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 autoclave prior to placement in the fume hood. All other materials (bottles, 50 mL tubes, etc.) must be sterilized with 70% ethanol solution. Always wear personal protective equipment at all times when working with biohazardous waste (lab coat, gloves, long hair tied back, etc.).
<ol>
	<li>Enzymatic digestion solution (Final solution: 200 mL)
	<ol>
		<li>Pipette 180 mL of HBSS-/- into a 500 mL beaker, add sterile stirring magnet and place on stirring plate at room temperature.</li>
		<li>Weigh out and add the following to the HBSS-/- solution slowly and sequentially: 20 mL of FBS, 400 mg Collagenase 2 ([final] = 2 mg/mL), 20 mg Soy bean trypsin inhibitor ([final] = 0.1mg/mL), 30 mg DNase 1 ([final] = 0.15 mg/mL), and 200 mg BSA ([final] = 1 mg/mL)</li>
		<li>Set the stirring to medium speed, and allow the mixture to stir for 10-20 min (cover with tin foil or paraffin film while stirring).</li>
		<li>Pour the mixed solution into a 250 mL glass bottle using a plastic funnel and place in the fume hood.</li>
		<li>Pass the solution through a plastic top filtration unit (0.22 &#956;m membrane filter, 500 mL), aliquot 20 mL into 50 mL tubes (10 tubes total) and store at -20 &#176;C until use.</li>
	</ol>
	</li>
	<li>RPMI 1640 Media (2% washing solution and 10% complete growth medium)
	<ol>
		<li>Combine RPMI 1640 and FBS in a glass bottle in the fume hood.</li>
		<li>For 2% FBS solution, combine 490 mL of RPMI 1640 and 10 mL FBS.</li>
		<li>For 10% FBS solution, combine 450 mL RPMI 1640 and 50 mL FBS.</li>
		<li>Pipette 500 &#956;L of normocin into the 500 mL glass bottle from the previous step containing RPMI media and FBS (50 mg/mL stock, 0.05 mg/mL working concentration).</li>
		<li>Pass media through a 0.22 &#956;m membrane filter and store in a 500 mL glass bottle at 4 &#176;C until use.</li>
	</ol>
	</li>
	<li>30 min prior to starting the experiment, place a 20 mL aliquot of frozen enzymatic digestion solution in a 37 &#176;C bead bath, place the 2% FBS and the 10% FBS RPMI 1640 media in a 37 &#176;C bead bath, turn on the rocking water bath and set to 37 &#176;C, 2 g, and set a temperature controlled centrifuge to 4 &#176;C.</li>
</ol>
2. Collection of Decidual Tissue from Term Placental Membranes
<ol>
	<li>Place bottles with HBSS+/+, HBSS-/- and five 50 mL tubes in a rack under the fume hood. Pipette 25 mL of HBSS+/+ in one tube.</li>
	<li>Using gloved hands, take the term placenta out from the container used for transporting it from the operating room theatre. Place the placenta on the diaper pad (maternal side up) and spread the fetal membranes out using scissors and forceps.
	<ol>
		<li>Lay overlapping diaper pads on the fume hood.</li>
		<li>Use a separate diaper to flip the placenta so the maternal side is face up.</li>
		<li>Find the point of membrane rupture and make incisions to allow the membrane to unfold and lay flat on the fumehood surface. 
		NOTE: Once the membrane is pulled back from the placenta, the decidua will be face up resting on the chorion layer, with the amnion layer at the back.</li>
	</ol>
	</li>
	<li>Using a plastic cell scraper, carefully scrape the decidual tissue off the chorion and place in the 50 mL tube containing 25 mL HBSS+/+.</li>
	<li>Scrape the membrane with moderate pressure. Do not apply too much pressure as it may result in chorion contamination. Collect small blood clots as well, as these contain some decidual cells. 
	Caution: After decidual collection, placenta must be packaged in a biohazard plastic bin and frozen in a -20 &#176;C freezer prior to appropriate disposal (according to your institutional rules). Bloody diaper pads must be wrapped in a seal-tight plastic bag and disposed of in a biohazard safety box.</li>
</ol>
3. Washing and Enzymatic Digestion of the Decidual Tissue
<ol>
	<li>Wash the collected tissues by gently shaking the 50 mL tube by hand and passing it through a 250 &#956;m metal sieve (250 &#956;m, size 60 mesh) resting over a sterile specimen (urine) container.</li>
	<li>Repeat the wash twice with HBSS+/+ and twice with HBSS-/- in fresh tubes for each wash. (final washes with HBSS-/- allows for the removal of calcium and magnesium present in HBSS+/+, which would otherwise interfere with the following enzymatic digestion process). 
	NOTE. During the washing steps, chorion tissue contamination will be apparent as the decidual tissue is light pink in color and amorphous, while the chorion is white, dense, and stringy. Therefore, chorion contamination can be easily removed with forceps.</li>
	<li>Optionally, if the decidual tissue is thick, transfer it to a sterile 10 cm diameter petri dish and proceed to mince of the tissue with two opposing scalpels in the dish.</li>
	<li>Place the washed tissue in sterile 50 mL tube containing 100 mg of tissue/mL of enzymatic digestion solution (see Preparations).
	<ol>
		<li>As a reference point, ensure that the level of decidual tissue reaches the 5-10 mL mark on the 50 mL tube.</li>
