Neisseria gonorrhoeae is a common Gram-negative pathogen with increasing resistance to all recommended antibiotics. There is a critical need to improve the efficiency of the antibiotic hit discovery process to replenish the drug development pipeline. Here, we show that deep learning models can augment high-throughput screens to identify readily available molecules with narrow-spectrum activity against difficult-to-treat strains of N. gonorrhoeae. We phenotypically tested 38,650 small molecules for N. gonorrhoeae growth inhibition to train a predictive graph neural network (GNN) model. We benchmarked the model's performance against other architectures, including a large language model, and found that GNNs more accurately identify active, drug-like molecules that are structurally distinct from the training set and known antibiotics. Using the model to virtually screen ~6 million compounds, we identified 213 compounds for experimental validation and found that 83 (39%) inhibited N. gonorrhoeae growth. Two of these compounds were structurally dissimilar to existing antibiotics, maintained potency against multidrug-resistant N. gonorrhoeae strains in vitro, exhibited promising selectivity indices, and were rapidly bactericidal with low frequencies of resistance. Proteomic studies revealed their distinct mechanisms of action, with one compound targeting alanine racemase, an enzyme involved in the essential process of peptidoglycan synthesis. Furthermore, the compounds showed early promise in reducing N. gonorrhoeae titers in a human vagina-on-a-chip infection model and a mouse vaginal infection model. Our work establishes the deep learning-enabled discovery of selective antibacterial compounds against N. gonorrhoeae as a much-needed hit discovery tool to address the growing crisis of antimicrobial resistance for this pathogen.

Hormonal fluctuations in women have major impacts on organ physiology, contributing to well-recognized sex differences in disease susceptibility and manifestations, as well as therapeutic responses. Yet, most experimental studies and preclinical models fail to take sex-specific differences in tissue responses or changes in female hormones into consideration. In this Minireview, we highlight the importance of incorporating hormonal dynamics in experimental design and focus on emerging in vitro new approach methodologies (NAMs), including primary human cell cultures, organoids, and microfluidic human organ-on-a-chip (Organ Chip) models, which are being used to study hormone-dependent biology across multiple organ systems in a more clinically relevant way. Clinical data linking estrogen and progesterone signaling to sex-specific disease manifestations and the limitations of association-based studies are first reviewed. Then we describe how new human-relevant NAMs that can replicate female hormone-dependent exposures and responses in vitro may be used to dissect disease mechanisms and discuss each model's unique advantages and disadvantages. We also consider how these new approaches may be used as preclinical models for drug development, toxicity testing, and personalized medicine in the future.

The fallopian tube serves as a sperm reservoir, and it is the site where the oocytes become fertilized. Here, we describe development of an organ-on-a-chip microfluidic model of the fallopian tube (FT Chip) lined by primary human epithelial cells and stromal fibroblasts derived from the FT ampulla. Abundant tissue folds lined by hormone-responsive, epithelial cells resembling those seen in vivo formed on-chip, but not in epithelial organoids cultured in gel cultures. Comparative time-resolved analysis of human sperm versus oocyte-sized microparticles introduced into the epithelial channel in the presence of estradiol revealed that sperm movement was significantly reduced, while the oocyte-sized particles increased, relative to movements in acellular chips. When the non-hormonal contraceptive TDI-11861 was administered to the chip, dose-dependent inhibition of human sperm motility was detected. Thus, this FT Chip may offer a human preclinical tool to study FT physiology and assess the efficacy and mechanism of action of contraceptives. ### Competing Interest Statement D.E.I. holds equity in Emulate Inc., chairs its scientific advisory board, and is a member of its board of directors. Gates Foundation, [INV-056272]

