Mucus production, host-microbiome interactions, hormone sensitivity, and innate immune responses modeled in human cervix chips.

Modulation of the cervix by steroid hormones and commensal microbiome play a central role in the health of the female reproductive tract. Here we describe organ-on-a-chip (Organ Chip) models that recreate the human cervical epithelial-stromal interface with a functional epithelial barrier and production of mucus with biochemical and hormone-responsive properties similar to living cervix. When Cervix Chips are populated with optimal healthy versus dysbiotic microbial communities (dominated by Lactobacillus crispatus and Gardnerella vaginalis, respectively), significant differences in tissue innate immune responses, barrier function, cell viability, proteome, and mucus composition are observed that are similar to those seen in vivo. Thus, human Cervix Organ Chips represent physiologically relevant in vitro models to study cervix physiology and host-microbiome interactions, and hence may be used as a preclinical testbed for development of therapeutic interventions to enhance women's health.

Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip.

BACKGROUND: A dominance of non-iners Lactobacillus species in the vaginal microbiome is optimal and strongly associated with gynecological and obstetric health, while the presence of diverse obligate or facultative anaerobic bacteria and a paucity in Lactobacillus species, similar to communities found in bacterial vaginosis (BV), is considered non-optimal and associated with adverse health outcomes. Various therapeutic strategies are being explored to modulate the composition of the vaginal microbiome; however, there is no human model that faithfully reproduces the vaginal epithelial microenvironment for preclinical validation of potential therapeutics or testing hypotheses about vaginal epithelium-microbiome interactions. RESULTS: Here, we describe an organ-on-a-chip (organ chip) microfluidic culture model of the human vaginal mucosa (vagina chip) that is lined by hormone-sensitive, primary vaginal epithelium interfaced with underlying stromal fibroblasts, which sustains a low physiological oxygen concentration in the epithelial lumen. We show that the Vagina Chip can be used to assess colonization by optimal L. crispatus consortia as well as non-optimal Gardnerella vaginalis-containing consortia, and to measure associated host innate immune responses. Co-culture and growth of the L. crispatus consortia on-chip was accompanied by maintenance of epithelial cell viability, accumulation of D- and L-lactic acid, maintenance of a physiologically relevant low pH, and down regulation of proinflammatory cytokines. In contrast, co-culture of G. vaginalis-containing consortia in the vagina chip resulted in epithelial cell injury, a rise in pH, and upregulation of proinflammatory cytokines. CONCLUSION: This study demonstrates the potential of applying human organ chip technology to create a preclinical model of the human vaginal mucosa that can be used to better understand interactions between the vaginal microbiome and host tissues, as well as to evaluate the safety and efficacy of live biotherapeutics products. Video Abstract.

Modeling mucus physiology and pathophysiology in human organs-on-chips.

The surfaces of human internal organs are lined by a mucus layer that ensures symbiotic relationships with commensal microbiome while protecting against potentially injurious environmental chemicals, toxins, and pathogens, and disruption of this layer can contribute to disease development. Studying mucus biology has been challenging due to the lack of physiologically relevant human in vitro models. Here we review recent progress that has been made in the development of human organ-on-a-chip microfluidic culture models that reconstitute epithelial tissue barriers and physiologically relevant mucus layers with a focus on lung, colon, small intestine, cervix and vagina. These organ-on-a-chip models that incorporate dynamic fluid flow, air-liquid interfaces, and physiologically relevant mechanical cues can be used to study mucus composition, mechanics, and structure, as well as investigate its contributions to human health and disease with a level of biomimicry not possible in the past.

Nutritional deficiency in an intestine-on-a-chip recapitulates injury hallmarks associated with environmental enteric dysfunction.

Environmental enteric dysfunction (EED)-a chronic inflammatory condition of the intestine-is characterized by villus blunting, compromised intestinal barrier function and reduced nutrient absorption. Here we show that essential genotypic and phenotypic features of EED-associated intestinal injury can be reconstituted in a human intestine-on-a-chip lined by organoid-derived intestinal epithelial cells from patients with EED and cultured in nutrient-deficient medium lacking niacinamide and tryptophan. Exposure of the organ chip to such nutritional deficiencies resulted in congruent changes in six of the top ten upregulated genes that were comparable to changes seen in samples from patients with EED. Chips lined with healthy epithelium or with EED epithelium exposed to nutritional deficiencies resulted in severe villus blunting and barrier dysfunction, and in the impairment of fatty acid uptake and amino acid transport; and the chips with EED epithelium exhibited heightened secretion of inflammatory cytokines. The organ-chip model of EED-associated intestinal injury may facilitate the analysis of the molecular, genetic and nutritional bases of the disease and the testing of candidate therapeutics for it.

Establishment of physiologically relevant oxygen gradients in microfluidic organ chips.

In vitro models of human organs must accurately reconstitute oxygen concentrations and gradients that are observed in vivo to mimic gene expression, metabolism, and host-microbiome interactions. Here we describe a simple strategy to achieve physiologically relevant oxygen tension in a two-channel human small intestine-on-a-chip (Intestine Chip) lined with primary human duodenal epithelium and intestinal microvascular endothelium in parallel channels separated by a porous membrane while both channels are perfused with oxygenated medium. This strategy was developed using computer simulations that predicted lowering the oxygen permeability of poly-dimethylsiloxane (PDMS) chips in specified locations using a gas impermeable film will allow the cells to naturally decrease the oxygen concentration through aerobic respiration and reach steady-state oxygen levels <36 mm Hg (<5%) within the epithelial lumen. The approach was experimentally confirmed using chips with embedded oxygen sensors that maintained this stable oxygen gradient. Furthermore, Intestine Chips cultured with this approach supported formation of a villus epithelium interfaced with a continuous endothelium and maintained intestinal barrier integrity for 72 h. This strategy recapitulates in vivo functionality in an efficient, inexpensive, and scalable format that improves the robustness and translatability of Organ Chip technology for studies on microbiome as well as oxygen sensitivity.

Enteric Coronavirus Infection and Treatment Modeled With an Immunocompetent Human Intestine-On-A-Chip.

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PBMCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.