		<li>Use approximately 20 mL of enzymatic digestion solution (Prepared in step 1.1) to digest the total decidua collected from a whole term fetal membrane.</li>
	</ol>
	</li>
	<li>Seal the cap of the tube with the paraffin film (seal the tube tightly and wrap paraffin film around the cap and top of the tube) under the culture hood and incubate decidual tissue at 37 &#176;C for 20 min in a shaking water bath (145 rpm, 2 g).</li>
	<li>After incubation, remove the paraffin film and sterilize the surface of the tube containing digested tissue with 70% ethanol and bring under the fume hood. Shake the tube briefly by hand.</li>
	<li>Collect cell suspension through the metal sieve (250 &#956;m, size 60 mesh) into a new sterile specimen container. Dilute with an equal volume (20 mL) of RPMI + 10% FBS containing 0.1% normocin to stop enzymatic reaction. Proceed directly to centrifugation step 4.1.</li>
	<li>If necessary, place the remaining undigested tissue back into a new 50 mL tube with 20 mL of fresh enzymatic digestion solution and repeat digestion (20 min, 37 &#176;C, shaking water bath).</li>
	<li>If a second digestion is necessary, place the first tube with cell suspension on ice (cover with paraffin film to keep sterile). Repeat steps 3.5-3.6 and combine the two cells suspensions.</li>
</ol>
4. Generating a Single Cell Suspension
<ol>
	<li>Centrifuge the cell suspension (420 g, 4 &#176;C, 11 min).</li>
	<li>Remove the supernatant and resuspend the cells in 40 mL of RPMI + 2% FBS containing 0.1% normocin (&#34;wash buffer&#34;). 
	NOTE: Cell pellet will be loose and gelatinous due to red blood cell contamination, aspirate supernatant with caution. Removal of the upper phase with a manual pipette may be required.</li>
	<li>Repeat the centrifugation at 420 g at 4 &#176;C for 11 min.</li>
	<li>Carefully remove the supernatant (As mentioned in Note Step 4.2) and resuspend the cell pellet in 5 mL of wash buffer and add 35 mL of erythrocyte lysis buffer in the same tube. 
	NOTE: If the pellet is large or very bloody, it can be split into two tubes for the erythrocyte lysis step by adding 10 mL of wash buffer and dividing equally into two tubes and then adding 35 mL of Erythrocyte lysis buffer to each tube.</li>
	<li>Incubate on ice for 20 min. Briefly vortex tube(s) at the beginning and the end of the incubation to lyse the red blood cells.</li>
	<li>Centrifuge at 420 g for 11 mins at 4 &#176;C.</li>
	<li>Carefully, remove the supernatant and resuspend the pellet in 40 mL of wash buffer.</li>
	<li>Pass the cells through a 70 &#956;m nylon filter to remove cell clumps.</li>
	<li>Centrifuge at 420 g for 11 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 10 mL of complete medium (RPMI 10% + FBS containing 0.1% normocin).</li>
	<li>Count cells using the trypan blue dye-exclusion hemocytometer procedure as described below:
	<ol>
		<li>Under the culture hood gently pipette cell suspension up and down 3x in order to thoroughly mix before combining with trypan blue. In a 1.5 mL tube prepare cell suspension, diluted 1:2 (combine 20 &#956;L of trypan blue solution and 20 &#956;L of decidual cell suspension). Carefully mix trypan blue-cell solution by pipetting up and down a couple of times (this solution is not sterile).</li>
		<li>Place the hemocytometer on the stage of the microscope with glass coverslip on top. 
		NOTE: Hemocytometer is a microscope slide with grids on it to give nine large squares divided by triple lines. Each large square has an area of 1 mm2, and the depth of fluid in the chamber is 0.1 mm. Therefore, the volume of fluid that can fill each large square is 1 mm*1 mm*0.1 mm= 0.1 mm3= 10-4 mL.</li>
		<li>Slowly add 10 &#956;L of the trypan blue-cell mixture into the groove of the hemocytometer, allowing capillary action to disperse the cell mixture over the entire slide (stop before mixture fills well).</li>
		<li>View the cells under a microscope (at 10X magnification) and count all white/green cells that exclude trypan blue (these are the viable cells) in the four large outer squares of the hemocytometer. When counting cells that touch the line, count only those that touch the right and upper lines but not those touching the left and bottom lines. Do not count dark blue cells; blue color indicates that the cell is dead as trypan blue dye can easily penetrate through the plasma membrane into the cytoplasm.</li>
		<li>To calculate the number of viable cells in 1 mL of cell suspension (X): 
		<img alt="Equation" src="/files/ftp_upload/57443/57443eq1v2.jpg" /> 
		2 = dilution factor 
		10,000 = conversion factor (1 mL = 1 cm3 = 10,000*0.1 mm3)</li>
	</ol>
	</li>
	<li>Dilute decidual cells to a desirable final concentration in RPMI + 10% FBS (growth medium). 
	NOTE: For tissue culture plating, pipette 2*106 cells/well into a plastic 6-well plate, 10*106 cells into a 10-cm plastic plate or 75,000 cells to a glass coverslip.</li>
</ol>

