STUDY QUESTION Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues? SUMMARY ANSWER We present a three-dimensional (3D) physiologically relevant, organ-on-a-chip model of the uterine wall containing primary endometrial and myometrial cellular participants, generated from human uterine tissue. WHAT IS KNOWN ALREADY As a highly dynamic reproductive organ, the human uterus plays fundamental physiological roles in menstruation and childbirth. The endometrial–myometrial junction (EMJ) defines the interface between the inner mucosal layer (endometrium) and outer smooth muscle zone (myometrium) that comprises the uterine wall. The EMJ is implicit in several uterine pathologies of unknown aetiology, including adenomyosis and abnormally invasive placenta; however, despite this, no patient-derived in vitro models of the uterine wall containing all EMJ participants currently exist. STUDY DESIGN, SIZE, DURATION We employed microfluidic technology to characterize multiple miniaturized models of the uterine wall. Protocols were tested that included variations in the seeding order of endometrial and myometrial fractions, and the addition of a low viscosity extracellular matrix to influence cell behaviour. Ultimately, functional hormone responses of patient-derived uterine wall models were assessed. PARTICIPANTS/MATERIALS, SETTING, METHODS Endometrial (n = 9) and myometrial biopsies (n = 4) were enzymatically dissociated to create epithelial, stromal and myometrial cellular fractions. Cell suspensions were seeded into non-adhesive poly(dimethylsiloxane) microfluidic devices containing 5 × 5 microwell arrays. The fate of individual cell types was monitored in real-time using fluorescent tracers, and cell phenotype was characterized by immunocytochemistry. Model functionality was assessed by measuring Ca2+ responses to agonist stimulation, and both insulin-like growth factor binding protein 1 (IGFBP-1) and osteopontin secretion in response to hormone stimulation. MAIN RESULTS AND THE ROLE OF CHANCE When subjected to microfluidic culture in isolation, endometrial stromal cells and smooth muscle myocytes formed compact spheroids, whilst epithelial cells produced diffuse aggregates. Tri-cultures were established by sequential seeding of individual or combined cell fractions at various ratios. Regardless of the protocol, epithelial cells localized to the outer periphery of tri-culture spheroids, which varied in morphology across the protocols. Incorporation of 5% [v/v] Matrigel® improved the reproducibility of 3D aggregates which exhibited robust self-assembly of a stromal/smooth muscle core encased in epithelium. Exposure of tri-cultures to oestradiol, medroxyprogesterone acetate and cyclic adenosine monophosphate (cAMP) increased secretion of IGFBP-1, which indicates stromal decidualization, and enhanced epithelial cell osteopontin secretion. Stimulation with endothelin-1 induced Ca2+ signalling in myocytes. LIMITATIONS, REASONS FOR CAUTION Endometrial and myometrial tissue was collected from relatively few donors. Myometrial tissue was collected from pregnant donors, which may have influenced the myocyte phenotype. Furthermore, endometrial tissue sampling was from women not having a hysterectomy, thus may not include the deeper basalis region, which may limit the physiological mimicry of the final models. WIDER IMPLICATIONS OF THE FINDINGS Our novel approach to modelling the uterine wall in 3D captures all of the main cell types in a medium-throughput system, enabling the screening of hundreds of cultures in parallel from a single biopsy. This system shows great promise for examining the cellular interplay between physiological cues and EMJ pathologies, such as the impact of uterine peristalsis and cyclical hormones on the pathogenesis of adenomyosis. STUDY FUNDING/COMPETING INTEREST(S) C.B. was supported by an Organ-on-a-Chip Technologies Network Pump Priming Project grant. C.J.H. was supported by a Wellbeing of Women project grant (RG2137), SRI/Bayer and Wellcome Trust IFFS3. D.K.H. was supported by a Wellbeing of Women project grant (RG2137) and MRC clinical research training fellowship (MR/V007238/1). M.Z. is Director and Co-Founder of ScreenIn3D Limited. The other authors declare no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

