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 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'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 (<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. 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.

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