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This work was funded by the National Institute of Child Health and Human Development (R01HD093726 to F.E.D. and T32HD094699 to S.S.D. and E.J.Z.), the National Cancer Institute (F31CA257300 to S.S.D.), and startup funds from the Department of Obstetrics and Gynecology (to F.E.D.). We want to acknowledge Dr. Candace Tingen and the laboratory of Dr. Teresa Woodruff, as well as Dr. Jennifer Rowley, for their foundational studies and optimization of methods to isolate primary mouse ovarian somatic cells and characterization of these cells.

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<li>Dipali, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Self-organizing+ovarian+somatic+organoids+preserve+cellular+heterogeneity+and+reveal+cellular+contributions+to+ovarian+aging%5BTitle%5D)+AND+%22Aging+Cell%22%5BJournal%5D)">Self-organizing ovarian somatic organoids preserve cellular heterogeneity and reveal cellular contributions to ovarian aging.</a> Aging Cell. 25 (1), e70333(2026).</li>
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<li>Converse, A., Zaniker, E. J., Amargant, F., Duncan, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recapitulating+folliculogenesis+and+oogenesis+outside+the+body%3A+encapsulated+in+vitro+follicle+growth%5BTitle%5D)+AND+%22Biol+Reprod%22%5BJournal%5D)">Recapitulating folliculogenesis and oogenesis outside the body: encapsulated in vitro follicle growth.</a> Biol Reprod. 108 (1), 5-22 (2023).</li>
<li>Dean, M., Jin, V., Russo, A., Lantvit, D. D., Burdette, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exposure+of+the+extracellular+matrix+and+colonization+of+the+ovary+in+metastasis+of+fallopian-tube-derived+cancer%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Exposure of the extracellular matrix and colonization of the ovary in metastasis of fallopian-tube-derived cancer.</a> Carcinogenesis. 40 (1), 41-51 (2019).</li>
<li>Charalambous, C., Webster, A., Schuh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aneuploidy+in+mammalian+oocytes+and+the+impact+of+maternal+ageing%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">Aneuploidy in mammalian oocytes and the impact of maternal ageing.</a> Nat Rev Mol Cell Biol. 24 (1), 27-44 (2023).</li>
<li>Rowley, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low+molecular+weight+hyaluronan+induces+an+inflammatory+response+in+ovarian+stromal+cells+and+impairs+gamete+development+in+vitro%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Low molecular weight hyaluronan induces an inflammatory response in ovarian stromal cells and impairs gamete development in vitro.</a> Int J Mol Sci. 21 (3), 1036(2020).</li>
<li>Amargant, F., Vieira, C., Pritchard, M. T., Duncan, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Systemic+low-dose+anti-fibrotic+treatment+attenuates+ovarian+aging+in+the+mouse%5BTitle%5D)+AND+%22Geroscience%22%5BJournal%5D)">Systemic low-dose anti-fibrotic treatment attenuates ovarian aging in the mouse.</a> Geroscience. 47 (3), 3475-3495 (2024).</li>
</ol>

The authors have no conflicts of interest to disclose.

This paper describes methods to isolate primary mouse ovarian somatic cells from a stroma-enriched fraction of the ovary, in a manner that maintains the heterogeneity of the ovarian somatic compartment. Primary mouse ovarian somatic cells can be cultured in a 2D monolayer or can be utilized to generate ovarian somatic organoids using scaffold-free, agarose micromolds. Agarose micromolds contain 96 microwells for organoid formation and fit in a single well of a 24-well plate, making this a high-throughput method to genera...

