A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues
4 affiliations
- 1Department of Clinical Laboratory, Chengdu Women’s and Children’s Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan, China
- 2Department of Obstetrics and Gynecology, Chengdu Women’s and Children’s Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan, China
- 3Hunter Biotechnology, Inc., Hangzhou, Zhejiang, China
- 4Department of Operating Room, Chengdu Women’s and Children’s Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, Sichuan, China
Abstract
PurposePelvic organ prolapse (POP) significantly impairs women’s quality of life; however, the lack of reliable in vitro human-derived models has hindered the elucidation of its pathogenesis and the development of targeted therapies. This study aims to establish a reproducible protocol for generating patient-derived organoids from human vaginal wall and uterosacral ligament (USL) tissues and to define the boundaries of applicability of this model for POP research.MethodsVaginal wall and USL tissues were obtained from patients with POP (stage ≥ III, n = 3) and benign controls (n = 2). Each biological sample was tested in three independent technical replicates for two parallel culture approaches, including dissociated primary cells and digested tissue fragments. The tissues were processed and digested using a combination of collagenase II and DNase. Primary cells and digested tissue fragments were cultured in a three-dimensional (3D) Matrigel system with specialized organoid culture medium supplemented with growth factors, including Wnt3a, R-Spondin 1, and Noggin. Successful organoid formation was prospectively defined as the formation of discrete, three-dimensional spherical or cystic structures with a clearly visible border, sustained growth for at least 7 days under bright-field microscopy, and a diameter ≥100 µm. Organoid formation, growth, and morphology were monitored over time. Immunofluorescence staining was performed on the organoids generated from patients with POP to analyze their cell composition. KRT14 (epithelial), vimentin (fibroblast-related), α-SMA (myofibroblast/smooth muscle-related), VWF (endothelial), and SM22 (smooth muscle) were detected.ResultsUnder light microscopy, primary cells from benign control vaginal wall tissue formed organoids within the first week of culture. By Day 11, clear three-dimensional structures measuring approximately 100–500 μm in diameter were observed. In contrast, vaginal wall-derived organoids from patients with POP exhibited similar morphology but were fewer and less dense. When undigested tissue fragments were cultured for 48 h to form cell aggregates and then dispersed and reembedded in Matrigel, the organoid yield was significantly greater than that when the cultures were initiated with discrete primary cells alone, and organoid growth was maintained through the second passage. Both primary cells and tissue fragments from benign control USL generated organoids, whereas USL tissues from patients with POP did not form organoids under identical conditions. Immunofluorescence analysis of vaginal wall-derived organoids revealed expression of the epithelial marker KRT14, whereas vimentin, α-SMA, VWF, and SM22 were not detected. These findings indicate that these organoids were predominantly composed of epithelial cells.ConclusionWe present a preliminary protocol for generating vaginal wall-derived organoids from both patients with POP and benign controls, which may serve as a useful in vitro tool for studying epithelial biology in POP. Importantly, pathological USL tissues from patients with POP failed to form organoids under identical conditions, whereas benign control USL did; this negative result defines a current applicability boundary and indicates that the protocol is not yet suitable for generating organoids from pathological USL or for modeling USL-derived pathology. Further optimization and validation in larger cohorts are therefore needed before POP research can be broadly applied.
Related
- Risk prediction models for venous thromboembolism in the intensive care unit: a systematic review protocol assessing model performance, risk of bias, and clinical applicability.
- Malignant transformation of deep infiltrating endometriosis at the uterosacral ligament to endometrioid adenocarcinoma: a rare Case Report
- Whole-genome sequencing in mitral valve prolapse: an exploratory study on genes associated with mitral valve prolapse and cardiac arrhythmias
- Association Between Joint Hypermobility and Pelvic Organ Prolapse: A Hospital‐Based Cross‐Sectional Study
- Effects of vaginal structure and active contractile force on the spatiotemporal evolution of pelvic organ prolapse
BibTeX
@article{yu2026protocol,
title = {{A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues}},
author = {Xia Yu and Sihan Wu and Qin Tao and Xinyu Luo and Yanfeng Huang and Xiu Yang and Xiao Yang and Yonghong Lin},
journal = {Frontiers in Cell and Developmental Biology},
year = {2026},
month = oct,
doi = {10.3389/fcell.2026.1977193},
url = {https://doi.org/10.3389/fcell.2026.1977193},
note = {CC BY},
}
RIS
TY - JOUR
TI - A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues
AU - Xia Yu
AU - Sihan Wu
AU - Qin Tao
AU - Xinyu Luo
AU - Yanfeng Huang
AU - Xiu Yang
AU - Xiao Yang
AU - Yonghong Lin
PY - 2026
DA - 2026/10/23
JO - Frontiers in Cell and Developmental Biology
DO - 10.3389/fcell.2026.1977193
UR - https://doi.org/10.3389/fcell.2026.1977193
AB - PurposePelvic organ prolapse (POP) significantly impairs women’s quality of life; however, the lack of reliable in vitro human-derived models has hindered the elucidation of its pathogenesis and the development of targeted therapies. This study aims to establish a reproducible protocol for generating patient-derived organoids from human vaginal wall and uterosacral ligament (USL) tissues and to define the boundaries of applicability of this model for POP research.MethodsVaginal wall and USL tissues were obtained from patients with POP (stage ≥ III, n = 3) and benign controls (n = 2). Each biological sample was tested in three independent technical replicates for two parallel culture approaches, including dissociated primary cells and digested tissue fragments. The tissues were processed and digested using a combination of collagenase II and DNase. Primary cells and digested tissue fragments were cultured in a three-dimensional (3D) Matrigel system with specialized organoid culture medium supplemented with growth factors, including Wnt3a, R-Spondin 1, and Noggin. Successful organoid formation was prospectively defined as the formation of discrete, three-dimensional spherical or cystic structures with a clearly visible border, sustained growth for at least 7 days under bright-field microscopy, and a diameter ≥100 µm. Organoid formation, growth, and morphology were monitored over time. Immunofluorescence staining was performed on the organoids generated from patients with POP to analyze their cell composition. KRT14 (epithelial), vimentin (fibroblast-related), α-SMA (myofibroblast/smooth muscle-related), VWF (endothelial), and SM22 (smooth muscle) were detected.ResultsUnder light microscopy, primary cells from benign control vaginal wall tissue formed organoids within the first week of culture. By Day 11, clear three-dimensional structures measuring approximately 100–500 μm in diameter were observed. In contrast, vaginal wall-derived organoids from patients with POP exhibited similar morphology but were fewer and less dense. When undigested tissue fragments were cultured for 48 h to form cell aggregates and then dispersed and reembedded in Matrigel, the organoid yield was significantly greater than that when the cultures were initiated with discrete primary cells alone, and organoid growth was maintained through the second passage. Both primary cells and tissue fragments from benign control USL generated organoids, whereas USL tissues from patients with POP did not form organoids under identical conditions. Immunofluorescence analysis of vaginal wall-derived organoids revealed expression of the epithelial marker KRT14, whereas vimentin, α-SMA, VWF, and SM22 were not detected. These findings indicate that these organoids were predominantly composed of epithelial cells.ConclusionWe present a preliminary protocol for generating vaginal wall-derived organoids from both patients with POP and benign controls, which may serve as a useful in vitro tool for studying epithelial biology in POP. Importantly, pathological USL tissues from patients with POP failed to form organoids under identical conditions, whereas benign control USL did; this negative result defines a current applicability boundary and indicates that the protocol is not yet suitable for generating organoids from pathological USL or for modeling USL-derived pathology. Further optimization and validation in larger cohorts are therefore needed before POP research can be broadly applied.
SN - 2296-634X
ER -