This study was funded by NIEHS/NIH/NCATS UG3 grant (ES029073) and Northwestern Feinberg School of Medicine Bridge fund (JJK). We would like to acknowledge the Northwestern Pathology Core Facility for processing the fixed organoids for paraffin embedding. We would like to acknowledge the entire UG3 team including the Woodruff, Burdette, and Urbanek labs for the insightful discussions and collaborations.

Endometrial samples were collected from premenopausal women undergoing routine hysterectomy for benign uterine conditions at Northwestern University Prentice Women&#39;s Hospital, according to an Institutional Review Board-approved protocol. Written consent was obtained from all women included in the study.
1. Preparation of Agarose Molds
<ol>
	<li>Prior to beginning cell isolation, cast and equilibrate 1.5% agarose micro molds (Table of Materials) to house the organoids according to the manufacturer&#8217;s instructions.</li>
</ol>
2. Generation of Endometrial Organoids
<ol>
	<li>Harvest primary stromal and epithelial cells
	<ol>
		<li>In a biosafety cabinet using aseptic technique, prepare enzyme solution (2.5 mg/mL collagenase type I + 0.1 mg/mL DNase I in 10 mL of dispase [500 units]) at 37 &#176;C, and sterile-filter the solution using a 0.2 &#181;m syringe filter into a 15 mL conical tube.</li>
		<li>In a biosafety cabinet using aseptic technique, scrape off endometrium from the uterine biopsies and mince tissue under hood into very small pieces with a scalpel. 
		NOTE: Endometrial tissue that is at least 20 mm x 10 mm x 1 mm is required to generate sufficient numbers of organoids.</li>
		<li>Put freshly minced tissue into the enzyme solution in a 15 mL conical tube. Close the cap and wrap the top with a wax film (Table of Materials) to prevent contamination.</li>
		<li>Put the tube with tissue in a water bath or an incubator at 37 &#176;C for 30 min with gentle shaking (80&#8722;100 rpm).</li>
		<li>Stack a 100 &#181;m cell strainer on top of a 20 &#181;m cell strainer on a 50 mL conical tube. Filter the solution through the two strainers, and then rinse the 15 mL conical tube with 10 mL of Hank&#8217;s balanced salt solution (HBSS; Table of Materials) and put the wash through the strainer to ensure all cells are collected. 
		NOTE: The flow-through liquid contains stromal cells and red blood cells (~20 mL total). Epithelial cells are collected on the 20 &#181;m strainer. Chunks of undigested tissue remain on top of the 100 &#181;m strainer. Discard undigested tissue into a biohazard waste container.</li>
		<li>Invert the 20 &#181;m cell strainer from step 2.1.5 onto a new 50 mL conical tube and wash the epithelial cells off the strainer with 20 mL of organoid media (Table of Materials) supplemented with 1% pen/strep.</li>
		<li>Centrifuge the conical tubes from steps 2.1.5 and 2.1.6 at 500 x g for 5 min.</li>
		<li>With the collected stromal cells, remove the supernatant and resuspend the pellet with 10 mL of red blood cell lysis buffer.&#160; Incubate at 37 &#176;C for 10&#8722;15 min. Centrifuge the conical tube at 500 x g for 5 min, remove the supernatant, and resuspend the pellet with 200&#8722;300 &#181;L of organoid media (see step 2.2.2 for further instructions).</li>
		<li>With the collected epithelial cells, remove the supernatant and re-suspend with 100 &#181;L of organoid media (see step 2.2.2 for further instructions).</li>
	</ol>
	</li>
	<li>Seeding cells
	<ol>
		<li>After equilibrating the 1.5% agarose molds in wells (1 mold per well) of a 24-well plate, remove organoid media on the outside of the agarose molds and tilt the tissue culture plate so that the medium from the cell seeding chamber of the agarose dishes can also be carefully removed.</li>
		<li>View epithelial (from step 2.1.9) and stromal (from step 2.1.8) cell suspensions under the microscope.&#160; Add more organoid media to either the epithelial or stromal cell suspensions 100 &#181;L at a time, so that the suspensions are roughly equal in density. 
		NOTE: Epithelial cells will clump together, and it is not advisable to digest/trypsinize epithelial cells into single cell suspensions.</li>
		<li>Combine 1 part of stromal cells with 3 parts of epithelial cells by volume.</li>
		<li>Pipette 50 &#181;L (60,000 cells) of the combined cell suspension into the cell seeding chamber of the agarose mold.</li>
		<li>Once the agarose molds are filled with cells, carefully add 400 &#181;L of fresh organoid media into the well of the 24-well plate. 
		NOTE: The medium reaches just below the surface of the agarose dishes and will not cause the cells to float out of the molds. After 2&#8722;3 days, cells will settle and start to form organoids.</li>
		<li>Change medium every second day with 500 &#181;L of organoid media.</li>
		<li>After 2-3 days, change medium to one that is supplemented with 0.1 nM estradiol (E) and 0.8 nM testosterone (T) to promote organization of epithelial and stromal cells. 
		NOTE: Although organization of epithelial and stromal cells can occur with or without hormones, more organoids will organize in the presence of hormones.</li>
	</ol>
	</li>
</ol>
3. Harvesting Endometrial Organoids for Experiments
NOTE: After the experiment is done, endometrial organoids can be processed for histology or RNA analysis.The following steps describe how to process the organoids.
<ol>
	<li>Histology
	<ol>
		<li>Dissolve 1.5% agarose in phosphate-buffered saline (PBS) by boiling. Allow the liquid agarose to cool to approximately 50 &#176;C.</li>
		<li>Under a dissecting microscope, tilt the 24-well plate and carefully pipette out the medium from the outside of agarose mold. Carefully pipette out the medium from the interior of the agarose mold to avoid disrupting the organoids.</li>
		<li>Under a dissecting microscope, carefully pipette 70-75 &#181;L of warm (50 &#176;C) 1.5% agarose into the chamber of the agarose dish. Be careful not to disturb the organoids.</li>
		<li>Let cool at 4 &#176;C for 5 min.</li>
		<li>Add 4% paraformaldehyde (PFA) into each well of the 24 well plate and fix the entire sealed agarose mold containing organoids overnight at 4 &#176;C. Then store in 70% ethanol at 4 &#176;C until ready to process for paraffin embedding.</li>
	</ol>
	</li>
	<li>RNA isolation
	<ol>
		<li>Under a dissecting microscope, tilt the 24-well plate and carefully pipette out the medium from the chamber of the agarose mold.</li>
		<li>Forcefully pipette 1 mL of fresh organoid medium directly into the agarose mold so the organoids are flushed out of the microwells. Be careful not to create too many bubbles.</li>
		<li>Repeat the pipetting again with the same medium from step 3.2.2.</li>
		<li>Collect all the medium containing the organoids. Centrifuge at max speed to collect the organoids and proceed to RNA extraction.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Henriet, P., Gaide Chevronnay, H. P., Marbaix, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+endocrine+and+paracrine+control+of+menstruation.">The endocrine and paracrine control of menstruation.</a> Molecular and Cellular Endocrinology. 358 (2), 197-207 (2012).</li><li>Gellersen, B., Brosens, I. A., Brosens, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decidualization+of+the+human+endometrium:+mechanisms,+functions,+and+clinical+perspectives.">Decidualization of the human endometrium: mechanisms, functions, and clinical perspectives.</a> Seminars in Reproductive Medicine. 25 (6), 445-453 (2007).</li><li>Gellersen, B., Brosens, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclic+decidualization+of+the+human+endometrium+in+reproductive+health+and+failure.">Cyclic decidualization of the human endometrium in reproductive health and failure.</a> Endocrine Reviews. 35 (6), 851-905 (2014).</li><li>Li, Q., 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=The+antiproliferative+action+of+progesterone+in+uterine+epithelium+is+mediated+by+Hand2.">The antiproliferative action of progesterone in uterine epithelium is mediated by Hand2.</a> Science. 331 (6019), 912-916 (2011).</li><li>Wetendorf, M., DeMayo, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progesterone+receptor+regulates+implantation,+decidualization,+and+glandular+development+via+a+complex+paracrine+signaling+network.">The progesterone receptor regulates implantation, decidualization, and glandular development via a complex paracrine signaling network.</a> Molecular and Cellular Endocrinology. 357 (1-2), 108-118 (2012).</li><li>Boretto, M., 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=Development+of+organoids+from+mouse+and+human+endometrium+showing+endometrial+epithelium+physiology+and+long-term+expandability.">Development of organoids from mouse and human endometrium showing endometrial epithelium physiology and long-term expandability.</a> Development. 144 (10), 1775-1786 (2017).</li><li>Turco, M. Y., 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=Long-term,+hormone-responsive+organoid+cultures+of+human+endometrium+in+a+chemically+defined+medium.">Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium.</a> Nature Cell Biology. 19 (5), 568-577 (2017).</li><li>Kurita, T., 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+progesterone+receptors+mediate+the+inhibitory+effects+of+progesterone+on+estrogen-induced+uterine+epithelial+cell+deoxyribonucleic+acid+synthesis.">Stromal progesterone receptors mediate the inhibitory effects of progesterone on estrogen-induced uterine epithelial cell deoxyribonucleic acid synthesis.</a> Endocrinology. 139 (11), 4708-4713 (1998).</li><li>Kim, J. J., Kurita, T., Bulun, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progesterone+action+in+endometrial+cancer,+endometriosis,+uterine+fibroids,+and+breast+cancer.">Progesterone action in endometrial cancer, endometriosis, uterine fibroids, and breast cancer.</a> Endocrine Reviews. 34 (1), 130-162 (2013).</li><li>Bui, H. N., 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=Dynamics+of+serum+testosterone+during+the+menstrual+cycle+evaluated+by+daily+measurements+with+an+ID-LC-MS/MS+method+and+a+2nd+generation+automated+immunoassay.">Dynamics of serum testosterone during the menstrual cycle evaluated by daily measurements with an ID-LC-MS/MS method and a 2nd generation automated immunoassay.</a> Steroids. 78 (1), 96-101 (2013).</li><li>Wiwatpanit, T., Murphy, A. R., Lu, Z., Urbanek, M., Burdette, J. E., Woodruff, T. 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The authors have nothing to disclose.

