2020 (8)
The differentiation of human induced pluripotent stem cells (hiPSCs) into functional hepatocytes has the potential to solve the shortage of human primary liver cells and would be of use in drug screening. In this frame, we developed a hiPSCs maturation strategy in microfluidic biochips, using a liver-on-chip approach, a promising technology mimicking in vivo physiology. Hepato-like tissues differentiated in biochips presented advanced liver features, including ALB and CYP3A4 expressing cells. The metabolomics and transcriptomics profiles of hepato-likes cells differentiated either in biochips or Petri dishes were integrated to compare their functionalities. The multi-omics analysis revealed 41 metabolites and 302 genes differentially expressed. Overall, biochip environment lead to higher degree of hepatic differentiation demonstrated by an increase in the metabolic production of lipids, fatty acids and biliary acids …
Liver Sinusoidal Endothelial Cells (LSECs) are an important component of the liver as they compose the microvasculature which allows the supply of oxygen, blood, and nutrients. However, maintenance of those cells in-vitro remains challenging as they tend to rapidly lose some of their characteristics such as fenestration or as their immortalized counterparts present poor characteristics. In this work, human induced pluripotent stem cells (hiPSCs) have been differentiated toward an LSECs phenotype. After differentiation, the RNA quantification allowed to demonstrate high expression of specific vascular markers (CD31, CD144, and STAB2). Immunostaining performed on the cells were found to be positive for both Stabilin-1 and Stabilin-2. Whole transcriptome analysis performed with the nanoCAGE method further confirmed the overall vascular commitment of the cells. The gene expression profile revealed the …
Organic field‐effect transistors (OFETs) can be potentially employed to monitor cell activities for healthcare and medical treatment, because of their attractive properties such as ease of use, flexibility, and low‐cost manufacturing processes. Although current OFET‐based sensors are suitable for point‐of‐care testing, the establishment of real‐time monitoring methods is highly demanded to continuously monitor health conditions and/or biological cell activities. In this regard, we herein propose a microfluidic platform integrated with an extended‐gate type OFET for real‐time glucose monitoring. The detection mechanism of glucose depends on an artificial receptor phenylboronic acid and its boronate esterification. After the optimization of microfluidics for the OFET‐based sensor, we applied it to monitor glucose consumption and release in a model of pseudo liver cells. Random increase or decrease in changes of the …
Liver Sinusoidal Endothelial Cells (LSECs) are an important component of the liver as they compose the microvasculature which allows the supply of oxygen, blood, and nutrients. However, maintenance of these cells in vitro remains challenging as they tend to rapidly lose some of their characteristics such as fenestration or as their immortalized counterparts present poor characteristics. In this work, human induced pluripotent stem cells (hiPSCs) have been differentiated toward an LSEC phenotype. After differentiation, the RNA quantification allowed demonstration of high expression of specific vascular markers (CD31, CD144, and STAB2). Immunostaining performed on the cells was found to be positive for both Stabilin-1 and Stabilin-2. Whole transcriptome analysis performed with the nanoCAGE method further confirmed the overall vascular commitment of the cells. The gene expression profile revealed the …
Liver Sinusoidal Endothelial Cells (LSECs) are an important component of the liver as they compose the microvasculature which allows the supply of oxygen, blood, and nutrients. However, maintenance of these cells in vitro remains challenging as they tend to rapidly lose some of their characteristics such as fenestration or as their immortalized counterparts present poor characteristics. In this work, human induced pluripotent stem cells (hiPSCs) have been differentiated toward an LSEC phenotype. After differentiation, the RNA quantification allowed demonstration of high expression of specific vascular markers (CD31, CD144, and STAB2). Immunostaining performed on the cells was found to be positive for both Stabilin-1 and Stabilin-2. Whole transcriptome analysis performed with the nanoCAGE method further confirmed the overall vascular commitment of the cells. The gene expression profile revealed the …
The liver zonation is an important phenomenon characterized by a gradient of several functions along the liver acinus. However, this gradient remains difficult to reproduce in in‐vitro conditions, making the obtention of an in‐vitro method to recapitulate the liver zonation a challenging issue. In this study, we evaluated the spatial evolution of the transcriptome profile of human induced pluripotent stem cells (hiPSCs) differentiated toward hepatocytes‐like cells (HLCs) phenotype in a microfluidic biochip environment. Cells collected at the inlet of the biochip, where the oxygen concentration is higher, were identified by the expression of genes involved in metabolic pathways related to cellular reorganization and cell proliferation. Cells collected in the middle and at the outlet of the biochips, where oxygen concentrations are lower, were characterized by the upregulation of genes involved in cellular detoxification …
The capability to produce and maintain functional human adult hepatocytes remains one of the major challenges for the use of in-vitro models toward liver cell therapy and industrial drug-screening applications. Among the suggested strategies to solve this issue, the use of human-induced pluripotent stem cells (hiPSCs), differentiated toward hepatocyte-like cells (HLCs) is promising. In this work, we propose a 31-day long protocol, that includes a final 14-day long phase of oncostatin treatment, as opposed to a 7-day treatment which led to the formation of a hepatic tissue functional for CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP3A4. The production of albumin, as well as bile acid metabolism and transport, were also detected. Transcriptome profile comparisons and liver transcription factors (TFs) motif dynamics revealed increased expression of typical hepatic markers such as HNF1A and of important metabolic …
Organ-on-chip technology is a promising tool for investigating physiological in vitro responses in drug screening development, and in advanced disease models. Within this framework, we investigated the behavior of rat islets of Langerhans in an organ-on-chip model. The islets were trapped by sedimentation in a biochip with a microstructure based on microwells, and perfused for 5 days of culture. The live/dead assay confirmed the high viability of the islets in the biochips cultures. The microfluidic culture leads to upregulation of mRNA levels of important pancreatic islet genes: Ins1, App, Insr, Gcgr, Reg3a and Neurod. Furthermore, insulin and glucagon secretion were higher in the biochips compared to the Petri conditions after 5 days of culture. We also confirmed glucose-induced insulin secretion in biochips via high and low glucose stimulations leading to high/low insulin secretion. The high responsiveness of …
2019 (6)
In the present study, we evaluated the performance of different protocols for the hepatic differentiation of hiPSCs in microfluidic biochips. Strategies for complete and partial on‐chip differentiation were tested. Unlike full on‐chip differentiation, the transfer of iPSCs from Petri dishes to biochips during the differentiation process produced a heterogeneous tissue with enhanced hepatic features compared to control cultures in Petri dishes. The tissue in biochips was constituted of cells expressing either stabilin‐1 or albumin, while no stabilin‐1 was detected in controls. Functional analysis also revealed double production rate for albumin in biochips (about 2000ng/day/106cells). Besides, tissues obtained in biochips and controls exhibited the metabolism of a specific bile acid. Whole transcriptome analysis with nanoCAGE exhibited a differential expression of 302 genes between control and biochip cultures and higher …
The development of an in vitro functional liver zonation model is a major issue to reproduce physiological liver features. Oxygen concentration is one of the potential explanations of a primary regulating factor of zonation. In this frame, we investigated the oxygen gradient inside a microfluidic device containing rat hepatocyte cultures. The device integrated a platinum (Pt) (II) octaethylporphyrin sensor, allowing a 2D mapping of the oxygen concentration. After 3h of adhesion of the hepatocytes, the sensor indicated an intense oxygen depletion, leading to an oxygen shortage in the center of the device. After a 30 min perfusion of the culture medium, we monitored the formation of the oxygen gradient along the culture due to cellular respiration. The profile of the oxygen gradient was modulated and controlled by increasing either the perfusion flow rate or the device thickness. In addition, the oxygen gradient was time …
Human hepatocyte-like cells derived from human induced pluripotent stem cells (hiPSC) may provide an unlimited supply of cells for in vitro liver models. However, hiPSC differentiation remains a major challenge due to immaturity of the hepatocytes obtained and the high cost of differentiation protocols currently proposed. Here, we studied the efficacy of new protocol, with reduction of growth factors, for the generation of hepatocyte-like cells from hiPSC. We performed metabolomic and mRNA analysis by RTqPCR and nanoCAGE processing to identify and understand key metabolisms during differentiation. By reducing the change frequency of the culture medium in the new protocol, we successfully generated hepatocyte-like cells producing albumin, urea, and CYP3A4 positive. The metabolomic analysis successfully extracted both signatures, common and specific, for each differentiation step. Integrating the …
We investigated the human induced pluripotent stem cells (hiPSCs) during a sequential in vitro step-by-step differentiation into hepatocyte-like cells (HLCs) using nanoCAGE, a method for promoters, transcription factors, and transcriptome analysis. Specific gene clusters reflected the different steps of the hepatic differentiation. The proliferation step was characterized by a typical cell cycle and DNA replication. The hepatic endoderm and the HLC steps were marked by a common signature including cell interactions with extracellular matrix (ECM), lipoproteins and hepatic biomarkers (such as albumin and alpha-fetoprotein). The specific HLC profile was characterized by important transcription factors such as HIF1A, JUN, MAF, KLF6, BMP4 and with a larger expression of genes related to Wnt signaling, extracellular matrix, lipid metabolism, urea cycle, drugs, and solute transporters. HLC profile was also characterized …
We believe that “the simpler we are, the more complete we become” is a key concept of chemical sensing systems. In this work, a “turn-on” fluorescence chemosensor array relying on only two self-assembled molecular chemosensors with ability of both qualitative and quantitative detection of phosphorylated saccharides has been developed. The easy-to-prepare chemosensor array was fabricated by in situ mixing of off-the-shelf reagents (esculetin, 4-methylesculetin, and 3-nitrophenylboronic acid). The fluorescence-based saccharide sensing system was carried out using indicator displacement assay accompanied with photo-induced electron transfer (PeT) under various pH conditions. The simultaneous recognition of fourteen types of saccharides including glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) was achieved with a successful classification rate of 100%. We also succeeded in the …
Human induced pluripotent stem cells (hiPSCs) are potentially an invaluable source of cells for regenerative medicine, disease modeling and drug discovery. However, the differentiation of hiPSCs into fully functional hepatocytes remains a major challenge. Despite the importance of the information carried by metabolomes, the exploitation of metabolomics for characterizing and understanding hiPSC differentiation remains largely unexplored. Here, to increase knowledge of hiPSC maturation into mature hepatocytes, we investigated their metabolomics profiles during sequential step-by-step differentiation: definitive endoderm (DE), specification into hepatocytes (HB-pro (hepatoblast progenitors)), progenitor hepatocytes (Pro-HEP) and mature hepatocyte-like cells (HLCs). Metabolomics analysis illustrated a switch from glycolysis-based respiration in DE step to oxidative phosphorylation in HLCs step. DE was …
2018 (5)
An interesting γ-carbonyl effect permits the dual iron/boron-catalyzed direct nucleophilic substitution of functionalized primary allylic alcohols with a large variety of nucleophiles. The resulting substitution products are useful synthetic platforms for heterocycle synthesis, as illustrated in a ready access to tetrahydroisoindol-4-ones.
