�����ƽ��

Figure 1. GyneCult™ Endometrial Organoid Medium Allows You to Model Endometrial Biology and the Role of Estrogen and Progesterone

(A) The human endometrium (pink) is a hormone-regulated tissue that responds to the rising and ebbing levels of two critical sex steroid hormones, estrogen (E2) and progesterone (P4). At the beginning of the menstrual cycle, around Day 5, E2 levels steadily rise, causing a deepening of endometrial glands and leading to the thickening of the endometrium in the proliferative phase. Ovulation occurs after the peak of E2 levels, around Day 14 of the menstrual cycle. Following ovulation, the corpus luteum, a temporary collection of cells that forms on the ovary, produces increasing amounts of P4 and moderate levels of E2, initiating the secretory phase and maintaining the endometrial lining. As the corpus luteum degenerates near Day 28, E2 and P4 levels decline, triggering endometrial cell apoptosis and menstruation, during which the functional layer of the endometrium is shed. (B) In vivo, endometrial glands arise from the myometrium and are composed of both a glandular and luminal epithelium that contain secretory and ciliated cell types—both of which can be captured by organoids generated by GyneCult™ Endometrial Organoid Medium (EOM). Depending on the phase of the menstrual cycle, ciliated cells range from 1 - 20% of endometrial cells, peaking on Days 12 - 14, at ovulation. Figures adapted from publications by Critchley et al., 2020 and Chumduri and Turco, 2021 licensed under .

Figure 2. Overview of the GyneCult™ Endometrial Organoid Medium Workflow for the Robust Generation of EOs

(A) Workflow showing the generation of endometrial organoids (EOs). Cells are seeded in Matrigel® domes at Day 0 at 4000 cells per 20 μL Matrigel®, with up to 3 domes per well in a 24-well non-tissue culture-treated plate. The domes are overlaid in a submerged culture with 500 μL of complete GyneCult™ Endometrial Organoid Medium (EOM) supplemented with 0.2 nM estrogen to facilitate passaging. Full-medium changes in each well were performed every 3 - 4 days, until Day 10 when cultures are ready for passage. (B) Representative images of EO morphologies throughout the GyneCult™ EOM workflow. Organoids with visible lumens are observable under the microscope by Day 7. By Days 10 - 12, cilia beating may be observed under high magnification brightfield microscopy. Further characterization of the organoids to identify secretory or ciliated cells via immunocytochemical (ICC) staining can be performed after Day 10. Scale bar = 500 μm.

Figure 3. GyneCult™ Endometrial Organoid Medium Enables Robust Expansion of EOs Across Donor Samples, Including Epithelial-Sparse Starting Material

(A) Brightfield images of endometrial organoid (EO) cultures from 3 individual donors grown in GyneCult™ Endometrial Organoid Medium supplemented with 0.2 nM estrogen. Cultures were imaged at Seeding (Passage 0, Day 0) and at Passages 1 through 5 (P1 - P5), corresponding to 8 - 10 days post-seeding for each passage. Donors 1 and 3 provided epithelial-rich samples that generated robust organoid cultures in P1. Donor 2, however, provided epithelial-sparse material, as indicated by the presence of elongated cells (stromal) and a lack of epithelial organoids seen in P1 prior to subculture. Scale bar = 500 μm. (B) Cumulative doublings were recorded at each passage as a measure of proliferative capacity. Cultures derived from Donor 2 expanded at comparable rates to epithelial-rich samples, and all cultures achieved at least 10 cumulative doublings over five passages. These results demonstrate that GyneCult™ Endometrial Organoid Medium supports robust expansion of EOs, even from low-epithelial-content starting material.

Figure 4. GyneCult™ Endometrial Organoid Medium Enables Robust EO Formation from Diverse Primary Cell Sources

(A, B, D, E) Representative brightfield images of 2-week endometrial organoids (EOs) cultured in GyneCult™ Endometrial Organoid Medium supplemented with estrogen. Organoids were generated from a range of primary endometrial sources, including tissue sampled from (A) proliferative and (B) secretory endometrium, (D) endometriosis lesions, and from (E) menstrual effluent. (C) E-Cadherin (red) immunostaining confirms the epithelial identity of the organoids. These results demonstrate the versatility of GyneCult™ Endometrial Organoid Medium as a platform for modeling endometrial biology and diseases such as endometriosis. Images courtesy of Dr. Thomas Andrieu from the University of Bern. Scale bar = 100 μm.

Figure 5. EOs Cultured in GyneCult™ Endometrial Organoid Medium Maintain Major Cell Types and Hormone Responsiveness

Immunocytochemistry staining and brightfield images of endometrial organoids (EOs) cultured under distinct hormonal conditions corresponding to the four phases of the menstrual cycle over a 14-day period. A subset of organoids was fixed for imaging at each stage, while remaining cultures progressed to the next phase. (A, E, I) Cultures were maintained in early proliferative conditions (0.2 nM estrogen) for the first 4 days after sample seeding, and transitioned to (B, F, J) late proliferative conditions (1 nM estrogen) in the subsequent 3 days. On Day 7, proliferative phase cultures were harvested and fixed. The rest of the EOs were then cultured under (C, G, K) early secretory conditions (0.2 nM estrogen, 4 nM progesterone) for the next 3 days, and then transitioned to (D, H, L) late secretory (0.5 nM estrogen, 40 nM progesterone) conditions for the remaining 4 days of culture. EOs were stained for (A - D) progestagen-associated endometrial protein (PAEP; green) and acetylated tubulin (red), where apical acetylated-tubulin staining facing the lumen indicates ciliated cells. PAEP, a secreted protein, shows accumulation in the proliferative phases, followed by apical localization and a decrease in cytoplasmic levels in the late secretory phase, suggesting active secretion in organoid cultures. (E - H) Transcription factors PAX8 (green) and SOX9 (red) staining shows that GyneCult™ Endometrial Organoid Medium gives rise to organoid cultures that maintain their stem cell niches (PAX8+SOX9+ cells) and respond to hormones to differentiate into secretory cells (PAX8+SOX9-), while maintaining ciliated cells throughout culture.