Although the oocyte, or female gamete, is essential for giving rise to the next generation, somatic cell populations in the ovary play critical roles in gamete development, steroid hormone production, and ovarian homeostasis. Oocytes, surrounded by somatic granulosa and theca cells, form the functional units of the ovary known as follicles. Granulosa cells are physically connected to the oocyte by transzonal projections and provide metabolic and nutrient support to the oocyte in addition to regulating meiotic arrest1,2. Moreover, in response to gonadotropins, granulosa and theca cells produce steroid hormones, including estrogen, progesterone, and androgens, which have effects beyond the reproductive system, impacting the cardiovascular, musculoskeletal, and nervous systems, and thereby affecting overall female health3. Ovarian follicles develop within a heterogeneous ovarian stroma composed of vasculature, immune cells, several populations of interstitial fibroblasts and mesenchymal cells, as well as a highly ordered extracellular matrix (ECM)4. This stromal microenvironment provides physical and chemical cues that impact follicle growth, ovulation, and luteinization4.
Several methods exist for isolating and culturing gametes and follicles in vitro, and these methods have advanced our understanding of oogenesis and folliculogenesis5. However, the somatic compartment of the ovary is relatively understudied, in part, due to the lack of robust in vitro models. Current models of the ovarian somatic compartment are largely restricted to a single cell type, such as fibroblasts, ovarian surface epithelial, endothelial, immune, or granulosa cells6,7,8,9,10,11. The culture of broadly defined primary ovarian somatic cells in 2D favors macrophage populations over time, resulting in a loss of cellular heterogeneity12. Moreover, traditional two-dimensional (2D) monolayer culture does not allow for the diverse cell-cell and cell-matrix interactions that are present in the ovary. Ovarian tissue explants maintain the cellular and structural complexity of the native tissue but vary in composition depending on where within the ovary they were derived13,14.
Organoids are three-dimensional (3D), multi-cellular, miniaturized versions of organs or tissues that recapitulate in vivo organization and functions15,16. Organoids can be generated from stem cells or primary cells isolated from normal or diseased tissue15. In the context of the female reproductive system, organoids have been generated to model the endometrium, fallopian tube, cervix, and placenta17. However, with respect to the ovary, until recently, organoid models have been limited to a single cell type, such as ovarian surface epithelial or cancer cells, or have been generated from induced pluripotent stem cells to resemble follicle structures rather than the ovarian stroma17,18. Recent studies have generated and characterized organoids using heterogeneous populations of somatic cells isolated from mouse, rhesus macaque, and human ovarian tissue19,20,21. These organoid models have enabled investigation of cellular mechanisms underlying ovarian aging, as well as the effects of phthalate exposure on ECM composition19,21.
Given the critical importance of somatic cells, both in the follicle and stroma, to ovarian function, we describe methods for isolating somatic cells from a stroma-enriched fraction of mouse ovaries and for culturing them in vitro using either traditional monolayer culture or in an organoid model. Using scaffold-free agarose micromolds, ovarian somatic cells form organoids within 1&#8211;3 days. Organoids preserve key ovarian cell populations, including fibroblasts, macrophages, and steroidogenic cells, and can be cultured for up to 3 weeks with high viability. Ovarian somatic organoids are conducive to transcriptomic and histologic assessments, analysis of conditioned media, co-culture with other cell types, and compound screening methods. Overall, these organoids are a robust model for in vitro interrogation and modulation of the ovarian somatic compartment and can be applied to the study of ovarian development, aging, physiology, and pathology.