Dichlorodiphenyl-trichloroethane (DDT) and permethrin (PMT) are amongst most prevalent pesticides in the environment. Although their toxicity has been extensively studied, molecular mechanisms and metabolic effects remain unclear, including in liver where their detoxification occurs. Here, we used metabolomics, coupled to RT-qPCR analysis, to examine effects of DDT and PMT on hepatocytes cultivated in biochips. At 150 μM, DDT caused cell death, cytochrome P450 induction and modulation of estrogen metabolism. Metabolomics analysis showed an increase in some lipids and sugars after 6 h, and a decrease in fatty acids (tetradecanoate, octanoate and linoleate) after 24 h exposure. We also found a change in expression associated with genes involved in hepatic estrogen, lipid, and sugar metabolism. PMT at 150 μM perturbed lipid/sugar homeostasis and estrogen signaling pathway, between 2 and …
Human induced pluripotent stem (hiPS) cells represent a potential source of human cells for regenerative and personalized medicine applications. It is envisioned that hiPS cells could be an efficient tool for disease modelling and drug therapy testings. However, the successful derivation of hiPS cells to mature hepatocytes remains a challenge as far as the current proposed protocols lead to immature patterns and are very expensive (due to growth factors cost). In this work, we have compared two protocols of differentiation to produce hepatocyte-like cells. The first protocol is the conventional protocol adapted from Si-Tayeb et al. in 2010. By changing the frequency of culture medium exchange, we reduced the use of growth factors in the second protocol by 40%. Both protocols lead to hepatocyte-like characteristics including albumin (ALB) and cytochrome P450/3A4 (CYP3A4) expression in cells. Albumin production …
This paper presents a new hepatic culture device, in which oxygen concentration gradient formed by cellular respiration can be visualized as a two-dimensional optical image. An oxygen sensor layer containing Pt (II) octaethylporphyrin (PtOEP) was embedded at the bottom of the device, allowing visualization of oxygen concentration distribution over the cell culture area. Oxygen concentration was determined by analyzing the modulation of fluorescence intensity emitted by the sensor, which is negatively correlated to the concentration. The results in HepG2 cell culture showed that the oxygen gradient and local oxygen partial pressure could be regulated by controlling the perfusion flow rate in the device. The local and physiological oxygen gradient comparable to the in vivo liver lobules can successfully be reproduced in the device. Therefore, periportal and perivenous hepatic like-zones were successfully realized …
Despite the diversity of studies on pesticide toxicities, there is a serious lack of information concerning the toxic effect of pesticides mixtures. Dichlorodiphenyl‐trichloroethane (DDT) and permethrin (PMT) are among the most prevalent pesticides in the environment and have been the subject of several toxicological studies. However, there are no data on the toxicity of their mixtures. In this study, we used an approach combining cell culture in microfluidic biochips with gas chromatography–mass spectrometry metabolomics profiling to investigate the biomarkers of toxicity of DDT, PMT and their mixtures. All parameters observed indicated that no significant effect was observed in hepatocytes cultures exposed to low doses (15 μm) of DDT and PMT. Conversely, combined low doses induce moderate oxidative stress and cell death. The toxic signature of high doses of pesticides (150 μm) was illustrated by severe …
2017 (15)
We have compared the transcriptomic profiles of human induced pluripotent stem cells after their differentiation in hepatocytes like cells in plates and microfluidic biochips. The biochips provided a 3D and dynamic support during the cell differentiation when compared to the 2D static cultures in plates. The microarray have demonstrated the up regulation of important pathway related to liver development and maturation during the culture in biochips. Furthermore, the results of the transcriptomic profile, coupled with immunostaining, and RTqPCR analysis have shown typical biomarkers illustrating the presence of responders of biliary like cells, hepatocytes like cells, and endothelial like cells. However, the overall tissue still presented characteristic of immature and foetal patterns. Nevertheless, the biochip culture provided a specific micro-environment in which a complex multicellular …
The synthesis of pyrimidines is a very active area of research, due to the wide applicability of such compounds in pharmaceutical or coordination chemistry. The recent advances since the most recent reviews (2008/2009) are collected here, demonstrating the surge of creativity that this domain has experienced during the last seven years.
