Organ Chips Research Articles

Disrupting Tumour Niches: Advanced Strategies for Targeting the Tumour Microenvironment in Cancer Therapy

Abstract: The review paper provides an extensive overview of strategies for targeting the tumour microenvironment (TME) to enhance cancer therapy. It begins by underscoring the importance of profiling and comprehending the TME through advanced technologies like organ chips and artificial intelligence. The paper discusses multiple approaches to modulate the pro-tumour TME, including strategies for eliminating, normalizing, and targeting tumour cells. It delves into specific aspects such as cancer-associated fibroblasts, extracellular matrix, hypoxia, acidosis, neovascularisation, tumour-infiltrating T cells, the immune system, exosomes, tumour-associated neutrophils, and tumour angiogenesis. Emphasis is placed on the necessity of a multifaceted approach to effectively target the complex and dynamic TME, which plays a crucial role in tumour progression and therapeutic resistance. The conclusion highlights the significant impact of the TME on cancer therapy and identifies promising research and clinical application avenues. The paper underscores the shift in cancer treatment paradigms, advocating for strategies that address the intricate interactions within the TME to improve therapeutic outcomes.

A Pumpless, High‐Throughput Microphysiological System to Mimic Enteric Innervation of Duodenal Epithelium and the Impact on Barrier Function

AbstractEnteric neurons are critical in maintaining organ homeostasis within the small intestine, and their dysregulation are implicated in gastrointestinal disorders and neurodegenerative diseases. Most in vitro models lack enteric innervation, limiting basic discovery and disease modeling research. Here, a high‐throughput 3D microphysiological system (MPS), or organ chip is presented that supports a primary epithelial monolayer interfacing directly with encapsulated primary enteric neurons. The device features twelve 3D MPSs per device and gravity‐driven flow via a laboratory rocker to induce biomimetic shear stress on the epithelium culture and provide continuous nutrient presentation. Intestinal and neural tissue exhibited expected morphologies. Neural gene upregulation in the epithelium suggests RNA contamination from proximal enteric neurons extending neurites toward the epithelial monolayer. With the enteric nervous system (ENS), barrier integrity significantly increased for both TEER and permeability assays, a 1.25‐fold greater resistance and 10% lower permeability as compared to epithelium cultured alone. The presence of the ENS resulted in a significant (1.4‐fold) reduction in epidermal growth factor (EGF). Additionally, several key epithelial genes are compared between duodenal tissue and epithelial monolayers with and without neurons present. Results demonstrated changes in cytokine gene expression and WNT pathways, highlighting innervation is essential to create more biomimetic and physiologically relevant in vitro models.

Intestinal organ chips for disease modelling and personalized medicine.

Alterations in intestinal structure, mechanics and physiology underlie acute and chronic intestinal conditions, many of which are influenced by dysregulation of microbiome, peristalsis, stroma or immune responses. Studying human intestinal physiology or pathophysiology is difficult in preclinical animal models because their microbiomes and immune systems differ from those of humans. Although advances in organoid culture partially overcome this challenge, intestinal organoids still lack crucial features that are necessary to study functions central to intestinal health and disease, such as digestion or fluid flow, as well as contributions from long-term effects of living microbiome, peristalsis and immune cells. Here, we review developments in organ-on-a-chip (organ chip) microfluidic culture models of thehuman intestine that are lined by epithelial cells and interfaced with other tissues (such as stroma or endothelium), which can experience both fluid flow and peristalsis-like motions. Organ chips offer powerful ways to model intestinal physiology and disease states for various human populations and individual patients, and can be used to gain new insight into underlying molecular and biophysical mechanisms of disease. They can also be used as preclinical tools to discover new drugs and then validate their therapeutic efficacy and safety in the same human-relevant model.

Organ chips with integrated multifunctional sensors enable continuous metabolic monitoring at controlled oxygen levels

Despite remarkable advances in Organ-on-a-chip (Organ Chip) microfluidic culture technology, recreating tissue-relevant physiological conditions, such as the region-specific oxygen concentrations, remains a formidable technical challenge, and analysis of tissue functions is commonly carried out using one analytical technique at a time. Here, we describe two-channel Organ Chip microfluidic devices fabricated from polydimethylsiloxane and gas impermeable polycarbonate materials that are integrated with multiple sensors, mounted on a printed circuit board and operated using a commercially available Organ Chip culture instrument. The novelty of this system is that it enables the recreation of physiologically relevant tissue-tissue interfaces and oxygen tension as well as non-invasive continuous measurement of transepithelial electrical resistance, oxygen concentration and pH, combined with simultaneous analysis of cellular metabolic activity (ATP/ADP ratio), cell morphology, and tissue phenotype. We demonstrate the reliable and reproducible functionality of this system in living human Gut and Liver Chip cultures. Changes in tissue barrier function and oxygen tension along with their functional and metabolic responses to chemical stimuli (e.g., calcium chelation, oligomycin) were continuously and noninvasively monitored on-chip for up to 23 days. A physiologically relevant microaerobic microenvironment that supports co-culture of human intestinal cells with living Lactococcus lactis bacteria also was demonstrated in the Gut Chip. The integration of multi-functional sensors into Organ Chips provides a robust and scalable platform for the simultaneous, continuous, and non-invasive monitoring of multiple physiological functions that can significantly enhance the comprehensive and reliable evaluation of engineered tissues in Organ Chip models in basic research, preclinical modeling, and drug development.

Advances in gut-brain organ chips.

The brain and gut are sensory organs responsible for sensing, transmitting, integrating, and responding to signals from the internal and external environment. In-depth analysis of brain-gut axis interactions is important for human health and disease prevention. Current research on the brain-gut axis primarily relies on animal models. However, animal models make it difficult to study disease mechanisms due to inherent species differences, and the reproducibility of experiments is poor because of individual animal variations, which leads to a significant limitation of real-time sensory responses. Organ-on-a-chip platforms provide an innovative approach for disease treatment and personalized research by replicating brain and gut ecosystems in vitro. This enables a precise understanding of their biological functions and physiological responses. In this article, we examine the history and most current developments in brain, gut, and gut-brain chips. The importance of these systems for understanding pathophysiology and developing new drugs is emphasized throughout the review. This article also addresses future directions and present issues with the advancement and application of gut-brain-on-a-chip technologies.

Publisher's note

Substance use disorders (SUDs) are complex mental health conditions involving a problematic pattern of substance use. Challenges remain in understanding their neural mechanisms, which are likely to lead to improved SUD treatments. Human brain organoids, brain-like 3D in vitro cultures derived from human stem cells, show unique potential in recapitulating the response of a developing human brain to substances. Here, we review the recent progress in understanding SUDs using human brain organoid models focusing on neurodevelopmental perspectives. We first summarize the background of SUDs in humans. Moreover, we introduce the development of various human brain organoid models and then discuss current progress and findings underlying the abuse of substances like nicotine, alcohol, and other addictive drugs using organoid models. Furthermore, we review efforts to develop organ chips and microphysiological systems to engineer better human brain organoids for advancing SUD studies. Lastly, we conclude by elaborating on the current challenges and future directions of SUD studies using human brain organoids.

3D embedded bioprinting of large-scale intestine with complex structural organization and blood capillaries

