connecting human uterine fibroblast-seeded organ chips to pods and differentiation of vaginal epithelial cells for studying living vaginal microbiome

Vajinal mikrobiyomu incelemek için mikroakışkan vajina çipi

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.

Yazar Spotlight: Vajinal Çip Kullanarak Vajinal Mikrobiyom Etkileşimleri Üzerine Araştırmada Devrim Yaratmak

Bir İnsan Çip Üzerinde Vajinada Sağlıklı ve Disbiyotik Vajinal Mikro Ortamların Modellenmesi

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.

İnsan Uterin Fibroblast Tohumlu Organ Çiplerinin Bölmelere Bağlanması ve Canlı Vajinal Mikrobiyomun İncelenmesi için Vajinal Epitel Hücrelerinin Farklılaşması

Başlamak için, her biri 50 mililitre, bir fibroblast ortamı ve vajinal epitel ortamı olmak üzere ayrı 50 mililitrelik konik tüplere alın. Isıtılmış ortamı steril bir vakum altında beş dakika boyunca gazdan arındırın. Ardından, dinamik akış modülü için temiz tepsilerin CaviCide ve etanol ile dezenfekte edilmesi.Bölmeleri ambalajından çıkarın ve dinamik akış modülü tepsilerine yerleştirin. Apikal giriş rezervuarına iki mililitre gazı alınmış vajinal epitel ortamı pipetleyin. Ve bazal giriş rezervuarına üç mililitre gazı alınmış fibroblast ortamı ekleyin.Apikal çıkış rezervuarına 500 mikrolitre gazı alınmış vajinal epitel ortamı ve bazal çıkış rezervuarına 500 mikrolitre gazı alınmış fibroblast ortamı pipetleyin. Bölmeleri içeren tepsileri dinamik akış modülüne kaydırın. Şimdi, asal döngüyü iki kez çalıştırın.Dört ana döngüden sonra bir damlacık oluşmazsa, bölmenin çıkış rezervuarının içindeki bağlantı noktasıyla doğrudan temas edin. Bölmeleri elle astarlamak için ortamın kanaldaki rezervuar arasında akmasına izin vermek için ilgili ortamdan 200 mikrolitre pipetleyin. Daha sonra, insan uterus fibroblast tohumlu organ çiplerinden pipet uçlarını çıkarın.Her bir yonganın tüm bağlantı noktalarının üzerine ilgili ortamdan bir damlacık yerleştirin. Cipsleri bölmelere kaydırın ve bölmeleri tepsilere yerleştirin. Talaşların yüzeyindeki herhangi bir ortamı aspire edin, ardından her tepsiyi dinamik bir akış modülüne kaydırın.Daha sonra, modülde, üst ve alt kısmı sıvı olarak, apikal akışı saatte 15 mikrolitreye, bazal akışı saatte 30 mikrolitreye, germeyi% 0'a ve frekansı sıfır hertz'e ayarlayın. Modülde düzenleme döngüsünü çalıştırın ve gece boyunca akışa izin verin. Organ çip cihazında, farklılaşmadan sonra aşamalı olarak birden fazla hücre tabakası oluşturan sürekli bir vajinal epitel hücresi tabakası gözlendi.

connecting-human-uterine-fibroblast-seeded-organ-chips-to-pods

Research

JoVE Journal

Bioengineering

Connecting Human Uterine Fibroblast-Seeded Organ Chips to Pods and Differentiation of Vaginal Epithelial Cells for Studying Living Vaginal Microbiome

50 Görüntüleme.  Harvard University. Başlamak için, her biri 50 mililitre, bir fibroblast ortamı ve vajinal epitel ortamı olmak üzere ayrı 50 mililitrelik konik tüplere alın. Isıtılmış ortamı steril bir vakum altında beş dakika boyunca gazdan arındırın. Ardından, dinamik akış modülü için temiz tepsilerin CaviCide ve etanol ile dezenfekte edilmesi.Bölmeleri ambalajından çıkarın ve dinamik akış modülü tepsilerine yerleştirin. Apikal giriş rezervuarına iki mililitre gazı alınmış vajina...

Video: İnsan Uterin Fibroblast Tohumlu Organ Çiplerinin Bölmelere Bağlanması ve Canlı Vajinal Mikrobiyomun İncelenmesi için Vajinal Epitel Hücrelerinin Farklılaşması

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 ...

Biyomühendislik

Seeding Organ Chips with Human Uterine Fibroblasts (HUFs) for Studying Living Vaginal Microbiome

Canlı Vajinal Mikrobiyomu İncelemek için İnsan Rahim Fibroblastları (HUF'ler) ile Organ Çiplerinin Tohumlanması

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

Mikroaerofilik Koşullar Altında Canlı Vajinal Mikrobiyomu İncelemek için Farklılaşmış İnsan Vajinal Epitel Hücreleri ile Organ Çiplerinin Bakteriyel Aşılanması