In lab-on-a-chip (LoC) devices, microfluidic displacement of liquids is a key component. electrowetting on dielectric (EWOD) is a technique to move fluids, with the advantage of not requiring channels, pumps or valves. Fluids are discretized into droplets on microelectrodes and moved by applying an electric field via the electrodes to manipulate the contact angle. Micro-objects, such as biological cells, can be transported inside of these droplets. However, the design of conventional microelectrodes, made by standard micro-fabrication techniques, fixes the path of the droplets, and limits the reconfigurability of paths and thus limits the parallel processing of droplets. In that respect, thin film transistor (TFT) technology presents a great opportunity as it allows infinitely reconfigurable paths, with high parallelizability. We propose here to investigate the possibility of using TFT array devices for high throughput cell …
The lack of a reliable in vitro system to assess reprotoxicity is an emerging problem in the context of European law for Registration, Evaluation, Authorization and Restriction of Chemicals (REACH, 2007), as it requires a reduction in animal utilization for testing. Furthermore, in vitro reprotoxicological tests would be more relevant and greatly improved by integrating both hepatic metabolism and the blood–testis barrier. Here, we took advantage of an integrated insert in a dynamic microfluidic platform (IIDMP) to co‐cultivate hepatocytes in biochip and Sertoli cells in the bicameral chamber. This microfluidic tool has been previously demonstrated to be helpful in cell function and/or quality improvement. We demonstrate that permeability of the Sertoli barrier is reduced by dynamic coculture in our system. Exometabolomics analysis reveals that interactions between hepatocytes and Sertoli cells may have been mediated …
Thin-Film-Transistors Liquid-Crystal Display has become a standard in the field of displays. However, the structure of these devices presents interest not only in that field, but also for biomedical applications. One of the key components, called here TFT substrate, is a glass substrate with a dense and large array of thousands of transparent micro-electrodes that can be considered as a large scale multi-electrode array(s). Multi-electrode array(s) are widely used for in vitro electrical investigations on neurons and brain, allowing excitation, registration, and recording of their activity. However, the range of application of conventional multi-electrode array(s) is usually limited to some tens of cells in a homogeneous cell culture, because of a small area, small number and a low density of the micro-electrodes. TFT substrates do not have these limitations and the authors are currently studying the possibility to use TFT …
Microfluidic flow-focusing systems are simple and cheap devices to produce monodisperse emulsions. The objective of the study is to determine the flow conditions to create water-in-oil emulsions with flow- and pressure-driven techniques, the use of pressure controllers becoming more systematic, owing to their high precision and capability to generate flows within a large range of fluid properties. The challenge is to make the link between applied pressures and flow rates to be able to switch from pressure to flow-rate driven systems (or vice-versa). To reach this purpose, we have derived a simple model using the electronic–hydraulic analogy between fluid transport in microchannels and electron transport in electric circuit. Thanks to the model, we show that droplets are generated in both cases within exactly the same range of values of inlet-to-outlet pressure differences (and thus flow rates). A unique diagram …
Bayesian networks (BNs) are widely used graphical models usable to draw statistical inference about directed acyclic graphs. We presented here Graph_sampler a fast free C language software for structural inference on BNs. Graph_sampler uses a fully Bayesian approach in which the marginal likelihood of the data and prior information about the network structure are considered. This new software can handle both the continuous as well as discrete data and based on the data type two different models are formulated. The software also provides a wide variety of structure prior which can depict either the global or local properties of the graph structure. Now based on the type of structure prior selected, we considered a wide range of possible values for the prior making it either informative or uninformative. We proposed a new and much faster jumping kernel strategy in the Metropolis–Hastings algorithm …
A microfluidic liver biochip was coupled with a mass spectrometer to detect in real time the drug metabolism of hepatocytes. The hepatocytes were cultivated in the biochip for 35 h. The biochip was placed in a small-scale incubator in which the temperature and CO2 concentration were controlled. The biochip was connected serially to a mass spectrometer, a peristaltic pump and a culture medium reservoir. The injection in the mass spectrometer was performed every 10 min for 11 h. The metabolism of midazolam, phenacetin, omeprazole, dextromethorphan, repaglinide, rosuvastatin, tolbutamide and caffeine was investigated. We monitored the apparition of omeprazole sulfone, hydroxy omeprazole, repaglinide glucuronide, rosuvastatin lactone, dextrorphan, 1-hydroxy midazolam, 4-hydroxy midazolam, 1,4-hydroxy midazolam, paracetamol and 1,3-methylxanthine. Although these were observed …
The very efficient addition of α,α‐difluoro‐α‐(trimethylsilyl)acetamides to aldehydes, ketones and N‐(tert‐butanesulfinyl)imines is described. The reaction is promoted by a catalytic amount of tetra‐n‐butylammonium diphenyltrifluorosilicate (TBAT) and high yields, as well as very high stereoselectivities in the case of N‐(tert‐butanesulfinyl)imines, are achieved. The synthetic potential of this method is illustrated by the conversion of the resulting products to β‐hydroxy ketones, diols and β‐amino alcohols by addition of various Grignard reagents or reduction of the amide moiety.
