Analysis of Interleukin 8 Secretion by a Stem-Cell-Derived Human-Intestinal-Epithelial-Monolayer Platform.

In vitro models of the human intestinal epithelium derived from primary stem cells are much needed for the study of intestinal immunology in health and disease. Here, we describe an intestinal monolayer cultured on a porous membrane with accessible basal and apical surfaces for assay of intestinal cytokine production in response to stimuli. The system was composed of a differentiated, confluent epithelial monolayer derived from human primary stem cells obtained from small or large intestine. Interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) were the most abundant inflammatory cytokines produced by the intestinal epithelium. The epithelium from all five tested regions of the intestine preferentially secreted into the apical reservoir of the monolayer, with a 26-fold greater concentration of IL-8 present in the apical reservoir of the colonic monolayer relative to that in the basal reservoir. Upon application of tumor-necrosis factor α (TNF-α) to the basal surface of the colonic monolayer, the IL-8 concentration significantly increased in the basal, but not the apical, reservoir. A dose-dependent elevation of IL-8 in the basal reservoir was observed for TNF-α-stimulation of the monolayer but not for an organoid-based platform. To demonstrate the utility of the monolayer system, 88 types of dietary metabolites or compounds were screened for their ability to modulate IL-8 production in the basal reservoir of the intestinal monolayer in the absence or presence of TNF-α. No dietary metabolite or compound caused an increase in IL-8 in the basal reservoir in the absence of TNF-α. After addition of TNF-α to the monolayer, two compounds (butyrate and gallic acid) suppressed IL-8 production, suggesting their potential anti-inflammatory effects, whereas the dietary factor forskolin significantly increased IL-8 production. These results demonstrate that the described human-intestinal-monolayer platform has the potential for assays and screening of metabolites and compounds that alter the inflammatory response of the intestine.

Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts.

Sphingolipids play important roles in plasma membrane structure and cell signaling. However, their lateral distribution in the plasma membrane is poorly understood. Here we quantitatively analyzed the sphingolipid organization on the entire dorsal surface of intact cells by mapping the distribution of (15)N-enriched ions from metabolically labeled (15)N-sphingolipids in the plasma membrane, using high-resolution imaging mass spectrometry. Many types of control experiments (internal, positive, negative, and fixation temperature), along with parallel experiments involving the imaging of fluorescent sphingolipids--both in living cells and during fixation of living cells--exclude potential artifacts. Micrometer-scale sphingolipid patches consisting of numerous (15)N-sphingolipid microdomains with mean diameters of ∼200 nm are always present in the plasma membrane. Depletion of 30% of the cellular cholesterol did not eliminate the sphingolipid domains, but did reduce their abundance and long-range organization in the plasma membrane. In contrast, disruption of the cytoskeleton eliminated the sphingolipid domains. These results indicate that these sphingolipid assemblages are not lipid rafts and are instead a distinctly different type of sphingolipid-enriched plasma membrane domain that depends upon cortical actin.

Microfluidic gut-axis-on-a-chip models for pharmacokinetic-based disease models.

The low success rate of new drugs transitioning from animal testing to human clinical trials necessitates the development of more accurate and representative in vitro models. Recent advances in multi-organ-on-a-chip technology offer promising avenues for studying complex organ-organ interactions. Gut-liver-on-a-chip systems hold particular promise for mimicking the intricate interplay between the gut and liver, which play crucial roles in nutrient absorption, drug metabolism, detoxification, and immune response. Here, we discuss the key components of the gut-liver axis, including the gut epithelium, liver cells, gut microbiota, and their roles in the organ functions. We then explore the potential of gut-liver-on-a-chip models to replicate the intricate interactions between the two organs for pharmacokinetic studies and their expansion to more complicated multi-organ models. Finally, we provide perspectives and future directions for developing more physiologically relevant gut-liver-axis models for more efficient drug development, studying liver diseases, and personalizing treatment strategies.

Recent Advances in Gut- and Gut-Organ-Axis-on-a-Chip Models.

The human gut extracts nutrients from the diet while forming the largest barrier against the outer environment. In addition, the gut actively maintains homeostasis through intricate interactions with the gut microbes, the immune system, the enteric nervous system, and other organs. These interactions influence digestive health and, furthermore, play crucial roles in systemic health and disease. Given its primary role in absorbing and metabolizing orally administered drugs, there is significant interest in the development of preclinical in vitro model systems that can accurately emulate the intestine in vivo. A gut-on-a-chip system holds great potential as a testing and screening platform because of its ability to emulate the physiological aspects of in vivo tissues and expandability to incorporate and combine with other organs. This review aims to identify the key physiological features of the human gut that need to be incorporated to build more accurate preclinical models and highlights the recent progress in gut-on-a-chip systems and competing technologies toward building more physiologically relevant preclinical model systems. Furthermore, various efforts to construct multi-organ systems with the gut, called gut-organ-axis-on-a-chip models, are discussed. In vitro gut models with physiological relevance can provide valuable platforms for bridging the gap between preclinical and clinical studies.

A Microphysiological System with an Anaerobic Air-Liquid Interface and Functional Mucus Layer for Coculture of Intestinal Bacteria and Primary Human Colonic Epithelium.

Coculture of intestinal bacteria with primary human intestinal epithelium provides a valuable tool for investigating host-colon bacterial interactions and for testing and screening therapeutics. However, most current intestinal model systems lack key physiological features of the in vivo colon, such as both a proper oxygen microenvironment and a mucus layer. In this work, a new in vitro colonic microphysiological system is demonstrated with a cell-derived, functional mucus that closely resembles the in vivo colonic mucosa and apical microenvironment by employing an anaerobic air-liquid interface culture. The human primary colon epithelial cells in this new in vitro system exhibit high cell viability (>98%) with ≈100 µm thick functional mucus layer on average. Successful coculture of model anaerobic gut bacterial strains Lactobacillus rhamnosus GG and Anaerobutyricum hallii without loss in human cell viability demonstrates that this new model can be an invaluable tool for future studies of the impact of commensal and pathogenic bacteria on the colon.

In vitro co-culture of Clostridium scindens with primary human colonic epithelium protects the epithelium against Staphylococcus aureus.

A complex and dynamic network of interactions exists between human gastrointestinal epithelium and intestinal microbiota. Therefore, comprehending intestinal microbe-epithelial cell interactions is critical for the understanding and treatment of intestinal diseases. Primary human colonic epithelial cells derived from a healthy human donor were co-cultured with Clostridium scindens (C. scindens), a probiotic obligate anaerobe; Staphylococcus aureus (S. aureus), a facultative anaerobe and intestinal pathogen; or both bacterial species in tandem. The co-culture hanging basket platform used for these experiments possessed walls of controlled oxygen (O2) permeability to support the formation of an O2 gradient across the intestinal epithelium using cellular O2 consumption, resulting in an anaerobic luminal and aerobic basal compartment. Both the colonic epithelial cells and C. scindens remained viable over 48 h during co-culture. In contrast, co-culture with S. aureus elicited significant damage to colonic epithelial cells within 24 h. To explore the influence of the intestinal pathogen on the epithelium in the presence of the probiotic bacteria, colonic epithelial cells were inoculated sequentially with the two bacterial species. Under these conditions, C. scindens was capable of repressing the production of S. aureus enterotoxin. Surprisingly, although C. scindens converted cholic acid to secondary bile acids in the luminal medium, the growth of S. aureus was not significantly inhibited. Nevertheless, this combination of probiotic and pathogenic bacteria was found to benefit the survival of the colonic epithelial cells compared with co-culture of the epithelial cells with S. aureus alone. This platform thus provides an easy-to-use and low-cost tool to study the interaction between intestinal bacteria and colonic cells in vitro to better understand the interplay of intestinal microbiota with human colonic epithelium.

A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells.

Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.

Advanced Organotypic In Vitro Model Systems for Host-Microbial Coculture.

In vitro model systems have been advanced to recapitulate important physiological features of the target organ in vivo more closely than the conventional cell line cultures on a petri dish. The advanced organotypic model systems can be used as a complementary or alternative tool for various testing and screening. Numerous data from germ-free animal studies and genome sequencings of clinical samples indicate that human microbiota is an essential part of the human body, but current in vitro model systems rarely include them, which can be one of the reasons for the discrepancy in the tissue phenotypes and outcome of therapeutic intervention between in vivo and in vitro tissues. A coculture model system with appropriate microbes and host cells may have great potential to bridge the gap between the in vitro model and the in vivo counterpart. However, successfully integrating two species in one system introduces new variables to consider and poses new challenges to overcome. This review aims to provide perspectives on the important factors that should be considered for developing organotypic bacterial coculture models. Recent advances in various organotypic bacterial coculture models are highlighted. Finally, challenges and opportunities in developing organotypic microbial coculture models are also discussed.

A Platform for Co-Culture of Primary Human Colonic Epithelium With Anaerobic Probiotic Bacteria.

An in vitro platform was designed and optimized for the co-culture of probiotic anaerobic bacteria with a primary human colonic epithelium having a goal of assessing the anti-inflammatory impact of the probiotic bacteria. The device maintained a luminal O2 concentration at <1% while also supporting an oxygenated basal compartment at 10% for at least 72 h. Measurement of the transepithelial resistance of a confluent colonic epithelium showed high monolayer integrity while fluorescence assays demonstrated that the monolayer was comprised primarily of goblet cells and colonocytes, the two major differentiated cell subtypes of the colonic epithelium. High monolayer barrier function and viability were maintained during co-culture of the epithelium with the probiotic obligate anaerobe Anaerobutyricum hallii (A. hallii). Importantly the device supported a static co-culture of microbes and colonic epithelium mimicking the largely static or low flow conditions within the colonic lumen. A model inflamed colonic epithelium was generated by the addition of tumor necrosis factor-α (TNF-α) and lipopolysaccharide (LPS) to the basal and luminal epithelium sides, respectively. Co-culture of A. hallii with the LPS/TNF-α treated intestine diminished IL-8 secretion by ≥40% which could be mimicked by co-culture with the A. hallii metabolite butyrate. In contrast, co-culture of the inflamed epithelium with two strains of lactic acid-producing bacteria, Lactobacillus rhamnosus GG (LGG) and Bifidobacterium adolescentis (B. adolescentis), did not diminish epithelial IL-8 secretion. Co-culture with colonic epithelial cells from different donors demonstrated a consistent anti-inflammatory effect by A. hallii, but distinct responses to co-culture with LGG and B. adolescentis. The demonstrated system offers a simple and easily adopted platform for examining the physiologic impact of alterations in the intestinal epithelium that occur in the presence of probiotic bacteria and their metabolites.

Pure State v-Representability of Density Matrix Embedding Theory.

Density matrix embedding theory (DMET) formally requires the matching of density matrix blocks obtained from high-level and low-level theories, but this is sometimes not achievable in practical calculations. In such a case, the global band gap of the low-level theory vanishes, and this can require additional numerical considerations. We find that both the violation of the exact matching condition and the vanishing low-level gap are related to the assumption that the high-level density matrix blocks are noninteracting pure-state v-representable (NI-PS-V), which assumes that the low-level density matrix is constructed following the Aufbau principle. To relax the NI-PS-V condition, we develop an augmented Lagrangian method to match the density matrix blocks without referring to the Aufbau principle. Numerical results for the 2D Hubbard and hydrogen model systems indicate that, in some challenging scenarios, the relaxation of the Aufbau principle directly leads to exact matching of the density matrix blocks, which also yields improved accuracy.

Suspended Collagen Hydrogels to Replicate Human Colonic Epithelial Cell Interactions with Immune Cells.

The human colon plays a critical role in fluid and salt absorption and harbors the largest immune compartment. There is a widespread need for in vitro models of human colon physiology with its innate immune system. A method is described to produce a cassette with a network of struts supporting a suspended, non-chemically cross-linked collagen hydrogel scaffold compatible with the co-culture of primary gastrointestinal epithelium and migratory inflammatory cells. The epithelial monolayer cultured on the suspended collagen possesses a population of polarized and differentiated cells similar to that present in vivo. This epithelial layer displays proper barrier function with a transepithelial electrical resistance (TEER) ≥ 1,500 Ω cm2 and an apparent permeability ≤10-5 cm2 s-1 . Immune cells plated on the basal face of the scaffold transmigrated over a period of 24 h to the epithelial layer in response to epithelial production of IL-8 induced by luminal stimulation of Clostridium difficile Toxin A. These studies demonstrate that this in vitro platform possesses a functional primary colonic epithelial layer with an immune cell compartment capable of recruitment in response to pro-inflammatory cues coming from the epithelium.

Graph Transformer Networks: Learning meta-path graphs to improve GNNs.

Graph Neural Networks (GNNs) have been widely applied to various fields due to their powerful representations of graph-structured data. Despite the success of GNNs, most existing GNNs are designed to learn node representations on the fixed and homogeneous graphs. The limitations especially become problematic when learning representations on a misspecified graph or a heterogeneous graph that consists of various types of nodes and edges. To address these limitations, we propose Graph Transformer Networks (GTNs) that are capable of generating new graph structures, which preclude noisy connections and include useful connections (e.g., meta-paths) for tasks, while learning effective node representations on the new graphs in an end-to-end fashion. We further propose enhanced version of GTNs, Fast Graph Transformer Networks (FastGTNs), that improve scalability of graph transformations. Compared to GTNs, FastGTNs are up to 230× and 150× faster in inference and training, and use up to 100× and 148× less memory while allowing the identical graph transformations as GTNs. In addition, we extend graph transformations to the semantic proximity of nodes allowing non-local operations beyond meta-paths. Extensive experiments on both homogeneous graphs and heterogeneous graphs show that GTNs and FastGTNs with non-local operations achieve the state-of-the-art performance for node classification tasks. The code is available: https://github.com/seongjunyun/Graph_Transformer_Networks.

In vitro generation of self-renewing human intestinal epithelia over planar and shaped collagen hydrogels.

The large intestine, with its array of crypts lining the epithelium and diverse luminal contents, regulates homeostasis throughout the body. In vitro crypts formed from primary human intestinal epithelial stem cells on a 3D shaped hydrogel scaffold replicate the functional and architectural features of in vivo crypts. Collagen scaffolding assembly methods are provided, along with the microfabrication and soft lithography protocols necessary to shape these hydrogels to match the dimensions and density of in vivo crypts. In addition, stem-cell scale-up protocols are provided so that even ultrasmall primary samples can be used as starting material. Initially, these cells are seeded as a proliferative monolayer over the shaped scaffold and cultured as stem/proliferative cells to expand them and cover the scaffold surface with the crypt-shaped structures. To convert these immature crypts into fully polarized, functional units with a basal stem cell niche and luminal differentiated cell zone, stable, linear gradients of growth factors are formed across the crypts. This platform supports the formation of chemical gradients across the crypts, including those of growth and differentiation factors, inflammatory compounds, bile and food metabolites and bacterial products. All microfabrication and device assembly steps are expected to take 8 d, with the primary cells cultured for 12 d to form mature in vitro crypts.

Stem/Proliferative and Differentiated Cells within Primary Murine Colonic Epithelium Display Distinct Intracellular Free Ca2+ Signal Codes.

The second messenger, intracellular free calcium (Ca2+ ), acts to transduce mitogenic and differentiation signals incoming to the colonic epithelium. A self-renewing monolayer of primary murine colonic epithelial cells is formed over a soft, transparent hydrogel matrix for the scalable analysis of intracellular Ca2+ transients. Cultures that are enriched for stem/proliferative cells exhibit repetitive, high frequency (≈25 peaks h-1 ), and short pulse width (≈25 s) Ca2+ transients. Upon cell differentiation the transient frequency declines by 50% and pulse width widens by 200%. Metabolites and growth factors that are known to modulate stem cell proliferation and differentiation through Wnt and Notch signaling pathways, including CHIR-99021, N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), bone morphogenetic proteins (BMPs), and butyrate, also modulate Ca2+ oscillation patterns in a consistent manner. Increasing the stiffness of the supportive matrix from 200 Pa to 3 GPa shifts Ca2+ transient patterns toward those resembling differentiated cells. The ability to monitor Ca2+ oscillations with the spatial and temporal resolution offered by this platform, combined with its amenability to high-content screens, provides a powerful tool for investigating real-time communication within a wide range of primary tissues in addition to the colonic epithelium.

Hyperglycemia minimally alters primary self-renewing human colonic epithelial cells while TNFα-promotes severe intestinal epithelial dysfunction.

Hyperglycemia is thought to increase production of inflammatory cytokines and permeability of the large intestine. Resulting intestinal inflammation is then often characterized by excess secretion of tumor necrosis factor alpha (TNFα). Thus, hyperglycemia in hospitalized patients suffering from severe trauma or disease is frequently accompanied by TNFα secretion, and the combined impact of these insults on the intestinal epithelium is poorly understood. This study utilized a simple yet elegant model of the intestinal epithelium, comprised of primary human intestinal stem cells and their differentiated progeny, to investigate the impact of hyperglycemia and inflammatory factors on the colonic epithelium. When compared to epithelium cultured under conditions of physiologic glucose, cells under hyperglycemic conditions displayed decreased mucin-2 (MUC2), as well as diminished alkaline phosphatase (ALP) activity. Conditions of 60 mM glucose potentiated secretion of the cytokine IL-8 suggesting that cytokine secretion during hyperglycemia may be a source of tissue inflammation. TNFα measurably increased secretion of IL-8 and IL-1ß, which was enhanced at 60 mM glucose. Surprisingly, intestinal permeability and paracellular transport were not altered by even extreme levels of hyperglycemia. The presence of TNFα increased MUC2 presence, decreased ALP activity, and negatively impacted monolayer barrier function. When TNFα hyperglycemia and ≤30 mM glucose and were combined, MUC2 and ALP activity remained similar to that of TNFα alone, although synergistic effects were seen at 60 mM glucose. An automated image analysis pipeline was developed to assay changes in properties of the zonula occludens-1 (ZO-1)-demarcated cell boundaries. While hyperglycemia alone had little impact on cell shape and size, cell morphologic properties were extraordinarily sensitive to soluble TNFα. These results suggest that TNFα acted as the dominant modulator of the epithelium relative to glucose, and that control of inflammation rather than glucose may be key to maintaining intestinal homeostasis.

Microphysiological System Design: Simplicity Is Elegance.

Design parameters for microphysiological systems (MPS) are driven by the need for new tools to answer questions focusing on human physiology in a robust and reliable manner. Within this perspective, engineering benchmarks and principles are identified to guide the construction of new devices in the MPS field, with emphasis placed on the design principles common to all tissues, as well as those unique to a subset of tissues. Leading organ replica technologies that recapitulate various functions of the brain, heart, intestine, and lung are highlighted as examples that meet the identified benchmarks and standards, with current barriers for large scale production and commercialization discussed. To reach their full potential and achieve widespread use, MPS will have to be recognized officially by government agencies, and toward this end, considerations of MPS as a potential regulatory tool are presented.

Tunable layer-by-layer polyelectrolyte platforms for comparative cell assays.

We developed a cell-based assay based on the spin-assisted layer-by-layer (LbL) assembled polyelectrolyte matrix platforms. Three types of human breast epithelial cell lines including normal cells (184B5), noncancerous fibrocystic disease cells (MCF 10F), and metastatic cancerous cells (CAMA-1) were cultured, analyzed, and compared in parallel on various LbL-assembled polymer films. Poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) electrolyte polymers were used as the basic building units to form various LbL polyelectrolyte matrices. The mechanical rigidity, surface charge, and biorecognition property of the LbL platforms were controlled by tailoring the LbL surface, thermal cross-linking, and protein modification. Cellular phenotypic changes in adhesion, proliferation, and morphology on these LbL films were characterized and analyzed for the three different cell types. Our analysis results indicate that the cellular phenotype can be controlled by taking advantage of different surface charge, mechanical property, and biological modification (i.e., fibronectin in this case) of the LbL multilayer platforms. Importantly, cell phenotypical quantification results show that the cell spreading area per cell and optical density are useful parameters in distinguishing metastatic cancer cells from normal or fibrocystic disease cells on these LbL films. These LbL-based cell assay platforms have a potential for the development of various disease diagnostic cell assays.

Comparison of Tightening Screw Accuracy of Electronic Torque Drivers.

PURPOSE: To compare the tightening torque accuracy of three electronic torque drivers. MATERIALS AND METHODS: Three electronic torque drivers were assessed using two measurement methods-pure output torque (POT) and clinical output torque (COT). For both methods, assessments were performed at set torques of 10, 20, 30, and 40 Ncm, 10 times for each setting, with each driver tested. Appropriate statistical analysis was performed according to data distribution (ie, normal vs non-normal) (α = .05). RESULTS: POT was significantly higher than COT at a set torque of 30 Ncm (P < .001). CONCLUSION: In fastening an implant screw at 30 Ncm, the operator should also consider the output torque generated in the electronic torque driver.

Primary Cell-Derived Intestinal Models: Recapitulating Physiology.

The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field.

An in vitro intestinal platform with a self-sustaining oxygen gradient to study the human gut/microbiome interface.

An oxygen gradient formed along the length of colonic crypts supports stem-cell proliferation at the normoxic crypt base while supporting obligate anaerobe growth in the anoxic colonic lumen. Primary human colonic epithelial cells derived from human gastrointestinal stem cells were cultured within a device possessing materials of tailored oxygen permeability to produce an oxygen-depleted luminal (0.8% ± 0.1% O2) and oxygen-rich basal (11.1% ± 0.5% O2) compartment. This oxygen difference created a stable oxygen gradient across the colonic epithelial cells which remained viable and properly polarized. Facultative and obligate anaerobes Lactobacillus rhamnosus, Bifidobacterium adolescentis, and Clostridium difficile grew readily within the luminal compartment. When formed along the length of an in vitro crypt, the oxygen gradient facilitated cell compartmentalization within the crypt by enhancing confinement of the proliferative cells to the crypt base. This platform provides a simple system to create a physiological oxygen gradient across an intestinal mimic while simultaneously supporting anaerobe co-culture.