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. 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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. 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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. 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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&#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...

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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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

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

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 Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...