We have generated human endometrial organoids comprised of epithelial and stromal cells of the endometrium without the use of exogenous scaffold materials. While it has already been shown that primary endometrial epithelial cells can form organoids6,7, these cells were embedded in a gelatinous matrix of proteins secreted by mouse sarcoma cells (see Table of Materials) to help form spheroid-like structures. In addition, endometrial stromal cells were absent. We speculate that the stromal cells in our system, which is an essential supportive component of the endometrial tissue, provided the scaffold for epithelial cells to adhere to and paracrine signaling occurred between cell types. The organization of the epithelial cells around the stromal cells indicated an active interaction between the two cell types which provided the physiologic cues. As hormonal response of the endometrium relies on paracrine interactions between the epithelial and stromal cells4,8,9, our multicellular organoids provide an alternate, more complex mimic of the native tissue.
The organoids that were generated here were mostly somatic cells, although a small percent of cells was stem-like. We have not yet passaged these organoids for multiple generations and do not know if they would survive and propagate. In addition, endometrial organoids are heterogeneous in nature in that not all organoids contained the same number of epithelial, stromal or stem cells, even those originating from the same tissue. Sizes of the organoids also differed and while most cells formed spheroid like structures, loose cells within each microwell were observed. The presence of E and T appeared to promote organoid formation with the specific organization of epithelial cells lining the outside and stromal cells in the center. We have not tested whether E or T alone would promote organoid formation and what the optimal length of treatment would be. Additional testing of parameters and conditions would be beneficial to generate the ideal endometrial organoids suited for the experiment planned.
Medium is an important component of culturing organoids for promoting growth and survival. From our experience in working with endometrial cells, growth of epithelial cells in culture is the most challenging in contrast to stromal cells which propagate well. Different media were tested, including the organoid media of choice (Table of Materials), which is used to generate mammospheres and tumorspheres of breast cancer, which are of epithelial cell origin. Our tests revealed that this medium permitted organoids to maintain their structural integrity and viability over the course of 14&#8722;28 day cultures. The effect of this medium on stromal cells, however, needs additional investigation. Despite their survival and production of collagen in the organoid media, the proliferation rate observed in 3D culture was much less than in monolayers. The decreased proliferation may however be due to the 3D structure as well. Given that the organoid media used is commercially available, medium components remain unclear due to its proprietary nature.
In future studies, we envision increasing the complexity of these endometrial organoids to incorporate other cell types found in the endometrium, including endothelial and immune cells. This is just the beginning of engineering a complete endometrial mimic that can be used to study biology, function, disease, and to test drugs.