In lab-on-a-chip (LoC) devices, microfluidic displacement of liquids is a key component. electrowetting on dielectric (EWOD) is a technique to move fluids, with the advantage of not requiring channels, pumps or valves. Fluids are discretized into droplets on microelectrodes and moved by applying an electric field via the electrodes to manipulate the contact angle. Micro-objects, such as biological cells, can be transported inside of these droplets. However, the design of conventional microelectrodes, made by standard micro-fabrication techniques, fixes the path of the droplets, and limits the reconfigurability of paths and thus limits the parallel processing of droplets. In that respect, thin film transistor (TFT) technology presents a great opportunity as it allows infinitely reconfigurable paths, with high parallelizability. We propose here to investigate the possibility of using TFT array devices for high throughput cell …
A methodology allowing the direct preparation of β-amino-α,α-difluoroketones from the Ruppert–Prakash reagent (CF3TMS), acyltrimethylsilanes and N-Boc or N-(diphenylphosphinyl)imines is reported. The process, initiated by a catalytic amount of tetra-n-butylammonium difluorotriphenylsilicate (TBAT), involves the addition of CF3TMS to the acylsilane, followed by a Brook rearrangement and elimination of a fluoride anion. The latter promotes the addition of the resulting difluoroenoxysilane to the imine. The higher electrophilicity of the acylsilane compared to the imine allows the direct mixing of all the reagents in a three-component, one-pot process.
A microfluidic liver biochip was coupled with a mass spectrometer to detect in real time the drug metabolism of hepatocytes. The hepatocytes were cultivated in the biochip for 35 h. The biochip was placed in a small-scale incubator in which the temperature and CO2 concentration were controlled. The biochip was connected serially to a mass spectrometer, a peristaltic pump and a culture medium reservoir. The injection in the mass spectrometer was performed every 10 min for 11 h. The metabolism of midazolam, phenacetin, omeprazole, dextromethorphan, repaglinide, rosuvastatin, tolbutamide and caffeine was investigated. We monitored the apparition of omeprazole sulfone, hydroxy omeprazole, repaglinide glucuronide, rosuvastatin lactone, dextrorphan, 1-hydroxy midazolam, 4-hydroxy midazolam, 1,4-hydroxy midazolam, paracetamol and 1,3-methylxanthine. Although these were observed …
In lab-on-a-chip (LoC) devices, microfluidic displacement of liquids is a key component. electrowetting on dielectric (EWOD) is a technique to move fluids, with the advantage of not requiring channels, pumps or valves. Fluids are discretized into droplets on microelectrodes and moved by applying an electric field via the electrodes to manipulate the contact angle. Micro-objects, such as biological cells, can be transported inside of these droplets. However, the design of conventional microelectrodes, made by standard micro-fabrication techniques, fixes the path of the droplets, and limits the reconfigurability of paths and thus limits the parallel processing of droplets. In that respect, thin film transistor (TFT) technology presents a great opportunity as it allows infinitely reconfigurable paths, with high parallelizability. We propose here to investigate the possibility of using TFT array devices for high throughput cell …
Comme l’a rappelé Jean-Paul Bord, l’objectif de ce second colloque était d’interroger les évolutions de l’enseignement de la cartographie au regard de ceux de la géomatique, conformément aux missions de la Commission Enseignement du CFC. Le dynamisme de la seconde, à savoir la géomatique, avec la généralisation de la numérisation de l’information spatiale et l’accroissement considérable de sa production grâce à la multiplication des outils intégrant la géolocalisation, a pu donner à penser à un affaiblissement de la cartographie. Disposant d’un temps limité pour former les étudiants, les deux disciplines n’allaient-elles pas entrer en concurrence ? Ou au contraire se construire en complémentarité ? Assistons-nous à une hiérarchisation des deux disciplines, à minima dans une progression pédagogique de la cartographie vers la géomatique ? Enfin l’évolution des outils mobilisés pour produire des …
Thin-Film-Transistors Liquid-Crystal Display has become a standard in the field of displays. However, the structure of these devices presents interest not only in that field, but also for biomedical applications. One of the key components, called here TFT substrate, is a glass substrate with a dense and large array of thousands of transparent micro-electrodes that can be considered as a large scale multi-electrode array(s). Multi-electrode array(s) are widely used for in vitro electrical investigations on neurons and brain, allowing excitation, registration, and recording of their activity. However, the range of application of conventional multi-electrode array(s) is usually limited to some tens of cells in a homogeneous cell culture, because of a small area, small number and a low density of the micro-electrodes. TFT substrates do not have these limitations and the authors are currently studying the possibility to use TFT …
2016 (9)
Although microfluidics represents a promising technology for drug screening industry and toxicity tests, their industrial applications using cells are limited by drawbacks of the weakly mass production capability of the biochips. In this work, we report the fabrication of resistant fluorinated microfluidic devices using a material widely used in polymer industries. To build the microdevices, two patterned layers with precise and regular microchannels were developed by photocuring of perfluoropolyethers (PFPEs). These layers were successfully sealed by UV irradiation. Then, Liver HepG2/C3A and kidney MDCK cells were cultured in PFPE biochips. The growth, cell viability and basal metabolism of cells cultured in PFPE biochips were studied and compared with results obtained using polydimethylsiloxane (PDMS) biochips. The results have shown that the cells can attach to the biochip bottom, spread, and proliferate well …
The addition of various RCF2Li compounds to a wide range of carbonyl electrophiles (aldehydes, ketones, acylsilanes, esters, and lactones) is reported. The reaction proceeds at very low temperature under Barbier conditions through a Br/Li exchange from the corresponding RCF2Br compounds. In contrast with existing methods that use more stable, but less reactive, organometallic species, there is efficient addition to esters, lactones and poorly reactive ketones.
