-11058</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

To validate the efficiency and viability of the isolated cells, they were characterized by two methods: flow cytometry and immunocytochemistry (ICC). 4 cell populations were targeted; decidualized stromal cells were detected by the anti-vimentin antibody, pan-leukocyte marker CD45 was used to identify decidual immune cells, cytokeratin was used to detect epithelial/endothelial cells and finally, cytokeratin 7 was used to detect any potential trophoblast (chorion or placental) contaminatio...

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

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 ...

The overall goal of this cell isolation protocol is to develop a method for isolating primary decidual cells with high yield end cell viability. This method can help answer key questions in the field of human reproduction, such as the deciduous role in blastocyst implantation and labor initiation. It can also provide insight into various pregnancy related pathologies.The main advantage of this technique is that it provides a time and cost effective method for the isolation of primary decidual cells with high viability end yields. To begin, leave five 50 milliliter tubes on a rack and two bottles of HBSS and HBSS in the fume hood. Then pipette 25 milliliters of HBSS in the appropriately labeled tube.Next, lay overlapping diapers in the fume hood. Then, take the term placenta out of the container and place it in the fume hood. Position the placenta on the diaper pad with the maternal side up.If the placenta is maternal side down, use an additional diaper to flip the placenta. Look for the point of membrane rupture and make incisions using scissors and forceps to allow the membrane to unfold and lay flat on the fume hood surface. Then use a cell scraper to gently scrape the decidual tissue off of the chorion.Transfer the tissue to a 50 milliliter tube containing 25 milliliters of HBSS Remember to also collect small blood clots, as these also contain some decidual cells. Gently shake the 50 milliliter tube to wash the collected tissue, then pass the tissue through a 250 micrometer metal sieve. Repeat the washing process twice with HBSS followed by two washes with HBSS in fresh tubes.Chorion contamination is easily visible due to its threadlike shape and lighter color and can be removed after washing. After washing, place the tissue in a sterile 50 milliliter tube containing 20 milliliters of enzymatic digestion solution to digest the entire decidua obtained from the whole term fetal membrane. Use paraffin film to seal the cap of the tube.Then incubate the tube at 37 degrees Celsius in a shaking water bath. Set the timer for 20 minutes. Once the incubation is over, remove the paraffin film.Use 70%ethanol to sterilize the surface of the tube containing the digested tissue and place it under the fume hood. Shake the tube briefly. Next, pass the cell suspension through a metal sieve into a new sterile specimen container.Dilute the cell suspension with an equal volume of culture medium to stop the enzymatic reaction. Transfer the cell suspension to a new 50 milliliter tube. Then centrifuge the cells at 420 G for 11 minutes at four degrees Celsius.If required, place the remaining undigested tissue into a new 50 milliliter tube containing 20 milliliters of fresh enzymatic digestion solution and repeat the digestion process. If the digestion is performed a second time, leave the first tube with the cell suspension on ice. Once the second digestion is complete, combine both cell suspensions.After centrifugation, carefully aspirate the supernatant. A manual pipette may be required to remove the supernatant. Then, re-suspend the cell pellet in wash buffer.After washing the cells, repeat the centrifugation process. At the end of the centrifugation process, aspirate the supernatant and re-suspend the cell pellet in five milliliters of wash buffer and 35 milliliters of erythrocyte lysis buffer. In order to lyse the red blood cells, briefly vortex the tube at the beginning and at the end of the incubation.Then, incubate the cell suspension on ice for 20 minutes. After incubation, repeat the centrifugation process. Post-centrifugation, aspirate the supernatant and re-suspend the pellet in 40 milliliters of wash buffer, then pass the cells through a 70 micrometer nylon filter to exclude cell clumps.Repeat the centrifugation process. Aspirate the supernatant once centrifugation is done. Then re-suspend the cell pellet in 10 milliliters of culture medium.Count the number of cells with a hemocytometer using the Trypan blue dye exclusion method. In order to determine the cell type composition of the decidual cell population, a multi-color imaging flow cytometry gating strategy has been developed. The analysis shows exclusion of cell doublets and cell debris using serial gating.Once a debris-free single cell population is obtained, cell viability is determined using a fixable viability dye. The analysis demonstrates 80%viability of the total decidual cell population, indicated by the blue gating. Next, the isolated decidual cells are stained with fluorophore conjugated mouse monoclonal antibodies.This is to analyze the presence of stromal, immune, epithelial and trophoblast cell markers and the viability dye. These markers will determine the decidual cell types. Flow cytometry fluorescent images demonstrate a positively stained cell for all four markers and viability dye.These markers are then used to determine the percentage of each cell type in the decidual cell population. Interestingly, the Vimentin positive stromal cells constitute the highest population of cells, while there are virtually no trophoblast cells. These results demonstrate the purity and high viability of the isolated primary human decidual cells.Here, the dot plots obtained show the gating for the positive cell type populations. Fluorescent minus one controls are used to accurately gate the true positive decidual cell populations. Next, captured spinning disk confocal microscopy images are used to validate the flow cytometric results by showing that the decidual cell population is primarily composed of Vimentin positive stromal cells with no trophoblast cell contamination.Once mastered, this technique can be completed in three hours. Following this procedure, the decidual cells can be analyzed in a variety of ways to examine gene and protein expression, which can better help us understand the cellular changes occurring in pregnancy complications.

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