[Ru(dpp)3]Cl2-Embedded Oxygen Nano Polymeric Sensors: A Promising Tool for Monitoring Intracellular and Intratumoral Oxygen Gradients with High Quantum Yield and Long Lifetime.

Unraveling the intricacies between oxygen dynamics and cellular processes in the tumor microenvironment (TME) hinges upon precise monitoring of intracellular and intratumoral oxygen levels, which holds paramount significance. The majority of these reported oxygen nanoprobes suffer compromised lifetime and quantum yield when exposed to the robust ROS activities prevalent in TME, limiting their prolonged in vitro usability. Herein, the ruthenium-embedded oxygen nano polymeric sensor (Ru-ONPS) is proposed for precise oxygen gradient monitoring within the cellular environment and TME. Ru-ONPS (≈64±7 nm) incorporates [Ru(dpp)3]Cl2 dye into F-127 and crosslinks it with urea and paraformaldehyde, ensuring a prolonged lifetime (5.4 µs), high quantum yield (66.65 ± 2.43% in N2 and 49.80 ± 3.14% in O2), superior photostability (>30 min), and excellent stability in diverse environmental conditions. Based on the Stern-Volmer plot, the Ru-ONPS shows complete linearity for a wide dynamic range (0-23 mg L-1), with a detection limit of 10 µg mL-1. Confocal imaging reveals Ru-ONPS cellular uptake and intratumoral distribution. After 72 h, HCT-8 cells show 5.20±1.03% oxygen levels, while NIH3T3 cells have 7.07±1.90%. Co-culture spheroids display declining oxygen levels of 17.90±0.88%, 10.90±0.88%, and 5.10±1.18%, at 48, 120, and 216 h, respectively. Ru-ONPS advances cellular oxygen measurement and facilitates hypoxia-dependent metastatic research and therapeutic target identification.

Oxygen gradients dictate angiogenesis but not barriergenesis in a 3D brain microvascular model.

A variety of biophysical properties are known to regulate angiogenic sprouting, and in vitro systems can parse the individual effects of these factors in a controlled setting. Here, a three-dimensional brain microvascular model interrogates how variables including extracellular matrix composition, fluid shear stress, and radius of curvature affect angiogenic sprouting of cerebral endothelial cells. Tracking endothelial migration over several days reveals that application of fluid shear stress and enlarged vessel radius of curvature both attenuate sprouting. Computational modeling informed by oxygen consumption assays suggests that sprouting correlates to reduced oxygen concentration: both fluid shear stress and vessel geometry alter the local oxygen levels dictated by both ambient conditions and cellular respiration. Moreover, increasing cell density and consequently lowering the local oxygen levels yields significantly more sprouting. Further analysis reveals that the magnitude of oxygen concentration is not as important as its spatial concentration gradient: decreasing ambient oxygen concentration causes significantly less sprouting than applying an external oxygen gradient to the vessels. In contrast, barriergenesis is dictated by shear stress independent of local oxygen concentrations, suggesting that different mechanisms mediate angiogenesis and barrier formation and that angiogenic sprouting can occur without compromising the barrier. Overall, these results improve our understanding of how specific biophysical variables regulate the function and activation of cerebral vasculature, and identify spatial oxygen gradients as the driving factor of angiogenesis in the brain.

Study 3D Endothelial Cell Network Formation under Various Oxygen Microenvironment and Hydrogel Composition Combinations Using Upside-Down Microfluidic Devices.

Formation of 3D networks is a crucial process for endothelial cells during development of primary blood vessels under both normal and pathological conditions. In order to investigate effects of oxygen microenvironment and matrix composition on the 3D network formation, an upside-down microfluidic cell culture device capable of generating oxygen gradients is developed in this paper. In cell experiments, network formation of human umbilical vein endothelial cells (HUVECs) within fibrinogen-based hydrogels with different concentrations of hyaluronic acid (HA) is systematically studied. In addition, five different oxygen microenvironments (uniform normoxia, 5%, and 1% O2 ; oxygen gradients under normoxia and 5% O2 ) are also applied for the cell culture. The generated oxygen gradients are characterized based on fluorescence lifetime measurements. The experimental results show increased 3D cell network length when the cells are cultured under the oxygen gradients within the hydrogels with the HA addition suggesting their roles in promoting network formation. Furthermore, the formed networks tend to align along the direction of the oxygen gradients indicating the presence of gradient-driven cellular response. The results demonstrate that the developed upside-down microfluidic device can provide an advanced platform to investigate 3D cell culture under the controlled oxygen microenvironments for various biomedical studies in vitro.