The human endometrium lines the uterine cavity and serves as the first contact for the embryo during implantation. The endometrium is comprised of luminal and glandular epithelial cells, supportive stromal fibroblasts, endothelial cells and immune cells. Together, these cell types make up the endometrial tissue which is one of the most responsive tissues to sex steroid hormones1. The changes that occur during each menstrual cycle are striking. Appropriate growth and remodeling of the endometrium is required to allow for embryo implantation to occur. Aberrant response to estrogen and progesterone can result in a refractory endometrium that does not allow for successful establishment of pregnancy and can even result in diseases including endometrial neoplasia.
In order to study the hormone responses and essential changes that occur in the endometrium, cells from endometrial tissues excised from patients during surgery or endometrial biopsy have been propagated in cell culture. Endometrial stromal cells preferentially proliferate and propagate readily, and the process of differentiation induced by progesterone can be recapitulated in vitro. As a result, much has been learned during this differentiation process, termed decidualization2,3. The other major cell type in the endometrium, the luminal and glandular epithelial cells, however, do not grow well as traditional monolayers, losing polarity, becoming senescent, and having limited proliferative potential. As a result, less is known of their biology and their role in the human endometrium. As many neoplasia originate from the epithelial cells, mechanisms associated with hyperplasia or transformation to cancer cells remain to be fully defined. Furthermore, studies have established that hormone response involves the intimate paracrine actions between the epithelial and stromal cells of the endometrium4,5.
Recently, a three-dimensional (3D) organoid culture of endometrial epithelial cells was established by two independent groups6,7, which are the first reports of organoids formed from endometrial tissue. These organoids were comprised of endometrial epithelial cells embedded within a protein matrix (Table of Materials) and did not include an important hormonally responsive compartment of the endometrial endometrium, the stromal fibroblasts. As the matrix proteins can vary from lot to lot and can trigger signaling pathways that do not necessarily occur in the tissue, it would be ideal to replace the matrix proteins with components of the endometrium. In the current study, a protocol to generate scaffold-free human endometrial organoids of epithelial and stromal cells of the human endometrium is presented. The presence of stromal cells not only provides the support for epithelial cells but also provides the necessary paracrine actions that have been established to be important for endometrial hormone response4,8,9.
The new multicellular endometrial organoid offers a model system of the endometrium that is simple to generate and that incorporates both epithelial and stromal cells. These organoids can be used to study long-term hormonal changes and early events of disease such as tumorigenesis due to hormonal imbalance or exogenous insults. The complexity of these organoids could eventually be expanded to include other cell types, including endothelial and immune cells with possibly myometrial cells to truly mimic human tissue physiology.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:981px;" width="981">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Agarose HS, molecular biology grade</td>
			<td style="width:229px;">Denville Scientific</td>
			<td style="width:112px;">CA3510-6</td>
			<td style="width:320px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose molds</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td>Z764043</td>
			<td>(https://www.microtissues.com/)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Ammonium chloride (NH4Cl)</td>
			<td style="width:229px;">Amresco</td>
			<td style="width:112px;">0621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">&#946;-Estradiol</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">E2257</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Collagenase, Type II, powder</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td style="width:112px;">17101015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Dispase</td>
			<td style="width:229px;">Corning</td>
			<td>354235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">DNase I</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td>D4513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">EDTA</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:112px;">BP120-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Eosin Stain</td>
			<td style="width:229px;">VWR</td>
			<td style="width:112px;">95057-848</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Estrogen Receptor (SP1), rabbit monoclonal antibody</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td>RM-9101-S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Fluoroshield with DAPI, histology mounting medium</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">F6057</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hank&#39;s Balanced Salt Solution (HBSS)</td>
			<td style="width:229px;">Corning</td>
			<td style="width:112px;">21-022-CV</td>
			<td>1x without calcium, magnesium, and phenol red</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hematoxylin Stain Solution</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td style="width:112px;">3530-32</td>
			<td>Modified Harris formulation, mercury free</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Heparin solution</td>
			<td style="width:229px;">STEMCELL Technologies</td>
			<td style="width:112px;">07980</td>
			<td>added to MammoCult media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Hydrocortisone stock solution</td>
			<td style="width:229px;">STEMCELL Technologies</td>
			<td style="width:112px;">07925</td>
			<td>added to MammoCult media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Organoid media - Mammocult</td>
			<td style="width:229px;">STEMCELL Technologies</td>
			<td style="width:112px;">05620</td>
			<td>supplemented with 2 &#181;L/mL heparin and 5 &#181;L/mL hydrocortisone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Paraformaldehyde, 16% solution</td>
