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/pubmed?term=((The+endocrine+and+paracrine+control+of+menstruation%5BTitle%5D)+AND+%22Molecular+and+Cellular+Endocrinology%22%5BJournal%5D)">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/pubmed?term=((Decidualization+of+the+human+endometrium%3A+mechanisms%2C+functions%2C+and+clinical+perspectives%5BTitle%5D)+AND+%22Seminars+in+Reproductive+Medicine%22%5BJournal%5D)">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/pubmed?term=((Cyclic+decidualization+of+the+human+endometrium+in+reproductive+health+and+failure%5BTitle%5D)+AND+%22Endocrine+Reviews%22%5BJournal%5D)">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/pubmed?term=((The+antiproliferative+action+of+progesterone+in+uterine+epithelium+is+mediated+by+Hand2%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">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/pubmed?term=((The+progesterone+receptor+regulates+implantation%2C+decidualization%2C+and+glandular+development+via+a+complex+paracrine+signaling+network%5BTitle%5D)+AND+%22Molecular+and+Cellular+Endocrinology%22%5BJournal%5D)">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/pubmed?term=((Development+of+organoids+from+mouse+and+human+endometrium+showing+endometrial+epithelium+physiology+and+long-term+expandability%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">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/pubmed?term=((Long-term%2C+hormone-responsive+organoid+cultures+of+human+endometrium+in+a+chemically+defined+medium%5BTitle%5D)+AND+%22Nature+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Stromal+progesterone+receptors+mediate+the+inhibitory+effects+of+progesterone+on+estrogen-induced+uterine+epithelial+cell+deoxyribonucleic+acid+synthesis%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">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/pubmed?term=((Progesterone+action+in+endometrial+cancer%2C+endometriosis%2C+uterine+fibroids%2C+and+breast+cancer%5BTitle%5D)+AND+%22Endocrine+Reviews%22%5BJournal%5D)">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/pubmed?term=((Dynamics+of+serum+testosterone+during+the+menstrual+cycle+evaluated+by+daily+measurements+with+an+ID-LC-MS%2FMS+method+and+a+2nd+generation+automated+immunoassay%5BTitle%5D)+AND+%22Steroids%22%5BJournal%5D)">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. K., Kim, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaffold-free+endometrial+organoids+respond+to+excess+androgens+associated+with+polycystic+ovarian+syndrome%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Endocrinology+%26+Metabolism%22%5BJournal%5D)">Scaffold-free endometrial organoids respond to excess androgens associated with polycystic ovarian syndrome.</a> The Journal of Clinical Endocrinology & Metabolism. , (2019).</li>
</ol>

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 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;

<table><tbody><tr><td>Agarose HS, molecular biology grade</td><td>Denville Scientific</td><td>CA3510-6</td><td></td></tr><tr><td>Agarose molds</td><td>Sigma-Aldrich</td><td>Z764043</td><td>(https://www.microtissues.com/)</td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Amresco</td><td>0621</td><td></td></tr><tr><td>&amp;beta;-Estradiol</td><td>Sigma-Aldrich</td><td>E2257</td><td></td></tr><tr><td>Collagenase, Type II, powder</td><td>Thermo Fisher Scientific</td><td>17101015</td><td></td></tr><tr><td>Dispase</td><td>Corning</td><td>354235</td><td></td></tr><tr><td>DNase I</td><td>Sigma-Aldrich</td><td>D4513</td><td></td></tr><tr><td>EDTA</td><td>Fisher Scientific</td><td>BP120-1</td><td></td></tr><tr><td>Eosin Stain</td><td>VWR</td><td>95057-848</td><td></td></tr><tr><td>Estrogen Receptor (SP1), rabbit monoclonal antibody</td><td>Thermo Fisher Scientific</td><td>RM-9101-S</td><td></td></tr><tr><td>Fluoroshield with DAPI, histology mounting medium</td><td>Sigma-Aldrich</td><td>F6057</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution (HBSS)</td><td>Corning</td><td>21-022-CV</td><td>1x without calcium, magnesium, and phenol red</td></tr><tr><td>Hematoxylin Stain Solution</td><td>Thermo Fisher Scientific</td><td>3530-32</td><td>Modified Harris formulation, mercury free</td></tr><tr><td>Heparin solution</td><td>STEMCELL Technologies</td><td>07980</td><td>added to MammoCult media</td></tr><tr><td>Hydrocortisone stock solution</td><td>STEMCELL Technologies</td><td>07925</td><td>added to MammoCult media</td></tr><tr><td>Organoid media - Mammocult</td><td>STEMCELL Technologies</td><td>05620</td><td>supplemented with 2 &amp;micro;L/mL heparin and 5 &amp;micro;L/mL hydrocortisone</td></tr><tr><td>Paraformaldehyde, 16% solution</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td></td></tr><tr><td>Phosphate buffered saline, pH 7.4</td><td>Sigma-Aldrich</td><td>P3813</td><td></td></tr><tr><td>Progesterone Receptor, PgR 1294, unconjugated, culture supernatant</td><td>Agilent Technologies</td><td>M356801-2</td><td></td></tr><tr><td>protein matrix - Matrigel</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>Purified mouse anti-E-cadherin antibody</td><td>BD Biociences</td><td>610181</td><td>Clone 36</td></tr><tr><td>Recombinant anti-vimentin antibody [EPR3776]</td><td>Abcam</td><td>ab92547</td><td></td></tr><tr><td>RNA lysis and isolation kit</td><td>Zymo Research</td><td>R2060</td><td></td></tr><tr><td>Sodium bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S6014</td><td></td></tr><tr><td>Testosterone</td><td>Sigma-Aldrich</td><td>86500</td><td></td></tr><tr><td>Trichrome Stain</td><td>Abcam</td><td>ab150686</td><td></td></tr><tr><td>Wax film - Parafilm</td><td>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 ...