<table><tbody><tr><td>0.2 &amp;mu;m, Polyethersulfone syringe filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>MIDSCI</td><td>PR-MCT15</td><td></td></tr><tr><td>100 mL glass beaker</td><td>Fisherbrand</td><td>FB100100</td><td></td></tr><tr><td>100 mm Petri dishes</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>15 mL conical tubes</td><td>ThermoFisher Scientific</td><td>339651&amp;nbsp;</td><td></td></tr><tr><td>24-well plates (TC-treated)</td><td>Corning</td><td>353047</td><td></td></tr><tr><td>28-gauge insulin needles</td><td>Exel International</td><td>26027</td><td></td></tr><tr><td>35 mm Petri dishes</td><td>Corning</td><td>351008</td><td></td></tr><tr><td>3&amp;beta;-HSD antibody</td><td>Cosmo Bio</td><td>K0607</td><td>1:1000</td></tr><tr><td>4% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>1574100</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>45 mm watch glass</td><td>Electron Microscopy Sciences</td><td>7054345</td><td></td></tr><tr><td>50 mL concial tubes</td><td>ThermoFisher Scientific</td><td>339652&amp;nbsp;</td><td></td></tr><tr><td>60 mL sterile syringe</td><td>Air-Tite</td><td>ML60</td><td></td></tr><tr><td>60 mm TC-treated plates</td><td>Corning</td><td>353037</td><td></td></tr><tr><td>6-well plates (TC-treated)</td><td>Corning</td><td>353046</td><td></td></tr><tr><td>Agarose</td><td>Hoefer</td><td>GR140-500</td><td>Gel strength &gt;1200 g/cm&lt;sup&gt;2&lt;/sup&gt;; Gel Temperature 36 &amp;deg;C</td></tr><tr><td>Autoclave sterilization pouch</td><td>Fisherbrand</td><td>01-812-55</td><td></td></tr><tr><td>Biotinylated Goat Anti-Mouse IgG</td><td>Vector Laboratories</td><td>BA-9200-1.5</td><td>1:200</td></tr><tr><td>Biotinylated Goat Anti-Rabbit IgG</td><td>Vector Laboratories</td><td>PK-6101</td><td>1:200</td></tr><tr><td>Biotinylated Goat Anti-Rat IgG</td><td>Vector Laboratories</td><td>BA-9400-1.5</td><td>1:100</td></tr><tr><td>Bluing reagent</td><td>Fisherbrand</td><td>23-245681</td><td></td></tr><tr><td>Centrifuge with swing bucket rotor for 50 mL conical tubes</td><td>ThermoFisher Scientific</td><td>Sorvall X1R Pro-MD</td><td></td></tr><tr><td>Class II biosafety cabinet</td><td>The Baker Company</td><td>SG403A</td><td></td></tr><tr><td>Cleaved Caspase-3 (CC3) antibody</td><td>Cell Signaling Technology</td><td>9579T</td><td>1:250</td></tr><tr><td>CO2 incubator</td><td>ThermoFisher Scientific</td><td>Heracell VIOS 160i CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td></td></tr><tr><td>Collagenase, Type IV, powder</td><td>Gibco, Fisher Scientific</td><td>17-104-019</td><td></td></tr><tr><td>C-Series Mouse Cytokine Antibody Array 3 Kit</td><td>RayBiotech</td><td>AAM-CYT-3-8</td><td></td></tr><tr><td>Cytoseal XYL</td><td>Epredia</td><td>83124</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas</td><td>Millipore Sigma</td><td>DN25-100MG</td><td>Dnase I</td></tr><tr><td>Dissecting microscissors</td><td>World Precision Instruments</td><td>WPI-14003</td><td></td></tr><tr><td>Dissecting scissors</td><td>World Precision Instruments</td><td>WPI-15922</td><td></td></tr><tr><td>Dissection microscope</td><td>Leica</td><td>MZ9.5, S9 series</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Gibco, Fisher Scientific</td><td>14-190-250</td><td>pH 7.0&amp;ndash;7.3</td></tr><tr><td>Eosin</td><td>Fisherbrand</td><td>23-314631</td><td></td></tr><tr><td>F4/80 antibody</td><td>Bio-Rad</td><td>MCA497G</td><td>1:50</td></tr><tr><td>Fetal Bovine Serum, certified, heat inactivated, United States</td><td>Gibco, ThermoFisher Scientific</td><td>10082147</td><td></td></tr><tr><td>Fine-tipped dissecting forceps</td><td>World Precision Instruments</td><td>WPI-14098</td><td></td></tr><tr><td>FOXL2 antibody</td><td>Abcam</td><td>ab246511</td><td>1:200</td></tr><tr><td>Graefe Forceps</td><td>Fine Science Tools</td><td>1105010</td><td></td></tr><tr><td>Hematoxylin</td><td>EK Industries</td><td>EKI 47971GL</td><td></td></tr><tr><td>Hemocytometer</td><td>MedOne</td><td>DHCN015</td><td></td></tr><tr><td>Imaging System</td><td>ThermoFisher Scientific</td><td>EVOS FL Auto</td><td></td></tr><tr><td>IntestiCult Organoid Growth Medium (Mouse)</td><td>STEMCELL Technologies</td><td>06005</td><td>Basal medium, Supplements 1 and 2</td></tr><tr><td>Laminar flow hood</td><td>IVFtech</td><td>IVFtech sterile workstation</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>Gibco, Fisher Scientific</td><td>11415114</td><td></td></tr><tr><td>Microscope slides</td><td>Mercedes Scientific</td><td>MER 7255/90/WH/CC</td><td></td></tr><tr><td>MicroTissues 3D Petri Dish micro-mold spheroids</td><td>Millipore Sigma</td><td>Z764043-6EA</td><td>Silicone casts for agarose micromolds</td></tr><tr><td>Modified Davidson&#039;s Fixative</td><td>Electron Microscopy Sciences</td><td>6413350</td><td></td></tr><tr><td>OVCAR8-RFP cells</td><td></td><td></td><td>Gift from Joanna Burdette PhD, University of Illinois Chicago</td></tr><tr><td>Penicillin-Streptomycin</td><td>Millipore Sigma</td><td>P4333-100ML</td><td></td></tr><tr><td>Picrosirius Red staining solution</td><td>StatLab</td><td>STPSRPT</td><td></td></tr><tr><td>RPMI 1640 Medium (ATCC modification)</td><td>Gibco, Fisher Scientific</td><td>A1049101</td><td></td></tr><tr><td>Tabletop centrifuge</td><td>Eppendorf</td><td>Centrifuge 5430 R&amp;nbsp;</td><td></td></tr><tr><td>Trypan blue</td><td>ThermoFisher Scientific</td><td>T10282</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco, ThermoFisher Scientific</td><td>25300054</td><td></td></tr><tr><td>Vimentin antibody</td><td>Cell Signaling Technology</td><td>5741S</td><td>1:100</td></tr><tr><td>&amp;alpha;MEM, GlutaMAX Supplement, no nucleosides</td><td>Gibco, Fisher Scientific</td><td>32561102</td><td></td></tr></tbody></table>