			<td style="width:229px;">Electron Microscopy Sciences</td>
			<td style="width:112px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Penicillin-Streptomycin</td>
			<td style="width:229px;">Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Phosphate buffered saline, pH 7.4</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">P3813</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Progesterone Receptor, PgR 1294, unconjugated, culture supernatant</td>
			<td style="width:229px;">Agilent Technologies</td>
			<td>M356801-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">protein matrix - Matrigel</td>
			<td style="width:229px;">BD Biosciences</td>
			<td style="width:112px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Purified mouse anti-E-cadherin antibody</td>
			<td style="width:229px;">BD Biociences</td>
			<td style="width:112px;">610181</td>
			<td>Clone 36</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:320px;">Recombinant anti-vimentin antibody [EPR3776]</td>
			<td style="width:229px;">Abcam</td>
			<td style="width:112px;">ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNA lysis and isolation kit</td>
			<td style="width:229px;">Zymo Research</td>
			<td>R2060</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:320px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td style="width:112px;">S6014</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Testosterone</td>
			<td style="width:229px;">Sigma-Aldrich</td>
			<td>86500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Trichrome Stain</td>
			<td style="width:229px;">Abcam</td>
			<td style="width:112px;">ab150686</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:320px;">Wax film - Parafilm</td>
			<td style="width:229px;">VWR</td>
			<td>52858-000</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of multicellular human primary endometrial organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A schematic of the protocol is depicted in Figure 1. Uterine tissue was obtained from surgery after it was examined by pathologists. The endometrial lining was separated from the myometrium by scraping and the endometrial tissue was enzymatically digested to cells as outlined in the protocol. Epithelial and stromal cells were added into microwells in the agarose molds. After 7 days in culture, organoids were treated with E and T for an additional 7&#8722;14 days.
For histological processing, the agarose molds containing endometrial organoids were sealed with agarose followed by fixation with 4% PFA overnight. These molds were processed for standard paraffin embedding, sectioned and stained for histology, immunofluorescence or immunohistochemistry. The hematoxylin and eosin (H&#38;E) staining (Figure 2A) revealed a spheroid-like structure with a single layer of cells lining the outside and cells in the center. Cell-specific markers for endometrial epithelial (E-cadherin) and stromal cells (vimentin) revealed epithelial cells surrounding the organoid with stromal cells in the center (Figure 2B).
Markers of endometrial physiology confirmed that the endometrial organoids retained certain characteristics of the native tissue. Trichrome staining, which stains collagen blue and cells red, showed the presence of collagen within the center where stromal cells resided, demonstrating active production and secretion of collagen by stromal cells similar to the native tissue (Figure 3A). In addition, immunohistochemical staining revealed the presence of estrogen receptors (ER), androgen receptors (AR), and progesterone receptors (PR) in both the epithelial and stromal cells (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60384/60384fig1v2.jpg" /> 
Figure 1: Generation of scaffold-free 3D endometrial organoids from human primary endometrial cells. Uterine tissues were obtained from premenopausal endometrial tissues with benign pathology and the endometrial lining was excised from the tissue piece. Upon enzymatic digestion, endometrial epithelial and stromal cells were seeded into 1.5% agarose molds at a 3:1 ratio by volume and maintained in sex hormone-free medium for up to 7 days. Estradiol (E) and testosterone (T) were added to the 3D cultures to mimic the levels of E and T during the follicular phase of a menstrual cycle10 and to promote organization of the organoids.&#160;Adapted from Teerawat et al.,&#160;JCEM, (2019)11.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60384/60384fig2.jpg" /> 
Figure 2: Histology and immunofluorescent staining of cell specific markers on endometrial organoids.&#160;Organoids were observed after 14 days of stepwise hormone treatment mimicking the follicular phase of a normal menstrual cycle (0.1 nM E and 0.8 nM T for 7 days, 1 nM E and 0.8 nM T for an additional 6 days, followed by 1 nM E and 1.25 nM T for 1 day). (A) H&#38;E staining was done on paraffin embedded organoids. (B) Cell specific markers were assessed by immunofluorescent staining of E-cadherin (epithelial, red), vimentin (stromal, green) and 4&#8242;,6-diamidino-2-phenylindole (DAPI; nucleus, blue) which revealed structural organization of the cells in the organoids. Scale bar = 20 &#181;m. Adapted from Teerawat et al., JCEM,&#160;(2019)&#60;11.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60384/60384fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60384/60384fig3.jpg" /> 
Figure 3: Endometrial organoids exhibit characteristics of native tissue after 14 days of follicular phase hormone treatment. (A) Trichrome staining was done to visualize collagen (blue) and cells (red). Trichrome stain was performed on endometrial tissue (left panel) and endometrial organoids (Scale bars = 20 &#181;m, right panel). (B) Immunohistochemical staining for ER, AR and PR was done in endometrial tissue (Scale bar = 100 &#181;m bottom panels) and endometrial organoids (Scale bar = 20 &#181;m, top panels). Positive stain is shown in brown and hematoxylin stain solution was added as the counter stain shown in blue.&#160;Adapted from Teerawat et al.,&#160;JCEM,&#160;(2019)11.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60384/60384fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation of Multicellular Human Primary Endometrial Organoids at JoVE.com