Mapping the biological activities of filamentous oxygenic photogranules.

Oxygenic photogranules have been suggested as alternatives to activated sludge in wastewater treatment. Challenging for modeling photogranule-based processes is the heterogeneity of photogranule morphologies, resulting in different activities by photogranule type. The measurement of microscale-activities of filamentous photogranules is particularly difficult because of their labile interfaces. We present here an experimental and modeling approach to quantify phototrophic O2 production, heterotrophic O2 consumption, and O2 diffusion in filamentous photogranules. We used planar optodes for the acquisition of spatio-temporal oxygen distributions combined with two-dimensional mathematical modeling. Light penetration into the photogranule was the factor controlling photogranule activities. The spatial distribution of heterotrophs and phototrophs had less impact. The photosynthetic response of filaments to light was detectable within seconds, emphasizing the need to analyze dynamics of light exposure of individual photogranules in photobioreactors. Studying other recurring photogranule morphologies will eventually enable the description of photogranule-based processes as the interplay of interacting photogranule populations.

Transcapillary PO2 gradients in contracting muscles across the fibre type and oxidative continuum.

KEY POINTS: Within skeletal muscle the greatest resistance to oxygen transport is thought to reside across the short distance at the red blood cell-myocyte interface. These structures generate a significant transmural oxygen pressure (PO2 ) gradient in mixed fibre-type muscle. Increasing O2 flux across the capillary wall during exercise depends on: (i) the transmural O2 pressure gradient, which is maintained in mixed-fibre muscle, and/or (ii) elevating diffusing properties between microvascular and interstitial compartments resulting, in part, from microvascular haemodynamics and red blood cell distribution. We evaluated the PO2 within the microvascular and interstitial spaces of muscles spanning the slow- to fast-twitch fibre and high- to low-oxidative capacity spectrums, at rest and during contractions, to assess the magnitude of transcapillary PO2 gradients in rats. Our findings demonstrate that, across the metabolic rest-contraction transition, the transcapillary pressure gradient for O2 flux is: (i) maintained in all muscle types, and (ii) the lowest in contracting highly oxidative fast-twitch muscle. ABSTRACT: In mixed fibre-type skeletal muscle transcapillary PO2 gradients (PO2 mv-PO2 is; microvascular and interstitial, respectively) drive O2 flux across the blood-myocyte interface where the greatest resistance to that O2 flux resides. We assessed a broad spectrum of fibre-type and oxidative-capacity rat muscles across the rest-to-contraction (1 Hz, 120 s) transient to test the novel hypotheses that: (i) slow-twitch PO2 is would be greater than fast-twitch, (ii) muscles with greater oxidative capacity have greater PO2 is than glycolytic counterparts, and (iii) whether PO2 mv-PO2 is at rest is maintained during contractions across all muscle types. PO2 mv and PO2 is were determined via phosphorescence quenching in soleus (SOL; 91% type I+IIa fibres and CSa: ∼21 µmol min-1 g-1 ), peroneal (PER; 33% and ∼20 µmol min-1 g-1 ), mixed (MG; 9% and ∼26 µmol min-1 g-1 ) and white gastrocnemius (WG; 0% and ∼8 µmol min-1 g-1 ) across the rest-contraction transient. PO2 mv was higher than PO2 is in each muscle (∼6-13 mmHg; P < 0.05). SOL PO2 isarea was greater than in the fast-twitch muscles during contractions (P < 0.05). Oxidative muscles had greater PO2 isnadir (9.4 ± 0.8, 7.4 ± 0.9 and 6.4 ± 0.4; SOL, PER and MG, respectively) than WG (3.0 ± 0.3 mmHg, P < 0.05). The magnitude of PO2 mv-PO2 is at rest decreased during contractions in MG only (∼11 to 7 mmHg; time × (PO2 mv-PO2 is) interaction, P < 0.05). These data support the hypothesis that, since transcapillary PO2 gradients during contractions are maintained in all muscle types, increased O2 flux must occur via enhanced intracapillary diffusing conductance, which is most extreme in highly oxidative fast-twitch muscle.

