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

Microfluidic Vagina Chip for Studying the Vaginal Microbiome

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.

Author Spotlight: Revolutionizing Research on Vaginal Microbiome Interactions Using a Vaginal Chip

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

Бактериальная инокуляция органных чипов дифференцированными эпителиальными клетками влагалища человека для изучения живого микробиома влагалища в микроаэрофильных условиях

Для начала смешайте рассчитанное количество каждого бактериального штамма, чтобы в общей сложности получить примерно 5 миллионов колониеобразующих единиц на миллилитр. Центрифугируйте смесь при 7 000 г в течение семи минут при четырех градусах Цельсия. Затем осторожно пипетируйте надосадочную жидкость.Повторно суспендируйте гранулу в сбалансированном солевом растворе Хэнка с низкой буферной способностью и глюкозой. Теперь отделите чипы органов, содержащие дифференцированные клетки эпителия влагалища человека, от стручков. Заглушите выходное отверстие базального канала чипа наконечником для дозатора.Добавьте 200 микролитров среды для дифференцировки без антибиотиков на входное отверстие базального канала, не нажимая на поршень пипетки. Освободите наконечник дозатора от пипетки, чтобы среда свободно текла по каналу. Затем пипеткой 37 микролитров бактериального инокулята подается на вход в апикальный канал чипа, одновременно отсасывая из выходного отверстия.Потяните за кончик. Затем заткните входное и выходное отверстия апикального канала наконечниками для дозатора. Для контрольных чипов пипетку пипетку 37 микролитров сбалансированного солевого раствора Хэнка с низкой буферной способностью и глюкозой на входе апикального канала.После аспирации любых сред на поверхности стружки поместите стружку в 150-миллиметровую чашку Петри. Колонии L.crispatus и G.vaginalis были обнаружены в течение 48 часов после осаждения, что подтвердило, что как здоровые, так и дисбиотические бактерии прижились в чипах влагалища. pH влагалищных чипов, инокулированных L.crispatus, был аналогичен pH неинокулированных контрольных чипов, а при совместном культивировании с G.vagnalis у них наблюдалось значительное повышение pH.

Research

JoVE Journal

Bioengineering

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

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

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

Посев чипов органов фибробластами матки человека (HUF) для изучения живого микробиома влагалища

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

Соединение микросхем органов, засеянных фибробластами матки человека, со стручками и дифференцировка эпителиальных клеток влагалища для изучения живого микробиома влагалища