Can a microfluidic Organ Chip model of the human cervix replicate cervical conditions to evaluate sperm translocation, motility and the efficacy of non-hormonal contraceptives? Human Cervix Chip mimics the cervical microenvironment across the follicular and luteal phases, enabling real-time analysis of sperm translocation, motility and impact of non-hormonal contraceptives. Traditional contraceptive testing relies on in vivo and in vitro models, which often lack human physiological relevance and reproducibility. Ex vivo cervical mucus penetration assays are limited by variability, while animal models pose ethical concerns and species differences. Microfluidic Organ chips have emerged as powerful tools to replicate human reproductive tract conditions, offering controlled, scalable environments for studying sperm behavior and drug interactions. However, their application in contraceptive research remains underexplored. A human Cervix Chip capable of mimicking the cervical microenvironment could provide a robust platform for evaluating sperm motility, translocation, and non-hormonal contraceptive efficacy. This study used a microfluidic human Cervix that incorporated fluctuating levels of estrogen and progesterone to simulate the follicular and luteal phases of the menstrual cycle. The impact of different concentrations of non-hormonal contraceptives on sperm migration, cervical epithelial cells, and the cervical environment was assessed. The effects of healthy vaginal microbiome on contraceptive efficacy also were examined. Primary human cervical epithelial and fibroblast cells were cultured within a 2-channel microfluidic chip to replicate cervical architecture. Sperm from multiple donors were introduced into the Cervix Chip, and sperm motility and translocation were assessed using high-resolution imaging. The effects of non-hormonal contraceptive agents on sperm function were evaluated in real time, providing insights into their potential efficacy in altering sperm behavior within the cervical environment. Sperm translocation through the cervix-on-chip was significantly affected by non-hormonal contraceptive agent TDI-11861, a soluble adenylyl cyclase (sAC) inhibitor, demonstrating a substantial reduction in sperm motility under experimental conditions (p < 0.05). This effect was observed in Cervix Chips exposed to both follicular and luteal phase hormones, mimicking the natural hormonal fluctuations of the menstrual cycle. Additionally, sperm translocation and motility were assessed in the presence of a consortium of Lactobacillus crispatus, which mirrors the typical cervico-vaginal microbiome. The impact of TDI-11861 was dose-dependent with higher concentrations leading to significantly reduced sperm translocation (p < 0.05) and motility (p < 0.05), highlighting the potential of the Cervix Chip as a preclinical platform for evaluating the efficacy of non-hormonal contraceptive agents. These results underscore the ability of the human Cervix Chip to faithfully replicate key physiological aspects of the cervical environment, offering a promising tool for early-stage contraceptive screening. While the findings were consistent across multiple experimental replicates, further studies are required to explore the long-term effects of these agents, as well as their interaction with different microbial communities. Overall, these findings suggest the human Cervix Chip holds considerable potential in advancing contraceptive research with high translational relevance. The model does not fully capture contributions of immune cells, endothelium, or hormonal regulation over extended periods. Further validation with larger numbers of patient-derived samples and in vivo comparisons is necessary. This human Cervix Chip represents a scalable and ethical alternative for contraceptive research with potential applications in fertility studies and drug screening. It could facilitate the development of novel non-hormonal contraceptives with improved efficacy and safety profiles. No

This study explores the protective role of cervicovaginal mucus in maintaining vaginal health, particularly in relation to bacterial vaginosis (BV), using organ chip technology. By integrating human Cervix and Vagina Chips, we demonstrated that cervical mucus significantly reduces inflammation and epithelial damage caused by a dysbiotic microbiome commonly associated with BV. Proteomic analysis of the Vagina Chip, following exposure to mucus from the Cervix Chip, revealed differentially abundant proteins, suggesting potential biomarkers and therapeutic targets for BV management. Our findings highlight the essential function of cervical mucus in preserving vaginal health and underscore the value of organ chip models for studying complex interactions within the female reproductive tract. This research provides new insights into the mechanisms underlying vaginal dysbiosis and opens avenues for developing targeted therapies and diagnostic tools to enhance women’s reproductive health.

Cervical dysfunction, a major contributor to preterm labor and neonatal mortality, remains poorly understood due to the absence of physiologically relevant human models. Here, we show that a microfluidic human Endocervix Chip lined by primary endocervical epithelium interfaced with stromal cells and cultured under pregnancy-like hormonal conditions recapitulates key aspects of the biology of the endocervix, including formation of a mucus plug-like structure with antimicrobial properties. Culturing a dysbiotic cervico-vaginal microbiome on-chip increased secretion of pro-inflammatory cytokines observed in patients with preterm labor and enhanced production of matrix metalloproteinases (MMPs) that degrade stromal extracellular matrix (ECM). Perfusion with inflammatory cytokines at clinically relevant concentrations altered cervical mucus composition, upregulated prostaglandin-endoperoxide synthase 2 expression, increased MMP secretion, and reduced collagen production, which together drive dissolution of the stromal ECM and promote cervical ripening. Addition of circulating peripheral blood mononuclear cells (PBMCs) amplified these effects. Administration of a clinically approved drug for prevention of preterm labor that the Food and Drug Administration (FDA) recently deemed ineffective was also found to be inactive in the chip, while an approved therapeutic antagonist of the IL-1 receptor successfully protected against cervical dysfunction in this model. These findings demonstrate that IL-1 acts directly on human cervical tissues to promote changes associated with initiation of labor and that primary human endocervix chips may represent a useful preclinical model for studies on cervical dysfunction associated with preterm birth. One Sentence Summary Human endocervix chip modeling of cervical dysfunction suggests that IL-1 receptor antagonists may prevent preterm labor. ### Competing Interest Statement D.E.I. holds equity in Emulate, chairs its scientific advisory board and is a member of its board of directors. The remaining authors declare no competing interests. Bill & Melinda Gates Foundation, https://ror.org/0456r8d26, INV-035977

