A Microfluidic Chip Architecture Enabling a Hypoxic Microenvironment and Nitric Oxide Delivery in Cell Culture

A hypoxic (low oxygen level) microenvironment and nitric oxide paracrine signaling play important roles in the control of both biological and pathological cell responses. In this study, we present a microfluidic chip architecture for nitric oxide delivery under a hypoxic microenvironment in human embryonic kidney cells (HEK-293). The chip utilizes two separate, but interdigitated microfluidic channels. The hypoxic microenvironment was created by sodium sulfite as the oxygen scavenger in one of the channels. The nitric oxide microenvironment was created by sodium nitroprusside as the light-activated nitric oxide donor in the other channel. The solutions are separated from the cell culture by a 30 µm thick gas-permeable, but liquid-impermeable polydimethylsiloxane membrane. We show that the architecture is preliminarily feasible to define the gaseous microenvironment of a cell culture in the 100 µm and 1 mm length scales

A Microfluidic Chip Architecture Enabling a Hypoxic Microenvironment and Nitric Oxide Delivery in Cell Culture

A hypoxic (low oxygen level) microenvironment and nitric oxide paracrine signaling play important roles in the control of both biological and pathological cell responses. In this study, we present a microfluidic chip architecture for nitric oxide delivery under a hypoxic microenvironment in human embryonic kidney cells (HEK-293). The chip utilizes two separate, but interdigitated microfluidic channels. The hypoxic microenvironment was created by sodium sulfite as the oxygen scavenger in one of the channels. The nitric oxide microenvironment was created by sodium nitroprusside as the light-activated nitric oxide donor in the other channel. The solutions are separated from the cell culture by a 30 µm thick gas-permeable, but liquid-impermeable polydimethylsiloxane membrane. We show that the architecture is preliminarily feasible to define the gaseous microenvironment of a cell culture in the 100 µm and 1 mm length scales

A microfluidic oxygen sink to create a targeted cellular hypoxic microenvironment under ambient atmospheric conditions

Physiological oxygen levels within the tissue microenvironment are usually lower than 14%, in stem cell niches these levels can be as low as 0-1%. In cell cultures, such low oxygen levels are usually mimicked by altering the global culture environment either by O-2 removal (vacuum or oxygen absorption) or by N-2 supplementation for O-2 replacement. To generate a targeted cellular hypoxic microenvironment under ambient atmospheric conditions, we characterised the ability of the dissolved oxygen-depleting sodium sulfite to generate an in-liquid oxygen sink. We utilised a microfluidic design to place the cultured cells in the vertical oxygen gradient and to physically separate the cells from the liquid. We demonstrate generation of a chemical in-liquid oxygen sink that modifies the surrounding O-2 concentrations. O-2 level control in the sink-generated hypoxia gradient is achievable by varying the thickness of the polydimethylsiloxane membrane. We show that intracellular hypoxia and hypoxia response element-dependent signalling is instigated in cells exposed to the microfluidic in-liquid O-2 sink-generated hypoxia gradient. Moreover, we show that microfluidic flow controls site-specific microenvironmental kinetics of the chemical O-2 sink reaction, which enables generation of intermittent hypoxia/re-oxygenation cycles. The microfluidic O-2 sink chip targets hypoxia to the cell culture microenvironment exposed to the microfluidic channel architecture solely by depleting O-2 while other sites in the same culture well remain unaffected. Thus, responses of both hypoxic and bystander cells can be characterised. Moreover, control of microfluidic flow enables generation of intermittent hypoxia or hypoxia/re-oxygenation cycles. (C) 2018 Published by Elsevier Ltd on behalf of Acta Materialia Inc.Peer reviewe

Impact of Metabolic Regulators on the Expression of the Obesity Associated Genes FTO and NAMPT in Human Preadipocytes and Adipocytes

FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators.We assessed FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively.FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes

Le pH en image ! Suivi spatio-temporel du pH dans la rhizosphère à l’aide d’optodes planaires

International audienceThe soil is a complex, heterogeneous and dynamic environment, it is there that various and essential biogeochemical processes take place for the functionning of plants and their ecosystems. The study of soil-roots interactions in the different soil horizons is crucial to understand the dynamics of nutriments and carbon as well as the role played by micro-organisms associated to the rhizosphere. These interactions take into account processes at tiny scales and temporal dynamics that cannot be reached easily in situ. We present here a method of measure of the soil pH in situ, a key parameter of the soil-roots-micro-organisms interactions through an imaging technique called planar optodes. These optical and chemical sensors allow to follow over time without invasion the soil pH and its spatialization at millimeter or infra scale. We present an application of this new tool to follow in situ rhizophere pH of cereals and legumes.Le sol est un environnement complexe, hétérogène et dynamique, lieu de multiples processus biogéochimiques essentiels au fonctionnement des plantes et des écosystèmes. L'étude des interactions sol-racine dans les différents horizons du sol est cruciale pour appréhender la dynamique des nutriments et du carbone, ainsi que le rôle joué par les microorganismes associés au niveau de la rhizosphère. Ces interactions relèvent de processus à fines échelles et de dynamiques temporelles difficilement accessibles in situ. Nous présentons ici une méthode de mesure in situ du pH du sol, paramètre clé des interactions sol-racine-microorganisme, via une technique d'imagerie, les optodes planaires. Ces capteurs optiques et chimiques permettent un suivi temporel non invasif du pH du sol et sa spatialisation à l'échelle millimétrique, voire infra. Nous présentons une application de cet outil novateur au suivi in situ du pH de la rhizosphère de céréales et de légumineuses

A microfluidic oxygen sink to create a targeted cellular hypoxic microenvironment under ambient atmospheric conditions

Physiological oxygen levels within the tissue microenvironment are usually lower than 14%, in stem cell niches these levels can be as low as 0–1%. In cell cultures, such low oxygen levels are usually mimicked by altering the global culture environment either by O2 removal (vacuum or oxygen absorption) or by N2 supplementation for O2 replacement. To generate a targeted cellular hypoxic microenvironment under ambient atmospheric conditions, we characterised the ability of the dissolved oxygen-depleting sodium sulfite to generate an in-liquid oxygen sink. We utilised a microfluidic design to place the cultured cells in the vertical oxygen gradient and to physically separate the cells from the liquid. We demonstrate generation of a chemical in-liquid oxygen sink that modifies the surrounding O2 concentrations. O2 level control in the sink-generated hypoxia gradient is achievable by varying the thickness of the polydimethylsiloxane membrane. We show that intracellular hypoxia and hypoxia response element-dependentsignalling is instigated in cells exposed to the microfluidic in-liquid O2 sink-generated hypoxia gradient. Moreover, we show that microfluidic flow controls site-specific microenvironmental kinetics of the chemical O2 sink reaction, which enables generation of intermittent hypoxia/re-oxygenation cycles. The microfluidic O2 sink chip targets hypoxia to the cell culture microenvironment exposed to the microfluidic channel architecture solely by depleting O2 while other sites in the same culture well remain unaffected. Thus, responses of both hypoxic and bystander cells can be characterised. Moreover, control of microfluidic flow enables generation of intermittent hypoxia or hypoxia/re-oxygenation cycles. Statement of Significance: Specific manipulation of oxygen concentrations in cultured cells’ microenvironment is important when mimicking low-oxygen tissue conditions and pathologies such as tissue infarction or cancer. We utilised a sodium sulfite-based in-liquid chemical reaction to consume dissolved oxygen. When this liquid was pumped into a microfluidic channel, lowered oxygen levels could be measured outside the channel through a polydimethylsiloxane PDMS membrane allowing only for gaseous exchange. We then utilised this setup to deplete oxygen from the microenvironment of cultured cells, and showed that cells responded to hypoxia on molecular level. Our setup can be used for specifically removing oxygen from the cell culture microenvironment for experimental purposes and for generating a low oxygen environment that better mimics the cells’ original tissue environments.Peer reviewe

A patient-based model of RNA mis-splicing uncovers treatment targets in Parkinson's disease.

Parkinson's disease (PD) is a heterogeneous neurodegenerative disorder with monogenic forms representing prototypes of the underlying molecular pathology and reproducing to variable degrees the sporadic forms of the disease. Using a patient-based in vitro model of PARK7-linked PD, we identified a U1-dependent splicing defect causing a drastic reduction in DJ-1 protein and, consequently, mitochondrial dysfunction. Targeting defective exon skipping with genetically engineered U1-snRNA recovered DJ-1 protein expression in neuronal precursor cells and differentiated neurons. After prioritization of candidate drugs, we identified and validated a combinatorial treatment with the small-molecule compounds rectifier of aberrant splicing (RECTAS) and phenylbutyric acid, which restored DJ-1 protein and mitochondrial dysfunction in patient-derived fibroblasts as well as dopaminergic neuronal cell loss in mutant midbrain organoids. Our analysis of a large number of exomes revealed that U1 splice-site mutations were enriched in sporadic PD patients. Therefore, our study suggests an alternative strategy to restore cellular abnormalities in in vitro models of PD and provides a proof of concept for neuroprotection based on precision medicine strategies in PD