Generation of Multicellular Human Primary Endometrial Organoids

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

10.8K Views.  Northwestern University. A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

Video: Generation of Multicellular Human Primary Endometrial Organoids

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that limits the translation of basic brain research into clinical application. While induced pluripotent stem cells (iPSCs) have replaced the ethically questionable human embryonic stem cells, iPSC-based neuronal differentiation studies remain descriptive at the cellular level but fail to adequately provide the details that could be derived from a complex, 3D human brain tissue. 
This gap is now filled through the application of iPSC-derived, 3D brain organoids, "Brains in a dish," that model many features of complex human brain development. Here, a method for generating iPSC-derived, 3D brain organoids is described. The organoids can help with modeling autosomal recessive primary microcephaly (MCPH), a rare human neurodevelopmental disorder. A widely accepted explanation for the brain malformation in MCPH is a depletion of the neural stem cell pool during the early stages of human brain development, a developmental defect that is difficult to recreate or prove in vitro. 
To study MCPH, we generated iPSCs from patient-derived fibroblasts carrying a mutation in the centrosomal protein CPAP. By analyzing the ventricular zone of microcephaly 3D brain organoids, we showed the premature differentiation of neural progenitors. These 3D brain organoids are a powerful in vitro system that will be instrumental in modeling congenital brain disorders induced by neurotoxic chemicals, neurotrophic viral infections, or inherited genetic mutations.

Modeling human brain development has been hindered due to the unprecedented complexity of neural epithelial tissue. Here, a method for the robust generation of brain organoids to delineate early events of human brain development and to model microcephaly in vitro is described.