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Developmental Biology

10.9K 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

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.

This protocol discusses an approach for generating epithelial organoids from primary normal and tumor mammary tissue through differential centrifugation. Furthermore, instructions are included for three-dimensional culturing as well as immunofluorescent imaging of embedded organoids.

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Cancer Research

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 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 ...

Human Ovarian Epithelium Organoids: A Model for Tissue Repair and Drug Testing

Human Ovarian Surface Epithelium Organoids as a Platform to Study Tissue Regeneration

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound healing while restoring ovarian integrity. Additionally, the OSE may serve as the source of epithelial ovarian cancers. Although the OSE regenerative properties have been well studied in mice, understanding the precise mechanism of tissue repair in the human ovary remains hampered by limited access to human ovaries and suitable in vitro culture protocols. Tissue-specific organoids, miniaturized in vitro models replicating both structural and functional aspects of the original organ, offer new opportunities for studying organ physiology, disease modeling, and drug testing. 
Here, we describe a method to isolate primary human OSE (hOSE) from whole ovaries and establish hOSE organoids. We include a morphological and cellular characterization showing heterogeneity between donors. Additionally, we demonstrate the capacity of this culture method to evaluate hormonal effects on OSE-organoid growth over a 2-week period. This method may enable the discovery of factors contributing to OSE regeneration and facilitate patient-specific drug screenings for malignant OSE.

Author Spotlight: Advancing Human Ovarian Repair and Cancer Studies with Surface Epithelium Organoids

This protocol describes establishing three-dimensional (3D) tissue organoids from primary human ovarian surface epithelium (hOSE) cells. The protocol includes isolation of hOSE from freshly collected ovaries, cellular expansion of the hOSE, cryopreservation-thawing procedures, and organoid derivation. Immunofluorescence, quantitative analysis, and showcasing utility as a screening platform are included.

The ovarian surface epithelium (OSE), the outermost layer of the ovary, undergoes rupture during each ovulation and plays a crucial role in ovarian wound ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

Biology

Generation and Characterization of Murine Oral Mucosal Organoid Cultures

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, gingiva, lips, palate, and tongue. Its uppermost layer, the oral epithelium, is maintained by adult stem cells throughout life. Proliferation and differentiation of adult epithelial stem cells have been intensively studied using in vivo mouse models as well as two-dimensional (2D) feeder-cell based in vitro models. Complementary to these methods is organoid technology, where adult stem cells are embedded into an extracellular matrix (ECM)-rich hydrogel and provided with a culture medium containing a defined cocktail of growth factors. Under these conditions, adult stem cells proliferate and spontaneously form three-dimensional (3D) cell clusters, the so-called organoids. Organoid cultures were initially established from murine small intestinal epithelial stem cells. However, the method has since been adapted for other epithelial stem cell types. Here, we describe a protocol for the generation and characterization of murine oral mucosal organoid cultures. Primary epithelial cells are isolated from murine tongue tissue, embedded into an ECM hydrogel, and cultured in a medium containing: epidermal growth factor (EGF), R-spondin, and fibroblast growth factor (FGF) 10. Within 7 to 14 days of initial seeding, the resulting organoids can be passaged for further expansion and cryopreservation. We additionally present strategies for the characterization of established organoid cultures via 3D whole-mount imaging and gene-expression analysis. This protocol may serve as a tool to investigate oral epithelial stem cell behavior ex vivo in a reductionist manner.

We present a method for the generation and characterization of oral mucosal organoid cultures derived from the tongue epithelium of adult mice.

The mucous lining covering the inside of our mouth, the oral mucosa, is a highly compartmentalized tissue and can be subdivided into the buccal mucosa, ...

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 ...

Generation and Characterization of Rat Uterus Organoids from Rat Endometrial Epithelial Stem Cells

Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. While human and mouse endometrial organoids have been developed, research on rat endometrial organoids remains limited. Given that rats can better simulate certain endometrial pathologies, such as intrauterine adhesions, this study aimed to establish rat endometrial organoids. We present a detailed protocol for the isolation and culture of rat endometrial epithelial stem cells (reESCs) and the generation of rat endometrial organoids. Using a refined reESCs expansion medium, we successfully isolated and stably expanded reESCs, demonstrating their long-term culture potential. The reESC-generated organoids exhibited typical structural and functional characteristics of the endometrium, including hormone responsiveness. Our results showed that rat endometrial organoids could be cultured over a long term with stable proliferation, maintaining the glandular structure, cell polarity, and functional characteristics of the endometrial epithelium. This novel rat-derived endometrial organoid model provides a valuable platform for studying endometrial diseases and testing therapeutic interventions, with potential applications across various mammalian species.

Here, we present a protocol to isolate and culture rat endometrial epithelial stem cells (reESCs), generating rat endometrial organoids. This method facilitates in vitro studies of endometrial diseases, enabling gene editing and other cellular manipulations.

Endometrial organoids offer valuable insights into the development and pathophysiology of endometrial diseases and serve as platforms for drug testing. ...