A hands-free stool sampling system for monitoring intestinal health and disease.

Analysis of stool offers simple, non-invasive monitoring for many gastrointestinal (GI) diseases and access to the gut microbiome, however adherence to stool sampling protocols remains a major challenge because of the prevalent dislike of handling one's feces. We present a technology that enables individual stool specimen collection from toilet wastewater for fecal protein and molecular assay. Human stool specimens and a benchtop test platform integrated with a commercial toilet were used to demonstrate reliable specimen collection over a wide range of stool consistencies by solid/liquid separation followed by spray-erosion. The obtained fecal suspensions were used to perform occult blood tests for GI cancer screening and for microbiome 16S rRNA analysis. Using occult blood home test kits, we found overall 90% agreement with standard sampling, 96% sensitivity and 86% specificity. Microbiome analysis revealed no significant difference in within-sample species diversity compared to standard sampling and specimen cross-contamination was below the detection limit of the assay. Furthermore, we report on the use of an analogue turbidity sensor to assess in real time loose stools for tracking of diarrhea. Implementation of this technology in residential settings will improve the quality of GI healthcare by facilitating increased adherence to routine stool monitoring.

Potential Pitfalls in Wastewater Phosphorus Analysis and How to Avoid Them.

Due to the increasing adoption of nutrient discharge regulations, many research groups are stepping into new territory with phosphorus (P) measurements. Accurate reporting of P concentrations in effluent from novel wastewater treatment technologies is critical for protecting both environmental and human health. Analysis of P in wastewater is prone to pitfalls because of the (1) variety of chemical forms of P in wastewater (orthophosphate, condensed P, and organic P), (2) availability of different chemical assays for measuring different P forms, and (3) different conventions in the units for reporting P. Here, we present a case study highlighting how these pitfalls affect analysis and interpretation of P measurements. We show that, when used appropriately, commercially-available kits are indeed accurate tools for evaluating reactive P and total P concentrations. For both standard solutions and real wastewater, we systematically remove steps from the total P protocol to show how protocol deviations affect the results. While standard solutions are important for validating analytical methods, commercially-available wastewater standard solutions only contain P as orthophosphate (reactive P). We therefore demonstrate options for making a mixed-P standard solution containing acid-hydrolyzable and/or organic P compounds that can be used to validate both reactive P and total P assays.

Field testing of a household-scale onsite blackwater treatment system in Coimbatore, India.

4.2 billion people live without access to safely managed sanitation services. This report describes the field testing of an onsite prototype system designed to treat blackwater from a single flush toilet and reuse of the treated effluent for flushing. The system passes wastewater through a solid-liquid separator followed by settling tanks and granular activated carbon columns into an electrochemical reactor that oxidizes chloride salts from urine to generate chlorine to remove pathogens. The objectives of the study were to verify the functionality of the system (previously demonstrated in the laboratory) under realistic use conditions, to identify maintenance requirements, and to make a preliminary assessment of the system's user acceptability. The prototype was installed in a women's workplace and residential toilet block in Coimbatore, India, and tested over a period of 10 months. The treated water met stringent disinfection threshold for both E. coli and helminth eggs and produced a clear, colorless effluent that met or nearly met local and international discharge standards for non-sewered sanitation systems. The effluent had an average chemical oxygen demand of 81 mg/L, total suspended solids of 11 mg/L, and reduction of total nitrogen by 65%. These tests determined the recommended service lifetimes and maintenance intervals for key system components including the electrochemical cell, granular activated carbon columns, and solid-liquid separator. User feedback regarding the use of treated blackwater as flush water was positive. These findings will inform the design and implementation of next-generation systems currently under development.

Field testing of a household-scale onsite blackwater treatment system in South Africa.