Ratiometric Nanoparticle Probe Based on FRET-Amplified Phosphorescence for Oxygen Sensing with Minimal Phototoxicity.

Luminescent oxygen probes enable direct imaging of hypoxic conditions in cells and tissues, which are associated with a variety of diseases, including cancer. Here, a nanoparticle probe that addresses key challenges in the field is developed, it: i) strongly amplifies room temperature phosphorescence of encapsulated oxygen-sensitive dyes; ii) provides ratiometric response to oxygen; and iii) solves the fundamental problem of phototoxicity of phosphorescent sensors. The nanoprobe is based on 40 nm polymeric nanoparticles, encapsulating ≈2000 blue-emitting cyanine dyes with fluorinated tetraphenylborate counterions, which are as bright as 70 quantum dots (QD525). It functions as a light-harvesting nanoantenna that undergoes efficient Förster resonance energy transfer to ≈20 phosphorescent oxygen-sensitive platinum octaethylporphyrin (PtOEP) acceptor dyes. The obtained nanoprobe emits stable blue fluorescence and oxygen-sensitive red phosphorescence, providing ratiometric response to dissolved oxygen. The light harvesting leads to ≈60-fold phosphorescence amplification and makes the single nanoprobe particle as bright as ≈1200 PtOEP dyes. This high brightness enables oxygen detection at a single-particle level and in cells at ultra-low nanoprobe concentration with no sign of phototoxicity, in contrast to PtOEP dye. The developed nanoprobe is successfully applied to the imaging of a microfluidics-generated oxygen gradient in cancer cells. It constitutes a promising tool for bioimaging of hypoxia.

Skeletal muscle interstitial O2 pressures: bridging the gap between the capillary and myocyte.

The oxygen transport pathway from air to mitochondria involves a series of transfer steps within closely integrated systems (pulmonary, cardiovascular, and tissue metabolic). Small and finite O2 stores in most mammalian species require exquisitely controlled changes in O2 flux rates to support elevated ATP turnover. This is especially true for the contracting skeletal muscle where O2 requirements may increase two orders of magnitude above rest. This brief review focuses on the mechanistic bases for increased microvascular blood-myocyte O2 flux (V̇O2 ) from rest to contractions. Fick's law dictates that V̇O2 elevations driven by muscle contractions are produced by commensurate changes in driving force (ie, O2 pressure gradients; ΔPO2 ) and/or effective diffusing capacity (DO2 ). While previous evidence indicates that increased DO2 helps modulate contracting muscle O2 flux, up until recently the role of the dynamic ΔPO2 across the capillary wall was unknown. Recent phosphorescence quenching investigations of both microvascular and novel interstitial PO2 kinetics in health have resolved an important step in the O2 cascade between the capillary and myocyte. Specifically, the significant transmural ΔPO2 at rest was sustained (but not increased) during submaximal contractions. This supports the contention that the blood-myocyte interface provides a substantial effective resistance to O2 diffusion and underscores that modulations in erythrocyte hemodynamics and distribution (DO2 ) are crucial to preserve the driving force for O2 flux across the capillary wall (ΔPO2 ) during contractions. Investigation of the O2 transport pathway close to muscle mitochondria is key to identifying disease mechanisms and develop therapeutic approaches to ameliorate dysfunction and exercise intolerance.

Skeletal muscle microvascular and interstitial PO2 from rest to contractions.