Accurate reproduction of human intestinal structure and function in vitro is of great significance for understanding the development and disease occurrence of the gut. However, most in vitro studies are often confined to 2D models, 2.5D organ chips or 3D organoids, which cannot fully recapitulate the tissue architecture, microenvironment and cell compartmentalization found in vivo. Herein, a centimeter-scale intestine tissue that contains intestinal features, such as hollow tubular structure, capillaries and tightly connected epithelium with in vivo-like ring folds, crypt-villi, and microvilli is constructed by 3D embedding bioprinting. In our strategy, a novel photocurable bioink composed of methacrylated gelatin, methacrylated sodium alginate and poly (ethylene glycol) diacrylate is developed for the fabrication of intestinal model. The Caco-2 cells implanted in the lumen are induced by the topological structures of the model to derive microvilli, crypt-villi, and tight junctions, simulating the intestinal epithelial barrier. The human umbilical vein endothelial cells encapsulated within the model gradually form microvessels, mimicking the dense capillary network in the intestine. This intestine-like tissue, which closely resembles the structure and cell arrangement of the human gut, can act as a platform to predict the therapeutic and toxic side effects of new drugs on the intestine.

Surface Deformation of Biocompatible Materials: Recent Advances in Biological Applications.

The surface topography of substrates is a crucial factor that determines the interaction with biological materials in bioengineering research. Therefore, it is important to appropriately modify the surface topography according to the research purpose. Surface topography can be fabricated in various forms, such as wrinkles, creases, and ridges using surface deformation techniques, which can contribute to the performance enhancement of cell chips, organ chips, and biosensors. This review provides a comprehensive overview of the characteristics of soft, hard, and hybrid substrates used in the bioengineering field and the surface deformation techniques applied to the substrates. Furthermore, this review summarizes the cases of cell-based research and other applications, such as biosensor research, that utilize surface deformation techniques. In cell-based research, various studies have reported optimized cell behavior and differentiation through surface deformation, while, in the biosensor and biofilm fields, performance improvement cases due to surface deformation have been reported. Through these studies, we confirm the contribution of surface deformation techniques to the advancement of the bioengineering field. In the future, it is expected that the application of surface deformation techniques to the real-time interaction analysis between biological materials and dynamically deformable substrates will increase the utilization and importance of these techniques in various fields, including cell research and biosensors.

Lubricant-Coated Organ-on-a-Chip for Enhanced Precision in Preclinical Drug Testing.

In drug discovery, human organ-on-a-chip (organ chip) technology has emerged as an essential tool for preclinical testing, offering a realistic representation of human physiology, real-time monitoring, and disease modeling. Polydimethylsiloxane (PDMS) is commonly used in organ chip fabrication owing to its biocompatibility, flexibility, transparency, and ability to replicate features down to the nanoscale. However, the porous nature of PDMS leads to unintended absorption of small molecules, critically affecting the drug response analysis. Addressing this challenge, the precision drug testing organ chip (PreD chip) is introduced, an innovative platform engineered to minimize small molecule absorption while facilitating cell culture. This chip features a PDMS microchannel wall coated with a perfluoropolyether-based lubricant, providing slipperiness and antifouling properties. It also incorporates an ECM-coated semi-porous membrane that supports robust multicellular cultures. The PreD chip demonstrates its outstanding antifouling properties and resistance to various biological fluids, small molecule drugs, and plasma proteins. In simulating the human gut barrier, the PreD chip demonstrates highly enhanced sensitivity in tests for dexamethasone toxicity and is highly effective in assessing drug transport across the human blood-brain barrier. These findings emphasize the potential of the PreD chip in advancing organ chip-based drug testing methodologies.

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.

Lymph Node-on-Chip Technology: Cutting-Edge Advances in Immune Microenvironment Simulation.

Organ-on-a-chip technology is attracting growing interest across various domains as a crucial platform for drug screening and testing and is set to play a significant role in precision medicine research. Lymph nodes, being intricately structured organs essential for the body's adaptive immune responses to antigens and foreign particles, are pivotal in assessing the immunotoxicity of novel pharmaceuticals. Significant progress has been made in research on the structure and function of the lymphatic system. However, there is still an urgent need to develop prospective tools and techniques to delve deeper into its role in various diseases' pathological and physiological processes and to develop corresponding immunotherapeutic therapies. Organ chips can accurately reproduce the specific functional areas in lymph nodes to better simulate the complex microstructure of lymph nodes and the interactions between different immune cells, which is convenient for studying specific biological processes. This paper reviews existing lymph node chips and their design approaches. It discusses the applications of the above systems in modeling immune cell motility, cell-cell interactions, vaccine responses, drug testing, and cancer research. Finally, we summarize the challenges that current research faces in terms of structure, cell source, and extracellular matrix simulation of lymph nodes, and we provide an outlook on the future direction of integrated immune system chips.

Human implantable tertiary lymphoid organs (TLO) for solid tumor therapy: from organ chips to the clinic?

Abstract We previously created an in vitro human TLO model from patient-derived, circulating immune cells using organ-on-a-chip devices and 3D culture in extracellular matrix (ECM). B cells in these TLOs express activation induced cytidine deaminase, which is only expressed in lymphoid tissues and is required for class switching and somatic hypermutation (SHM). When challenged by vaccines, these TLO undergo SHM and produce antigen-specific antibodies and CD8 T cells. To model cancer associated TLOs, we integrated human pancreatic and lung cancer cell lines into these TLO Chips to understand how the immune context of hot and cold tumors differentially impacts TLO formation. The high PDL1/L2 expressing lung cancer cell line that displayed a “hot” phenotype in published murine studies induced high levels of cytokines and stimulated increased TLO formation in the lymphoid Organ Chip. In contrast, the cold, low PDL1/L2 expressing lung and pancreatic cell lines did not induce this cytokine signature or TLO formation. Importantly, TLO assembly correlated with increased B cell activation, anti-tumor CD8 activity, and tumor cell death. When the same mix of immune cells and ECM was injected in humanized mice bearing subcutaneous tumors from the hot lung cancer cell line, new lymphoid tissue containing dense aggregates of T and B cells was induced, showing for the first time that direct injection of synthetic TLOs at the tumor site may offer an alternative form of therapy for solid tumors.

3D bioprinting in otorhinolaryngology: from bench to clinical application

Three-dimensional (3D) bioprinting is a promising additive manufacturing technology that uses imaging data and computer-assisted deposition of biological materials or cells to reconstruct complex 3D structures accurately. This technology has progressed rapidly, in part because of its integration across multiple disciplines and combination with other technologies for clinical applications. Advances in experimental research and clinical applications related to otorhinolaryngology have led to the development of diagnostic and treatment methods based on 3D bioprinting, including the development of tissue engineering scaffolds, biosensors, organ chips, and organoids, surgical planning, graft construction, and medical education. Additionally, otorhinolaryngologists will be better equipped to treat tissue function defects with personalized printed graft implants. It is also expected that 3D printing can be used to build ideal in vitro models in the future to help solve existing research challenges. This article briefly introduces the relevant 3D bioprinting technologies and bioinks that can be used by otorhinolaryngologists and discusses their potential applications in otorhinolaryngology.

Volatile carbonyl metabolites analysis of nanoparticle exposed lung cells in an organ-on-a-chip system

The evaluation of nanoparticles (NPs) cytotoxicity is crucial for advancing nanotechnology and assessing environmental pollution. However, existing methods for NPs cytotoxicity evaluation suffer from limited accuracy and inadequate information content. In the study, we developed a novel detection platform that enables the identification of cellular carbonyl metabolites at the organ level. The platform is integrated with a cell co-culture lung organ chip (LOC) and a micropillar concentrator. Notably, our work represents the successful measurement of the amounts of cellular metabolites on LOC system. The volatile carbonyl metabolites (VCMs) generated by cells exposure to various types of NPs with different concentrations were captured and detected by high-resolution mass spectrometry (MS). Compared with conventional cell viability and reactive oxygen species (ROS) analysis, our method discerns the toxicological impact of NPs at low concentrations by analyzed VCM at levels as low as ppb level. The LOC system based metabolic gas detection confirmed that low concentrations of NPs have a toxic effect on the cell model, which was not reflected in the fluorescence detection, and the effect of NP material is more significant than the size effect. Furthermore, this method can distinguish different NPs acting on cell models through cluster analysis of multiple VCMs.

