Engineered bacterial therapeutics for detecting and treating CRC.

Despite an overall decrease in occurrence, colorectal cancer (CRC) remains the third most common cause of cancer deaths in the USA. Detection of CRC is difficult in high-risk groups, including those with genetic predispositions, with disease traits, or from certain demographics. There is emerging interest in using engineered bacteria to identify early CRC development, monitor changes in the adenoma and CRC microenvironment, and prevent cancer progression. Novel genetic circuits for cancer therapeutics or functions to enhance existing treatment modalities have been tested and verified in vitro and in vivo. Inclusion of biocontainment measures would prepare strains to meet therapeutic standards. Thus, engineered bacteria present an opportunity for detection and treatment of CRC lesions in a highly sensitive and specific manner.

Tiny Organs, Big Impact: How Microfluidic Organ-on-Chip Technology Is Revolutionizing Mucosal Tissues and Vasculature.

Organ-on-chip (OOC) technology has gained importance for biomedical studies and drug development. This technology involves microfluidic devices that mimic the structure and function of specific human organs or tissues. OOCs are a promising alternative to traditional cell-based models and animals, as they provide a more representative experimental model of human physiology. By creating a microenvironment that closely resembles in vivo conditions, OOC platforms enable the study of intricate interactions between different cells as well as a better understanding of the underlying mechanisms pertaining to diseases. OOCs can be integrated with other technologies, such as sensors and imaging systems to monitor real-time responses and gather extensive data on tissue behavior. Despite these advances, OOCs for many organs are in their initial stages of development, with several challenges yet to be overcome. These include improving the complexity and maturity of these cellular models, enhancing their reproducibility, standardization, and scaling them up for high-throughput uses. Nonetheless, OOCs hold great promise in advancing biomedical research, drug discovery, and personalized medicine, benefiting human health and well-being. Here, we review several recent OOCs that attempt to overcome some of these challenges. These OOCs with unique applications can be engineered to model organ systems such as the stomach, cornea, blood vessels, and mouth, allowing for analyses and investigations under more realistic conditions. With this, these models can lead to the discovery of potential therapeutic interventions. In this review, we express the significance of the relationship between mucosal tissues and vasculature in organ-on-chip (OOC) systems. This interconnection mirrors the intricate physiological interactions observed in the human body, making it crucial for achieving accurate and meaningful representations of biological processes within OOC models. Vasculature delivers essential nutrients and oxygen to mucosal tissues, ensuring their proper function and survival. This exchange is critical for maintaining the health and integrity of mucosal barriers. This review will discuss the OOCs used to represent the mucosal architecture and vasculature, and it can encourage us to think of ways in which the integration of both can better mimic the complexities of biological systems and gain deeper insights into various physiological and pathological processes. This will help to facilitate the development of more accurate predictive models, which are invaluable for advancing our understanding of disease mechanisms and developing novel therapeutic interventions.

The Versatility and Diagnostic Potential of VOC Profiling for Noninfectious Diseases.

A variety of volatile organic compounds (VOCs) are produced and emitted by the human body every day. The identity and concentration of these VOCs reflect an individual's metabolic condition. Information regarding the production and origin of VOCs, however, has yet to be congruent among the scientific community. This review article focuses on the recent investigations of the source and detection of biological VOCs as a potential for noninvasive discrimination between healthy and diseased individuals. Analyzing the changes in the components of VOC profiles could provide information regarding the molecular mechanisms behind disease as well as presenting new approaches for personalized screening and diagnosis. VOC research has prioritized the study of cancer, resulting in many research articles and reviews being written on the topic. This review summarizes the information gained about VOC cancer studies over the past 10 years and looks at how this knowledge correlates with and can be expanded to new and upcoming fields of VOC research, including neurodegenerative and other noninfectious diseases. Recent advances in analytical techniques have allowed for the analysis of VOCs measured in breath, urine, blood, feces, and skin. New diagnostic approaches founded on sensor-based techniques allow for cheaper and quicker results, and we compare their diagnostic dependability with gas chromatography- and mass spectrometry-based techniques. The future of VOC analysis as a clinical practice and the challenges associated with this transition are also discussed and future research priorities are summarized.