KEY POINTS: Oxygen pressure gradients across the microvascular walls are essential for oxygen diffusion from blood to tissue cells. At any given flux, the magnitude of these transmural gradients is proportional to the local resistance. The greatest resistance to oxygen transport into skeletal muscle is considered to reside in the short distance between red blood cells and myocytes. Although crucial to oxygen transport, little is known about transmural pressure gradients within skeletal muscle during contractions. We evaluated oxygen pressures within both the skeletal muscle microvascular and interstitial spaces to determine transmural gradients during the rest-contraction transient in anaesthetized rats. The significant transmural gradient observed at rest was sustained during submaximal muscle contractions. Our findings support that the blood-myocyte interface provides substantial resistance to oxygen diffusion at rest and during contractions and suggest that modulations in microvascular haemodynamics and red blood cell distribution constitute primary mechanisms driving increased transmural oxygen flux with contractions. ABSTRACT: Oxygen pressure (PO2) gradients across the blood-myocyte interface are required for diffusive O2 transport, thereby supporting oxidative metabolism. The greatest resistance to O2 flux into skeletal muscle is considered to reside between the erythrocyte surface and adjacent sarcolemma, although this has not been measured during contractions. We tested the hypothesis that O2 gradients between skeletal muscle microvascular (PO2 mv ) and interstitial (PO2 is ) spaces would be present at rest and maintained or increased during contractions. PO2 mv and PO2 is   were determined via phosphorescence quenching (Oxyphor probes G2 and G4, respectively) in the exposed rat spinotrapezius during the rest-contraction transient (1 Hz, 6 V; n = 8). PO2 mv was higher than PO2 is in all instances from rest (34.9 ± 6.0 versus 15.7 ± 6.4) to contractions (28.4 ± 5.3 versus 10.6 ± 5.2 mmHg, respectively) such that the mean PO2 gradient throughout the transient was 16.9 ± 6.6 mmHg (P < 0.05 for all). No differences in the amplitude of PO2 fall with contractions were observed between the microvasculature and interstitium (10.9 ± 2.3 versus 9.0 ± 3.5 mmHg, respectively; P > 0.05). However, the speed of the PO2 is fall during contractions was slower than that of PO2 mv (time constant: 12.8 ± 4.7 versus 9.0 ± 5.1 s, respectively; P < 0.05). Consistent with our hypothesis, a significant transmural gradient was sustained (but not increased) from rest to contractions. This supports that the blood-myocyte interface is the site of a substantial PO2 gradient driving O2 diffusion during metabolic transients. Based on Fick's law, elevated O2 flux with contractions must thus rely primarily on modulations in effective diffusing capacity (mainly erythrocyte haemodynamics and distribution) as the PO2 gradient is not increased.

Directional Migration of MDA-MB-231 Cells Under O2/pH Gradients.

We hypothesized that cancer cells actively migrate toward intratumor microvessels, guided by tissue gradients of metabolic substrates (such as O2) and/or metabolites (such as CO2/H+). To test this hypothesis, we developed an in vitro model in which cellular energy metabolism establishes gradients of O2/nutrient/metabolite in monolayer cells cultured in a conventional culture dish. When gradients of O2 ranging from 3% to ~0% were produced, MDA-MB-231 cells located at 300, 500 and 1500 µm downstream in the gradient demonstrated significant directional migrations (Rayleigh z test). We also found a similar directionality in cell migration at the same location even when the initial O2 level in the O2 gradient was raised from 3% to 21%. Interestingly, such directionalities were no longer demonstrated when the cell density was lowered from 1.8 × 106 to 0.9 × 106 cells/ml. In the former, the magnitude of the extracellular pH gradient in regions 300 and 500 µm downstream in the gradient was significantly larger. Thus, the direction of cell migrations appeared to depend on the gradient of extracellular pH rather than on O2.

Computational Simulation of Tumor Hypoxia Based on In Vivo Microvasculature Assessed in a Dorsal Skin Window Chamber.

