Name: generation of multicellular human primary endometrial organoids
Uploaded: 2019-10-04T15:00:00
Description: Watch this Scientific Journal Video about Generation of Multicellular Human Primary Endometrial Organoids at JoVE.com

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 acc...

<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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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 w...

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;

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			<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>
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		<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.
...

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 ...

The endometrium is a tissue that changes every month in response to hormones. Hormone imbalance can prevent embryo attachment and also cause diseases like cancer. Our protocol rebuilds the endometrium so that it can be studied outside of the woman's body in a physiological way.The main advantage of this technique is that it provides a 3D structure of cells comprised of two hormonally responsive cell types, the epithelial and stroma cells that specifically organize and behave like they do in the body. Since the organoids can better mimic tissue behavior, they can eventually be used to test different drugs. The endometrial organoids can be used to study direct effects of currently known risk factors for cancer, including obesity and polycystic ovarian syndrome.Our organoids emphasize the importance of paracrine actions between two cell types. It's important to start with an adequate amount of tissue. Getting the right density of epithelial and stromal cells can be tricky.Also, the organoids are quite small and can be difficult to visualize with IHC staining. Visualization of this procedure is important because there are some steps that will be much easier to understand after seeing them. These include getting the right tissue layer of the uterus, getting the right density of cells to combine, and seeding the agarose molds.Before beginning cell isolation, cast and equilibrate 1.5%agarose micro-molds according to the manufacturer's instructions to house the organoids. In a biosafety cabinet, use aseptic techniques to prepare an enzyme solution at 37 degrees Celsius and sterile filter the solution using a 0.2 micrometer syringe filter into a 15-milliliter conical tube. Using a scalpel, scrape off endometrium from the uterine biopsies and mince the tissue into very small pieces.Place the freshly minced tissue into the 15-milliliter conical tube containing the enzyme solution. Close the cap and wrap the top of the tube with a wax film to prevent contamination. Place the tube into a water bath or incubator at 37 degrees Celsius for 30 minutes with gently shaking.After this, stack a 100-micron cell strainer on top of a 20-micron cell strainer on a 50-milliliter conical tube. Filter the solution through the two strainers. Rinse the 15-milliliter conical tube with HBSS and put this wash through the strainer to ensure that all cells are collected.Then, invert the 20-micron cell strainer onto a new 50-milliliter conical tube and wash the epithelial cells off the strainer with 20 milliliters of organoid media supplemented with 1%penicillin and streptomycin. Centrifuge the conical tubes at 500 G for five minutes. For the collected stromal cells, remove the supernatant and resuspend the pellet in 10 milliliters of red blood cell lysis buffer.Incubate at 37 degrees Celsius for 10 to 15 minutes. Centrifuge these cells at 500 G for five minutes. Remove the supernatant and resuspend the pellet with 200 to 300 microliters of organoid media.For collected epithelial cells, remove the supernatant and resuspend the pellet in 100 microliters of organoid media. After the 1.5%agarose molds are equilibrated, remove the organoid media on the outside of the agarose molds and tilt the tissue culture plate so that the medium from cell seeding chamber of the agarose dishes can also be carefully removed. View the epithelial and stromal cell suspensions under a microscope and add more organoid media to the suspensions, making sure to add only 100 microliters at a time so that the suspension are roughly equal in density.Then combine one part of the stromal cells with three parts of the epithelial cells by volume. Pipette 50 microliters of the combined cell suspension into the cell seeding chamber of the agarose mold. Once the agarose molds are filled with cells, carefully add 400 microliters of fresh organoid media into the wells of the 24-well plate.Incubate at 37 degrees Celsius with 5%carbon dioxide, making sure to change the medium every second day with 500 microliters of organoid media. After seven days, change the medium to one that is supplemented with 0.1 nanomolar estradiol and 0.8 nanomolar testosterone to promote organization of the epithelial and stromal cells. First, dissolve 1.5%agarose in PBS by boiling.Place the liquid agarose into a beaker of hot water and allow it to cool to approximately 50 degrees Celsius. Under a dissecting microscope, tilt the 24-well plate and carefully pipette out the medium from the outside of the agarose mold. Then carefully pipette out the medium from the interior of the agarose mold to avoid disrupting the organoids.Carefully add between 70 and 75 microliters of warm, 1.5%agarose into the chamber of the agarose dish, being careful not to disturb the organoids. Let the plate cool at four degrees Celsius for five minutes. After this, add 4%paraformaldehyde into each well of the 24-well plate and fix the entire sealed agarose mold overnight at four degrees Celsius.The next day, store the plate in 70%ethanol at four degrees Celsius until ready to process for paraffin embedding. To begin RNA isolation, view the plate under a dissecting microscope. Tilt the plate and carefully pipette out the medium from the chamber of the agarose mold.Forcefully pipette one milliliter of fresh organoid medium directly into the agarose mold so that the organoids are flushed out of the microwells, being careful not to create too many bubbles. Repeat this forceful pipetting process once using the same medium. After this, collect all of the medium containing the organoids.Centrifuge at 500 G to collect the organoids, and proceed to RNA extraction. In this study, human endometrial organoids comprised of epithelial and stromal cells of the endometrium are generated without the use of exogenous scaffold materials. For histological processing, the agarose molds containing the endometrial organoids are sealed with agarose, followed by fixation with 4%paraformaldehyde overnight.These molds are processed for standard paraffin embedding sectioned and stained for histology, immunofluorescence, or immunohistochemistry. The hematoxylin in eosin staining reveals a spheroid-like structure with a single layer of cells lining the outside and cells in the center. Cell-specific markers for endometrial, epithelial, and stromal cells reveal epithelial cells surrounding the organoid with stromal cells in the center.Markers of endometrial physiology confirm that the endometrial organoids retained certain characteristics of the native tissue. Tri-chrome staining, which stains collagen blue and cells red, shows 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. In addition, immunohistochemical staining reveals the presence of estrogen receptors, androgen receptors, and progesterone receptors in both the epithelial and stromal cells.It's important to plate the cells densely so that we have enough organoids for downstream experiments. This will take some practice. Endometrial organoids can be cultured under different conditions and then harvested for experiments like immunohistochemistry or immunofluorescence staining to study protein expression or harvested for RNA to study gene expression.Our lab investigates how the endometrium responds to altered hormone levels, which can lead to endometrial hyperplasia and cancer. We can use these organoids for long-term treatments of hormones and add pathological conditions such as excess testosterone found in polycystic ovarian syndrome, or signals from adipocytes to study obesity-induced endometrial changes. Human tissues are considered to be biohazardous, and it will be important to take appropriate precautions.

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