Duong Vi, Pooladanda Venkatesh, Mitchell Caroline
Department of Obstetrics, Gynecology and Reproductive Biology, Division of Urogynecology, Massachusetts General Hospital;
Massachusetts General Hospital/Vincent Center for Reproductive Biology/Harvard Medical School.
J Vis Exp. 2024 Nov 22(213). doi: 10.3791/65864.
Pelvic organ prolapse is a disorder that seriously impacts the quality of life of women. It occurs when muscles and ligaments weaken and cause pelvic organs to drop lower in the pelvis, creating a bulge in the vagina. Surgery to correct pelvic organ prolapse has been a mainstay treatment. Recently, there has been growing interest in studying the tissue composition of patients with prolapse at the cellular level. There is currently little consensus regarding the effect of donor or patient age on cell-based therapies. Current published protocols for vaginal fibroblast isolation either concentrate on premenopausal tissue or neglect to comment on the age of donor tissue. Most existing protocols use animal models. The consistency of human vaginal tissue is denser than the tissues used in most protocols. In this study, human vaginal tissue was obtained primarily from older donors, which likely contributed to the failure of existing protocols. The aim of this study is to describe a standard protocol for reliably acquiring human vaginal fibroblasts, regardless of donor age and menopausal status. Results were reproduced using tissue from nine separate donors who underwent pelvic organ prolapse surgery. Six patients were postmenopausal, with the oldest donor being 78 years old. The median age of the tissue donors was 59. Here, we describe a reliable method for generating a fibroblast-enriched single-cell suspension using a combination of enzymatic and mechanical dissociation and cell suspension pooling of multiple vaginal biopsies from a single donor. Reliable isolation of human vaginal primary fibroblasts may be useful in the study of pelvic organ prolapse as well as microbiome-host interactions.
盆腔器官脱垂是一种严重影响女性生活质量的疾病。当肌肉和韧带变弱并导致盆腔器官在盆腔中位置下移，从而在阴道内形成凸起时，就会发生这种疾病。矫正盆腔器官脱垂的手术一直是主要的治疗方法。最近，人们对在细胞水平研究脱垂患者的组织组成越来越感兴趣。目前，关于供体或患者年龄对基于细胞的治疗方法的影响，几乎没有达成共识。目前已发表的阴道成纤维细胞分离方案要么专注于绝经前组织，要么没有提及供体组织的年龄。大多数现有方案使用动物模型。人类阴道组织的致密程度高于大多数方案中使用的组织。在本研究中，人类阴道组织主要取自老年供体，这可能是现有方案失败的原因。本研究的目的是描述一种标准方案，用于可靠地获取人类阴道成纤维细胞，而不考虑供体年龄和绝经状态。使用接受盆腔器官脱垂手术的9个不同供体的组织重现了结果。6名患者已绝经，年龄最大的供体为78岁。组织供体的中位年龄为59岁。在此，我们描述了一种可靠的方法，通过酶解和机械解离相结合以及将来自单个供体的多个阴道活检组织的细胞悬液合并，来生成富含成纤维细胞的单细胞悬液。可靠地分离人类阴道原代成纤维细胞可能有助于盆腔器官脱垂以及微生物组与宿主相互作用的研究。