Malignant growth usually leads to the depletion of oxygen (O2) supply in most solid tumors. Hypoxia can cause resistance to standard radiotherapy, some chemotherapy and immunotherapy. Furthermore, it can also trigger malignant progression by modulating gene expression and inducing genetic instability. The relationship between microvasculature, perfusion and tumor hypoxia has been intensively studied and many computational simulations have been developed to model tissue O2 transport. Usually simplified 2D phantoms are used to investigate tumor hypoxia and it is assumed that vessels are perpendicular to the region of interest and randomly distributed across the domain. Such idealistic topology overlooks vascular heterogeneity and is not accurate enough to approximate real scenarios. In addition, experimental verification of the spatial gradient of computational simulations is not directly feasible. Realistic vasculature obtained from fluorescence imaging imported as geometry for partial differential equations solving did not receive necessary attention so far. Therefore, we established a computational simulation of in vivo conditions using experimental data obtained from dorsal skin window chamber tumor preparations in nude rats for the verification of computational results. Tumor microvasculature was assessed by fluorescence microscopy. Since the conventional finite difference method can hardly satisfy the real measurements, we established a finite element method (FEM) for the experimental data in this study. Realistic 2D tumor microvasculature was reconstructed by segmenting fluorescence images and then translated into FEM topology. O2 distributions and the O2 gradients were obtained by solving reaction-diffusion equations. The simulation results show that the development of tumor hypoxia is greatly influenced by the irregular architecture and function of microvascular networks.

Vascular patterning sets the stage for macro and micro hepatic architecture.

Background The liver is a complex organ with a variety of tissue components that require a precise architecture for optimal function of metabolic and detoxification processes. As a result of the delicate orchestration required between the various hepatic tissues, it is not surprising that impairment of hepatic function can be caused by a variety of factors leading to chronic liver disease. Results Despite the growing rate of chronic liver disease, there are currently few effective treatment options besides orthotopic liver transplantation. Better therapeutic options reside in the potential for genetic and cellular therapies that promote progenitor cell activation aiding de novo epithelial and vascular regeneration, cell replacement, or population of bioartificial hepatic devices. In order to explore this area of new therapeutic potential, it is crucial to understand the factors that promote hepatic function through regulating cell identities and tissue architecture. Conclusions In this commentary, we review the signals regulating liver cell fates during development and regeneration and highlight the importance of patterning the hepatic vascular systems to set the groundwork for the macro and micro hepatic architecture of the epithelium.

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-µm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 µM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 µM across the cell, or ∼0.6 µM/µm. Deeply deoxygenated (5-15 µM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues.

Ex Vivo Evaluation of Combination Immunotherapy Using Tumor-Microenvironment-on-Chip.

Combination immunotherapy has emerged as a promising strategy to address the challenges associated with immune checkpoint inhibitor (ICI) therapy in breast cancer. The efficacy of combination immunotherapy hinges upon the intricate and dynamic nature of the tumor microenvironment (TME), characterized by cellular heterogeneity and molecular gradients. However, current methodologies for drug screening often fail to accurately replicate these complex conditions, resulting in limited predictive capacity for treatment outcomes. Here, a tumor-microenvironment-on-chip (TMoC), integrating a circulation system and ex vivo tissue culture with physiological oxygen and nutrient gradients, is described. This platform enables spatial infiltration of cytotoxic CD8+ T cells and their targeted attack on the tumor, while preserving the high complexity and heterogeneity of the TME. The TMoC is employed to assess the synergistic effect of five targeted therapy drugs and five chemotherapy drugs in combination with immunotherapy, demonstrating strong concordance between chip and animal model responses. The TMoC holds significant potential for advancing drug development and guiding clinical decision-making, as it offers valuable insights into the complex dynamics of the TME.

Looking deeper into the effects of scouring and aeration on membrane aerated biofilms: Analysis of nitrogen conversion, oxygen profiles and nitrous oxide emissions.