Abstract 6791: Investigation of sex hormone effects in an organ chip model of endothelial barrier dysfunction in the triple negative breast cancer microenvironment

Abstract Inflammation and pathological vasculature are common features of the tumor microenvironment (TME). Sex hormones potently modulate vascular physiology and immune cell functions, and there are established correlations with the outcomes of triple negative (TNBC) despite the defining feature of receptor negativity. Circulating estrogens and androgens have been hypothesized as drivers of TNBC through the actions exerted by receptor-positive cells in the TME. Here, we present an organ chip model of the TNBC microenvironment for quantifying the impact of configurable TME variables such as the ECM composition and the relative densities and phenotypes of tumor cells, cancer associated fibroblasts, and myeloid cells on endothelial barrier function. After establishing baselines of vascular permeability in the presence and absence of TNBC cells, we tested the effect of estradiol, estrone, and dihydrotestosterone on barrier permeability. Multilayer polydimethylsiloxane (PDMS) organ chips were fabricated using standard soft lithography. The layers of the device were separated with semi-permeable membranes that allow exchange of soluble factors. Once assembled, the upper channel was loaded with female (XX) human umbilical vein endothelial cells. Collagen hydrogels were injected into the tissue layer and the devices were cultured on a rocker to induce endothelial barrier alignment via application of an average shear stress of 5 dynes/cm2. We optimized rocking conditions by assessing endothelial layer alignment, junctional organization, and permeability. TNBC cells and XX mammary carcinoma fibroblasts were added to the hydrogel to model the effects of TME-derived signals on endothelial barrier permeability. 40 kDa FITC- Dextran was chosen to assay permeability as it can show leakage due to active disruption of the barrier. Staining for F-actin and ZO-1 allowed for quantification of junctional organization. Sex hormone modulation of proinflammatory gene expression was assessed by RT-qPCR. The presence of TNBC cells significantly increased transport of 40kDa FITC-Dextran across the endothelial barrier, disrupted intercellular junction quality, and induced the expression of inflammatory adhesion molecules including ICAM-1. Our results demonstrate that sex hormones modulate TNBC-associated changes in endothelial cell bioenergetics, junctional organization, and macromolecular barrier permeability. Ongoing work will investigate the effects of age-matched XX monocyte derived macrophages on barrier function in the model. This organ chip platform provides an assay for measuring altered vascular inflammation and permeability in the TNBC microenvironment and may provide a venue for investigating vascular-normalizing effects of various candidate therapeutics. Citation Format: Delia Carlino, Ashley Martier, Yasmin Maurice, G. Wills Kpeli, Matthew Burow, Mark Mondrinos. Investigation of sex hormone effects in an organ chip model of endothelial barrier dysfunction in the triple negative breast cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6791.

Organ-on-chip technology's development and usages: A comprehensive review

Organ-on-a-chip technology is a new development technology that has made great contributions to the field of biomedicine-medicine, which began to be studied in 1950 but has not achieved effective results. With the development of technology and the passage of time, the first successful organ-on-a-chip products were introduced in 1990. Organ-on-chip technology has broken through the limitations of human drug testing and pathological research, solved the problem of unsatisfactory efficacy after using animal simulation, and greatly promoted the development of pathology and pharmacology. Organ-on-a-chip refers to the cell culture of human organs on a chip to simulate the function and characteristics of human organs, providing a new way for disease treatment and drug research and development. The researchers began by trying to grow cells on artificial substrates to mimic the structure and function of human organs. Organ-on-a-chip technology can process cell cultures while improving the environment for human drug testing and pathology research, truly simulating and performing experiments on humans. Organ-on-a-chip is a cell culture analog that utilizes a microfluidic device in which microchannels are used to guide and manipulate the solution. In recent years, with the development of bio print technology, the research of organ chips has entered a new stage. Bioprinting allows cells and biomaterials to be arranged in predetermined patterns and structures to build tissues and organs with specific functions. At present, the research of organ chips can already help us complete the test drug and case analysis, the development time of this technology is very short, and there is still a lot of development and exploration space in the future.