<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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin+11+advances+progesterone-induced+decidualization+of+human+endometrial+stromal+cells.">Interleukin 11 advances progesterone-induced decidualization of human endometrial stromal cells.</a> Molecular and Human Reproduction. 8, 636-643 (2002).</li><li>Wu, W. -. X., Brooks, J., Glasier, A. F., McNeilly, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+decidualization+and+prolactin+mRNA+and+production+at+different+stages+of+human+pregnancy.">The relationship between decidualization and prolactin mRNA and production at different stages of human pregnancy.</a> Society for Endocrinology. 14, 255-261 (1995).</li><li>Bell, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+secretion+of+protein+by+the+endometrium+and+decidua.">Synthesis and secretion of protein by the endometrium and decidua.</a> Implantation: Biology and Clinical Aspects. , 95-118 (1988).</li><li>Croy, B. A., Chantakru, S., Esadeg, S., Ashkar, A. A., Wei, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decidual+natural+killer+cells:+key+regulators+of+placental+development.">Decidual natural killer cells: key regulators of placental development.</a> Journal of Reproductive Immunology. 57, 151-168 (2002).</li><li>Smarason, A. K., Gunnarsson, A., Alfredsson, J. H., Valdimarsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monocytosis+and+monocytic+infiltration+of+decidua+in+early+pregnancy.">Monocytosis and monocytic infiltration of decidua in early pregnancy.</a> Journal of Clinical and Laboratory Immunology. 21, 1-5 (1986).</li><li>Daiter, E., Pampfer, S., Yeung, Y. G., Barad, D., Stanley, E. R., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+colony-+stimulating+factor-1+in+the+human+uterus+and+placenta.">Expression of colony- stimulating factor-1 in the human uterus and placenta.</a> Journal of Clinical Endocrinology and Metabolism. 74, 850-858 (1992).</li><li>Casey, M. L., Cox, S. M., Beutler, B., Milewich, L., MacDonald, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cachectin/tumor+necrosis+factor-alpha+formation+in+human+decidua.+Potential+role+of+cytokines+in+infection-induced+preterm+labor.">Cachectin/tumor necrosis factor-alpha formation in human decidua. Potential role of cytokines in infection-induced preterm labor.</a> Journal of Clinical Investigation. 83, 430-436 (1989).</li><li>Lala, P. K., Kennedy, T. G., Parhar, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+lymphocyte+alloreactivity+by+early+gestational+human+decidua.+II.+Characterization+of+the+suppressor+mechanisms.">Suppression of lymphocyte alloreactivity by early gestational human decidua. II. Characterization of the suppressor mechanisms.</a> Cellular Immunology. 127, 368-381 (1988).</li><li>Shynlova, O., Nedd-Roderique, T., Li, Y., Dorogin, A., Nguyen, T., Lye, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infiltration+of+myeloid+cells+into+decidua+is+a+critical+early+event+in+the+labour+cascade+and+post-partum+uterine+remodelling.">Infiltration of myeloid cells into decidua is a critical early event in the labour cascade and post-partum uterine remodelling.</a> Journal of Cellular and Molecular Medicine. 17, 311-324 (2013).</li><li>Osman, I., Young, A., Ledingham, M. A., Thomson, A. J., Jordan, F., Greer, I. A., Norman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukocyte+density+and+pro-inflammatory+cytokine+expression+in+human+fetal+membranes,+decidua,+cervix+and+myometrium+before+and+during+labour+at+term.">Leukocyte density and pro-inflammatory cytokine expression in human fetal membranes, decidua, cervix and myometrium before and during labour at term.</a> Molecular Human Reproduction. 9, 41-45 (2003).</li><li>Farine, T., Lye, S. J., Shynlova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+maternal+leukocytes+are+activated+in+response+to+cytokines+secreted+by+uterine+tissues+of+pregnant+women.">Peripheral maternal leukocytes are activated in response to cytokines secreted by uterine tissues of pregnant women.</a> Journal of Cellular and Molecular Immunology. 14, 635-638 (2017).</li><li>Hamilton, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+infiltrate+the+human+and+rat+decidua+during+term+and+preterm+labor:+evidence+that+decidual+inflammation+precedes+labor.">Macrophages infiltrate the human and rat decidua during term and preterm labor: evidence that decidual inflammation precedes labor.