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 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.
The ovary is considered one of the most dynamic organs in the body, undergoing constant cycles of wound healing and remodeling throughout the reproductive lifespan of the individual. A main player involved in the regeneration of ovarian tissue after each ovulatory cycle is the ovarian surface epithelium (OSE)1. The OSE is a mesothelium-derived single layer containing flat, cuboidal, and columnar epithelial cells that cover the entire ovarian surface2. Prior to ovulation, the ovarian stromal tissue on the surface of the ovulatory follicle undergoes proteolytic disruption to allow the release of the cumulus-oocyte complex. The wounded area, known as the ovulatory stigma, is then repaired, with complete closure of the ovarian surface achieved in less than 72 hours in mice3. The highly efficient capacity of the OSE to proliferate and close the ovulatory wound highlights the putative existence of a resident stem cell population4. Due to the limited availability of human ovaries from donors of reproductive age, most of the knowledge on the mechanisms of OSE repair comes from animal models. However, species-specific features hamper the translation from animal-based ovarian research to humans5.
In vitro studies have predominantly used 2-dimensional (2D) cell culture of human OSE, whereby cells grew in a monolayer attached to the surface of a culture plate, due to its cost-effectiveness and easy culture6,7,8. Nonetheless, this approach has limitations replicating the complexity of the ovarian tissue dynamics9. In this regard, 3D cell culture platforms with a special focus on ovarian organoids have revolutionized ovarian research10. Tissue organoids are miniaturized in vitro representations of the organ they are derived from, exhibiting 3D self-organization capacity and mimicking key functions and structures of their in vivo counterparts11. This technology offers the possibility to shed light on fundamental questions regarding development, regeneration, and tissue repair in the human ovary10. In recent years, researchers have also applied knowledge on ovarian organoids for the generation of patient-specific ovarian cancer (OC) organoids for disease modeling and personalized medicine12,13,14.
Based on different methods used for the generation of mouse OSE organoids and fallopian tube (FT) organoids15,16 as well as human OSE organoids12 and FT organoids17, we describe here a protocol for the derivation of human OSE organoids from human ovaries with potential applications in OSE regeneration studies. This protocol efficiently isolates primary OSE cells from whole human ovaries and includes a step-by-step description of 2D cell expansion and 3D hOSE organoid generation. The hOSE organoids displayed (donor-specific) variability in morphology and growth, highlighting their utility for personalized studies. Additionally, this protocol includes hOSE organoid maintenance, passaging, and immunofluorescence within the same culture plate. Furthermore, it provides a description of the different morphology that hOSE organoids can adopt and characterizes changes in immunophenotype during culture. Lastly, it showcases utility by investigating the influence of environmental cues, such as ovarian hormones, on hOSE organoid formation and growth based on hOSE organoid number and size.
The application of hOSE organoid technology will enhance our understanding of the ovary, with a specific emphasis on the mechanisms responsible for its remarkable regenerative capacity. As 3D human ovarian models continue to evolve, the reliance on animal models in ovarian research will decrease, leading to innovative therapies in the field of regenerative medicine18.
3D organoid technology is emerging as an indispensable tool for medical research. On the one hand, this in vitro platform offers the possibility to study fundamental mechanistic questions about tissue regeneration, wound healing, and development18. On the other hand, 3D organoids derived from patient samples allow personalized medicine studies, including diagnostics, drug testing, and cell therapy12,13,14,37,38. In the field of ovarian research, the hOSE has gained substantial interest since its implication as the origin of epithelial ovarian carcinomas39. Although it is thought that most high-grade serous ovarian carcinoma (HGSOC), one of the most common epithelial ovarian cancers, arises from the fallopian tubes40, current research on mice 3D ovarian organoids has proposed a potential dual origin of HGSOC from OSE and fallopian tube15,16.
Here, we described a protocol for the derivation of hOSE 3D organoids and outlined its application to bring novel mechanistic knowledge in ovarian tissue regeneration. This protocol includes a step-by-step method to isolate primary hOSE cells from human ovaries and generate 3D hOSE organoids. To ensure efficient hOSE organoid derivation, it is crucial to minimize ovarian manipulation. Due to its location on the ovarian surface and monolayer organization, the hOSE is prone to damage and loss during oophorectomy and organ manipulation. For this reason, we have favored an enzymatic and scraping method applied to the whole ovary to isolate hOSE2,8. In the present protocol, a mild enzymatic treatment was applied to disrupt the hOSE intercellular connections, followed by gentle scraping of the ovarian surface.
Comparing 2D with 3D hOSE culture, it is important to note that despite the initial high proliferation rate of hOSE cells in 2D culture, their cellular characteristics altered due to EMT, suggesting that the applied 2D culture conditions are not suitable to maintain an epithelial morphology. By contrast, 3D hOSE organoids could be passaged at least 4 times without signs of senescence. The OSE_3D organoid culture media used was based on that used by Kopper and colleagues for the derivation of OC and healthy hOSE organoids12 and by Kessler and colleagues for the derivation of human FT organoids17. The main difference was the replacement of human Wnt3a and R-Spondin-1 conditioned media by commercially available recombinant proteins to facilitate reproducibility.
Immunofluorescence techniques typically involve removing the tissue sample from the culture plate and processing it for paraffin- or cryo-sectioning. When working with very small structures, the risk of losing them during sample processing is high. In this protocol, the derivation of hOSE organoids takes place in cell culture plates that allow for direct microscopy imaging without the need to remove the hOSE organoids from the BME matrix. Furthermore, the whole-mount immunofluorescence method used here, described by Rezanejad and colleagues for pancreatic ductal organoids41, enabled in situ observation of protein localization within morphologically intact organoids. We demonstrated that, when performing this immunofluorescence protocol on hOSE organoids derived in multi-well chambered slides, there is highly efficient antibody penetration with a very low background signal.
Although most of the hOSE organoids derived using this method lacked CDH1 expression, some CDH1+ hOSE organoids formed, reaching larger sizes compared to CDH1- hOSE organoids. The expression of CDH1 has been associated with neoplastic hOSE phenotypes2,35. The ovaries used for hOSE isolation were donated by healthy transmasculine donors of reproductive age (27.1 ± 5 years old). These donors were under testosterone treatment for a period of 38 ± 15 months prior to oophorectomy. We cannot discard the possibility that the CDH1+ hOSE cells on the ovarian surface could be attributed to the testosterone treatment. Although androgen treatment has been linked to ovarian changes, such as anovulation42, hyperplasia of the cortical area43, and increased cortical stiffness44, the general ovarian pathology remains benign while using testosterone45.
In summary, this protocol highlights the potential of generating hOSE 3D organoids to decode mechanistic questions about ovarian tissue regeneration. Importantly, this method could also be applied for the detection of malignant cells present in ovarian biopsies from patients at risk of cancer development. Collectively, this method supports potential applications of this innovative in vitro platform for both fundamental ovarian function studies and clinical applications for individualized medical treatments.
The authors have nothing to disclose.
We would like to thank all patients who donated tissue for this study, the members of the Chuva de Sousa Lopes group for useful discussions, and I. De Poorter for designing the cartoons used in Figure 1. This research was funded by the European Research Council, grant number ERC-CoG-2016- 725722 (OVOGROWTH) for J.S.D.V. and S.M.C.d.S.L.; and the Novo Nordisk Foundation (reNEW), grant number NNF21CC0073729 for J.S.D.V. and S.M.C.d.S.L.
0.05% Trypsin/EDTA | Invitrogen | 25200-056 | |
12-well Culture Plate | Corning | 3336 | Sterile |
15 mL tubes | Greiner | 188271 | Sterile |
28cm Cell Scraper | Greiner Bio-One | 541070 | |
50 mL tubes | Greiner | 227261 | Sterile |
60 mm Petri dish | Greiner Bio-One | 628160 | |
A83-01 | Stem Cell Technologies | 72024 | |
Advanced DMEM/F12 | Gibco | 12634-010 | |
B27 supplement (50x) | ThermoFisher Scientific | 17504-044 | |
Bead bath | M714 | ||
Bovine serum albumin (BSA) | Sigma Aldrich | 10735086001 | |
Cell Dissociation Buffer | ThermoFisher Scientific | 13151014 | |
Cryo-container "Mr. Frosty" | BD Falcom | 479-3200 | |
DMEM Medium | ThermoFisher Scientific | 41966-029 | |
Donkey anti-Goat IgG Alexa Fluor 647 | Invitrogen | A-21447 | |
Donkey anti-Mouse IgG Alexa Fluor 488 | Invitrogen | A-21202 | |
Donkey anti-Mouse IgG Alexa Fluor 647 | Invitrogen | A-31571 | |
Donkey anti-Rabbit IgG Alexa Fluor 488 | Invitrogen | A-21206 | |
Donkey anti-Rabbit IgG Alexa Fluor 594 | Invitrogen | A-21207 | |
Donkey anti-Sheep IgG Alexa Flour 647 | Invitrogen | A-21448 | |
Fetal Bovine Serum (FBS) | ThermoFisher Scientific | A4736401 | |
Follicle Stimulating Hormone (FSH) | Sigma Aldrich | F4021 | |
Forskolin | Peprotech | 6652995 | |
Glutamax (100x) | Gibco | 35050-038 | |
Goat anti-CDH2 (N/R-cadherin) | Santa Cruz | SC-1502 | Mesenchymal Cells; Wong et al 1999 (human)25 |
Goat anti-PODXL (podocalyxin of GP135) | R&D Systems | AF1658 | Apical Polarity; Bryant et al 2014 (canine)21 |
Goat anti-Rat IgG Alexa Fluor 555 | Invitrogen | A-21434 | |
hEGF | R&D Systems | 263-EG | |
HEPES | Gibco | 15630-056 | |
Hydrocortisone | Sigma Aldrich | H0888 | |
Insulin-Transferrin-Selenium-Ethanolamine (ITS-X; 100x) | ThermoFisher Scientific | 51500-056 | |
Liberase DH Research Grade | Sigma Aldrich | A4736401 | |
Luna-II cell counter | Logos Biosystems | L40001 | |
Matrigel | Sigma Aldrich | 354277 | |
McCoy’s 5A Medium | ThermoFisher Scientific | 26600-023 | |
Mouse anti-ITGB1 (integrin beta 1) | Santa Cruz | SC-53711 | Basolateral Polarity; Bryant et al 2014 (canine)21 |
Mouse anti-KRT8 (cytokeratin 8) | Santa Cruz | SC-101459 | OSE Cells; Kopper et al 2019 (human)12 |
Mouse anti-VIM (vimentin) | Abcam | AB0809 | Mesenchymal Cells; Abedini et al 2020 (mouse)19 |
Mycozap Plus-CL | Lonza | V2A-2011 | |
N-Acetyl-L-cysteine | Sigma Aldrich | A9165 | |
Nicotinamide | Sigma Aldrich | N0636-100G | |
OVITRELLE-Choriogonadotropin alfa (hCG) | Merk | G03GA08 | |
Progesterone (P4) | Sigma Aldrich | P8783 | |
Rabbit anti-ACTA2 (alpha smooth muscle actin) | Abcam | AB5694 | Mesenchymal Cells; Abedini et al 2020 (mouse)19 |
Rabbit anti-CDH1 (E-cadherin) | Cell Signaling | CST 3195S | Epithelial Cells; Wong et al 1999 (human)25 |
Rabbit anti-LGR5 | Abcam | AB75850 | OSE Progenitor Cells; Flesken-Nikitin et al 2013 (mouse)22 |
Rabbit anti-YAP | Cell Signaling | 14074S | Proliferative OSE; Wang et al 2022 (mouse)24 |
Rat anti-CD44 PE-conjugated | eBioscience | 12-0441-81 | OSE Progenitor Cells; Bowen et al 2009 (human)20 |
Recombinant Human Heregulinβ-1 | Peprotech | 100-03 | |
Recombinant Human Noggin | Peprotech | 120-10C | |
Recombinant Human Wnt3a | R&D Systems | 5036-WN-010 | |
Recombinant Rspondin-1 | Peprotech | 120-38 | |
Red blood cells lysis buffer | eBiosciences | 00-4333-57 | |
Revitacell Supplement (100x) | ThermoFisher Scientific | A26445-01 | |
RNAse free DNAse | Qiagen | 79254 | |
SB-431542 | Tocris Bioscience | 1624/10 | |
Sheep anti-COL1A1 (pro-collagen 1 alpha 1) | R&D Systems | AF6220 | Mesenchymal Cells; Hosper et al 2013 (human)23 |
Y-27632 | StemCell Technologies | 72304 | |
β-Estradiol (E2) | Sigma-Aldrich | E8875 | |
μ-Slide 18-well culture plate | Ibidi | 8181 | Sterile |
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