Bacterial vaginosis (BV) is a vaginal infection caused by an imbalance in the vaginal microbiome characterized by a decrease in healthy bacteria dominated by Lactobacillus crispatus along with a concomitant increase in dysbiotic bacteria, such as Gardnerella . Current treatments commonly fail to fully eradicate BV, and the lack of more effective therapies is due in part to the absence of relevant human models. Here, we used a human vagina-on-a-chip (Vagina Chip) microfluidic culture device that has been previously shown to faithfully recapitulate the human vaginal microenvironment as well as the inflammatory and injurious effects of G. vaginalis to test the therapeutic efficacy of a consortium of L. crispatus alone or in combination with endolysin BNT331, which specifically targets and lyses Gardnerella . These studies revealed that when L. crispatus was added alone, it suppressed inflammation even though it failed to engraft or displace the G. vaginalis bacteria. In contrast, BNT331 effectively killed Gardnerella in dysbiotic Vagina Chip. Importantly, the combined administration of both treatments resulted in restoration of a healthier vaginal microenvironment, as indicated by higher engraftment of L. crispatus on-chip, inhibition of G. vaginalis , and a reduction in inflammation. Similar effects of this combined treatment were observed when administered to Vagina Chips infected with vaginal swab samples from BV patients. These data suggest that combination of a live biotherapeutic product composed of a L. crispatus consortium with a potent antimicrobial agent that targets G. vaginalis , such as BNT331, may offer an effective therapeutic strategy for patients with BV. One Sentence Summary Endolysin BNT331 combined with Lactobacillus crispatus reduces bacterial load and suppresses inflammation in a human vagina chip model of bacterial vaginosis. ### Competing Interest Statement D.E.I. holds equity in Emulate, chairs its scientific advisory board and is a member of its board of directors. L.C. and L.P.T. are employees of BioNTech and inventors on several patent applications related to BNT331. CM has a financial interest in Ancilia Biosciences, a company developing a new class of Live Biotherapeutics and other bacterial products. The interests of CM were reviewed and are managed by MGH and Mass General Brigham in accordance with their conflict-of-interest policies. CM is on the Scientific Advisory Board of Concerto Biosciences and has served as a consultant for Freya Biosciences. The remaining authors declare no competing interests. Bill and Melinda Gates Foundation, INV-035977

ABSTRACT Background The cervicovaginal mucus that coats the upper surface of the vaginal epithelium is thought to serve as a selective barrier that helps to clear pathogens, however, its role in modulating the physiology and pathophysiology of the human vagina is poorly understood. Bacterial vaginosis (BV), a common disease of the female reproductive tract that increases susceptibility to sexually transmitted infections, pelvic inflammatory disease, infertility, preterm birth, and both maternal and neonatal infections is characterized by the presence of a wide array of strict and facultative anaerobes, often including Gardnerella vaginalis . Objective To assess the role of cervical mucus in preventing dysbiosis-associated complications and preserving vaginal health. Study Design To better understand the role of cervicovaginal mucus in vaginal health, we used human organ-on-a-chip (Organ Chip) microfluidic culture technology to analyze the effects of cervical mucus produced in a human Cervix Chip when transferred to a human Vagina Chip BV model. Both chips are lined by primary human organ-specific (cervical or vaginal) epithelium interfaced with organ-specific stromal fibroblasts. Results Our data show that mucus-containing effluents from Cervix Chips protect Vagina Chips from inflammation and epithelial cell injury caused by co-culture with dysbiotic microbiome containing G. vaginalis . Proteomic analysis of proteins produced by the Vagina Chip following treatment with the Cervix Chip mucus also revealed a collection of differentially abundant proteins that may contribute to the vaginal response to dysbiotic microbiome, which could represent potential diagnostic biomarkers or therapeutic targets for management of BV. Conclusions This study highlights the importance of cervical mucus in control of human vaginal physiology and pathophysiology, and demonstrates the potential value of Organ Chip technology for studies focused on health and diseases of the female reproductive tract.

