This work was supported by grants from the Research Foundation-Flanders (FWO G.0856.13N) and the Research Council of the KU Leuven (OT/13/113). K.D.C. is funded by the FWO Belgium. A.H. is funded by OT/13/113. We would like to thank the Cell Imaging Core facility of the KU Leuven (http://gbiomed.kuleuven.be/english/corefacilities/microscopy/cic/cic.htm) for the use of the confocal microscope.

AlexaFluor
All animal experiments for this study were approved by the ethical review committee of the KU Leuven (Belgium) (Project P174/2013). Mice were housed under standard conditions, with ad libitum access to food pellets and tap water, and kept under controlled conditions (23 &#177;1.5 &#176;C, relative humidity 40 - 60%, 12/12 light/dark cycle).
1. Mice
<ol>
	<li>Use commercially available mouse strain (i.e. C57Bl/6, female 8 - 12 weeks old). Typically, 4 - 5 mice will yield an appropriate amount of cells for further experiments.</li>
	<li>Inject intact mice subcutaneously with 100 &#181;L of the 17&#946;-estradiol (E2) solution (100 ng/100 &#181;L) for 3 consecutive d prior to the isolation in order to standardize the phase of the estrous cycle of the animals and to induce proliferation of endometrial cells. The need for checking the cycle phase of the mice before starting the protocol is avoidable because of the 3 d&#39; estradiol treatment.</li>
</ol>
2. Preparations
<ol>
	<li>Make a solution of 100 ng/100 &#181;L E2 in arachis oil. For the injections of five mice (as described in 1.2), an amount of 1.5 mL is needed.
	<ol>
		<li>Dissolve 1 mg E2 in 1 mL ethanol to have a stock solution of 1 mg/mL.</li>
		<li>Dilute this stock solution 1:1,000 in arachis oil.</li>
	</ol>
	</li>
	<li>Make 500 mL of Hank&#39;s Balanced Salt Solution (HBBS) 1x complemented with 100 U/mL penicillin and 100 &#181;g/mL streptomycin (further referred to as HBSS+).</li>
	<li>Make 500 mL of filtered Mouse Endometrial Stromal Cell (MESC) medium to culture MESC: Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F12 (DMEM/F12) with phenol red, L-glutamine and 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid) (HEPES) containing 10% FBS, 0.5 &#181;g/mL amphotericin B, and 100 &#181;g/mL gentamicin (further referred to as MESC medium).</li>
	<li>Make 500 mL of filtered 2% MESC medium to culture MESC&#160;during decidualization: DMEM/F12 with phenol red, L-glutamine and HEPES containing 2% FBS, 0.5 &#181;g/mL amphotericin B, and 100 &#181;g/mL gentamicin.</li>
	<li>Prepare 500 mL of filtered Mouse Endometrial Epithelial Cell (MEEC) medium: DMEM containing 10% FBS, 0.5 &#181;g/mL amphotericin B, 100 &#181;g/mL gentamicin, 25% MCDB-105 medium, and 5 &#181;g/mL insulin (further referred to as MEEC medium).</li>
	<li>Prepare Poly-L-Lysine (PLL) coated coverslips for MESC culture.
	<ol>
		<li>Dissolve 25 mg of PLL in 250 mL of distilled water. Filter the solution using a 0.22 &#181;m filter.</li>
		<li>Add sterile coverslips in a 12-well plate. Add 300 &#181;L of dissolved PLL on the coverslips.</li>
		<li>Remove the solution after 30 min of incubation. The plates can be stored at 4 &#176;C until further use (up to 1 - 2 months).</li>
	</ol>
	</li>
	<li>Prepare a 10 mg/mL stock solution of collagenase type IA and store aliquots of 300 &#181;L at -20 &#176;C. Filter before use.</li>
</ol>
3. Preparations Required 24 h before Isolation
<ol>
	<li>Prepare collagen coated coverslips for MEEC culture.
	<ol>
		<li>Prepare a 0.02 M acetic acid solution in distilled water (1 mL per coverslip).</li>
		<li>Add 150 &#181;L collagen in 12 mL of 0.02 M acetic acid to obtain a final concentration of 50 &#181;g/mL.</li>
		<li>Add sterile coverslips in a 12-well plate.</li>
		<li>Add 1 mL of the collagen solution (see 3.1.2).</li>
		<li>Incubate O/N (minimum 12 h) at 37 &#176;C. 
		During isolation:</li>
		<li>Remove the collagen solution off the coverslips by suction and let it air-dry in a sterile environment (in the laminar flow cabinet).</li>
		<li>Wash the coverslips twice with Phosphate-buffered Saline (PBS).</li>
	</ol>
	</li>
	<li>Prepare a 2.5% pancreatin solution by adding 0.25 g pancreatin in a 15 mL canonical tube containing 10 mL of HBSS+. Shake (200 rpm) the solution at 37 &#176;C for 1 h to increase the solubility. Store at 4 &#176;C.</li>
</ol>
4. Isolation and Culture of Mouse Endometrial Epithelial Cells (MEEC) and Mouse Endometrial Stromal Cells (MESC)
<ol>
	<li>Add 25 mg of trypsin in a 15 mL tube containing 2.5% pancreatin solution (see 3.2) to end up with a final solution containing 0.25% trypsin (further referred to as MESC digestion mix).</li>
	<li>Isolation of uteri
	<ol>
		<li>Check the cycle phase of the animals with a vaginal smear examination.
		<ol>
			<li>Restrain the animal and flush the vagina gently with 50 &#181;L PBS 3 - 5 times.</li>
			<li>Collect the final flush on a glass slide.</li>
			<li>Examine the material under a bright field microscope using a 10X or 20X objective.</li>
			<li>Only use mice that are in the estrus phase. This stage is characterized by the presence of cornified epithelial cells in the vaginal lavage (Figure 1).</li>
		</ol>
		</li>
		<li>Euthanize the animals using an appropriate method such as cervical dislocation or CO2-induced narcosis.</li>
		<li>Spray the carcasses with 70% ethanol in order to generate a sterile environment.</li>
		<li>Using sterile surgical tools, open the abdomen and push the intestines aside to visualize both uterine horns.</li>
		<li>Grab the uterine horn at the most distal end under the fallopian tube. Dissect the uterine horns from the fallopian tube and remove adipose and connective tissue. Cut out the horn at the distal end above the uterine body and place it in a 35 mm Petri dish with 3 mL of HBSS+.
		<ol>
			<li>Repeat this step for the other horn and for the other animals until all uterine horns are collected.</li>
		</ol>
		</li>
		<li>Clean the uterine horn (in HBSS+) further under a stereoscope and remove all residual adipose or connective tissue.</li>
		<li>Cut the uterine horns open longitudinally to expose the uterine lumen and replace the horn in a new Petri dish with fresh HBSS+.</li>
		<li>Transfer all uterine horns to the 15 mL tube containing pancreatin and trypsin.</li>
		<li>Incubate horizontally for 60 min at 4 &#176;C on an orbital shaker (50 rpm).</li>
		<li>Incubate horizontally for 45 min at 23 &#176;C (RT), without shaking.</li>
		<li>Incubate horizontally for 15 min at 37 &#176;C (water bath), without shaking.</li>
		<li>Meanwhile make the MESC digestion mix by dissolving 300 &#181;L of the 1 mg/mL collagenase stock in 2.7 mL of 0.05% Trypsin-Ethylenediaminetetraacetic Acid (EDTA) solution. 
		NOTE: From now on, further isolation steps are performed in a sterile environment under a laminar flow cabinet.</li>
	</ol>
	</li>
	<li>Mouse Endometrial Epithelial Cell (MEEC) Culture
	<ol>
		<li>After 2 h of incubation, carefully pour away the supernatant solution.</li>
		<li>Transfer the uteri into a Petri dish containing cold MEEC medium and incubate for 5 min in order to inactivate trypsin activity.</li>
		<li>Transfer the uteri to a 15 mL tube containing 3 mL of cold HBSS+.</li>
		<li>Vortex for 10 s to release the epithelial sheets.</li>
		<li>Rinse the uteri in a clean Petri dish with 3 mL HBSS+.</li>
		<li>Repeat step 4.3.2 -&#160;4.3.4 to obtain a total of three suspensions containing epithelial sheets.</li>
		<li>Transfer the uteri to the MESC digestion mix and incubate for 30 min at 37 &#176;C while shaking (200 rpm) (go to step 4.4 for further isolation of MESC).</li>
		<li>Recover the epithelial sheets by pipetting the three cell suspensions gently on a 100 &#181;m nylon mesh in order to remove tissue debris.</li>