Innovations that enable cost-effective and resource-conserving treatment of human waste are required for the 4.2 billion people in the world who currently lack safe and reliable sanitation services. Onsite treatment and reuse of blackwater is one strategy towards this end, greatly reducing the need to transport wastewater over long distances either via sewers or trucks. Here, we report on the field testing of a prototype onsite blackwater treatment system conducted over a period of 8 months. The system was connected to a women's toilet in a public communal ablution block located in an informal settlement near Durban, South Africa. Liquid waste was treated by separation and diversion of large solids, settling of suspended solids, and filtration through activated carbon prior to disinfection by electrochemical oxidation. System performance was monitored daily by measurement of chemical and physical water quality parameters onsite and confirmed by periodic detailed analysis of chemical and biological parameters at an offsite lab. Daily monitoring of system performance indicated that the effluent had minimal color and turbidity (maximum 90 Pt/Co units and 6.48 NTU, respectively), and consistent evolution of chlorine as blackwater passed through the system. Weekly offsite analysis confirmed that the system consistently inactivated pathogens (E. coli and coliforms) and reduced chemical oxygen demand and total suspended solids to meet ISO 30500 category B standards. Significant reductions in total nitrogen load were also observed, though these reductions often fell short of the 70% reduction required by ISO 30500. No significant reduction in total phosphorus was observed. Maintenance requirements were identified, and the resilience of the system to restart following a prolonged shutdown was demonstrated, but significant improvements are required in the design of the solid/liquid separation mechanism for application of this system in a wiping culture.

Remediation of suspended solids and turbidity by improved settling tank design in a small-scale, free-standing toilet system using recycled blackwater.

Our research is focused on the development of decentralized waste water treatment technologies enabling onsite water reuse. Accumulation of solids with recycling of treated blackwater increases the energy required for disinfection with an electrochemical process. We hypothesized that improving the preprocess settling of blackwater by increasing the tortuosity of the liquid flow path would reduce this energy demand by reducing particle-associated chemical oxygen demand (COD). This approach successfully reduced the total suspended solids and turbidity in the process liquid accumulated per user-day equivalent. A modest reduction in the apparent steady-state accumulation of COD was also observed, likely because of the retention of COD associated with larger particles in the settling tanks. Interestingly, these improvements did not improve the energy efficiency of the electrochemical disinfection process, as predicted. These observations suggest that improving the energy efficiency of electrochemical disinfection will require remediation of dissolved COD.

A granular activated carbon/electrochemical hybrid system for onsite treatment and reuse of blackwater.

Over 1/3 of the global population lacks access to improved sanitation, leading to disease, death, and impaired economic development. Our group is working to develop rapidly deployable, cost-effective, and sustainable solutions to this global problem that do not require significant investments in infrastructure. Previously, we demonstrated the feasibility of a toilet system that recycles blackwater for onsite reuse as flush water, in which the blackwater is electrochemically treated to remove pathogens due to fecal contamination. However, this process requires considerable energy (48-93 kJ/L) to achieve complete disinfection of the process liquid, and the disinfected liquid retains color and chemical oxygen demand (COD) in excess of local discharge standards, negatively impacting user acceptability. Granular activated carbon (GAC) efficiently reduces COD in concentrated wastewaters. We hypothesized that reduction of COD with GAC prior to electrochemical treatment would both improve disinfection energy efficiency and user acceptability of the treated liquid. Here we describe the development and testing of a hybrid system that combines these technologies and demonstrate its ability to achieve full disinfection with improved energy efficiency and liquid quality more suitable for onsite reuse and/or discharge.

Electrochemical disinfection of repeatedly recycled blackwater in a free-standing, additive-free toilet.

Decentralized, energy-efficient waste water treatment technologies enabling water reuse are needed to sustainably address sanitation needs in water- and energy-scarce environments. Here, we describe the effects of repeated recycling of disinfected blackwater (as flush liquid) on the energy required to achieve full disinfection with an electrochemical process in a prototype toilet system. The recycled liquid rapidly reached a steady state with total solids reliably ranging between 0.50 and 0.65% and conductivity between 20 and 23 mS/cm through many flush cycles over 15 weeks. The increase in accumulated solids was associated with increased energy demand and wide variation in the free chlorine contact time required to achieve complete disinfection. Further studies on the system at steady state revealed that running at higher voltage modestly improves energy efficiency, and established running parameters that reliably achieve disinfection at fixed run times. These results will guide prototype testing in the field.

Enhanced H2O2 Production at Reductive Potentials from Oxidized Boron-Doped Ultrananocrystalline Diamond Electrodes.

This work investigates the surface chemistry of H2O2 generation on a boron-doped ultrananocrystalline diamond (BD-UNCD) electrode. It is motivated by the need to efficiently disinfect liquid waste in resource constrained environments with limited electrical power. X-ray photoelectron spectroscopy was used to identify functional groups on the BD-UNCD electrode surfaces while the electrochemical potentials of generation for these functional groups were determined via cyclic voltammetry, chronocoulometry, and chronoamperometry. A colorimetric technique was employed to determine the concentration and current efficiency of H2O2 produced at different potentials. Results showed that preanodization of an as-grown BD-UNCD electrode can enhance the production of H2O2 in a strong acidic environment (pH 0.5) at reductive potentials. It is proposed that the electrogeneration of functional groups at oxidative potentials during preanodization allows for an increased current density during the successive electrolysis at reductive potentials that correlates to an enhanced production of H2O2. Through potential cycling methods, and by optimizing the applied potentials and duty cycle, the functional groups can be stabilized allowing continuous production of H2O2 more efficiently compared to static potential methods.

An optically transparent membrane supports shear stress studies in a three-dimensional microfluidic neurovascular unit model.

We report a microfluidic blood-brain barrier model that enables both physiological shear stress and optical transparency throughout the device. Brain endothelial cells grown in an optically transparent membrane-integrated microfluidic device were able to withstand physiological fluid shear stress using a hydrophilized polytetrafluoroethylene nanoporous membrane instead of the more commonly used polyester membrane. A functional three-dimensional microfluidic co-culture model of the neurovascular unit is presented that incorporates astrocytes in a 3D hydrogel and enables physiological shear stress on the membrane-supported endothelial cell layer.

High-throughput cardiac safety evaluation and multi-parameter arrhythmia profiling of cardiomyocytes using microelectrode arrays.

Microelectrode arrays (MEAs) recording extracellular field potentials of human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) provide a rich data set for functional assessment of drug response. The aim of this work is the development of a method for a systematic analysis of arrhythmia using MEAs, with emphasis on the development of six parameters accounting for different types of cardiomyocyte signal irregularities. We describe a software approach to carry out such analysis automatically including generation of a heat map that enables quick visualization of arrhythmic liability of compounds. We also implemented signal processing techniques for reliable extraction of the repolarization peak for field potential duration (FPD) measurement even from recordings with low signal to noise ratios. We measured hiPS-CM's on a 48 well MEA system with 5minute recordings at multiple time points (0.5, 1, 2 and 4h) after drug exposure. We evaluated concentration responses for seven compounds with a combination of hERG, QT and clinical proarrhythmia properties: Verapamil, Ranolazine, Flecainide, Amiodarone, Ouabain, Cisapride, and Terfenadine. The predictive utility of MEA parameters as surrogates of these clinical effects were examined. The beat rate and FPD results exhibited good correlations with previous MEA studies in stem cell derived cardiomyocytes and clinical data. The six-parameter arrhythmia assessment exhibited excellent predictive agreement with the known arrhythmogenic potential of the tested compounds, and holds promise as a new method to predict arrhythmic liability.

Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor ß1.

Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)ß1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFß1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.

Effects of flow configuration on bone tissue engineering using human mesenchymal stem cells in 3D chitosan composite scaffolds.

Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds.

A biomimetic multicellular model of the airways using primary human cells.

Microfluidic cell cultures enable investigation of complex physiological tissue properties and functionalities. For convenience, they are often implemented with immortalized cell lines, but primary cells more closely approximate the in vivo biology. Our aim was to develop a biomimetic microfluidic model of the human airway using all primary cells. The model is comprised of airway epithelial cells cultured at an air-liquid interface, lung fibroblasts and polarized microvascular endothelial cells, respectively positioned in three vertically stacked, individually accessible compartments separated by nanoporous membranes. We report device fabrication, a gravity fed microfluidic system, and culture medium able to support functional co-cultures of all three primary human cell types. As characterized by imaging and permeability measurements, airway epithelial cells in microfluidic devices displayed mucociliary differentiation and barrier function. Subjacent fibroblasts and microvascular endothelial cells were added under conditions enabling co-culture for at least 5 days. Microfluidic airway models based on primary human cells in a relevant biomimetic configuration will improve physiological relevance and will enable novel disease modeling and drug development studies.