Endothelial dysfunction, prevalent in cardiovascular diseases (CVDs) and linked to conditions like diabetes, hypertension, obesity, renal failure, or hypercholesterolemia, is characterized by diminished nitric oxide (NO) bioavailability-a key signaling molecule for vascular homeostasis. Current two-dimensional (2D) in vitro studies on NO synthesis by endothelial cells (ECs) lack the crucial laminar shear stress, a vital factor in modulating the NO-generating enzyme, endothelial nitric oxide synthase (eNOS), under physiological conditions. Here we developed a tracer-based metabolomics approach to measure NO-specific metabolites with mass spectrometry (MS) and show the impact of fluid flow on metabolic parameters associated with NO synthesis using 2D and 3D platforms. Specifically, we tracked the conversion of stable-isotope labeled NO substrate L-Arginine to L-Citrulline and L-Ornithine to determine eNOS activity. We demonstrated clear responses in human coronary artery endothelial cells (HCAECs) cultured with 13C6, 15N4-L-Arginine, and treated with eNOS stimulator, eNOS inhibitor, and arginase inhibitor. Analysis of downstream metabolites, 13C6, 15N3 L-Citrulline and 13C5, 15N2 L-Ornithine, revealed distinct outcomes. Additionally, we evaluated the NO metabolic status in static 2D culture and 3D microvessel models with bidirectional and unidirectional fluid flow. Our 3D model exhibited significant effects, particularly in microvessels exposed to the eNOS stimulator, as indicated by the 13C6, 15N3 L-Citrulline/13C5, 15N2 L-Ornithine ratio, compared to the 2D culture. The obtained results indicate that the 2D static culture mimics an endothelial dysfunction status, while the 3D model with a unidirectional fluid flow provides a more representative physiological environment that provides a better model to study endothelial dysfunction.

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.

Summary: Background: Sulfadoxine-pyrimethamine (SP) antimalarial therapy has been suggested to potentially increase the birth weight of infants in pregnant women in sub-Saharan Africa, independently of malarial infection. Here, we utilized female intestinal organoid-derived cells cultured within microfluidic Organ Chips to investigate whether SP could directly impact intestinal function and thereby improve the absorption of essential fats and nutrients crucial for fetal growth. Methods: Using a human organ-on-a-chip model, we replicated the adult female intestine with patient organoid-derived duodenal epithelial cells interfaced with human intestinal endothelial cells. Nutrient-deficient (ND) medium was perfused to simulate malnutrition, resulting in the appearance of enteric dysfunction indicators such as villus blunting, reduced mucus production, impaired nutrient absorption, and increased inflammatory cytokine secretion. SP was administered to these chips in the presence or absence of human peripheral blood mononuclear cells (PBMCs). Findings: Our findings revealed that SP treatment effectively reversed multiple intestinal absorptive abnormalities observed in malnourished female Intestine Chips, as validated by transcriptomic and proteomic analyses. SP also reduced the production of inflammatory cytokines and suppressed the recruitment of PBMCs in ND chips. Interpretation: Our results indicate that SP could potentially increase birth weights by preventing enteric dysfunction and suppressing intestinal inflammation. This underscores the potential of SP as a targeted intervention to improve maternal absorption, subsequently contributing to healthier fetal growth. While SP treatment shows promise in addressing malabsorption issues that can influence infant birth weight, we did not model pregnancy in our chips, and thus its usefulness for treatment of malnourished pregnant women requires further investigation through clinical trials. Funding: The Bill and Melinda Gates Foundation, and the Wyss Institute for Biologically Inspired Engineering at Harvard University, and the HDDC Organoid Core of the P30 DK034854.