中文翻译：

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微流体阵列中子宫壁的功能性、患者来源的 3D 三重培养模型


研究问题 包含子宫壁主要细胞成分的功能性体外模型能否由患者组织的细胞生成？总结答案 我们提出了一个子宫壁的三维 （3D） 生理相关器官芯片模型，其中包含由人类子宫组织生成的原代子宫内膜和子宫肌层细胞参与者。已知的 作为一个高度动态的生殖器官，人类子宫在月经和分娩中起着基本的生理作用。子宫内膜-子宫肌层交界处 （EMJ） 定义了构成子宫壁的内粘膜层（子宫内膜）和外平滑肌区（子宫肌层）之间的界面。EMJ 隐含于几种病因不明的子宫病变中，包括子宫腺肌病和异常浸润性胎盘;然而，尽管如此，目前尚不存在包含所有 EMJ 参与者的患者来源的子宫壁体外模型。研究设计、大小、持续时间我们采用微流体技术来表征子宫壁的多个微型模型。测试了方案，其中包括子宫内膜和子宫肌层组分接种顺序的变化，以及添加低粘度细胞外基质以影响细胞行为。最终，评估了患者来源的子宫壁模型的功能性激素反应。参与者/材料、设置、方法 子宫内膜 （n = 9） 和子宫肌层活检 （n = 4） 被酶解离以产生上皮、基质和子宫肌层细胞组分。将细胞悬液接种到包含 5 × 5 微孔阵列的非粘附性聚（二甲基硅氧烷）微流控装置中。 使用荧光示踪剂实时监测单个细胞类型的命运，并通过免疫细胞化学表征细胞表型。通过测量 Ca2 + 对激动剂刺激的反应，以及胰岛素样生长因子结合蛋白 1 （IGFBP-1） 和响应激素刺激的骨桥蛋白分泌来评估模型功能。主要结果和机会的作用 当单独进行微流控培养时，子宫内膜基质细胞和平滑肌肌细胞形成致密的球状体，而上皮细胞产生弥漫性聚集体。通过以不同比例连续接种单个或组合细胞组分来建立三重培养物。无论方案如何，上皮细胞都定位于三培养球体的外围，其形态在不同方案中有所不同。掺入 5% [v/v] Matrigel® 提高了 3D 聚集体的可重复性，其表现出包裹在上皮中的基质/平滑肌核心的稳健自组装。三培养物暴露于雌二醇、醋酸甲羟孕酮和环磷酸腺苷 （cAMP） 增加了 IGFBP-1 的分泌，这表明基质蜕膜化，并增强了上皮细胞骨桥蛋白的分泌。内皮素-1 刺激诱导心肌细胞中的 Ca2+ 信号传导。局限性，谨慎的原因 从相对较少的供体中收集子宫内膜和子宫肌层组织。从怀孕供体收集子宫肌层组织，这可能影响了肌细胞表型。此外，子宫内膜组织取样来自未进行子宫切除术的女性，因此可能不包括较深的基底区域，这可能会限制最终模型的生理模拟。 研究结果的更广泛意义 我们在 3D 中对子宫壁进行建模的新方法在中等通量系统中捕获了所有主要细胞类型，从而能够从单次活检中平行筛选数百种培养物。该系统在检查生理线索和 EMJ 病理之间的细胞相互作用方面显示出巨大的前景，例如子宫蠕动和周期性激素对子宫腺肌病发病机制的影响。研究资金/利益争夺 C.B. 得到了 Organ-on-a-Chip Technologies Network Pump Priming Project 资助的支持。C.J.H. 得到了女性福祉项目资助 （RG2137）、SRI/拜耳和惠康信托基金 IFFS3 的支持。DKH 得到了女性福祉项目资助 （RG2137） 和 MRC 临床研究培训奖学金 （MR/V007238/1） 的支持。M.Z. 是 ScreenIn3D Limited 的董事兼联合创始人。其他作者声明没有利益冲突。试验注册号 N/A。