human primary ovarian surface epithelial cell isolation for obtaining organoids for in vitro tissue studies

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

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.

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

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

Our research focuses on understanding the ovarian function in humans. Due to the still present knowledge gap in female reproductive health, as well as the increasing need for new technology to treating fertility, we aim to study ovarian processes, such as folliculogenesis, or tissue regeneration in order to promote the development of new clinical protocols. In recent years, we have witnessed great advances in 3D tissue culture protocols.These developments have enabled the formation of, for instance, artificial ovaries, which are follicle like structures, capable of supporting the maturation of an all site full image. Additionally, we are also seeing how organoids or tumoroids, which are miniaturized version of an organ or a tissue, we are seeing how they are paving the way for further studies on ovarian biology and diseases. Access to ovarian samples coming from donors in reproductive age still represents a big challenge.On top of that, results obtain when using this material show a high inter donor viability, most likely due to factors such as differences in donor's age, metabolic state, and endocrine function. Altogether, this heterogeneity makes it difficult to reproduce results and to translate the protocols into the clinic. Despite that the ovaries stand out as one of the most dynamic and changing organs within the body it's regenerative mechanisms are still very poorly understood.And actually, most of the knowledge obtained so far still comes from animal models. With our OSC organoid protocol, we provide a platform to study the role of the human OSC layer in ovarian tissue regeneration and wound healing. Compared to others our protocol has been designed based on the use of non-pathological samples.This allow its application in developmental studies that are trying to find answers to fundamental mechanistic questions about the ovarian function in humans.

Human Primary Ovarian Surface Epithelial Cell Isolation for Obtaining Organoids for In Vitro Tissue Studies

To begin, transport human ovaries to the lab in 0.9%sodium chloride, or similar sterile saline solution on ice. Rinse ovaries with fresh, cold saline solution in a Petri dish. Transfer each ovary to a 50 milliliter conical tube, and add two to three milliliters of digestion medium to cover the ovary.Place the tube in a bead bath, tempered at 37 degrees Celsius for 30 minutes. Then carefully transfer the ovary into a 60-millimeter Petri dish containing 10 milliliters of collection medium. Using a cell scraper, scuff the ovarian surface containing the human ovarian surface epithelial cells, gently.Wash the scraper in the medium before scraping at least two more times. Transfer the human ovarian surface epithelial cells in the collection medium into a 15-milliliter tube. Spin down the cells at 240G for five minutes.If the pellet shows a red color indicative of contamination with red blood cells, resuspend the pellet in one milliliter of red blood cell lysis buffer. Then, add four milliliters of PBS with calcium and magnesium, and centrifuge at 240G for five minutes. Cryo-preserve the human ovarian surface epithelial cell pellet for future culture use.

human-primary-ovarian-surface-epithelial-cell-isolation-for-obtaining

Research

JoVE Journal

Developmental Biology

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.

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 ...

Developing Human Ovarian Epithelium Monolayer and Organoids for Studying Regeneration and Drug Effects

To begin, thaw cryopreserved human ovarian surface epithelial cells. Mix the cells with 10 milliliters of washing medium. After centrifuging the cells, resuspend pellet in two milliliters of ovarian surface epithelium or OSE 2D medium and transfer it into two wells of a 12-well plate.Add one milliliter of extra OSE 2D medium into the well and culture at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 72 hours before the first media renewal. Employ Trypan Blue staining to count freshly isolated or cryopreserved thawed live human ovarian surface epithelial cells with an automated cell counter. Mix the desired number of cells in one milliliter of ice cold OSE organoid basic medium, and centrifuge the tube at 240 G for five minutes.Using a pipette, resuspend the cell pellet in ice cold undiluted basement membrane extract solution to obtain 10, 000 cells per 10 microliters. Add 10 microliter droplets of OSE basement membrane extract solution per well in a pre-warmed multi-well chambered slide and place the plate upside down at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 15 minutes. After the gel solidifies, add 100 microliters of OSE 3D medium and culture at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide.After removing the spent medium, add 100 microliters of ice cold advanced DMEM F12 to each well. Scrape the bottom of the wells with a P1000 pipette tip to detach the gel droplets and transfer each floating gel droplet to a 1.5 milliliter tube. Centrifuge the tube at 240 G for five minutes.After discarding the supernatant, resuspend the pellet in 300 microliters of cell dissociation buffer and place the tubes in a pre-warmed bead bath at 37 degrees Celsius for 5 to 10 minutes. Next, add 300 microliters of human OSE organoid basic medium, and centrifuge at 240 G for five minutes. Finally, resuspend the pellet in the desired volume of undiluted basement membrane extract solution to reseed them at a suitable ratio for culturing.Primary human OSE cells exhibited cobblestone-like morphology up to passage three, but showed signs of senescence thereafter. Human OSE organoids from freshly isolated OSE cells grew larger than those from 2D expanded OSE cells after 14 days in culture. Various morphologies were observed in 3D OSE organoids, including cystic, monolayer, multilayered, and non-lumenized cell clusters.