Permethrin, a pyrethroid insecticide, is suspected to induce neuronal and hormonal disturbances in humans. The widespread exposure of the populations has been confirmed by the detection of the urinary metabolites of permethrin in biomonitoring studies. Permethrin is a chiral molecule presenting two forms, the cis and the trans isomers. Because in vitro studies indicated a metabolic interaction between the trans and cis isomers of permethrin, we adapted and calibrated a PBPK model for trans- and cis-permethrin separately in rats. The model also describes the toxicokinetics of three urinary metabolites, cis- and trans-3-(2,2 dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis- and trans-DCCA), 3-phenoxybenzoic acid (3-PBA) and 4′OH-phenoxybenzoic acid (4′-OH-PBA). In vivo experiments performed in Sprague–Dawley rats were used to calibrate the PBPK model in a Bayesian framework. The …
We have integrated in vitro and in silico data to describe the toxicity of chloroacetaldehyde (CAA) on renal cells via its production from the metabolism of ifosfamide (IFO) by hepatic cells. A pharmacokinetic (PK) model described the production of CAA by the hepatocytes and its transport to the renal cells. A system biology model was coupled to the PK model to describe the production of reactive oxygen species (ROS) induced by CAA in the renal cells. In response to the ROS production, the metabolism of glutathione (GSH) and its depletion were modeled by the action of an NFE2L2 gene‐dependent pathway. The model parameters were estimated in a Bayesian context via Markov Chain Monte Carlo (MCMC) simulations based on microfluidic experiments and literature in vitro data. Hepatic IFO and CAA in vitro intrinsic clearances were estimated to be 1.85 x 10‐9 μL s–1 cell–1 and 0.185 x 10‐9 μL s–1 cell–1 …
Capsules consist of droplets enclosed by a membrane with shear resistant properties especially when fabricated by interfacial cross-linking. In many applications, the protection and release of the internal medium need to be strictly controlled. It is possible to tune the membrane mechanical properties by changing the physico-chemical conditions of the fabrication process, but a good control of the production requires their characterization, which is a scientific challenge, since the objects are a few tens of microns in size at most. One advantageous approach is to resort to microfluidic techniques. We study the transient response of capsules having a cross-linked human serum albumin (HSA) membrane, as they flow through a sudden expansion. We determine the characteristic time scales of the capsule relaxation and compare them to the ones predicted by a full numerical model of the relaxation of a capsule flowing in …
A microfluidic technique is used to characterize the mechanical behavior of capsules that are produced in a two-step process: first, an emulsification step to form droplets, followed by a cross-linking step to encapsulate the droplets within a thin membrane composed of cross-linked proteins. The objective is to study the influence of the capsule size and protein concentration on the membrane mechanical properties. The microcapsules are fabricated by cross-linking of human serum albumin (HSA) with concentrations from 15 to 35 % (w/v). A wide range of capsule radii (∼40–450 μm) is obtained by varying the stirring speed in the emulsification step. For each stirring speed, a low threshold value in protein concentration is found, below which no coherent capsules could be produced. The smaller the stirring speed, the lower the concentration can be. Increasing the concentration from the threshold value and …
In this work, we have investigated the potential of perfluoropolyether (PFPE) polymers for use in biomaterial applications, especially in cell culture and tissue engineering. PFPE substrates were synthesized by the photocuring of liquid PFPE urethane dimethacrylate. These surfaces were then modified by ECM protein coatings and microstructuration, to promote cell adhesion and migration. The surface properties of PFPE and PDMS (used as a reference) samples were studied by static contact angle measurements and AFM imaging. Both polymer surfaces were hydrophobic, having sessile air–water contact angles superior to 100°. Collagen and fibronectin coatings were found to change the wettability of PFPE and PDMS samples. The biological testing of substrates was done using a liver organotypic culture to evaluate the migration and density of liver cells. The results over seven days of culture demonstrated that …
Human primary hepatocytes were cultivated in a microfluidic bioreactor and in Petri dishes for 13 days. mRNA kinetics in biochips showed an increase in the levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6, HNF4a, SULT1A1, UGT1A1 mRNA related genes when compared with post extraction levels. In addition, comparison with Petri dishes showed higher levels of CYP2B6, CYP2C19, CYP2C8, CYP3A4, CYP1A2, CYP2D6 related genes at the end of culture. Functional assays illustrated a higher urea and albumin production over the period of culture in biochips. Bioreactor drug metabolism (midazolam and phenacetin) was not superior to the Petri dish after 2 days of culture. The CYP3A4 midazolam metabolism was maintained in biochips after 13 days of culture, whereas it was almost undetectable in Petri dishes. This led to a 5000‐fold higher value of the metabolic ratio in the biochips …
A methodology allowing the one‐pot preparation of difluorinated aldols directly from Ruppert–Prakash reagent, acyltrimethylsilanes and aldehydes is reported. The process, initiated by a catalytic amount of an ammonium salt, involves the addition of (trifluoromethyl)trimethylsilane to the acylsilane, followed by a Brook rearrangement and elimination of a fluoride anion that promotes the subsequent aldol reaction. An efficient racemic reaction catalyzed by tetrabutylammonium difluorotriphenylsilicate is described, as well as our first efforts towards an asymmetric version.