Promising preclinical models for lung cancer research-lung cancer organoids: a narrative review.

Traditional cell line models are the commonly used preclinical models for lung cancer research. However, cell lines cannot recapitulate the complex tumor heterogeneity and cannot mimic the microenvironment of human cancer. Recently, 3D multicellular in vitro self-assembled models called "organoids" have been developed at a fast pace in the field of research, which can mimic the actual primary tumor. At present, several studies have reported on protocols of lung cancer organoids (LCOs) generation, and using LCOs can provide novel insight into the basic and translational research of lung cancer. However, the establishment of the LCO models remains challenging due to the complexity of lung cancer and the immaturity of organoid technology, so it is necessary to understand the influences of different methodologies on LCO generation and review the applications and limitations of LCO models. In this review, we searched the literature in the recent ten years in the field of LCOs. We summarized the methodology, the problems, and the solutions in the LCOs generation, its application and limitations, as well as proposing future challenges and perspectives. Currently, LCOs are successfully generated via exploring the methodology by the researchers. Though there are still challenges in clinical application, LCOs are applied in some cancer studies including investigation of anti-cancer treatment response in vitro, modeling tumor immune microenvironment, and construction of organ chips, which are forging a promising path towards precision medicine.

Addressing the ADME Challenges of Compound Loss in a PDMS-Based Gut-on-Chip Microphysiological System.

Microphysiological systems (MPSs) are promising in vitro technologies for physiologically relevant predictions of the human absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates. However, polydimethylsiloxane (PDMS), a common material used in MPSs, can both adsorb and absorb small molecules, thereby compromising experimental results. This study aimed to evaluate the feasibility of using the PDMS-based Emulate gut-on-chip to determine the first-pass intestinal drug clearance. In cell-free PDMS organ-chips, we assessed the loss of 17 drugs, among which testosterone was selected as a model compound for further study based on its substantial ad- and absorptions to organ chips and its extensive first-pass intestinal metabolism with well-characterized metabolites. A gut-on-chip model consisting of epithelial Caco-2 cells and primary human umbilical vein endothelial cells (HUVECs) was established. The barrier integrity of the model was tested with reference compounds and inhibition of drug efflux. Concentration-time profiles of testosterone were measured in cell-free organ chips and in gut-on-chip models. A method to deduce the metabolic clearance was provided. Our results demonstrate that metabolic clearance can be determined with PDMS-based MPSs despite substantial compound loss to the chip. Overall, this study offers a practical protocol to experimentally assess ADME properties in PDMS-based MPSs.

Bioprinted research models of urological malignancy.

Urological malignancy (UM) is among the leading threats to health care worldwide. Recent years have seen much investment in fundamental UM research, including mechanistic investigation, early diagnosis, immunotherapy, and nanomedicine. However, the results are not fully satisfactory. Bioprinted research models (BRMs) with programmed spatial structures and functions can serve as powerful research tools and are likely to disrupt traditional UM research paradigms. Herein, a comprehensive review of BRMs of UM is presented. It begins with a brief introduction and comparison of existing UM research models, emphasizing the advantages of BRMs, such as modeling real tissues and organs. Six kinds of mainstream bioprinting techniques used to fabricate such BRMs are summarized with examples. Thereafter, research advances in the applications of UM BRMs, such as culturing tumor spheroids and organoids, modeling cancer metastasis, mimicking the tumor microenvironment, constructing organ chips for drug screening, and isolating circulating tumor cells, are comprehensively discussed. At the end of this review, current challenges and future development directions of BRMs and UM are highlighted from the perspective of interdisciplinary science.

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.