Modeling collective cell behavior in cancer: Perspectives from an interdisciplinary conversation.

Collective cell behavior contributes to all stages of cancer progression. Understanding how collective behavior emerges through cell-cell interactions and decision-making will advance our understanding of cancer biology and provide new therapeutic approaches. Here, we summarize an interdisciplinary discussion on multicellular behavior in cancer, draw lessons from other scientific disciplines, and identify future directions.

Review of linear and nonlinear models in breath analysis by Cyranose 320.

Analysis of volatile organic compounds (VOCs) in breath specimens has potential for point of care (POC) screening due to ease of sample collection. While the electronic nose (e-nose) is a standard VOC measure across a wide range of industries, it has not been adopted for POC screening in healthcare. One limitation of the e-nose is the absence of mathematical models of data analysis that yield easily interpreted findings at POC. The purposes of this review were to (1) examine the sensitivity/specificity results from studies that analyzed breath smellprints using the Cyranose 320, a widely used commercial e-nose, and (2) determine whether linear or nonlinear mathematical models are superior for analyzing Cyranose 320 breath smellprints. This systematic review was conducted according to the guidelines of the Preferred Reporting Items for Systematic Review and Meta-Analyses using keywords related to e-nose and breath. Twenty-two articles met the eligibility criteria. Two studies used a linear model while the rest used nonlinear models. The two studies that used a linear model had a smaller range for mean of sensitivity and higher mean (71.0%-96.0%;M= 83.5%) compared to the studies that used nonlinear models (46.9%-100%;M= 77.0%). Additionally, studies that used linear models had a smaller range for mean of specificity and higher mean (83.0%-91.5%;M= 87.2%) compared to studies that used nonlinear models (56.9%-94.0%;M= 76.9%). Linear models achieved smaller ranges for means of sensitivity and specificity compared to nonlinear models supporting additional investigations of their use for POC testing. Because our findings were derived from studies of heterogenous medical conditions, it is not known if they generalize to specific diagnoses.

Causative Role of Anoxic Environment in Bacterial Regulation of Human Intestinal Function.

Introduction: Life on Earth depends on oxygen; human tissues require oxygen signaling, whereas many microorganisms, including bacteria, thrive in anoxic environments. Despite these differences, human tissues and bacteria coexist in close proximity to each other such as in the intestine. How oxygen governs intestinal-bacterial interactions remains poorly understood. Methods: To address to this gap, we created a dual-oxygen environment in a microfluidic device to study the role of oxygen in regulating the regulation of intestinal enzymes and proteins by gut bacteria. Two-layer microfluidic devices were designed using a fluid transport model and fabricated using soft lithography. An oxygen-sensitive material was integrated to determine the oxygen levels. The intestinal cells were cultured in the upper chamber of the device. The cells were differentiated, upon which bacterial strains, a facultative anaerobe, Escherichia coli Nissle 1917, and an obligate anaerobe, Bifidobacterium Adolescentis, were cultured with the intestinal cells. Results: The microfluidic device successfully established a dual-oxygen environment. Of particular importance in our findings was that both strains significantly upregulated mucin proteins and modulated several intestinal transporters and transcription factors but only under the anoxic-oxic oxygen gradient, thus providing evidence of the role of oxygen on bacterial-epithelial signaling. Conclusions: Our work that integrates cell and molecular biology with bioengineering presents a novel strategy to engineer an accessible experimental system to provide tailored oxygenated environments. The work could provide new avenues to study intestine-microbiome signaling and intestinal tissue engineering, as well as a novel perspective on the indirect effects of gut bacteria on tissues including tumors. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-022-00735-x.