		<li>Centrifuge the collected cell suspension at 500 x g for 5 min.</li>
		<li>Resuspend the pellet in 12 mL of MEEC medium and mix well.</li>
		<li>Settle the solution for 5 min in order to separate remaining MESC using gravity sedimentation.</li>
		<li>Remove carefully the upper 2 mL by using a 5 mL pipette.</li>
		<li>Centrifuge the cell suspension at 500 x g for 5 min.</li>
		<li>Resuspend in MEEC medium by gently pipetting up and down.</li>
		<li>Plate the cells on collagen-coated coverslips in the desired density for further experiments (Figure 2a).</li>
		<li>Incubate the cells at 37 &#176;C in a 5% CO2 incubator for a minimum of 12 h. 
		NOTE: For immunostaining or functional experiments like fluorescent calcium imaging, it is advised to plate the cells directly on the coverslips, while seeding in a 6-well plate is recommended when RNA or protein isolation is desired. Isolation of mouse endometrial stromal cells is divided into two rounds as the purity of the cells after only one digestion can be insufficient. The uteri are digested twice for an optimal cell recuperation and purity. In both rounds the technical interventions are identical. Cells collected from both rounds can be kept for further experiments, as desired by the researcher.</li>
	</ol>
	</li>
	<li>Mouse Endometrial Stromal Cell (MESC) Culture: 1st Round
	<ol>
		<li>Before the start of the first digestion round, prepare a second digestion mix by dissolving 300 &#181;L of the 1 mg/mL collagenase stock in 2.7 mL 0.05% Trypsin-EDTA solution. This mix is needed in step 4.4.8.</li>
		<li>Prepare three small Petri dishes with cold (4 &#176;C) HBSS+ and 3 x 15 mL tubes with 3 mL of MESC medium (tube X1, X2 and X3).</li>
		<li>After 30 min of incubation, shake the uteri-containing trypsin solution gently for 10 s. MESC cells will detach from the uterine tissue and will remain in the trypsin solution.</li>
		<li>Transfer the uteri to the first Petri dish containing 3 mL cold (4&#176;C) HBSS+ and rinse well.</li>
		<li>Add 3 mL of MESC medium to the tube originally containing the uterine horns (tube in step 4.4.3) to inhibit trypsin activity.</li>
		<li>Transfer the uteri from the Petri dish with cold (4 &#176;C) HBSS+ to tube X1 containing 3 mL of MESC medium and shake gently for 10 s.</li>
		<li>Repeat steps 4.4.4 (transfer to a Petri dish with cold (4 &#176;C) HBSS+) and 4.4.6 (transfer to tube X2 and X3) twice. 
		NOTE: Finally, this protocol will result in 4 cell suspensions: one tube containing the trypsin solution and 3 tubes with MESC medium containing MESC (tubes X1, X2 and X3).</li>
		<li>Transfer uteri in the new MESC digestion, as described in step 4.4.1 and incubate for 30 min at 37 &#176;C, vertically.</li>
		<li>Collect the stromal cells by passing the four cell suspensions through a 40 &#181;m nylon mesh. Rinse the mesh with an additional 5 mL of MESC.</li>
		<li>Centrifuge the cell suspension at 500 x g for 7 min.</li>
		<li>Resuspend the pellet in MESC and plate on PLL-coated coverslips for further experiments with the desired density.</li>
		<li>Incubate for a minimum of 4 - 6 h or O/N at 37 &#176;C in a 5% CO2 incubator.</li>
	</ol>
	</li>
	<li>Mouse Endometrial Stromal Cell (MESC) Culture: 2nd&#160;Round
	<ol>
		<li>Prepare three small Petri dishes with cold (4 &#176;C) HBSS+ and 3 x 15 mL tubes with 3 mL of MESC medium (Tubes Y1, Y2 and Y3).</li>
		<li>Repeat steps 4.4.3 - 4.4.7.</li>
		<li>Transfer the last cell suspension together with the uteri to a 40 &#181;m nylon mesh.</li>
		<li>Collect the stromal cells by passing the content of tube Y1, Y2 and Y3 through the nylon mesh. Rinse the mesh with an additional 5 mL of MESC.</li>
		<li>Centrifuge the cell suspension at 500 x g for 7 min.</li>
		<li>Resuspend the pellet in MESC medium and plate on PLL-coated coverslips for further experiments (Figure 2b).</li>
		<li>Incubate for a minimum of 4 - 6 h at 37 &#176;C with 5% CO2. 
		NOTE: For immunostaining or functional experiments like fluorescent calcium imaging, it is advised to plate the cells on coverslips, while seeding in a 6-well plate is recommended when RNA or protein isolation is desired.</li>
	</ol>
	</li>
</ol>
5. Vimentin/Cytokeratin Double Staining for Validation of Pure MEEC and MESC Cultures
<ol>
	<li>Seed the cells on coverslips.</li>
	<li>Wash 3 x 5 min with PBS containing calcium and magnesium on an orbital shaker (50 rpm).</li>
	<li>Fix the cells with 4% paraformaldehyde (PFA) (CAUTION!) for 10 min, not on shaker.</li>
	<li>Wash 3x with PBS without calcium and magnesium on shaker (50 rpm).</li>
	<li>Permeabilize the cells with 0.2% Triton X-100 for 10 min on a shaker (50 rpm).</li>
	<li>Wash 3x with PBS on a shaker.</li>
	<li>Block with 5% goat serum (GS) in PBS for 2 h on a shaker (50 rpm)</li>
	<li>Incubate cells with monoclonal rabbit anti-human vimentin (1:500) and monoclonal mouse anti-human pancytokeratin (1:1,000) at 4 &#176;C on a shaker (50 rpm). Antibodies are diluted in PBS with 0.5% GS.</li>
	<li>Wash 3x with PBS on a shaker (50 rpm).</li>
	<li>Incubate cells with secondary antibodies (1:1,000 in 0.5% GS) Alexa Fluor 488-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG on a shaker.</li>
	<li>Wash 3x with PBS on a shaker.</li>
	<li>Mount slides with aqueous mounting medium containing DAPI and dry O/N.</li>
	<li>Seal the slides with nail polish and keep on 4 &#176;C for further use (Figure 2a, b).</li>
</ol>
6. In Vitro Decidualization in a Coculture System
NOTE: Four to five animals are required to establish a coculture system in a 24-well plate. Continue with the following protocol in a sterile environment, preferably under a laminar flow cabinet.
<ol>
	<li>Prepare the cell culture inserts during the centrifugation and/or incubation steps of the epithelial cell isolation (as described in 4.3) in an additional 24-well plate.
	<ol>
		<li>Add 200 - 400 &#181;L of MESC medium into the desired amount of wells in the 24-well plate.</li>
		<li>Place the inserts in the prefilled 24-wells using sterile forceps.</li>
	</ol>
	</li>
	<li>Resuspend the MEEC pellet (step 4.3.14) in MEEC medium and divide over the inserts (working volume: 150 - 300 &#181;L per insert). Culture the cells at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Seed the stromal cells in another 24-well plate (compatible with the cell culture inserts) after the second round of stromal cell isolation (step 4.5.6). Use a working volume of 400 &#181;L cell suspension per well.</li>
	<li>Allow the stromal cells to attach for minimum 4 - 6 h at 37 &#176;C in a 5% CO2 incubator.</li>
	<li>Transfer the cell culture inserts covered with epithelial cells from the first 24-well plate into the second 24-well plate covered by stromal cells. Use sterilized forceps or touch the inserts only at their hanging foot.</li>
	<li>Culture the cells at 37 &#176;C in 5% CO2 incubator for a period of 3 - 5 d until the epithelial cells form a monolayer and the stromal cells achieve 80 - 90% confluency. Additionally, use the Transepithelial Electrical Resistance (TEER) to monitor the epithelial monolayer by use of a Volt/Ohm epithelial meter as described by Grant et al.7. Values of 800-1,000/well were sufficient to consider the epithelial monolayer confluent.</li>
	<li>If necessary, replace the medium every 2 - 3 d using glass pipettes or fine pipet tips. Start with the replacement of the medium of the stromal cells (seeded at the bottom), before replacing the medium in the inserts. It is recommended to use preheated cell culture medium at 37 &#176;C.</li>
