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

Summary

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.

Abstract

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.

Introduction

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 arrest^1,2. Moreover, in response to gonadotropins, granulosa and theca cells produce steroid hormones,....

Protocol

The use of animals to develop this protocol was in accordance with the Institutional Animal Care and Use Committee at Northwestern University and the National Institutes of Health Guidelines for Care and Use of Laboratory Animals (Northwestern University Animal Welfare Assurance Number: A3283-01; IACUC Protocol ID: IS00013082_IM12). Manufacturer information and catalog numbers for all materials, including silicone casts for agarose micromolds, media, and supplements, are given in the Table of Materials. An overview of the protocol steps is presented in Figure 1. All protocol steps should be performed using appropriate per....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 μm, Polyethersulfone syringe filter	Corning	431229
1.5 mL microcentrifuge tubes	MIDSCI	PR-MCT15
100 mL glass beaker	Fisherbrand	FB100100
100 mm Petri dishes	Corning	351029
15 mL conical tubes	ThermoFisher Scientific	339651
24-well plates (TC-treated)	Corning	353047
28-gauge insulin needles	Exel International	26027
35 mm Petri dishes	Corning	351008
3β-HSD antibody	Cosmo Bio	K0607	1:1000
4% Paraformaldehyde	Electron Microscopy Sciences	1574100
40 µm cell strainer	Corning	431750
45 mm watch glass	Electron Microscopy Sciences	7054345
50 mL concial tubes	ThermoFisher Scientific	339652
60 mL sterile syringe	Air-Tite	ML60
60 mm TC-treated plates	Corning	353037
6-well plates (TC-treated)	Corning	353046
Agarose	Hoefer	GR140-500	Gel strength >1200 g/cm2; Gel Temperature 36 °C
Autoclave sterilization pouch	Fisherbrand	01-812-55
Biotinylated Goat Anti-Mouse IgG	Vector Laboratories	BA-9200-1.5	1:200
Biotinylated Goat Anti-Rabbit IgG	Vector Laboratories	PK-6101	1:200
Biotinylated Goat Anti-Rat IgG	Vector Laboratories	BA-9400-1.5	1:100
Bluing reagent	Fisherbrand	23-245681
Centrifuge with swing bucket rotor for 50 mL conical tubes	ThermoFisher Scientific	Sorvall X1R Pro-MD
Class II biosafety cabinet	The Baker Company	SG403A
Cleaved Caspase-3 (CC3) antibody	Cell Signaling Technology	9579T	1:250
CO2 incubator	ThermoFisher Scientific	Heracell VIOS 160i CO2 Incubator
Collagenase, Type IV, powder	Gibco, Fisher Scientific	17-104-019
C-Series Mouse Cytokine Antibody Array 3 Kit	RayBiotech	AAM-CYT-3-8
Cytoseal XYL	Epredia	83124
Deoxyribonuclease I from bovine pancreas	Millipore Sigma	DN25-100MG	Dnase I
Dissecting microscissors	World Precision Instruments	WPI-14003
Dissecting scissors	World Precision Instruments	WPI-15922
Dissection microscope	Leica	MZ9.5, S9 series
DPBS, no calcium, no magnesium	Gibco, Fisher Scientific	14-190-250	pH 7.0–7.3
Eosin	Fisherbrand	23-314631
F4/80 antibody	Bio-Rad	MCA497G	1:50
Fetal Bovine Serum, certified, heat inactivated, United States	Gibco, ThermoFisher Scientific	10082147
Fine-tipped dissecting forceps	World Precision Instruments	WPI-14098
FOXL2 antibody	Abcam	ab246511	1:200
Graefe Forceps	Fine Science Tools	1105010
Hematoxylin	EK Industries	EKI 47971GL
Hemocytometer	MedOne	DHCN015
Imaging System	ThermoFisher Scientific	EVOS FL Auto
IntestiCult Organoid Growth Medium (Mouse)	STEMCELL Technologies	06005	Basal medium, Supplements 1 and 2
Laminar flow hood	IVFtech	IVFtech sterile workstation
Leibovitz's L-15 Medium	Gibco, Fisher Scientific	11415114
Microscope slides	Mercedes Scientific	MER 7255/90/WH/CC
MicroTissues 3D Petri Dish micro-mold spheroids	Millipore Sigma	Z764043-6EA	Silicone casts for agarose micromolds
Modified Davidson's Fixative	Electron Microscopy Sciences	6413350
OVCAR8-RFP cells			Gift from Joanna Burdette PhD, University of Illinois Chicago
Penicillin-Streptomycin	Millipore Sigma	P4333-100ML
Picrosirius Red staining solution	StatLab	STPSRPT
RPMI 1640 Medium (ATCC modification)	Gibco, Fisher Scientific	A1049101
Tabletop centrifuge	Eppendorf	Centrifuge 5430 R
Trypan blue	ThermoFisher Scientific	T10282
Trypsin-EDTA (0.05%), phenol red	Gibco, ThermoFisher Scientific	25300054
Vimentin antibody	Cell Signaling Technology	5741S	1:100
αMEM, GlutaMAX Supplement, no nucleosides	Gibco, Fisher Scientific	32561102