Measuring and regulating oxygen levels in microphysiological systems: design, material, and sensor considerations.

As microfabrication techniques and tissue engineering methods improve, microphysiological systems (MPS) are being engineered that recapitulate complex physiological and pathophysiological states to supplement and challenge traditional animal models. Although MPS provide unique microenvironments that transcend common 2D cell culture, without proper regulation of oxygen content, MPS often fail to provide the biomimetic environment necessary to activate and investigate fundamental pathways of cellular metabolism and sub-cellular level. Oxygen exists in the human body in various concentrations and partial pressures; moreover, it fluctuates dramatically depending on fasting, exercise, and sleep patterns. Regulating oxygen content inside MPS necessitates a sensitive biological sensor to quantify oxygen content in real-time. Measuring oxygen in a microdevice is a non-trivial requirement for studies focused on understanding how oxygen impacts cellular processes, including angiogenesis and tumorigenesis. Quantifying oxygen inside a microdevice can be achieved via an array of technologies, with each method having benefits and limitations in terms of sensitivity, limits of detection, and invasiveness that must be considered and optimized. This article will review oxygen physiology in organ systems and offer comparisons of organ-specific MPS that do and do not consider oxygen microenvironments. Materials used in microphysiological models will also be analyzed in terms of their ability to control oxygen. Finally, oxygen sensor technologies are critically compared and evaluated for use in MPS.

Simple Design for Membrane-Free Microphysiological Systems to Model the Blood-Tissue Barriers.

Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ·cm-2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.

A new microphysiological system shows hypoxia primes human ISCs for interleukin-dependent rescue of stem cell activity.

Background & Aims: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or conditions like Inflammatory Bowel Disease (IBD) where immune cell infiltration produces 'inflammatory hypoxia', a chronic condition that starves the mucosa of oxygen. Epithelial regeneration after ischemia and IBD suggests intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of acute and chronic hypoxia on human ISC (hISC) properties have not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs isolated from healthy human tissues. We then test the hypothesis that some inflammation-associated interleukins protect hISCs during prolonged hypoxia. Methods: hISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72hrs. Viability, HIF1α response, transcriptomics, cell cycle dynamics, and hISC response to cytokines were evaluated. Results: The novel MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs remain viable until 72hrs and exhibit peak HIF1α at 24hrs. hISCs lose stem cell activity at 24hrs that recovers at 48hrs of hypoxia. Hypoxia increases the proportion of hISCs in G1 and regulates hISC capacity to respond to multiple inflammatory signals. Hypoxia induces hISCs to upregulate many interleukin receptors and hISCs demonstrate hypoxia-dependent cell cycle regulation and increased organoid forming efficiency when treated with specific interleukins. Conclusions: Hypoxia primes hISCs to respond differently to interleukins than hISCs in normoxia through a transcriptional response. hISCs slow cell cycle progression and increase hISC activity when treated with hypoxia and specific interleukins. These findings have important implications for epithelial regeneration in the gut during inflammatory events.

Hypoxia Primes Human ISCs for Interleukin-Dependent Rescue of Stem Cell Activity.

BACKGROUND AND AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation in which immune cell infiltration produces inflammatory hypoxia starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting that intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins (ILs). METHODS: hISCs were exposed to <1.0% oxygen in the MPS for 6, 24, 48, and 72 hours. Viability, hypoxia-inducible factor 1a (HIF1a) response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF stabilizers and inhibitors were screened to evaluate HIF-dependent responses. RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72 hours and exhibit peak HIF1a at 24 hours. hISC activity was reduced at 24 hours but recovered at 48 hours. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 IL receptors. Prolyl hydroxylase inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24 hours of hypoxia. CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of ILs that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.

Towards Wearable Health Monitoring Devices.

Humans have searched far beyond our planet to understand the fundamental principles and mechanisms of life [...].

In-Line Microelectrode Arrays for Impedance Mapping of Microphysiological Systems.

Herein, a 60-electrode array is fabricated down the length of a microchamber for analysis of a microphysiological system. The electrode array is fabricated by standard photolithographic, metallization, and etching techniques. Permutations of 2-wire impedance measurements (10 Hz to 1 MHz) are made along the length of the microchannel using a multiplexer, Gamry potentiostat, and custom Labview code. An impedance "heat map" is created via custom algorithms. Spatial resolution and mapping capabilities are exhibited using conductive NaCl solutions and 2D cell culture.

Wearable multiplexed biosensor system toward continuous monitoring of metabolites.

Comprehensive metabolic panels are the most reliable and common methods for monitoring general physiology in clinical healthcare. Translation of this clinical practice to personal health and wellness tracking requires reliable, non-invasive, miniaturized, ambulatory, and inexpensive systems for continuous measurement of biochemical analytes. We report the design and characterization of a wearable system with a flexible sensor array for non-invasive and continuous monitoring of human biochemistry. The system includes signal conditioning, processing, and transmission parts for continuous measurement of glucose, lactate, pH, and temperature. The system can operate three discrete electrochemical cells. The system draws 15 mA under continuous operation when powered by a 3.7 V 150 mAh battery. The analog front-end of the electrochemical cells has four potentiostats and three multiplexers for multiplexed and parallel readout from twelve working electrodes. Utilization of redundant working electrodes improves the measurement accuracy of sensors by averaging chronoamperometric responses across the array. The operation of the system is demonstrated in vitro by simultaneous measurement of glucose and lactate, pH, and skin temperature. In benchtop measurements, the sensors are shown to have sensitivities of 26.31 µA mM-1·cm-2 for glucose, 1.49 µA mM-1·cm-2 for lactate, 54 mV·pH-1 for pH, and 0.002 °C-1 for temperature. With the custom wearable system, these values were 0.84 ± 0.03 mV µM-1·cm-2 or glucose, 31.87 ± 9.03 mV mM-1·cm-2 for lactate, 57.18 ± 1.43 mV·pH-1 for pH, and 63.4 µV·°C-1 for temperature. This miniaturized wearable system enables future evaluation of temporal changes of the sweat biomarkers.

Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides.

The impact of next-generation biorecognition elements (ligands) will be determined by the ability to remotely control their binding activity for a target biomolecule in complex environments. Compared to conventional mechanisms for regulating binding affinity (pH, ionic strength, or chaotropic agents), light provides higher accuracy and rapidity, and is particularly suited for labile targets. In this study, we demonstrate a general method to develop azobenzene-cyclized peptide ligands with light-controlled affinity for target proteins. Light triggers a cis/trans isomerization of the azobenzene, which results in a major structural rearrangement of the cyclic peptide from a non-binding to a binding configuration. Critical to this goal are the ability to achieve efficient photo-isomerization under low light dosage and the temporal stability of both cis and trans isomers. We demonstrated our method by designing photo-switchable peptides targeting vascular cell adhesion marker 1 (VCAM1), a cell marker implicated in stem cell function. Starting from a known VCAM1-binding linear peptide, an ensemble of azobenzene-cyclized variants with selective light-controlled binding were identified by combining in silico design with experimental characterization via spectroscopy and surface plasmon resonance. Variant cycloAZOB[G-VHAKQHRN-K] featured rapid, light-controlled binding of VCAM1 (KD,trans/KD,cis ∼ 130). Biotin-cycloAZOB[G-VHAKQHRN-K] was utilized to label brain microvascular endothelial cells (BMECs), showing co-localization with anti-VCAM1 antibodies in cis configuration and negligible binding in trans configuration.

Alkylsubstituted thienothiophene semiconducting materials: structure-property relationships.

A family of conjugated polymers with fused structures consisting of three to five thiophene rings and with the same alkyl side chains has been synthesized as a means to understand structure-property relationships. All three polymers showed well-extended conjugation through the polymer backbone. Ionization potentials (IP) ranged from 5.15 to 5.21 eV; these large values are indicative of their excellent oxidative stability. X-ray diffraction and AFM studies suggest that the polymer with the even number of fused thiophene rings forms a tight crystalline structure due to its tilted side chain arrangement. On the other hand, the polymers with the odd number of fused thiophene rings packed more loosely. Characterization in a field-effect transistor configuration showed that the mobility of the polymer with the even number of rings is 1 order of magnitude higher than its odd-numbered counterparts. Through this structure-property study, we demonstrate that proper design of the molecules and properly arranged side chain positions on the polymer backbone can greatly enhance polymer electronic properties.

Integrated phosphorescence-based photonic biosensor (iPOB) for monitoring oxygen levels in 3D cell culture systems.

Physiological processes, such as respiration, circulation, digestion, and many pathologies alter oxygen concentration in the blood and tissue. When designing culture systems to recapitulate the in vivo oxygen environment, it is important to integrate systems for monitoring and controlling oxygen concentration. Herein, we report the design and engineering of a system to remotely monitor and control oxygen concentration inside a device for 3D cell culture. We integrate a photonic oxygen biosensor into the 3D tissue scaffold and regulate oxygen concentration via the control of purging gas flow. The integrated phosphorescence-based oxygen biosensor employs the quenching of palladium-benzoporphyrin by molecular oxygen to transduce the local oxygen concentration in the 3D tissue scaffold. The system is validated by testing the effects of normoxic and hypoxic culture conditions on healthy and tumorigenic breast epithelial cells, MCF-10A cells and BT474 cells, respectively. Under hypoxic conditions, both cell types exhibited upregulation of downstream target genes for the hypoxia marker gene, hypoxia-inducible factor 1α (HIF1A). Lastly, by monitoring the real-time fluctuation of oxygen concentration, we illustrated the formation of hypoxic culture conditions due to limited diffusion of oxygen through 3D tissue scaffolds.

Tetrathienoacene copolymers as high mobility, soluble organic semiconductors.

Increasing the rigidity of the thiophene monomer through the use of an alkyl-substituted core that consists of four fused thiophene rings is shown to be a promising route toward high-performance organic semiconductors. We report on a dialkylated tetrathienoacene copolymer that can be deposited from solution to yield ordered films with a short pi-pi distance of 3.76 A and with a field-effect hole mobility that exceeds 0.3 cm2/V.s. This polymer enables simple transistor fabrication at relatively low temperatures, which is particularly important for the realization of large-area, mechanically flexible electronics.

Importance of C(2) symmetry for the device performance of a newly synthesized family of fused-ring thiophenes.

We investigated the relationship between molecular structure and field-effect hole mobility in a family of fused-ring polythiophene copolymers that we designed recently. The results suggest that a repeat unit that possesses a C(2) axis perpendicular to the conjugation plane is important to achieve a high-mobility. Our finding is supported by a review of literature data: Many polymer semiconductors showing a hole or electron mobility >0.1 cm(2)/V.s feature a repeat unit with C(2) symmetry; however exceptions have been found from some push - pull polymer structures.