2015 (13)
The use of soft materials as substrate for neural probes aims at achieving better compliance with the surrounding neurons while maintaining minimal rejection. Many strategies have emerged to enable such probes to penetrate the cortex, among which the use of resorbable polymers. We performed several tests involving two resorbable polymers considered most promising: polyethylene glycol (PEG) and silk fibroin (SF) from Bombyx Mori silkworms. Our coating method provides a repeatable, uniform structure optimized for a stress-reduced insertion of a parylene-C neural probe. Standard compression tests as well as in vitro and in vivo insertion assessments show that both SF and PEG-coated probes are stiff enough to avoid the buckling effect during insertion in the cortex. However, with a buckling force of 300 mN and a mechanical holding in vitro of tens of minutes, we assess silk fibroin to be more reliable for …
A new in vitro microfluidic platform (integrated insert dynamic microfluidic platform, IIDMP) allowing the co‐culture of intestinal Caco‐2 TC7 cells and of human primary hepatocytes was used to test the absorption and first‐pass metabolism of two drugs: phenacetin and omeprazole. The metabolism of these drugs by CYP1A2, CYP2C19 and CYP3A4 was evaluated by the calculation of bioavailabilities and of intrinsic clearances using a pharmacokinetic (PK) model. To demonstrate the usefulness of the device and of the PK model, predictions were compared with in vitro and in vivo results from the literature. Based on the IIDMP experiments, hepatic in vivo clearances of phenacetin and omeprazole in the IIDMP were predicted to be 3.10 ± 0.36 and 1.46 ± 0.25 ml/min/kg body weight, respectively. This appeared lower than the in vivo observed data with values ranging between 11.9–19.6 and 5.8–7.5 ml/min/kg body …
We have integrated in vitro and in silico information to investigate acetaminophen (APAP) and its metabolite N-acetyl-p-benzoquinone imine (NAPQI) toxicity in liver biochip. In previous works, we observed higher cytotoxicity of HepG2/C3a cultivated in biochips when exposed to 1 mM of APAP for 72 h as compared to Petri cultures. We complete our investigation with the present in silico approach to extend the mechanistic interpretation of the intracellular kinetics of the toxicity process. For that purpose, we propose a mathematical model based on the coupling of a drug pharmacokinetic model (PK) with a systemic biology model (SB) describing the reactive oxygen species (ROS) production by NAPQI and the subsequent glutathione (GSH) depletion. The SB model was parameterized using (i) transcriptomic data, (ii) qualitative results of time lapses ROS fluorescent curves for both control and 1-mM APAP …
In vitro metabolism of permethrin, a pyrethroid insecticide, was assessed in primary human hepatocytes. In vitro kinetic experiments were performed to estimate the Michaelis–Menten parameters and the clearances or formation rates of the permethrin isomers (cis- and trans-) and three metabolites, cis- and trans-3-(2,2 dichlorovinyl)-2,2-dimethyl-(1-cyclopropane) carboxylic acid (cis- and trans-DCCA) and 3-phenoxybenzoic acid (3-PBA). Non-specific binding and the activity of the enzymes involved in permethrin’s metabolism (cytochromes P450 and carboxylesterases) were quantified. Trans-permethrin was cleared more rapidly than cis-permethrin with a 2.6-factor (25.7 ± 0.6 and 10.1 ± 0.3 μL/min/106 cells respectively). A 3-factor was observed between the formation rates of DCCA and 3-PBA obtained from trans- and cis-permethrin. For both isomers, the rate of formation of DCCA was higher than the one of 3 …
Reducing the differentiation period for obtaining an in vitro intestinal barrier model is required to reduce the duration and cost for drug screening assays. In this frame, the Caco-2/TC7 subclone differentiation state was investigated from day 0 (D0) to day 32 (D32). As such, the expression of 45 genes (including cell junction, cell polarization, cell functionality, drug transport and metabolism genes) was followed throughout the 32 days. In parallel, the monolayer polarization and the formation of the cellular junctions were characterized by the immuno-staining of occludin, claudin-1 and actin proteins. The cell monolayer permeability was analyzed via transepithelial electric resistance measurements and paracellular transport of Lucifer Yellow. The P-gp efflux efficiency was assessed by rhodamine 123 transport. Alkaline phosphate activity was quantified to assess the cell differentiation. Three stages of differentiation were …
Bayesian networks (BNs) are widely used graphical models usable to draw statistical inference about network structures. We present here Graph sampler a fast free C language software for structural inference on BNs.Graph sampler uses a fully Bayesian approach in which the marginal likelihood of the data (the data prior predictive distribution) and prior information about the network structure are considered. Inference on continuously valued BNs is treated as a Gaussian regression problem where the regressands are children nodes and the regressors their parents. Normal-gamma or Zellner likelihoods can then be used. For the discrete case a Dirichlet-multinomial model is available. A Metropolis-Hastings algorithm with fast jump kernel is used to sample graphs from their joint posterior distribution. The source C code distributed is very compact, fast, uses low memory and disk storage. We also reviewed the key features and working environment of other available software. As an illustration we compared the performance of our code to that of Structmcmc, a R package, using simulated data sets for graphs of 5 to 120 nodes.