Abstract Study question What is the underlying cause of infertility associated with bacterial vaginosis (BV)? Summary answer We provide the first evidence to suggest that BV-associated infertility could be the result of sperm dysfunction induced by exposure to poor cervical microbiome. What is known already Infertility is a global health concern, impacting 186 million individuals worldwide. In 25% of infertile couples the underlying cause remains unexplained. The complex interactions between the host mucosal epithelium, microbiome, and other microenvironmental factors play a significant role in health and disease of the reproductive tract. BV affects 30% of reproductive-aged women and is linked to overgrowth of pathogenic Gardnerella vaginalis bacteria. BV has a strong association with infertility, but it is not clear whether the resulting changes in the vaginal microbiome play a causal role, and as such BV screening is not currently included in infertility evaluation or treatment Study design, size, duration Female reproductive tract protective factors such as cervical mucus helps sperm retain their ability to fertilize for days compared to hours in suspension. We previously leveraged human organ-on-a-chip microfluidic culture technology to develop a physiological in vitro model of human cervix (Cervix Chip) that is lined with primary cervical epithelium interfaced with stromal fibroblasts. The epithelium forms a functional tissue barrier and produces mucus with compositional, biophysical, and hormone-responsive properties similar to the living cervix. Participants/materials, setting, methods We used 2D culture of sperm and bacteria as a screening and validated the effect of the identified bacteria on physiological in vitro Cervix Chip. A BV consortium containing E2 and E4 Gardnerella vaginalis bacteria was co-cultured with the Cervix Chip for 48 hours before adding human sperm samples. The motility of fluorescently labeled sperm was non-invasively evaluated in live chips using time-lapse imaging and quantitatively analyzed to obtain the percentage of motile sperm. Main results and the role of chance We initially analyzed the effects of bacterial co-cultur L. crispatus (optimal) or G. vaginalis (suboptimal) bacteria on sperm motility using a conventional 2D culture system. We observed a significant drop in sperm motility within 2 hours when exposed to G. vaginalis compared to L. crispatus or the control condition (no bacteria). We have established sperm staining and live tracking on Cervix Chip. Motile sperm was tracked on Cervix Chip for more than 7 days compared to 12 hours in suspension. We then tested this effect on-chip and observed that co-culture of live sperm with dysbiotic Cervix Chip (infected with G. vaginalis) resulted in a significant reduction in the sperm motility within 24 hours after exposure compared to the sperm exposed to the chips without bacteria. The dysbiotic Cervix Chip also exhibited a disease phenotype with elevated levels of proinflammatory cytokines including IL-1α, IL-1β, IL-6, IL-8, and TNF-α in the effluents of the chip's epithelial channel as well as significantly reduced thickness of the cervical mucus layer that correlated with reduced sperm motility. This finding is consistent with the observation that successful fertility is often associated with the quality of cervical mucus, which can be compromised in the BV dysbiotic condition. Limitations, reasons for caution Primary cervical epithelium cells used in the study were sourced from a single donor. Frozen sperm samples prepared for intrauterine insemination were used in the study Wider implications of the findings Our study suggests that infertility related to BV may be caused by sperm dysfunction, and BV screening should be considered in patients with unexplained infertility. We propose a model that can be used to identify dysbiotic conditions in the female reproductive tract that are unfavorable to sperm motility. Trial registration number N/A

Endothelial dysfunction is a common denominator in cardiovascular diseases (CVDs) associated with diabetes, hypertension, obesity, renal failure or hypercholesterolemia. In these disease states, circulating adverse metabolic or hemostatic risk factors drive the progression of inflammation, thrombosis, platelet activation and atherosclerosis. A hallmark of endothelial dysfunction is the reduced bioavailability of nitric oxide (NO), a signaling molecule essential for vascular homeostasis. Numerous studies have focused on NO synthesis by endothelial cells (ECs) using in vitro cultures to understand the pathophysiology of endothelial dysfunction. A limitation of these studies is that the expression of the NO-generating enzyme, endothelial nitric oxide synthase (eNOS), in physiological conditions is modulated by the exposure of the ECs to laminar shear stress, a stimulus that is clearly lacking in most two-dimensional (2D) cultures.Here we developed a tracer-based metabolomics approach to measure NO-specific metabolites with mass spectrometry (MS) and show the impact of unidirectional fluid flow on metabolic parameters associated with NO synthesis using 2D and three-dimensional (3D) platforms. Specifically, we tracked the conversion of stable-isotope labeled NO substrate L-Arginine to L-Citrulline and L-Ornithine to determine eNOS activity. We demonstrated that when human coronary artery endothelial cells (HCAECs) cultured in media containing 13C6,15N4-L-Arginine treated with eNOS stimulator – vascular endothelial growth factor (VEGF), eNOS inhibitor – L-NAME and arginase inhibitor - S-(2- boronoethyl)-L-cysteine (BEC), their downstream metabolites - 13C6,15N3 L-Citrulline and 13C5,15N2 L- Ornithine showed clear responses as measured using Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). In this study, we also assessed the NO metabolic status of a static 2D culture, a 3D microvessel model with bidirectional flow, and our 3D model with unidirectional fluid flow generated by a microfluidic pump. Compared to 2D culture, our 3D model showed significant effects in the control and microvessels exposed to VEGF when Citrulline/Ornithine ratio was analyzed. The obtained result indicates that the 2D static culture mimics more endothelial dysfunction status. Our detection method and 3D model with a unidirectional fluid flow provides a more representative physiological environment that exhibits perfect model to study endothelial dysfunction.### Competing Interest StatementThe authors have declared no competing interest.