This study investigated the effects of aeration and scouring strategies on the performance of Membrane Aerated Biofilm Reactors (MABRs) and the distribution of oxygen and nitrous oxide in the biofilm. Four flat sheet MABRs were operated with synthetic feed under different conditions: two with intermittent aeration (iMABR) and two with continuous aeration (cMABR). Scouring was induced by bubbling dinitrogen gas through the reactor bulk at low and high frequencies (LF and HF). In the iMABRs, a partial nitritation biofilm initially developed, but the biofilm adapted to the aeration strategy over time and became nitrifying. The cMABRs directly developed a nitrifying biofilm without a significant phase of partial nitritation. Limiting oxygen availability improved the overall performance with regards to total nitrogen (TN) removal by providing a better environment for anaerobic ammonium oxidation (Anammox) while limiting complete nitrification. Oxygen profiles were measured in the iMABR over time at different biofilms depths, showing that intermittent aeration led to various oxygen concentrations and temporal variations in the oxygen availabilities at different depths of the biofilm. Also, N2O emissions from the MABRs differed greatly between the different systems, but still remained lower compared to other reactor configurations for nitrogen removal, making the MABR technology a worthy alternative. The results showed large differences between the operating strategies of the MABRs and can help to gain more insight into the specific properties of MABRs for nitrogen removal.

Studying sprouting angiogenesis under combination of oxygen gradients and co-culture of fibroblasts using microfluidic cell culture model.

Sprouting angiogenesis is an essential process for expanding vascular systems under various physiological and pathological conditions. In this paper, a microfluidic device capable of integrating a hydrogel matrix for cell culture and generating stable oxygen gradients is developed to study the sprouting angiogenesis of endothelial cells under combinations of oxygen gradients and co-culture of fibroblast cells. The endothelial cells can be cultured as a monolayer endothelium inside the device to mimic an existing blood vessel, and the hydrogel without or with fibroblast cells cultured in it provides a matrix next to the formed endothelium for three-dimensional sprouting of the endothelial cells. Oxygen gradients can be stably established inside the device for cell culture using the spatially-confined chemical reaction method. Using the device, the sprouting angiogenesis under combinations of oxygen gradients and co-culture of fibroblast cells is systematically studied. The results show that the oxygen gradient and the co-culture of fibroblast cells in the hydrogel can promote sprouting of the endothelial cells into the hydrogel matrix by altering cytokines in the culture medium and the physical properties of the hydrogel. The developed device provides a powerful in vitro model to investigate sprouting angiogenesis under various in vivo-like microenvironments.

Bioengineered 3D Tissue Model of Intestine Epithelium with Oxygen Gradients to Sustain Human Gut Microbiome.

The human gut microbiome is crucial to hosting physiology and health. Therefore, stable in vitro coculture of primary human intestinal cells with a microbiome community is essential for understanding intestinal disease progression and revealing novel therapeutic targets. Here, a three-dimensional scaffold system is presented to regenerate an in vitro human intestinal epithelium that recapitulates many functional characteristics of the native small intestines. The epithelium, derived from human intestinal enteroids, contains mature intestinal epithelial cells and possesses selectively permeable barrier functions. Importantly, by properly positioning the scaffolds cultured under normal atmospheric conditions, two physiologically relevant oxygen gradients, a proximal-to-distal oxygen gradient along the gastrointestinal (GI) tract, and a radial oxygen gradient across the epithelium, are distinguished in the tissues when the lumens are faced up and down in cultures, respectively. Furthermore, the presence of the low oxygen gradients supported the coculture of intestinal epithelium along with a complex living commensal gut microbiome (including obligate anaerobes) to simulate temporal microbiome dynamics in the native human gut. This unique silk scaffold platform may enable the exploration of microbiota-related mechanisms of disease pathogenesis and host-pathogen dynamics in infectious diseases including the potential to explore the human microbiome-gut-brain axis and potential novel microbiome-based therapeutics.

Establishment of a Modular Anaerobic Human Intestine Chip.

It is impossible to analyze human-specific host-microbiome interactions using animal models and existing in vitro methods fail to support survival of human cells in direct contact with complex living microbiota for extended times. Here we describe a protocol for culturing human organ-on-a-chip (Organ Chip) microfluidic devices lined by human patient-derived primary intestinal epithelium in the presence of a physiologically relevant transluminal hypoxia gradient that enables their coculture with hundreds of different living aerobic and anaerobic bacteria found within the human gut microbiome. This protocol can be adapted to provide different levels of oxygen tension to facilitate coculturing of microbiome from different regions of gastrointestinal tract, and the same system can be applied with any other type of Organ Chip. This method can help to provide further insight into the host-microbiome interactions that contribute to human health and disease, enable discovery of new microbiome-related diagnostics and therapeutics, and provide a novel approach to advanced personalized medicine.