The SARS-CoV-2 S1 Spike Protein Promotes MAPK and NF-kB Activation in Human Lung Cells and Inflammatory Cytokine Production in Human Lung and Intestinal Epithelial Cells.

The coronavirus disease 2019 (COVID-19) pandemic began in January 2020 in Wuhan, China, with a new coronavirus designated SARS-CoV-2. The principal cause of death from COVID-19 disease quickly emerged as acute respiratory distress syndrome (ARDS). A key ARDS pathogenic mechanism is the "Cytokine Storm", which is a dramatic increase in inflammatory cytokines in the blood. In the last two years of the pandemic, a new pathology has emerged in some COVID-19 survivors, in which a variety of long-term symptoms occur, a condition called post-acute sequelae of COVID-19 (PASC) or "Long COVID". Therefore, there is an urgent need to better understand the mechanisms of the virus. The spike protein on the surface of the virus is composed of joined S1-S2 subunits. Upon S1 binding to the ACE2 receptor on human cells, the S1 subunit is cleaved and the S2 subunit mediates the entry of the virus. The S1 protein is then released into the blood, which might be one of the pivotal triggers for the initiation and/or perpetuation of the cytokine storm. In this study, we tested the hypothesis that the S1 spike protein is sufficient to activate inflammatory signaling and cytokine production, independent of the virus. Our data support a possible role for the S1 spike protein in the activation of inflammatory signaling and cytokine production in human lung and intestinal epithelial cells in culture. These data support a potential role for the SARS-CoV-2 S1 spike protein in COVID-19 pathogenesis and PASC.

Patient-derived pancreatic cancer-on-a-chip recapitulates the tumor microenvironment.

The patient population suffering from pancreatic ductal adenocarcinoma (PDAC) presents, as a whole, with a high degree of molecular tumor heterogeneity. The heterogeneity of PDAC tumor composition has complicated treatment and stalled success in clinical trials. Current in vitro techniques insufficiently replicate the intricate stromal components of PDAC tumor microenvironments (TMEs) and fail to model a given tumor's unique genetic phenotype. The development of patient-derived organoids (PDOs) has opened the door for improved personalized medicine since PDOs are derived directly from patient tumors, thus preserving the tumors' unique behaviors and genetic phenotypes. This study developed a tumor-chip device engineered to mimic the PDAC TME by incorporating PDOs and stromal cells, specifically pancreatic stellate cells and macrophages. Establishing PDOs in a multicellular microfluidic chip device prolongs cellular function and longevity and successfully establishes a complex organotypic tumor environment that incorporates desmoplastic stroma and immune cells. When primary cancer cells in monoculture were subjected to stroma-depleting agents, there was no effect on cancer cell viability. However, targeting stroma in our tumor-chip model resulted in a significant increase in the chemotherapy effect on cancer cells, thus validating the use of this tumor-chip device for drug testing.

Investigation of insulin resistance through a multiorgan microfluidic organ-on-chip.

The development of hepatic insulin resistance (IR) is a critical factor in developing type 2 diabetes (T2D), where insulin fails to inhibit hepatic glucose production but retains its capacity to promote hepatic de novo lipogenesis leading to hyperglycemia and hypertriglyceridemia. Improving insulin sensitivity can be effective in preventing and treating T2D. However, selective control of glucose and lipid synthesis has been difficult. It is known that excess white adipose tissue is detrimental to insulin sensitivity, whereas brown adipose tissue transplantation can restore it in diabetic mice. However, challenges remain in our understanding of liver-adipose communication because the confounding effects of hypothalamic regulation of metabolic function cannot be ruled out in previous studies. There is a lack ofin vitromodels that use primary cells to study cellular-crosstalk under insulin resistant conditions. Building upon our previous work on the microfluidic primary liver and adipose organ-on-chips, we report for the first time, the development of an integrated insulin resistant liver-adipose (white and brown) organ-on-chip. The design of the microfluidic device was carried out using computational fluid dynamics; the experimental studies were conducted by carrying out detailed biochemical analysis RNA-seq analysis on both cell types. Further, we tested the hypothesis that brown adipocytes (BAC) regulated both hepatic insulin sensitivity and de novo lipogenesis. Our results show that BAC effectively restored insulin sensitivity and supressed hepatic glucose production and de novo lipogenesis suggesting that the experimental platform could be useful for identifying potential therapeutics to treat IR and diabetes.