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales.

Abstract New vaccine candidates evaluated in animals can lead to unpredicted toxicities or poor efficacy in human clinical trials due to species-specific differences in immune responses. To address this problem, we developed the human ectopic lymphoid follicle (LF) chip containing primary human immune cells, including autologous blood B and T lymphocytes and antigen presenting cells, cultured in a three-dimensional extracellular matrix (ECM) gel within an organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing activation-induced cytidine deaminase and exhibit plasma cell differentiation upon activation. Increased basal lymphocyte proliferation is observed in the human LF chip as compared to conventional 2D cultures lacking ECM or perfusion. The human LF chips demonstrated improved antibody responses to split virion influenza vaccination compared to conventional planar 2D cultures, which were enhanced by the addition of a squalene-in-water emulsion (SWE) adjuvant, and this was accompanied by increases in LF size and number. To extend this work, we modelled intramuscular vaccination by co-culturing monocytes with muscle cells in the presence of different vaccines and adjuvants. We found that presence of muscle cells can promote cytokine production, antigen uptake and monocyte activation. Seeding these monocytes into the LF chips with autologous T and B cells could promote cytokine production and LF formation, which extends the LF chips usage for testing intramuscular vaccination. Supported by grants from the Defense Advanced Research Projects Agency under Cooperative Agreement Number W911NF-12-2-0036, the National Institutes of Health grant UG3HL141797, Bill and Melinda Gates Foundation and the Wyss Institute for Biologically Inspired Engineering.

ABSTRACT 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 of the L. crispatus consortia 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.

Lymphoid follicles (LFs) are responsible for generation of adaptive immune responses in secondary lymphoid organs and form ectopically during chronic inflammation. A human model of ectopic LF formation will provide a tool to understand LF development and an alternative to non-human primates for preclinical evaluation of vaccines. Here, it is shown that primary human blood B- and T-lymphocytes autonomously assemble into ectopic LFs when cultured in a 3D extracellular matrix gel within one channel of a two-channel organ-on-a-chip microfluidic device. Superfusion via a parallel channel separated by a microporous membrane is required for LF formation and prevents lymphocyte autoactivation. These germinal center-like LFs contain B cells expressing Activation-Induced Cytidine Deaminase and exhibit plasma cell differentiation upon activation. To explore their utility for seasonal vaccine testing, autologous monocyte-derived dendritic cells are integrated into LF Chips. The human LF chips demonstrate improved antibody responses to split virion influenza vaccination compared to 2D cultures, which are enhanced by a squalene-in-water emulsion adjuvant, and this is accompanied by increases in LF size and number. When inoculated with commercial influenza vaccine, plasma cell formation and production of anti-hemagglutinin IgG are observed, as well as secretion of cytokines similar to vaccinated humans over clinically relevant timescales.

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.

The endothelial cells from the microvasculature are key drivers and targets of inflammatory and thrombotic processes in microvascular diseases. The study of bioactive lipids in inflammatory processes has been largely based on two-dimensional endothelial cell cultures. Three-dimensional microvessels-on-a-chip provides an opportunity to monitor the inflammatory phenotype of human microvessels in a more physiological-relevant environment. This protocol describes the culture of endothelial cells as three-dimensional microvessels in the OrganoPlate. The microvessels are treated with tumor necrosis factor alpha to induce inflammation. The collection of samples from the microvessels is optimized for measuring bioactive lipids with liquid chromatography-mass spectrometry, providing a more informative metabolic readout as compared with functional assays.