Department of Biomedical Engineering, University of Strathclyde, Wolfson Centre, Glasgow, UK.
Department of Electronic and Electrical Engineering, Centre for Microsystems & Photonics, University of Strathclyde, Glasgow, UK.
Hum Reprod. 2024 Nov 1;39(11):2537-2550. doi: 10.1093/humrep/deae214.
Can a functional in vitro model, containing the main cellular components of the uterine wall, be generated from cells derived from patient tissues?
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.
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.
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.
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.
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能否从患者组织来源的细胞中生成含有子宫壁主要细胞成分的功能性体外模型？
我们提出了一种三维（3D）生理相关的子宫壁器官芯片模型，其中包含源自人子宫组织的主要子宫内膜和子宫肌层细胞参与者。
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重要的生理作用。子宫内膜-子宫肌层交界处（EMJ）定义了子宫壁的内层黏膜层（子宫内膜）和外层平滑肌区（子宫肌层）之间的界面。EMJ 隐含在几种病因不明的子宫壁病理学中，包括子宫腺肌病和异常侵袭性胎盘；然而，尽管如此，目前还没有包含所有 EMJ 参与者的患者来源的子宫壁体外模型。
研究设计、规模、持续时间：我们采用微流控技术对多种微型子宫壁模型进行了特征描述。测试了包括改变子宫内膜和子宫肌层分数的播种顺序，以及添加低粘度细胞外基质以影响细胞行为的变化的方案。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：从酶解的子宫内膜（n=9）和子宫肌层活检（n=4）中创建上皮、基质和子宫肌层细胞分数。将细胞悬浮液播种到含有 5×5微井阵列的非粘附聚二甲基硅氧烷微流控装置中。使用荧光示踪剂实时监测各个细胞类型的命运，并通过免疫细胞化学对细胞表型进行特征描述。通过测量对激动剂刺激的 Ca2+反应以及对激素刺激的胰岛素样生长因子结合蛋白 1（IGFBP-1）和骨桥蛋白分泌来评估模型功能。
当在微流控培养中单独培养时，子宫内膜基质细胞和平滑肌肌细胞形成紧密的球体，而上皮细胞产生弥散的聚集物。通过以不同比例顺序播种单个或组合细胞分数来建立三细胞培养物。无论方案如何，上皮细胞都定位于三细胞球体的外周边缘，而不同方案的球体形态各异。加入 5%[v/v]Matrigel®可改善 3D 聚集物的重现性，使其具有基质/平滑肌核心包裹在上皮中的稳健自组装。将三细胞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刺激诱导肌细胞中的 Ca2+信号。
局限性、谨慎的原因：子宫内膜和子宫肌层组织取自相对较少的供体。子宫肌层组织取自妊娠供体，这可能影响肌细胞表型。此外，子宫内膜组织采样来自未接受子宫切除术的女性，因此可能不包括较深的基底层，这可能限制最终模型的生理模拟。
我们建立子宫壁 3D 模型的新方法捕获了所有主要细胞类型的介质通量系统，能够从单个活检并行筛选数百种培养物。该系统为研究生理线索与 EMJ 病理学之间的细胞相互作用提供了巨大的潜力，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/竞争利益：C.B. 得到了器官芯片技术网络启动项目拨款的支持。C.J.H. 得到了 Wellbeing of Women 项目拨款（RG2137）、SRI/Bayer 和 Wellcome Trust IFFS3 的支持。D.K.H. 得到了 Wellbeing of Women 项目拨款（RG2137）和 MRC 临床研究培训奖学金（MR/V007238/1）的支持。M.Z. 是 ScreenIn3D 有限公司的董事和联合创始人。其他作者没有利益冲突。
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