bacterial inoculation of organ chips with differentiated human vaginal epithelial cells for studying living vaginal microbiome under microaerophilic conditions

用于研究阴道微生物组的微流体阴道芯片

A vaginal microbiome dominated by Lactobacillus spp. that helps to maintain an acidic microenvironment plays an important role in maintaining female reproductive health1. However, at times there can be a change in the composition of microbial communities that comprise the microbiome, which results in an increase in the diversity of vaginal bacteria. These dysbiotic changes, which often result in a switch from a Lactobacillus-dominated state to one dominated by more&#160;diverse anaerobic bacterial species (e.g., Gardnerella vaginalis), are associated with various diseases of the reproductive system, such as bacterial vaginosis, atrophic vaginitis, urinary tract infection, vulvovaginal candidiasis, urethritis, and chorioamnionitis2,3,4,5. These diseases, in turn, increase a woman&#39;s chances of acquiring sexually transmitted diseases and pelvic inflammatory disease6,7,8,9. They also pose a higher risk for pre-term birth and miscarriages in pregnant women10,11,12 and have also been implicated in infertility13,14,15,16.
Although efforts have been made to model vaginal dysbiosis using vaginal epithelial cells cultured in static, two-dimensional (2D) culture systems17,18, they do not effectively mimic the physiology and complexity of the vaginal microenvironment19. Animal models also have been used to study vaginal dysbiosis; however, their menstrual phases and host-microbiome interactions differ greatly from that in humans, and thus, the physiological relevance of results from these studies remains unclear19,20,21. To counteract these issues, organoids and Transwell insert models of human vaginal tissue also have been used to study host-pathogen interactions in the FRT19,22,23,24. But because these are static cultures, they can only support co-culture of human cells with living microbes for a short period of time (&#60;16-24 h), and they lack many other potentially important physical features of the human vaginal microenvironment, such as mucus production and fluid flow22.
Organ Chips are three-dimensional (3D) microfluidic culture systems that contain one or more parallel hollow microchannels lined by living cells cultured under dynamic fluid flow. The two-channel chips enable the recreation of organ-level tissue-tissue interfaces by culturing different cell types (e.g., epithelium and stromal fibroblasts or epithelium and vascular endothelium) on opposite sides of a porous membrane that separates the two parallel channels (Figure 1). Both tissues can be independently exposed to fluid flow, and they can also experience microaerobic conditions to enable co-culture with a complex microbiome25,26,27,28. This approach was recently leveraged to develop a human Vagina Chip lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen and enables co-culture with healthy and dysbiotic microbiomes for at least 3 days in vitro29. It was demonstrated that the Vagina Chip could be used to study colonization by optimal (healthy) L. crispatus consortia and detect inflammation and injury caused by non-optimal (non-healthy) G. vaginalis containing consortia.&#160;Here, we describe in detail the methods that are used to create the human Vagina Chip as well as to establish healthy and dysbiotic bacterial communities on-chip.

作者聚焦：使用阴道芯片彻底改变阴道微生物组相互作用的研究

在人类阴道芯片中模拟健康和生态失调的阴道微环境

We successfully developed a human vagina chip that supports spontaneous differentiation of squamous stratified vaginal epithelium, forms a strong barrier, responds to hormones, and generates a microbiome supporting oxygen gradients. 2D and organoid cultures are currently used to study vaginal dysbiosis, but they aren't able to mimic the physiological complexities of the vaginal microenvironment due to their static nature. Animal models too can't model the same hormonal changes present in the human menstrual cycle, and the vaginal microbiome across animal species and humans differ.We set out to explore whether organ chip technology can be used to develop a preclinical model of human vagina microbiome interactions, which could potentially be used for discovery and assessment of potential microbiome-based therapeutics. The vagina chip allows for dynamic fluid flow across the vaginal epithelium and stroma, which prolongs its co-culturing with vaginal microbial consortia and maintains a physiologically relevant microenvironment. The human vagina-on-a-chip is an exciting tool that can be used to study varying diseases of the female reproductive tract and validate new biotherapeutics.

用分化的人阴道上皮细胞细菌接种器官芯片在微需氧条件下研究活的阴道微生物组

首先，将每种细菌菌株的计算量混合，使每毫升总共达到约 500 万个菌落形成单位。将混合物以 7， 000 G 在 4 摄氏度下离心 7 分钟。接下来，小心地用移液液出上清液。将沉淀重新悬浮在缓冲能力和葡萄糖含量低的 Hank 平衡盐溶液中。现在，从豆荚中分离出含有分化的人阴道上皮细胞的器官芯片。用移液器吸头塞住芯片基底通道的出口。将 200 μL 无抗生素分化培养基加入基底通道入口，而不向下推移液器柱塞。从移液器中松开移液器吸头，让培养基自由流过通道。接下来，将 37 微升的细菌接种物移液到芯片的顶端通道入口，同时从出口吸出。拉动尖端。然后，用移液器吸头插入顶端通道入口和出口。对于对照芯片，将 37 微升缓冲容量低和葡萄糖的 Hank 平衡盐溶液移液到顶端通道入口。吸出芯片表面的任何介质后，将芯片放入 150 毫米培养皿中。在接种后 48 小时内检测到 L.crispatus 和 G.vaginalis 的菌落，证实健康和生态失调细菌都植入了阴道芯片中。接种了 L.crispatus 的阴道芯片的 pH 值与未接种的对照芯片的 pH 值相似，当与 G.vaginalis 共培养时，它们的 pH 值显着升高。

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Research

JoVE Journal

Bioengineering

Bacterial Inoculation of Organ Chips with Differentiated Human Vaginal Epithelial Cells for Studying Living Vaginal Microbiome Under Microaerophilic Conditions

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Modeling Healthy and Dysbiotic Vaginal Microenvironments in a Human Vagina-on-a-Chip

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy reproductive system may lead to life-threatening diseases, infertility, or adverse outcomes during pregnancy. One barrier in the field is that there has been a dearth of preclinical, experimental models that faithfully mimic the physiology and pathophysiology of the FRT. Current in vitro and animal models do not fully recapitulate the hormonal changes, microaerobic conditions, and interactions with the vaginal microbiome. The advent of Organ-on-a-Chip (Organ Chip) microfluidic culture technology that can mimic tissue-tissue interfaces, vascular perfusion, interstitial fluid flows, and the physical microenvironment of a major subunit of human organs can potentially serve as a solution to this problem. Recently, a human Vagina Chip that supports co-culture of human vaginal microbial consortia with primary human vaginal epithelium that is also interfaced with vaginal stroma and experiences dynamic fluid flow has been developed. This chip replicates the physiological responses of the human vagina to healthy and dysbiotic microbiomes. A detailed protocol for creating human Vagina Chips has been described in this article.

This article describes a protocol for creating a microfluidic vagina-on-a-chip (Vagina Chip) culture device that enables the study of human host interactions with a living vaginal microbiome under microaerophilic conditions. This chip can be used as a tool to investigate vaginal diseases as well as to develop and test potential therapeutic countermeasures.

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy ...