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

ヒト卵巣上皮オルガノイド:組織修復および薬物試験のモデル

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.

著者スポットライト:表面上皮オルガノイドによるヒト卵巣修復およびがん研究の進歩

組織再生研究のプラットフォームとしてのヒト卵巣表面上皮オルガノイド

In vitro組織研究のためのオルガノイドを得るためのヒト初代卵巣表面上皮細胞の単離

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

Research

JoVE Journal

Developmental Biology

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

131 ビュー. まず、ヒトの卵巣を0.9%塩化ナトリウム、または氷上の同様の滅菌生理食塩水で実験室に輸送します。ペトリ皿で新鮮な冷たい生理食塩水で卵巣をすすぎます。各卵巣を50ミリリットルの円錐管に移し、卵巣を覆うために2〜3ミリリットルの消化培地を追加します。チューブをビーズバスに入れ、摂氏37度で30分間焼き戻します。次に、卵巣を...

ビデオ: In vitro組織研究のためのオルガノイドを得るためのヒト初代卵巣表面上皮細胞の単離

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.

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 ...