Organ-Chip Models: Opportunities for Precision Medicine in Pancreatic Cancer.

Pancreatic Ductal Adenocarcinoma (PDAC) is an expeditiously fatal malignancy with a five-year survival rate of 6-8%. Conventional chemotherapeutics fail in many cases due to inadequate primary response and rapidly developing resistance. This treatment failure is particularly challenging in pancreatic cancer because of the high molecular heterogeneity across tumors. Additionally, a rich fibro-inflammatory component within the tumor microenvironment (TME) limits the delivery and effectiveness of anticancer drugs, further contributing to the lack of response or developing resistance to conventional approaches in this cancer. As a result, there is an urgent need to model pancreatic cancer ex vivo to discover effective drug regimens, including those targeting the components of the TME on an individualized basis. Patient-derived three-dimensional (3D) organoid technology has provided a unique opportunity to study patient-specific cancerous epithelium. Patient-derived organoids cultured with the TME components can more accurately reflect the in vivo tumor environment. Here we present the advances in organoid technology and multicellular platforms that could allow for the development of "organ-on-a-chip" approaches to recapitulate the complex cellular interactions in PDAC tumors. We highlight the current advances of the organ-on-a-chip-based cancer models and discuss their potential for the preclinical selection of individualized treatment in PDAC.

Towards an Insulin Resistant Adipose Model on a Chip.

INTRODUCTION: Adipose tissue and adipocytes are primary regulators of insulin sensitivity and energy homeostasis. Defects in insulin sensitivity of the adipocytes predispose the body to insulin resistance (IR) that could lead to diabetes. However, the mechanisms mediating adipocyte IR remain elusive, which emphasizes the need to develop experimental models that can validate the insulin signaling pathways and discover new mechanisms in the search for novel therapeutics. Currently in vitro adipose organ-chip devices show superior cell function over conventional cell culture. However, none of these models represent disease states. Only when these in vitro models can represent both healthy and disease states, they can be useful for developing therapeutics. Here, we establish an organ-on-chip model of insulin-resistant adipocytes, as well as characterization in terms of insulin signaling pathway and lipid metabolism. METHODS: We differentiated, maintained, and induced insulin resistance into primary adipocytes in a microfluidic organ-on-chip. We then characterized IR by looking at the insulin signaling pathway and lipid metabolism, and validated by studying a diabetic drug, rosiglitazone. RESULTS: We confirmed the presence of insulin resistance through reduction of Akt phosphorylation, Glut4 expression, Glut4 translocation and glucose uptake. We also confirmed defects of disrupted insulin signaling through reduction of lipid accumulation from fatty acid uptake and elevation of glycerol secretion. Testing with rosiglitazone showed a significant improvement in insulin sensitivity and fatty acid metabolism as suggested by previous reports. CONCLUSIONS: The adipose-chip exhibited key characteristics of IR and can serve as model to study diabetes and facilitate discovery of novel therapeutics.

A novel standalone microfluidic device for local control of oxygen tension for intestinal-bacteria interactions.

The intestinal environment is unique because it supports the intestinal epithelial cells under a normal oxygen environment and the microbiota under an anoxic environment. Due to importance of understanding the interactions between the epithelium and the microbiota, there is a strong need for developing representative and simple experimental models. Current approaches do not capture the partitioned oxygen environment, require external anaerobic chambers, or are complex. Another major limitation is that with the solutions that can mimic this oxygen environment, the oxygenation level of the epithelial cells is not known, raising the question whether the cells are hypoxic or not. We report standalone microfluidic devices that form a partitioned oxygen environment without the use of an external anaerobic chamber or oxygen scavengers to coculture intestinal epithelial and bacterial cells. By changing the thickness of the device cover, the oxygen tension in the chamber was modulated. We verified the oxygen levels using several tests: microscale oxygen sensitive sensors which were integrated within the devices, immunostaining of Caco-2 cells to determine hypoxia levels, and genetically encoded bacteria to visualize the growth. Collectively, these methods monitored oxygen concentrations in the devices more comprehensively than previous reports and allowed for control of oxygen tension to match the requirements of both intestinal cells and anaerobic bacteria. Our experimental model is supported by the mathematical model that considered diffusion of oxygen into the top chamber. This allowed us to experimentally determine the oxygen consumption rate of the intestinal epithelial cells under perfusion.

A 3D human adipose tissue model within a microfluidic device.

An accurate in vitro model of human adipose tissue could assist in the study of adipocyte function and allow for better tools for screening new therapeutic compounds. Cell culture models on two-dimensional surfaces fall short of mimicking the three-dimensional in vivo adipose environment, while three-dimensional culture models are often unable to support long-term cell culture due, in part, to insufficient mass transport. Microfluidic systems have been explored for adipose tissue models. However, current systems have primarily focused on 2D cultured adipocytes. In this work, a 3D human adipose microtissue was engineered within a microfluidic system. Human adipose-derived stem cells (ADSCs) were used as the cell source for generating differentiated adipocytes. The ADSCs differentiated within the microfluidic system formed a dense lipid-loaded mass with the expression of adipose tissue genetic markers. Engineered adipose tissue showed a decreased adiponectin secretion and increased free fatty acid secretion with increasing shear stress. Adipogenesis markers were downregulated with increasing shear stress. Overall, this microfluidic system enables the on-chip differentiation and development of a functional 3D human adipose microtissue supported by the interstitial flow. This system could potentially serve as a platform for in vitro drug testing for adipose tissue-related diseases.

Establishment and characterization of a primary murine adipose tissue-chip.

Better experimental models are needed to enhance our understanding of metabolic regulation which is seen in obesity and metabolic disorders, such as type 2 diabetes. In vitro models based on microfluidics enable physiological representations of tissues with several advantages over conventional culture systems, such as perfused flow to better mimic the physiological environment. Although cell lines such as 3T3-L1 have been incorporated in microfluidic devices, murine primary preadipocytes have not been differentiated and maintained for long-term monitoring in these culture systems. We describe the differentiation of these cells into white adipose depots on a perfused microfluidic chip. We compare the effects of shear flow on these cells, and show with a direct comparison of high/low shear conditions that direct shear is detrimental to the viability of preadipocytes. We further develop a dual-chamber microfluidic chip that enables perfusion while at the same time protects the cells from direct fluidic shear. We show that the dual-layer microfluidic device enables long-term culture of cells and allows stimulation of cells through perfusion-we can culture, differentiate, and maintain the differentiated adipose tissue for over multiple weeks in the device. Both triglycerides and lipolytic glycerol production increased significantly by several folds during differentiation. After successful differentiation, the adipocytes had upregulated expression of leptin and adiponectin, which are important makers of the final stage of adipogenic differentiation. In conclusion, the dual-layer microfluidic device incorporated with primary adipocytes improves the understanding of adipose differentiation under dynamic conditions and is positioned to serve as a disease model for studying obesity and other metabolic disorders.

Novel Microfluidic Colon with an Extracellular Matrix Membrane.

Collagen is a key element of basal lamina in physiological systems that participates in cell and tissue culture. Its function is for cell maintenance and growth, angiogenesis, disease progression, and immunology. The goal of our present study was to integrate a micrometer resolution membrane that is synthesized out of rat-tail type I collagen in a microfluidic device with apical and basolateral chambers. The collagen membrane was generated by lyophilization. In order to evaluate the compatibility of the resulting membrane with organs-on-chips technology, it was sandwiched between layers of polydimethylsiloxane (PDMS) that had been prepared by replica molding, and the device was used to culture human colon caco 2 cells on the top of the membrane. Membrane microstructure, transport, and cell viability in the organs-on-chips were observed to confirm the suitability of our resulting membrane. Through transport studies, we compared diffusion of two different membranes: Transwell and our resulting collagen membrane. We found that mass transport of 40 kDa dextran was an order of magnitude higher through the collagen membrane than that through the Transwell membrane. Human colon caco 2 cells were cultured in devices with no, Transwell, or ECM membrane to evaluate the compatibility of cells on the ECM membrane compared to the other two membranes. We found that caco 2 cells cultured on the collagen membrane had excellent viability and function for extended periods of time compared to the other two devices. Our results indicate a substantial improvement in establishing a physiological microenvironment for in vitro organs-on-chips.

Real-time measurement of cholesterol secreted by human hepatocytes using a novel microfluidic assay.

The use of microfluidics has become widespread in recent years because of the use of lesser resources such as small size, low volume of reagents, and physiological representation of mammalian cells. One of the advantages of microfluidic-based cell culture is the ability to perfuse culture media which tends to improve cellular health and function. Although measurement of cellular function conventionally is carried out using well-plates and plate readers, these approaches are insufficient to carry out in-line analysis of perfused cell cultures because of mismatch between volumes and sensitivity. We report the development of a novel microfluidic device and assay that is carried out under perfusion, in-line to measure the cholesterol secreted from a human hepatocyte tissue-chip. The heart of the assay is the unique implementation of enzymatic chemistry that is carried out on a polystyrene bead. Using this approach, we successfully measured cholesterol secreted by the perfused human hepatocytes.

A microfluidic in-line ELISA for measuring secreted protein under perfusion.

Recent progress in the development of microfluidic microphysiological systems such as 'organs-on-chips' and microfabricated cell culture is geared to simulate organ-level physiology. These tissue models leverage microengineering technologies that provide capabilities of presenting cultured cells with input signals in a more physiologically relevant context such as perfused flow. Proteins that are secreted from cells have important information about the health of the cells. Techniques to quantify cellular proteins include mass spectrometry to ELISA (enzyme-linked immunosorbent assay). Although our capability to perturb the cells in the microphysiological systems with varying inputs is well established, we lack the tools to monitor in-line the cellular responses. User intervention for sample collection and off-site is cumbersome, causes delays in obtaining results, and is especially expensive because of collection, storage, and offline processing of the samples, and in many case, technically impractical to carry out because of limitated sample volumes. To address these shortcomings, we report the development of an ELISA that is carried out in-line under perfusion within a microfluidic device. Using this assay, we measured the albumin secreted from perfused hepatocytes without and under stimulation by IL-6. Since the method is based on a sandwich ELISA, we envision broad application of this technology to not just organs-on-chips but also to characterizing the temporal release and measurement of soluble factors and response to drugs.

Long-term maintenance of a microfluidic 3D human liver sinusoid.

The development of long-term human organotypic liver-on-a-chip models for successful prediction of toxic response is one of the most important and urgent goals of the NIH/DARPA's initiative to replicate and replace chronic and acute drug testing in animals. For this purpose, we developed a microfluidic chip that consists of two microfluidic chambers separated by a porous membrane. The aim of this communication is to demonstrate the recapitulation of a liver sinusoid-on-a-chip, using human cells only for a period of 28 days. Using a step-by-step method for building a 3D microtissue on-a-chip, we demonstrate that an organotypic in vitro model that reassembles the liver sinusoid microarchitecture can be maintained successfully for a period of 28 days. In addition, higher albumin synthesis (synthetic) and urea excretion (detoxification) were observed under flow compared to static cultures. This human liver-on-a-chip should be further evaluated in drug-related studies.