The contrasting roles of nitric oxide drive microbial community organization as a function of oxygen presence.

Microbial assemblages are omnipresent in the biosphere, forming communities on the surfaces of roots and rocks and within living tissues. These communities can exhibit strikingly beautiful compositional structures, with certain members reproducibly occupying particular spatiotemporal microniches. Despite this reproducibility, we lack the ability to explain these spatial patterns. We hypothesize that certain spatial patterns in microbial communities may be explained by the exchange of redox-active metabolites whose biological function is sensitive to microenvironmental gradients. To test this, we developed a simple community consisting of synthetic Pseudomonas aeruginosa strains with a partitioned denitrification pathway: a strict consumer and strict producer of nitric oxide (NO), a key pathway intermediate. Because NO can be both toxic or beneficial depending on the amount of oxygen present, this system provided an opportunity to investigate whether dynamic oxygen gradients can tune metabolic cross-feeding and fitness outcomes in a predictable fashion. Using a combination of genetic analysis, controlled growth environments, and imaging, we show that oxygen availability dictates whether NO cross-feeding is deleterious or mutually beneficial and that this organizing principle maps to the microscale. More generally, this work underscores the importance of considering the double-edged and microenvironmentally tuned roles redox-active metabolites can play in shaping microbial communities.

Discrepancies on the Role of Oxygen Gradient and Culture Condition on Mesenchymal Stem Cell Fate.

Over the past few years, mesenchymal stem (or stromal) cells (MSCs) have garnered enormous interest due to their therapeutic value especially for their multilineage differentiation potential leading to regenerative medicine applications. MSCs undergo physiological changes upon in vitro expansion resulting in expression of different receptors, thereby inducing high variabilities in therapeutic efficacy. Therefore, understanding the biochemical cues that influence the native local signals on differentiation or proliferation of these cells is very important. There have been several reports that in vitro culture of MSCs in low oxygen gradient (or hypoxic conditions) upregulates the stemness markers and promotes cell proliferation in an undifferentiated state, as hypoxia mimics the conditions the progenitor cells experience within the tissue. However, different studies report different oxygen gradients and culture conditions causing ambiguity in their interpretation of the results. In this progress report, it is aimed to summarize recent studies in the field with specific focus on conflicting results reported during the application of hypoxic conditions for improving the proliferation or differentiation of MSCs. Further, it is tried to decipher the factors that can affect characteristics of MSC under hypoxia and suggest a few techniques that could be combined with hypoxic cell culture to better recapitulate the MSC tissue niche.

Gradient Hydrogels.

Gradient hydrogels represent a pivotal and expanding direction of three-dimensional cell culture. Since various types of gradients play an important role in physiological and pathological processes in vivo, recreation of these gradients in vitro allows a better understanding of cellular behavior, intercellular and cell-matrix interactions. Moreover, gradient hydrogels can advance the creation of functionally improved and physiologically relevant tissue engineered constructs. Another application of gradient hydrogels is the optimization of the 3D in vitro microenvironment (e.g., in terms of hydrogel stiffness or concentration of adhesion ligands). Tunable hydrogels provide researchers with a versatile toolbox to manufacture such gradients in vitro. In this chapter different types of in vivo and in vitro gradients in hydrogels will be presented. Equipment and methods for various gradient fabrications will be discussed. Furthermore, methods of gradient characterizations in hydrogels will be reported. As one of the most recent developments, the influence of low oxygen concentration on cells, as well as the creation and characterization of oxygen gradients in hydrogels will be described. In the last part, achievements in the creation of multiple combinatorial gradients will be presented. The aim of this chapter is to give the reader an overview on existing techniques and biological importance of gradient hydrogels in basic science as well as in applied research.