The restricted availability of suitable in vitro models that can reliably represent complex human brain development is a significant bottleneck that ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

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 ...

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

This protocol describes the production of a mouse extrahepatic bile duct 3-dimensional organoid system. These biliary organoids can be maintained in culture to study cholangiocyte biology. Biliary organoids express markers of both progenitor and biliary cells and are composed of polarized epithelial cells.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have ...

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed in order to create testicular organoids. Many of these methods rely upon extracellular matrix (ECM) to promote de novo tissue assembly, however, there are differences between methods in terms of biomimetic morphology and function of tissues. Moreover, there are few direct comparisons of published methods. Here, a direct comparison is made by studying differences in organoid generation protocols, with provided outcomes. Four archetypal generation methods: (1) 2D ECM-free, (2) 2D ECM, (3) 3D ECM-free, and (4) 3D ECM culture are described. Three primary benchmarks were used to assess the testicular organoid generation. These are cellular self-assembly, inclusion of major cell types (Sertoli, Leydig, germ, and peritubular cells), and appropriately compartmentalized tissue architecture. Of the four environments tested, 2D ECM and 3D ECM-free cultures generated organoids with internal morphologies most similar to native testes, including the de novo compartmentalization of tubular versus interstitial cell types, the development of tubule-like-structures, and an established long-term endocrine function. All methods studied utilized unsorted, primary murine testicular cell suspensions and used commonly accessible culture resources. These testicular organoid generation techniques provide a highly accessible and reproducible toolkit for research initiatives into testicular organogenesis and physiology in vitro.

Here, four methods for generating testicular organoids from primary neonatal murine testicular cells are described i.e., extracellular matrix (ECM) and ECM-free 2D and 3D culture environments. These techniques have multiple research applications and are especially useful for studying testicular development and physiology in vitro.

Testicular organoids provide a tool for studying testicular development, spermatogenesis, and endocrinology in vitro. Several methods have been developed ...

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is powered by local progenitor cells and renders taste function prone to disruption by a multitude of medical treatments, which in turn severely impacts the quality of life. Thus, studying this process in the context of drug treatment is vital to understanding if and how taste progenitor function and TRC production are affected. Given the ethical concerns and limited availability of human taste tissue, mouse models, which have a taste system similar to humans, are commonly used. Compared to in vivo methods, which are time-consuming, expensive, and not amenable to high throughput studies, murine lingual organoids can enable experiments to be run rapidly with many replicates and fewer mice. Here, previously published protocols have been adapted and a standardized method for generating taste organoids from taste progenitor cells isolated from the circumvallate papilla (CVP) of adult mice is presented. Taste progenitor cells in the CVP express LGR5 and can be isolated via EGFP fluorescence-activated cell sorting (FACS) from mice carrying an Lgr5EGFP-IRES-CreERT2 allele. Sorted cells are plated onto a matrix gel-based 3D culture system and cultured for 12 days. Organoids expand for the first 6 days of the culture period via proliferation and then enter a differentiation phase, during which they generate all three taste cell types along with non-taste epithelial cells. Organoids can be harvested upon maturation at day 12 or at any time during the growth process for RNA expression and immunohistochemical analysis. Standardizing culture methods for production of lingual organoids from adult stem cells will improve reproducibility and advance lingual organoids as a powerful drug screening tool in the fight to help patients experiencing taste dysfunction.

The protocol presents a method for culturing and processing lingual organoids derived from taste stem cells isolated from the posterior taste papilla of adult mice.

The sense of taste is mediated by taste buds on the tongue, which are composed of rapidly renewing taste receptor cells (TRCs). This continual turnover is ...

Generation of Multicellular Human Primary Endometrial Organoids

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Chapters in this video

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Generation of iPSC-derived Human Brain Organoids to Model Early Neurodevelopmental Disorders

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

Generation of Organoids from Mouse Extrahepatic Bile Ducts

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Extra Cellular Matrix-Based and Extra Cellular Matrix-Free Generation of Murine Testicular Organoids

Generation and Culture of Lingual Organoids Derived from Adult Mouse Taste Stem Cells