Magnetic resonance contrast and biological effects of intracellular superparamagnetic iron oxides on human mesenchymal stem cells with long-term culture and hypoxic exposure.

BACKGROUND AIMS: Human mesenchymal stem cells (hMSCs) have gained interest for treatment of stroke injury. Using in vitro culture, the purpose of this study was to investigate the long-term detectability of hMSCs by magnetic resonance imaging (MRI) after transfection with a superparamagnetic iron oxide (SPIO) and evaluate the effects of SPIO on cellular activity, particularly under an ischemic environment. METHODS: hMSCs were exposed to low doses of SPIOs. After a short incubation period, cells were cultured for additional 1, 7 and 14 d to evaluate proliferation, colony formation and multilinear potential. Labeled cells were imaged and evaluated in agarose to quantify R2 and R2∗ contrast at each time point. Cells were placed in a low-oxygen, low-serum environment and tested for cytotoxicity. In addition, labeled cells were transplanted into an ischemic stroke model and evaluated with ex vivo MRI and histology. RESULTS: Cellular events such as proliferation and differentiation were not affected at any of the exposures tested when cultured for 14 d. The low iron exposure and short incubation time are sufficient for detectability with MRI. However, the higher iron dosage results in higher calcification and cytotoxicity under in vitro ischemic conditions. Transplantation of the hMSCs labeled with an initial exposure of 22.4 µg of Fe showed excellent retention of contrast in stroke-induced rats. CONCLUSIONS: Although SPIO labeling is stable for long-term MRI detection and has limited effects on the multilineage potential of hMSCs, high-dose SPIO labeling may affect hMSC survival under serum and oxygen withdrawal.

High yield fabrication of multilayer polydimethylsiloxane [corrected] devices with freestanding micropillar arrays.

A versatile method to fabricate a multilayer polydimethylsiloxane (PDMS) device with micropillar arrays within the inner layer is reported. The method includes an inexpensive but repeatable approach for PDMS lamination at high compressive force to achieve high yield of pillar molding and transfer to a temporary carrier. The process also enables micropillar-containing thin films to be used as the inner layer of PDMS devices integrated with polymer membranes. A microfluidic cell culture device was demonstrated which included multiple vertically stacked flow channels and a pillar array serving as a cage for a collagen hydrogel. The functionality of the multilayer device was demonstrated by culturing collagen-embedded fibroblasts under interstitial flow through the three-dimensional scaffold. The fabrication methods described in this paper can find applications in a variety of devices, particularly for organ-on-chip applications.

Erratum: Publisher's Note: "High yield fabrication of multilayer polydimethylsiloxane devices with freestanding micropillar arrays" [Biomicrofluidics 7, 056503 (2013)].

[This corrects the article on p. 056503 in vol. 7.].

Perfusion conditioning of hydroxyapatite-chitosan-gelatin scaffolds for bone tissue regeneration from human mesenchymal stem cells.

Tissue-engineered bone grafts require an osteogenic cell source and a scaffold capable of supporting tissue regeneration. Hydroxyapatite (H), chitosan (C), and gelatin (G), when combined, produce a biomimetic scaffold with a chemical similarity to the main structural components of natural bone tissue. In this study a phase-separation technique was used to produce a porous 3D HCG scaffold, containing a network of cross-linked chitosan and gelatin fibrils coated in hydroxyapatite, with pore size readily controlled by freezing temperature. The HCG scaffolds were then seeded with human mesenchymal stem cells (hMSCs), using a depth filtration system after preconditioning with serum-containing medium for 7 days under either static or perfusion conditions. The effects of static and perfusion media preconditioning on protein adsorption, surface morphology, hMSC attachment, proliferation and osteogenic differentiation were examined. Perfusion preconditioning, as opposed to static preconditioning, enhances adsorption of ECM proteins, which in turn promotes hMSC proliferation and osteogenic differentiation. The results demonstrate the importance of convective flow in modulating the 3D HCG microenvironment and highlight its profound influence on 3D construct development.