	<li>Prepare the decidualization medium to induce in vitro decidualization before starting the experiment. Use a working volume of 400 &#181;L per well of stromal cells.
	<ol>
		<li>Prepare the following stock solutions and store aliquots at -20 &#176;C.
		<ol>
			<li>Prepare 250 &#181;M medroxyprogesterone 17-acetate (MPA) in ethanol.</li>
			<li>Prepare 100 mM stock solution 8-Bromoadenosine 3&#8242;,5&#8242;- cAMP in ultrapure water.</li>
		</ol>
		</li>
		<li>Make the decidual medium containing 1 &#181;M MPA and 0.5 mM cAMP in MESC medium containing 2% FBS.</li>
	</ol>
	</li>
	<li>Gently remove the medium from the wells and add the decidual medium onto the stromal cells. If a control condition is needed, use the MESC medium containing 2% FBS.</li>
	<li>Remove the medium from the cell culture inserts and add 300 &#181;L of MEEC medium onto the cells.</li>
	<li>Incubate the cells for 5 d in an incubator at 37 &#176;C in 5% CO2.</li>
	<li>After the fifth day, assess the extent of decidualization in stromal cells by RT-qPCR (see below).</li>
	<li>Validate the viability of the epithelial cells using the Resazurin-based viability reagent.
	<ol>
		<li>Prepare a 1:10 solution of viability reagent in MEEC medium.</li>
		<li>Transfer the cell culture inserts into a new 24-well plate.</li>
		<li>Add 150 - 300 &#181;L of the viability reagent - MEEC solution onto the cells.</li>
		<li>Incubate at 37 &#176;C in 5% CO2 for at least 4 - 5 h or overnight and assess cell viability by observing a color shift (blue to purple/pink). 
		NOTE: Decidualization can also be evaluated using a mouse prolactin-specific ELISA, as preferred by the researcher.</li>
	</ol>
	</li>
</ol>
7.Validation of Decidualization by qRT-PCR
NOTE: For validation of the decidualization by qRT-PCR, it is recommended to perform all test-conditions in duplicate in order to receive a sufficient amount of cells for RNA analysis. 
Quantitative RT-PCR is performed as described previously in De Clercq et al.8. 
In brief:
<ol>
	<li>Isolate the total RNA using a commercial RNA isolation kit.
	<ol>
		<li>Assess the quantity and quality of the RNA using commercial methods according to manufacturer&#39;s protocol, respectively.</li>
	</ol>
	</li>
	<li>Synthesize cDNA, according to manufacturer&#39;s protocol starting from 1 &#181;g of total RNA.</li>
	<li>Use a commercial kit according to manufacturer&#39;s protocol to carry out the qRT-PCR reaction.
	<ol>
		<li>Run all samples in triplicate.</li>
		<li>Make use of the commercial TaqMan Master Mix and specific TaqMan probes for the desired housekeeping genes and prolactin (prl8a2) to carry out the reaction.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cummings, A. M., Yochim, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+uterus+in+preparation+for+gestation:+a+model+for+the+action+of+progesterone.">Differentiation of the uterus in preparation for gestation: a model for the action of progesterone.</a> J Theor Biol. 106 (3), 353-374 (1984).</li><li>Jabbour, H. N., Kelly, R. W., Fraser, H. M., Critchley, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+regulation+of+menstruation.">Endocrine regulation of menstruation.</a> Endocr Rev. 27 (1), 17-46 (2006).</li><li>Wang, X., Matsumoto, H., Zhao, X., Das, S. K., Paria, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+signals+direct+the+formation+of+tight+junctional+permeability+barrier+in+the+decidualizing+stroma+during+embryo+implantation.">Embryonic signals direct the formation of tight junctional permeability barrier in the decidualizing stroma during embryo implantation.</a> J Cell Sci. 117 (Pt 1), 53-62 (2004).</li><li>Zygmunt, M., Herr, F., Munstedt, K., Lang, U., Liang, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+and+vasculogenesis+in+pregnancy.">Angiogenesis and vasculogenesis in pregnancy.</a> Eur J Obstet Gynecol Reprod Biol. 110 Suppl 1, S10-S18 (2003).</li><li>Power, M. L., Schulkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The evolution of the human placenta. , (2012).</li><li>Finn, C. 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+initiation+of+the+decidual+cell+reaction+in+the+uterus+of+the+aged+mouse.">The initiation of the decidual cell reaction in the uterus of the aged mouse.</a> J Reprod Fertil. 11 (3), 423-428 (1966).</li><li>Grant, K. S., Wira, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mouse+uterine+stromal+cells+on+epithelial+cell+transepithelial+resistance+(TER)+and+TNFalpha+and+TGFbeta+release+in+culture.">Effect of mouse uterine stromal cells on epithelial cell transepithelial resistance (TER) and TNFalpha and TGFbeta release in culture.</a> Biol Reprod. 69 (3), 1091-1098 (2003).</li><li>De Clercq, K., 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=Functional+expression+of+transient+receptor+potential+channels+in+human+endometrial+stromal+cells+during+the+luteal+phase+of+the+menstrual+cycle.">Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.</a> Hum Reprod. 30 (6), 1421-1436 (2015).</li><li>Pieper, F. R., 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=Regulation+of+vimentin+expression+in+cultured+epithelial+cells.">Regulation of vimentin expression in cultured epithelial cells.</a> Eur J Biochem. 210 (2), 509-519 (1992).</li><li>Greaves, E., 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=A+novel+mouse+model+of+endometriosis+mimics+human+phenotype+and+reveals+insights+into+the+inflammatory+contribution+of+shed+endometrium.">A novel mouse model of endometriosis mimics human phenotype and reveals insights into the inflammatory contribution of shed endometrium.</a> Am J Pathol. 184 (7), 1930-1939 (2014).</li><li>Pierro, E., 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=Stromal-epithelial+interactions+modulate+estrogen+responsiveness+in+normal+human+endometrium.">Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium.</a> Biol Reprod. 64 (3), 831-838 (2001).</li><li>Kim, M. R., 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=Progesterone-dependent+release+of+transforming+growth+factor-beta1+from+epithelial+cells+enhances+the+endometrial+decidualization+by+turning+on+the+Smad+signalling+in+stromal+cells.">Progesterone-dependent release of transforming growth factor-beta1 from epithelial cells enhances the endometrial decidualization by turning on the Smad signalling in stromal cells.</a> Mol Hum Reprod. 11 (11), 801-808 (2005).</li><li>Chen, J. C., 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=Coculturing+human+endometrial+epithelial+cells+and+stromal+fibroblasts+alters+cell-specific+gene+expression+and+cytokine+production.">Coculturing human endometrial epithelial cells and stromal fibroblasts alters cell-specific gene expression and cytokine production.</a> Fertil Steril. 100 (4), 1132-1143 (2013).</li><li>Renaud, J., Martinoli, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+Insert+Co-culture+System+of+Two+Cellular+Types+in+the+Absence+of+Cell-Cell+Contact.">Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact.</a> J Vis Exp. (113), (2016).</li><li>Arnold, J. T., Kaufman, D. G., Seppala, M., Lessey, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endometrial+stromal+cells+regulate+epithelial+cell+growth+in+vitro:+a+new+co-culture+model.">Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model.</a> Hum Reprod. 16 (5), 836-845 (2001).</li><li>Park, D. W., 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=A+well-defined+in+vitro+three-dimensional+culture+of+human+endometrium+and+its+applicability+to+endometrial+cancer+invasion.">A well-defined in vitro three-dimensional culture of human endometrium and its applicability to endometrial cancer invasion.</a> Cancer Lett. 195 (2), 185-192 (2003).</li></ol>

The authors have nothing to disclose.

Decidualization is the progesterone-dependent differentiation of endometrial stromal cells into round secreting decidual cells. In human, this process occurs spontaneously during the luteal phase of the menstrual cycle and is initiated in the stromal cells surrounding the vascular cells to form the predecidua. However, in rodents, the presence of a blastocyst is imperative to induce decidualization. The fact that decidualization only occurs after contact with the endometrial epithelial cells implies that important factors are secreted by the epithelial cells to induce decidualization in the underlying stroma. Although this difference in the initiation of the decidualization process should be acknowledged, the fact that human decidualization becomes more robust upon embryo implantation suggests that similar mechanisms could be involved. In vitro cell cultures are often used to study the effect of specific molecules during the process of decidualization. Nevertheless, the availability and amount of human tissue that can be collected is often limited and accompanied by variations, e.g.,&#160;cycle phase, number of pregnancies and presence of gynecological diseases like endometriosis or adenomyosis. Many of these issues can be prevented by the use of murine tissue that is easily accessible and cycle phase independent. Another strong advantage of using murine tissue is the opportunity to use genetically modified rodents and the possibility to setup a large number of experiments. At the moment, many different isolation protocols have been described in literature with high variability in the age of the animals and the period in the estrus phase at the moment of use.
This study presents a new method for primary endometrial co-cultures of stromal and epithelial cells in which advantage was taken of a standardized estrous cycle by a daily injection of estrogen prior the isolation. Furthermore, estrogen is known to induce proliferation in epithelial and stromal cells which further increases the yield per isolation. The isolation protocol described in this paper was based on Grant et al.7, however, further optimization steps were included to increase the yield, purity, and viability of the different cell cultures. First, HBSS was always supplemented with antibiotics to avoid contamination of the primary culture. During the isolation of MEEC, a temperature adaptation was done (15 min at 37 &#176;C) to increase the harvest of the epithelial cells during the first digestion. Next, an additional step was included to temper enzymatic activity after trypsin-digestion by adding medium containing FBS to inhibit its activity. Furthermore, MEECs were passed through a filter of which the mesh was larger than what is commonly described (100 &#181;m) as they often come in clusters and cell sheets. Moreover, contamination by stromal cells was reduced by performing an additional step in which MEECs and MESCs were separated based on gravity sedimentation. Finally, MEECs were seeded on collagen-coated coverslips to increase the attachment of cells to glass coverslips, as it became clear that attachment to uncoated or PLL-coated glass coverslips was insufficient. During the isolation of the MESCs, collagenase (1 mg/mL) was supplemented to improve digestion. Furthermore, this digestion step was performed in duplicate to avoid contamination of remaining MEECs. All these additional steps resulted in pure and viable cultures of homogenous cell populations.
The purity of the cell population was evaluated with immunostaining and qRT-PCR using vimentin as a stromal marker and cytokeratin as an epithelial marker. Clearly, an opposite expression pattern of vimentin and cytokeratin was observed in MEEC and MESC. However, it can be noticed that the relative mRNA expression of vimentin and cytokeratin is similar in the MEEC cultures. Similar results are published by other research groups, in which epithelial cells in culture acquire vimentin expression9. More important is the difference in protein expression between vimentin and cytokeratin as was observed in the immunohistological stainings of the different cell populations. Double immunostaining showed that EECs were positive for cytokeratin and ESC were positive for vimentin. The use of these markers in immunostaining is an established method and standardly used to indicate the cell types10.
Although a lot of research has been performed on the decidualization of MESC, it remains possible that the presence of epithelial cells in this model may alter the results of decidualization. Firstly, it has been shown that if MEEC grow to a monolayer in the presence of MESC-conditioned medium or in a co-culture setting, the monolayer has an improved epithelial cell transepithelial electrical resistance (TEER), resulting from factors secreted by MESC7. Moreover, Pierro et al.11 provided evidence that epithelial proliferation, influenced by 17&#946;-estradiol, might be mediated by factors secreted from the stromal cells, and alternatively, epithelial cells can release factors that modulate stromal decidualization12,13. Overall, it is clear that the epithelial-stromal co-culture environment provides a more physiological situation that allows for paracrine interaction and communication. The method of culturing two different cell types using an insert co-culture system was described earlier14,15 and was adapted for the use of murine primary cells. By the detection of prolactin mRNA levels as a read-out for decidualization, the described technique has a very reproducible read-out for decidualization available and allows thereby the study of the epithelial-stromal relationship during decidualization. Paracrine signals mediated from MEEC might alter the extent of decidualization in the stromal cells. Moreover, the model allows for specific modulation of the epithelial side without directly affecting the stromal cells. Therefore, this co-culture of MEEC and MESC offers a perfect tool to evaluate the effect of hormones, cytokines, etc. on epithelial cells with a read-out on stromal decidualization. Another advantage of this co-culture system is that it allows researchers to investigate the involvement of a specific protein in the decidualization-process in either the epithelial or stromal compartment by using cells isolated from transgenic animals. Furthermore, this technique will be very helpful to investigate blastocyst adhesion to evaluate whether paracrine factors secreted by epithelial cells in the presence of a blastocyst are sufficient to induce decidualization of the underlying stromal cells. An important step in trophoblast invasion correlated to extracellular matrix degradation, which is mediated by the action of proteolytic enzymes. The proposed technique can be applied as an in vitro model to investigate the cross-talk among the blastocyst, the epithelial cells and the supporting stroma.
However, at the moment little is known about the origin of the epithelial cells which can be as well luminal as glandular. Therefore, further characterization of the genetic and molecular profile of the epithelial cells is required. In addition, the described method is limited in the available knowledge about the polarization of the epithelial cells. At the moment, the polarization of epithelial cells was not taken into account. However, differences in expression of membrane proteins are described in apical and basal membranes. Inappropriate polarization of the epithelial layer could have an impact on the paracrine interaction between epithelial and stromal cells. However, methods to obtain polarized epithelial cells has been described and could be included in the described technique15,16.
Altogether, the described protocol proposes a technique to successfully establish primary cell cultures of mouse endometrial epithelial and stromal cells. This technique results in pure cell cultures as confirmed by qRT-PCR and immunostaining of vimentin and cytokeratin. Ultimately, an epithelial and stromal co-culture was introduced that allows for the investigation of paracrine signals mediated by either MEEC and/or MESC in the decidualization process.

The human endometrium is the inner lining of the uterus and undergoes monthly cycles of breakdown and repair as a preparation for possible pregnancies. It consists of a single layer of epithelial cells lining the uterine lumen and the underlying stroma which varies in thickness according to fluctuations of ovarian hormones estrogen and progesterone. During the luteal phase, the postovulatory progesterone surge will induce differentiation of endometrial stromal cells into larger, round decidual cells, a process called decidualization1,2. In human, this phenomenon occurs spontaneously to form the predecidua, but becomes more pronounced upon embryo implantation. A functional consequence of decidualization is that the uterus transiently becomes receptive to embryo implantation. This interval is labeled as the &#39;window of implantation&#39;. During the early period of embryo implantation, the decidualized endometrial stromal cells surrounding the implanting embryo will provide a barrier to prevent the passage of harmful substances to the embryo3. During pregnancy, this decidua will provide nutrients for the developing fetus before placentation has taken place, and will later form the maternal part of the placenta4.
Ethical and practical considerations limit the design of human studies to investigate the process of decidualization and have prompted the use of animal models. Being a member of the hemochorial placentation group, the endometrium of rodents will also undergo decidualization prior to placentation5. In rodents, however, the initiation of the decidualization process depends on the presence of blastocysts in the uterine lumen, indicating that an extra stimulus is necessary to trigger the decidual responses6. This implies an intricate communication between the endometrial epithelial cells that have direct contact with the blastocyst, and the underlying stromal cells. Nevertheless, decidualization can be artificially induced using stimuli as mechanical scratching or the instillation of oil in a hormonally primed uterus. Although in vivo studies using artificial decidualization models have allowed us to improve our insights in the complex signaling process of decidualization, mechanistic in-depth studies, e.g., investigation of the indispensable communication between epithelial and stromal cells, are limited. Therefore, in vitro decidualization studies can be used to manipulate important pathways and study fundamental processes. To this end, primary endometrial stromal cell cultures can be set up from human or rodent endometrium. Employing rodents consents the use of genetically modified animals to investigate the role of a specific protein of interest during the decidualization process. However, immature, prepuberty mice are often used in order to avoid complications of the estrous cycle, while in other cases uteri are collected from all phases of the estrous cycle, which results in a heterogeneity in the cultures. Moreover, the process of decidualization has been studied in different protocols, e.g.,&#160;by (i) supplementing hormones (estrogen, progesterone or cyclic adenosine monophosphate (cAMP)) to the culturing medium, or by (ii) isolation of decidual cells from mice at day 4.5 of (pseudo)pregnancy, which demand additional need for plug checking and vasectomized males. These limitations demonstrate the necessity of a standardized method to study in vitro decidualization. In this study, a physiological model to examine in vitro decidualization starting from adult, non(pseudo)pregnant mice will be presented. In this method, daily injection of 17&#946;-estradiol (E2) will induce proliferation of endometrial cells, resulting in an increased yield of homogenous and pure cell populations. Furthermore, the use of a coculturing system allows the study of the epithelial-stromal crosstalk and the ability to manipulate the process of decidualization on different levels.

<table><tbody><tr><td>ThinCert Tissue culture insert for 24 well plates; 1 &amp;micro;m pore size; transparant</td><td>Greiner Bio-one</td><td>662610</td><td>Inserts are also provided for other multiwell plates and/or other pore sizes. Other suppliers: Merck Millipore.&lt;br/&gt; https://shop.gbo.com/en/row/articles/catalogue/article/&lt;br/&gt;0110_0110_0090_0030/13408/&amp;nbsp;</td></tr><tr><td>Nunc Cell culture treated 24 well plates</td><td>Thermo Scientific</td><td>142475</td><td>http://www.thermofisher.com/order/catalog/product/142475</td></tr><tr><td>8-Bromoadenosine 3&amp;prime;,5&amp;prime;-cyclic monophosphate sodium salt</td><td>Sigma Aldrich</td><td>B7880</td><td>Prepare a stock solution of 100 mM in Milli Q water. Store aliquots at -20 &amp;deg;C.&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/b7880?lang=en&amp;amp;region=BE&amp;nbsp;&lt;br/&gt;</td></tr><tr><td>Medroxyprogesterone 17-acetate</td><td>Sigma Aldrich</td><td>M1629</td><td>Prepare a stock solution of 250 &amp;micro;M in ethanol. Store aliquots at -20 &amp;deg;C.&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/m1629?lang=en&amp;amp;region=BE&amp;nbsp;&lt;br/&gt;</td></tr><tr><td>Arachis oil</td><td>Supermarket</td><td></td><td>Standard cooking arachis oil that can be found in every supermarket is used</td></tr><tr><td>PrestoBlue cell viability reagent</td><td>Thermo Scientific</td><td>A13261</td><td>https://www.thermofisher.com/order/catalog/product/A13261?ICID=cvc-prestoblue-c1t1</td></tr><tr><td>HBSS 1x, no calcium, no magnesium, no phenol red</td><td>Gibco</td><td>14175-046</td><td>Store in fridge as long as possible. Use cold HBSS+ during the protocol.&lt;br/&gt; https://www.thermofisher.com/order/catalog/product/14175046&amp;nbsp;&lt;br/&gt;</td></tr><tr><td>17-&amp;beta; Estradiol</td><td>Sigma Aldrich</td><td>E8875</td><td>Prepare a stock solution of 1 mg/mL in EtOH before diluting in arachis oil (1:1,000).&lt;br/&gt;http://www.sigmaaldrich.com/catalog/product/sigma/e8875?lang=en&amp;amp;region=BE</td></tr><tr><td>Cell medium filter Stericup, 0.22 &amp;micro;m, polyethersulfone, 500 mL, radio-sterilized</td><td>Merck Millipore</td><td>SCGPU05RE</td><td>http://www.merckmillipore.com/BE/en/product/Stericup-GP%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-500%C2%A0mL%2C-radio-sterilized,MM_NF-SCGPU05RE</td></tr><tr><td>Penicillin-Streptomycin (5,000 U/mL)</td><td>Gibco</td><td>15070-063</td><td>https://www.thermofisher.com/order/catalog/product/15070063?ICID=search-15070-063</td></tr><tr><td>DMEM/F12, phenol red, L-glutamine, HEPES</td><td>Gibco</td><td>11330-032</td><td>https://www.thermofisher.com/order/catalog/product/11330057?ICID=search-11330-057</td></tr><tr><td>Fetal Bovine Serum, qualified, heat inactivated, E.U.-approved, South America Origin</td><td>Gibco</td><td>10500-056</td><td>https://www.thermofisher.com/order/catalog/product/10500064?ICID=search-10500-064</td></tr><tr><td>Amphotericin B</td><td>Gibco</td><td>15290-018</td><td>https://www.thermofisher.com/order/catalog/product/15290018?ICID=search-15290-018</td></tr><tr><td>Gentamicin (50 mg/mL)</td><td>Gibco</td><td>15750-037</td><td>https://www.thermofisher.com/order/catalog/product/15750037?ICID=search-15750-037</td></tr><tr><td>DMEM, low glucose, pyruvate, no glutamine, no phenol red</td><td>Sigma Aldrich</td><td>D5921</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/d5921?lang=en&amp;amp;region=BE</td></tr><tr><td>MCDB-105</td><td>Sigma Aldrich</td><td>M6395</td><td>Dilute in Milli Q water, adjust pH to 7 with NaOH and filter before use. Store in the dark.&lt;br/&gt; http://www.sigmaaldrich.com/catalog/product/sigma/m6395?lang=en&amp;amp;region=BE&amp;nbsp;&lt;br/&gt;</td></tr><tr><td>Insulin from bovine pancreas</td><td>Sigma Aldrich</td><td>I1882</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/i1882?lang=en&amp;amp;region=BE</td></tr><tr><td>Coverslips, glass, 18 mm &amp;Oslash;</td><td>Glaswarenfabrik Karl Hecht</td><td>1001/18</td><td>http://www.hecht-assistent.de/187/?L=1&amp;amp;tx_rmproducts_pi1[g]=937&lt;br/&gt;&amp;amp;tx_rmproducts_pi1[p]=3504&lt;br/&gt;&amp;amp;tx_rmproducts_pi1[v]=20088&lt;br/&gt;&amp;amp;cHash=abd12065683fe74b00eaa&lt;br/&gt;5e562824d06</td></tr><tr><td>Poly-L-lysine</td><td>Sigma Aldrich</td><td>P2636</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p2636?lang=en&amp;amp;region=BE</td></tr><tr><td>Collagenase type IA from Clostridium histolyticum</td><td>Sigma Aldrich</td><td>C2674</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/c2674?lang=en&amp;amp;region=BE</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco</td><td>14190-094</td><td>https://www.thermofisher.com/order/catalog/product/14190094?ICID=search-14190-094</td></tr><tr><td>Trypsin from bovine pancreas</td><td>Sigma Aldrich</td><td>T7409</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/t7409?lang=en&amp;amp;region=BE</td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco</td><td>25300-054</td><td>Warm to room temperature before use.&lt;br/&gt; https://www.thermofisher.com/order/catalog/product/25300054?ICID=search-25300-054&amp;nbsp;&lt;br/&gt;</td></tr><tr><td>Cell strainer, 40 &amp;micro;m mesh, disposable</td><td>BD Falcon</td><td>352340</td><td>https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680</td></tr><tr><td>Cell strainer, 100 &amp;micro;m mesh, disposable</td><td>BD Falcon</td><td>352360</td><td>https://www.fishersci.com/shop/products/falcon-cell-strainers-4/p-48680</td></tr><tr><td>Millex-GP Syringe Filter Unit, 0.22&amp;nbsp;&amp;micro;m, polyethersulfone, 33&amp;nbsp;mm, gamma sterilized</td><td>Merck Millipore</td><td>SLGP033RS</td><td>https://www.merckmillipore.com/BE/en/product/Millex-GP-Syringe-Filter-Unit%2C-0.22%C2%A0%C2%B5m%2C-polyethersulfone%2C-33%C2%A0mm%2C-gamma-sterilized,MM_NF-SLGP033RS?bd=1</td></tr><tr><td>Acetic acid (glacial) 100%</td><td>Merck Millipore</td><td>1.00063</td><td>https://www.merckmillipore.com/BE/en/product/Acetic-acid-%28glacial%29-100%25,MDA_CHEM-100063</td></tr><tr><td>Collagen I</td><td>Corning&amp;nbsp;</td><td>354236</td><td>From rat tail.&lt;br/&gt; https://catalog2.corning.com/LifeSciences/en-BR/Shopping/ProductDetails.aspx?categoryname=&amp;amp;&lt;br/&gt;productid=354236%28&lt;br/&gt;Lifesciences%29#&amp;nbsp;&lt;br/&gt;</td></tr><tr><td>Pancreatin from porcine pancreas</td><td>Sigma Aldrich</td><td>P3292</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p3292?lang=en&amp;amp;region=BE</td></tr><tr><td>35 mm Tissue Culture Dishes, Polystyrene, Sterile</td><td>BD Falcon</td><td>353001</td><td>https://us.vwr.com/store/catalog/product.jsp?product_id=4675630</td></tr><tr><td>Ethanol absolute AnalaR NORMAPUR ACS, Reag. Ph. Eur. analytical reagent</td><td>VWR Chemicals</td><td>20821296</td><td>https://uk.vwr.com/store/catalog/product.jsp?catalog_number=20821.310DP</td></tr><tr><td>monoclonal rabbit anti-human vimentin (D21H3)&amp;nbsp;</td><td>Cell Signalling Tech</td><td>#5741</td><td>Use 1/500.&lt;br/&gt; http://www.cellsignal.com/products/primary-antibodies/vimentin-d21h3-xp-rabbit-mab/5741&lt;br/&gt;</td></tr><tr><td>monoclonal mouse anti-human pan cytokeratin&amp;nbsp;</td><td>Sigma Aldrich</td><td>C2562</td><td>Use 1/1,000.&lt;br/&gt;http://www.sigmaaldrich.com/catalog/product/sigma/c2562?lang=en&amp;amp;region=BE</td></tr><tr><td>Paraformaldehyde Solution, 4% in PBS</td><td>Affymetrix</td><td>19943</td><td>http://www.affymetrix.com/catalog/130621/USB/Paraformaldehyde+Solution+4+percent+in+PBS#1_1</td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100</td><td>http://www.sigmaaldrich.com/catalog/product/SIAL/X100?lang=en&amp;amp;region=BE&amp;amp;gclid=&lt;br/&gt;Cj0KEQjwhN-6BRCJsePgxru9iIw&lt;br/&gt;BEiQAI8rq&lt;br/&gt;879lESeuIU4N4AEQRLNEXj4f&lt;br/&gt;8z65KkD-q8Xr35sQDRgaAp-k&lt;br/&gt;8P8HAQ</td></tr><tr><td>Goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/g9023?lang=en&amp;amp;region=BE</td></tr><tr><td>Goat anti-rabbit Alexa Fluor 488</td><td>Thermo Scientific</td><td>A-11034</td><td>http://www.thermofisher.com/order/genome-database/searchResults?query=488&amp;amp;&lt;br/&gt;resultPage=1&amp;amp;&lt;br/&gt;resultsPerPage=15&amp;amp;&lt;br/&gt;autocomplete=&amp;amp;&lt;br/&gt;searchMode=keyword&amp;amp;&lt;br/&gt;productTypeSelect=antibody&amp;amp;&lt;br/&gt;targetTypeSelect&lt;br/&gt;=antibody_secondary&amp;amp;&lt;br/&gt;species=ltechall&amp;amp;keyword=&lt;br/&gt;488#filters=genericsort2:\%22Capra%20hircus\%22;;generic6:^\%22Oryctolagus%20cuniculus\%22$;;conjugates:^\%22Alexa%20Fluor&amp;amp;reg;%20488\%22$;;</td></tr><tr><td>Goat anti-mouse Alexa Fluor 594</td><td>Thermo Scientific</td><td>A-11032</td><td>http://www.thermofisher.com/order/genome-database/searchResults?query=594&amp;amp;&lt;br/&gt;resultPage=1&amp;amp;&lt;br/&gt;resultsPerPage=15&amp;amp;&lt;br/&gt;autocomplete=&amp;amp;&lt;br/&gt;priorSearchTerms=&amp;amp;&lt;br/&gt;searchMode=keyword&amp;amp;&lt;br/&gt;productTypeSelect=antibody&amp;amp;&lt;br/&gt;targetTypeSelect=&lt;br/&gt;antibody_secondary&amp;amp;&lt;br/&gt;species=ltechall&amp;amp;&lt;br/&gt;keyword=594#filters=genericsort2:\%22Capra%20hircus\%22;;generic6:^\%22Mus%20musculus\%22$;;</td></tr><tr><td>VECTASHIELD mounting medium with DAPI</td><td>Vector Laboratories</td><td>H-1200</td><td>http://vectorlabs.com/vectashield-mounting-medium-with-dapi.html</td></tr><tr><td>DPBS, with calcium and magnesium</td><td>Gibco</td><td>14040174</td><td>https://www.thermofisher.com/order/catalog/product/14040133</td></tr></tbody></table>

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.

General Overview of Protocol
Figure 1A illustrates a general overview of the protocol. A daily injection of E2 (100 ng) for three consecutive days was used to synchronize the animals and standardize the phase of the estrous cycle. The estrous cycle can be assessed by vaginal lavages and is divided into the proestrus, estrus, diestrus, and metestrus phase. The cycle phase can be designated according to the ratio of desquamated cells in the lavage, which are subjected to hormonal changes. Increasing levels of estrogen will make the animals evolve to the estrus phase. This phase can easily be detected by the presence of exclusively cornified epithelial cells and the absence of leukocytes (Figure 1B). On day 4, uteri of animals that are in estrus phase are collected and MEEC and MESC are isolated (Figure 1C). Decidualization can be induced by adding 0.5 mM cAMP and 1 &#181;M MPA to the 2% FBS medium during an incubation period of 5 d.
Quality Control of MEEC and MESC Cultures
The purity of the primary cell cultures can be assessed by the difference in vimentin and cytokeratin expression. Vimentin is an intermediate filament that is expressed in mesenchymal cells, hence in the endometrial stromal cells. Cytokeratin is an intermediate filament found in the cytoskeleton of epithelial tissue. Figure 2A shows the mRNA expression level of vimentin and cytokeratin in MEEC and MESC assessed using qRT-PCR. Vimentin levels are high in MESC and low in MEEC (2.2x higher, p &#60; 0.001), while cytokeratin levels are high in MEEC and low in MESC (36x higher, p &#60; 0.001). A vimentin/cytokeratin double staining was performed and indicated that stromal cells express vimentin whereas epithelial cells express cytokeratin (Figure 2B, C). The absence of primary antibody was used as a negative control for the immunostaining (Figure 2D, E). These immunohistological stainings show that both endometrial cell cultures (MEEC and MESC) have a purity of up to 90%.
Decidualization in MEEC/MESC Cocultures
After isolation, mouse endometrial and stromal cells were seeded in a co-culture system to mimic the endometrial environment. Cells were cultured until the epithelial cells formed a monolayer in the cell culture insert (TEER values of 800 - 1,000/well), and the stromal cells reached a sub-confluent state. Decidualization was induced in the stromal cells with the application of 0.5 mM cAMP and 1 &#181;M MPA. After five days of incubation, prolactin mRNA levels were assessed in the stromal cells by qRT-PCR as an indication for decidualization (Figure 3A). Prolactin levels were highly expressed in cells subjected to the hormones and nondetectable in cells incubated with the control medium (p &#60; 0.01).
Since epithelial cells are hard to visualize inside the cell culture inserts, a viability assay was performed immediately after the five-day incubation period (Figure 3B). A mix of normal growth medium and a viability reagent was added to the wells and incubated for a period of minimal 8 - 12 h. The color shift from blue to bright pink indicated the presence of viable epithelial cells. Note that the color shift will only occur when viable cells are present.
<img alt="Figure 1" src="/files/ftp_upload/55168/55168fig1.jpg" /> 
Figure 1: General Overview of the Protocol. (A) Scheme of the isolation protocol starting with three consecutive days of estrogen injections. On day 3, the necessary preparations should be prepared. On day 4, estrus phase is evaluated (indicated by star) by performing a vaginal lavage. MESC and MEEC are isolated using different digestion steps. On day 6, or when cells are confluent, decidualization can be induced in the co-culture system by adding cAMP and MPA to the medium and an incubation period of five days (day 6 - 11). (B) Representative picture of the estrus phase characterized by the presence of cornified epithelial cells in the vaginal lavage (magnification 10X). (C) Representative picture of MESC and MEEC (magnification 20X, scale bar: 100 &#181;m). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55168/55168fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55168/55168fig2.jpg" /> 
Figure 2: Immunostaining and Quantitative RT-PCR for Vimentin and Cytokeratin in MEEC and MESC. (A) Messenger RNA levels of vimentin and cytokeratin expression to indicate the purity of the cultures. RNA levels were relatively quantified to the geometric mean of housekeeping genes ACTB and TBP. Data were shown as mean &#177; SEM. Vimentin/cytokeratin double staining on MESC cultures (B) and MEEC cultures (C). Insert in the left shows a 63X magnification view. Negative control with primary antibody omitted for MESC (D) and MEEC (E) (Scale bar: 50 &#181;m).&#160;***: p &#60;0.001 with Two-way ANOVA with Bonferroni correction for multiple testing. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55168/55168fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 3" src="/files/ftp_upload/55168/55168fig3.jpg" /> 
Figure 3: Validation of Decidualization via RT-PCR. (A) mRNA levels of prolactin expression in stromal cells to evaluate decidualization. RNA levels were relatively quantified to the geometric mean of housekeeping genes PGK1 and TBP. The&#160;fold change in cells cultured in control or decidual medium is shown as mean &#177;SEM. (B) Illustrative pictures of stromal cells before (top) and after (bottom) decidualization stimulus. The insert on the right illustrates a magnification of a decidualized stromal cell. Decidualization is characterized by the presence of multinucleated cells, indicated by black arrows. (C) Representative images of the assessment of epithelial cell viability in the insert after the assay. Pictures were taken immediately after the administration of the viability reagent (Prestoblue) (0 h) and the following morning (ON).&#160;**: p &#60;0.01 with Mann-Whitney U test. ON: overnight. Scale bar: 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55168/55168fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization at JoVE.com