</a> Biology of Reproduction. 86, 39 (2011).</li><li>Casey, M. L., Cox, S. M., Word, A., Macdonald, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokines+and+infection-induced+preterm+labour.">Cytokines and infection-induced preterm labour.</a> Reprodution Fertility and Development. 2, 499-510 (1990).</li><li>Yellon, S. M., Mackler, A. M., Kirby, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+leukocyte+traffic+and+activation+in+parturition.">The role of leukocyte traffic and activation in parturition.</a> Journal of the Society for Gynecologic Investigation. 10, 323-338 (2003).</li><li>Gomez-Lopez, N., StLouis, D., Lehr, M. S., Sanchez-Rodriguez, E. N., Arenas-Hernandez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+cells+in+term+and+preterm+labor.">Immune cells in term and preterm labor.</a> Cellular &amp; Molecular Immunology. 11, 571-581 (2014).</li><li>Xu, Y., Plazyo, O., Romero, R., Hassan, S. S., Gomez-Lopez, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+leukocytes+from+the+human+maternal-fetal+interface.">Isolation of leukocytes from the human maternal-fetal interface.</a> Journal of Visualized Experiments. 99, (2015).</li><li>Trundley, T., Gardner, L., Northfield, J., Moffett, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+isolation+of+cells+from+the+human+fetal-maternal+interface.">Methods for isolation of cells from the human fetal-maternal interface.</a> Methods in Molecular Medicine. 122, 109-122 (2006).</li><li>Jividen, K., Movassagh, M. J., Jazaeri, A., Li, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+methods+for+establishing+primary+human+endometrial+stromal+cells+from+hysterectomy+specimens.">Two methods for establishing primary human endometrial stromal cells from hysterectomy specimens.</a> Journal of Visualized experiments. 87, (2014).</li><li>Pelekanos, R. A., Sardesai, V. S., Futrega, K., Lott, W. B., Kuhn, M., Doran, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+mesenchymal+stem/stromal+cells+derived+from+human+placenta+tissue.">Isolation and expansion of mesenchymal stem/stromal cells derived from human placenta tissue.</a> Journal of Visualized experiments. 112, (2016).</li><li>De Clercq, K., Hennes, A., Vrien, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+endometrial+epithelial+and+stromal+cells+for+in+vitro+decidualization.">Isolation of mouse endometrial epithelial and stromal cells for in vitro decidualization.</a> Journal of Visualized Experiments. 121, (2017).</li><li>Zhang, J., Shynlova, O., Sabras, S., Bang, A., Briollais, L., Lye, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunophenotyping+and+activation+status+of+maternal+and+peripheral+blood+leukocytes+during+pregnancy+and+labour,+both+term+and+preterm.">Immunophenotyping and activation status of maternal and peripheral blood leukocytes during pregnancy and labour, both term and preterm.</a> Journal of Cellular and Molecular Medicine. 10, 2386-2402 (2017).</li></ol>

The authors have nothing to disclose

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&amp;rsquo;s balanced salt solution with calcium and magnesium</td><td>Prepared in facility (LTRI)</td><td></td><td></td></tr><tr><td>Hank&amp;rsquo;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&amp;amp;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...

Watch this Scientific Journal Video about Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae at JoVE.com

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 ...

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Research

JoVE Journal

Medicine

14.1K Views.  University of Toronto. 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.

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

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 ...

Genetics

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Developmental Biology

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Biology

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Bioengineering

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Immunology and Infection

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

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

Summary

Explore More Videos

Chapters in this video

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Isolation of Leukocytes from the Human Maternal-fetal Interface

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay