{
  "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.",
  "abstract_shown": true,
  "affiliations": [
    "Department 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",
    "Department 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",
    "Hunter Biotechnology, Inc., Hangzhou, Zhejiang, China",
    "Department 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"
  ],
  "article_type": null,
  "arxiv": null,
  "author_total": 8,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Xia Yu"
    },
    {
      "affs": [
        1
      ],
      "name": "Sihan Wu"
    },
    {
      "affs": [
        1
      ],
      "name": "Qin Tao"
    },
    {
      "affs": [
        1
      ],
      "name": "Xinyu Luo"
    },
    {
      "affs": [
        2
      ],
      "name": "Yanfeng Huang"
    },
    {
      "affs": [
        3
      ],
      "name": "Xiu Yang"
    },
    {
      "affs": [
        1
      ],
      "name": "Xiao Yang"
    },
    {
      "affs": [
        1
      ],
      "name": "Yonghong Lin"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/dx2c0hakbajjc.bib",
  "cited_by": 0,
  "doi": "10.3389/fcell.2026.1977193",
  "fulltext_url": "https://www.frontiersin.org/articles/10.3389/fcell.2026.1977193/full",
  "id": "dx2c0hakbajjc",
  "issns": [
    "2296-634X"
  ],
  "landing_url": "https://doi.org/10.3389/fcell.2026.1977193",
  "language": "en",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "journal",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "lcc",
      "QH301-705.5"
    ],
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      "openalex",
      "2745|Pelvic floor disorders treatments"
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  "openalex": "W7223316023",
  "pdf_url": "https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2026.1977193/pdf",
  "pmcid": null,
  "pmid": null,
  "published": "2026-10-23",
  "publisher": "Frontiers Media S.A.",
  "ris": "https://research.draconitic.com/paper/dx2c0hakbajjc.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T13:43:48Z",
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      "url": "https://doi.org/10.3389/fcell.2026.1977193"
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  "subjects": [
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      "field": "medicine",
      "origin": "openalex:2745|Pelvic floor disorders treatments",
      "subfield": "general"
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  ],
  "title": "A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues",
  "title_text": "A protocol for vaginal wall-derived organoids from women with pelvic organ prolapse and benign controls: limited applicability to pathological uterosacral ligament tissues",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T13:43:48Z",
  "url": "https://research.draconitic.com/paper/dx2c0hakbajjc",
  "venue": "Frontiers in Cell and Developmental Biology",
  "versions": []
}