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.

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. ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

24.0K Views.  Katholieke Universiteit Leuven (KU Leuven). 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.

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

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 ...

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 ...

Medicine

Generation of a Mouse Artificial Decidualization Model with Ovariectomy for Endometrial Decidualization Research

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of decidualization leads to various endometrial disorders, such as infertility, recurrent miscarriage, and preterm birth. The development and use of the endometrial decidualization model in reproductive studies have been a highlight for reproductive researchers for a long time. The mouse has been extensively used in studying reproduction and decidualization. There are three well-established mouse models regarding decidualization, namely natural pregnancy decidualization (NPD), artificial decidualization (AD), and in vitro decidualization (IVD). Among them, AD is considered a reliable model for mouse decidualization, which is easy to implement and close to NPD. This paper focuses on a modified method of the generation and application process of the mouse artificial decidualization model with ovariectomy to avoid ovarian effects, which can obtain highly reproducible results with small within group variances. This method provides a good and reliable animal model for the study of endometrial decidualization.

Here, we describe the method of generating an artificial decidualization model using the ovariectomized mouse, a classic endometrial decidualization experiment in the research field of endometrial decidualization.

Endometrial decidualization is a unique differentiation process of the endometrium, closely related to menstruation and pregnancy. Impairment of ...

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

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

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

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

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The development of novel therapies and diagnostic tools for endometriosis has been limited due in part to challenges in studying the disease. Outside of primates, few mammals menstruate, and none develop spontaneous endometriosis. Rodent models are popular but require artificial induction of endometriosis, with many utilizing either immunocompromised mice or surgically induced disease. Recently, more attention has been given to models involving intraperitoneal injection. We present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified system that relies on microscopic quantification in lieu of subjective grading. In this model, we perform hormonal stimulation of donor mice, intraperitoneal injection, systematic abdominal survey and tissue harvest, and histologic quantification that can be performed and verified at any time after necropsy. This model requires minimal resources and training; does not require expertise by lab technicians in murine survival surgery or in the identification of gross endometriotic lesions; can be used in immunocompromised, immunocompetent, and/or mutant mice; and reliably creates endometriotic lesions that are histologically consistent with human endometriotic disease.

Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The ...

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

Biology

Evaluating Embryo Adhesion in an In Vitro Co-Culture Model with Ishikawa Cells

Establishment of an Embryo Implantation Model In Vitro

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic biological research, drug development, and screening. This paper presents a simple, rapid, and highly efficient in vitro model for embryo implantation. In this protocol, we first introduce mouse blastocyst acquisition and human endometrial adenocarcinoma cells (Ishikawa)&#160;preparation for implantation, followed by the co-culture method for mouse embryos and Ishikawa cells. Finally, we conducted a study to assess the impact of varying concentrations of 17-&#946;-estradiol (E2) and progesterone (P4) on embryo adhesion rates based on this model. Our findings revealed that high concentrations of E2 significantly reduced embryo adhesion, whereas the addition of progesterone could restore the adhesion rate. This model offers a simple and fast platform for evaluating and screening molecules involved in the adhesion process, such as cytokines, drugs, and transcription factors controlling implantation and endometrial receptivity.

Author Spotlight: Advancing Therapeutic Strategies for Improving Pregnancy Rates by Analyzing Embryo-Endometrium Interactions

This protocol describes methodologies to establish simple and efficient embryo implantation in vitro model for evaluating the relevant molecules affecting the embryo implantation process.

Embryo implantation is the first step in the establishment of a successful pregnancy. An in vitro model for embryo implantation is critical for basic ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

3D-Printed Scaffold Model to Investigate Embryo Implantation Mechanisms

Co-culture Model Using Two Types of Adherent Cell Lines

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications between various cell types within the decidua are crucial for regulating endometrial receptivity; however, the molecular mechanisms mediating this interaction are still unclear. In this regard, a model to study the implantation process is needed to establish a comprehensive in vitro model that can recapitulate the biology of endometrial epithelium-stroma interaction. This model is composed of regular cell-culture plates and a matching scaffold, which is generated by three-dimensional (3D) printing from low-cost materials. Here, we detail a set of protocols for model construction, cell preparation, cell seeding, cell culture, observation, and evaluation. Furthermore, we have included representative results with cells exhibiting good growth conditions under the microscope. This study aimed to develop in vitro models that would mimic the interaction between endometrial stromal cells and epithelial cells, as well as between trophoblast cells and endometrial cells.

Author Spotlight: In Vitro Endometrial Models to Study Epithelial-Stroma and Embryo Interactions

The protocol shows a novel in vitro experimental model that can recapitulate the biology of two kinds of adherent cell lines with a three-dimensional (3D)-printed scaffold. The construction of this model and operating procedures, from cell preparation and cell culture to analysis and evaluation, are described.

Embryo implantation is affected by the interactions among different cell types in the mother-embryo interface. The direct and indirect communications ...

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

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