probing the ovarian microenvironment: methods for isolation, 2d culture, and organoid culture of mouse ovarian somatic cells

Probing the Ovarian Microenvironment: Methods for Isolation, 2D Culture, and Organoid Culture of Mouse Ovarian Somatic Cells

The ovary consists of heterogeneous populations of somatic cells, both within the follicle and the surrounding stroma, which are critical to support ovarian function and for the generation of high-quality gametes. We report methods for isolating somatic cells from mouse ovaries, including endothelial, epithelial, steroidogenic, stromal, and immune cells. When these primary ovarian somatic cells are plated and cultured in a traditional 2D culture system, the cellular heterogeneity, organization, as well as cell-cell and cell-matrix interactions typically found in the ovary are lost. Thus, we also describe how to generate mouse ovarian somatic organoids using a scaffold-free approach. These organoids self-assemble, maintain diverse cell populations, and produce extracellular matrix and secreted factors, including cytokines. Organoids can be utilized for co-culture experiments and can be maintained in culture for at least 3 weeks with high viability. Overall, these models enable interrogation of ovarian physiology and pathology from the somatic cell perspective.

To determine the cellular composition of ovarian somatic organoids, we performed immunohistochemistry of organoid tissue sections after 6 days of culture using antibodies against key ovarian cell types. Organoids contained vimentin-positive fibroblasts, F4/80-positive macrophages, as well as steroidogenic cell populations (Figure 6B)19. We utilized an antibody against Foxl2 to mark granulosa and granulosa-lutein cells, in addition to an antibody against 3β-HSD to mark...

We describe methods for isolating mouse ovarian somatic cells from a stroma-enriched fraction of the ovary and for generating mouse ovarian somatic organoids using a scaffold-free approach.

The ovary consists of heterogeneous populations of somatic cells, both within the follicle and the surrounding stroma, which are critical to support ...

probing-ovarian-microenvironment-methods-for-isolation-2d-culture

Research

JoVE Journal

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Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to quantify follicles in cultured ovaries of young mice without serial sectioning. Using whole organ immunofluorescence and tissue clearing, physical sectioning is replaced with optical sectioning. This method of sample preparation and visualization maintains organ integrity and facilitates automated quantification of specific cells.

Whole Mount Immunofluorescence and Follicle Quantification of Cultured Mouse Ovaries

Ovarian tissue cultures can be used as models of follicle development, ovulation, and follicle atresia and indicate regulatory mechanisms of dynamic ovarian processes.

Ovarian Tissue Culture to Visualize Phenomena in Mouse Ovary

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Bovine Ovarian Cortex Tissue Culture

This protocol offers a systematic framework for the establishment of ovarian cancer organoids from different disease stages and addresses the challenges of patient-specific variability to increase yield and enable robust long-term expansion for subsequent applications. It includes detailed steps for tissue processing, seeding, adjusting media requirements, and immunofluorescence staining.

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

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.

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

This protocol details the development of high-grade serous ovarian carcinoma (HGSOC) mouse models in vitro and in vivo. From the derivation of murine fallopian tube organoids, their genetic modification to recapitulate human HGSOC genetics, and their introduction in the ovarian bursa of syngeneic mice for in vivo development of tumors.

Development of Mouse-Derived Organoid Lines from Fallopian Tube Epithelial Cells for High Grade Serous Ovarian Carcinoma Modeling

We describe methods to culture ovarian follicles in a novel scaffold-free agarose mold that complement in vitro gametogenesis.

Production and Use of Customizable Agarose Molds for Scaffold-Free Mouse Ovarian Follicle Culture

Northwestern University, Chicago.  We describe methods for isolating mouse ovarian somatic cells from a stroma-enriched fraction of the ovary and for generating mouse ovarian somatic organoids using a scaffold-free approach.

Probing the Ovarian Microenvironment: Methods for Isolation, 2D Culture, and Organoid Culture of Mouse Ovarian Somatic Cells

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