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

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.

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

Başlamak için, her bir bakteri suşunun hesaplanan miktarını mililitre başına yaklaşık 5 milyon koloni oluşturan birime kadar karıştırın. Karışımı 7.000 G'de dört santigrat derecede yedi dakika santrifüjleyin. Ardından, süpernatanı dikkatlice pipetleyin.Peletleri, düşük tamponlama kapasitesi ve glikoz ile Hank'in Dengeli Tuz Çözeltisinde yeniden süspanse edin. Şimdi, farklılaşmış insan vajinal epitel hücrelerini içeren organ çiplerini bölmelerden ayırın. Çipin bazal kanalının çıkışını bir pipet ucuyla takın.Pipet pistonunu aşağı itmeden bazal kanal girişine 200 mikrolitre antibiyotik içermeyen farklılaşma ortamı ekleyin. Pipet ucunu pipetten serbest bırakarak ortamın kanaldan serbestçe akmasına izin verin. Daha sonra, çıkıştan aspire ederken 37 mikrolitre bakteri aşısını çipin apikal kanal girişine pipetleyin.Ucu çekin. Ardından, apikal kanal girişini ve çıkışını pipet uçlarıyla tıkayın. Kontrol çipleri için, apikal kanal girişine düşük tamponlama kapasitesine sahip 37 mikrolitre Hank'in Dengeli Tuz Çözeltisi ve glikozu pipetleyin.Çipin yüzeyindeki herhangi bir ortamı aspire ettikten sonra, cipsleri 150 milimetrelik bir Petri kabına yerleştirin. L. crispatus ve G. vaginalis kolonileri, kaplamadan sonraki 48 saat içinde tespit edildi ve hem sağlıklı hem de disbiyotik bakterilerin vajina çiplerine aşılandığını doğruladı. L. crispatus ile aşılanan vajina çiplerinin pH'ı, aşılanmamış kontrol çiplerininkine benzerdi ve G. vaginalis ile birlikte kültürlendiğinde, önemli ölçüde artmış pH yaşadılar.

bacterial-inoculation-organ-chips-with-differentiated-human-vaginal

Research

JoVE Journal

Bioengineering

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

63 Görüntüleme.  Harvard University. Başlamak için, her bir bakteri suşunun hesaplanan miktarını mililitre başına yaklaşık 5 milyon koloni oluşturan birime kadar karıştırın. Karışımı 7.000 G'de dört santigrat derecede yedi dakika santrifüjleyin. Ardından, süpernatanı dikkatlice pipetleyin.Peletleri, düşük tamponlama kapasitesi ve glikoz ile Hank'in Dengeli Tuz Çözeltisinde yeniden süspanse edin. Şimdi, farklılaşmış insan vajinal epitel hücrelerini içeren organ çiplerini bölmelerden ay...

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

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

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

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