The use of soft materials as substrate for neural probes aims at achieving better compliance with the surrounding neurons while maintaining minimal rejection. Many strategies have emerged to enable such probes to penetrate the cortex, among which the use of resorbable polymers. We performed several tests involving two resorbable polymers considered most promising: polyethylene glycol (PEG) and silk fibroin (SF) from Bombyx Mori silkworms. Our coating method provides a repeatable, uniform structure optimized for a stress-reduced insertion of a parylene-C neural probe. Standard compression tests as well as in vitro and in vivo insertion assessments show that both SF and PEG-coated probes are stiff enough to avoid the buckling effect during insertion in the cortex. However, with a buckling force of 300 mN and a mechanical holding in vitro of tens of minutes, we assess silk fibroin to be more reliable for …
Thanks to the beneficial effect of fluorine substitution on the pharmacokinetic properties of molecules, an ever increasing number of marketed drugs incorporate a fluorine atom into their structure. As a consequence, the synthesis of fluorinated molecules has become a very active research field. Among the numerous approaches, fluorinated enol ethers are valuable building blocks that allow the introduction of a fluoro- or difluoromethyl group through a wide variety of reactions. The present review lists different methods for their preparation and sums up their numerous synthetic applications.
A methodology allowing the preparation of aldols featuring a fluorinated stereogenic center is reported. The corresponding fluoroenolates are formed in situ from stable β,β‐difluoro‐α‐(trimethylsilyl)alcohols, through a base‐mediated process involving a Brook rearrangement followed by a fluoride elimination, and are directly added to aromatic aldehydes. Two different sets of conditions were disclosed. The first one involves the stoichiometric addition of potassium tert‐butoxide (t‐BuOK) while the second is based on the use of a catalytic amount of an ammonium phenoxide. The latter opens the way for a catalytic and asymmetric version of this Brook/elimination/aldol reaction (BEAR) sequence.
A synthesis of difluorinated α-C-galactosylceramides analogs featuring an extra hydroxy group in 1′-position is reported. These compounds were prepared according to unprecedented and unusual methodologies that were previously reported by the authors on simpler substrates. Unfortunately, all four compounds, which feature different lipidic chain lengths, failed to activate iNKT cells. The question whether it is due to the anomeric oxygen/CF2 transposition or to the presence of the extra OH group remains unanswered.
Conception of new pyrimidylmethylamine (pyrma) ligands and their corresponding Pd(II) complexes has been described. Both symmetrical and non-symmetrical ligands were prepared and subjected to complexation. Two different coordination modes, Pd(N,N)– or Pd(C,N,N)–pyrma, have been evidenced depending on the substitution of the pyrimidine ring and the nature or the shape of the additional pendant arm. In a non-symmetrical pyrimidine series, the substituent-induced discrimination of each heterocyclic nitrogen atom provoked regio-controlled coordination to the metal center. The molecular structure of pyrma–Pd(II) complexes in the solution state has been elucidated thanks to combined NMR experiments and DFT calculations. This study highlights the potency of 15N and 13C NMR spectroscopy for the elucidation of the regio-selective coordination to the Pd(II) in the pyrma-based complex series. DFT …
This paper presents the design and performance of a single-ended transimpedance amplifier (TIA) for gigabit passive optical networks implementing orthogonal frequency division multiplexing modulation format. The circuit is realized using an industrial GaAs integrated technology. Low power consumption and small chip area are the main challenges in the TIA design. On-wafer characterization in terms of S-parameters, noise figure, gain compression, and intermodulation are presented and compared to simulated results showing a good agreement. The TIA is linked with a broadband PIN photodiode and introduced in a radio over fiber system. The optical link is simulated in a microwave software and characterized in terms of error vector magnitude by varying the radio frequency input power and the laser bias current.
We have integrated in vitro and in silico information to investigate acetaminophen (APAP) and its metabolite N-acetyl-p-benzoquinone imine (NAPQI) toxicity in liver biochip. In previous works, we observed higher cytotoxicity of HepG2/C3a cultivated in biochips when exposed to 1 mM of APAP for 72 h as compared to Petri cultures. We complete our investigation with the present in silico approach to extend the mechanistic interpretation of the intracellular kinetics of the toxicity process. For that purpose, we propose a mathematical model based on the coupling of a drug pharmacokinetic model (PK) with a systemic biology model (SB) describing the reactive oxygen species (ROS) production by NAPQI and the subsequent glutathione (GSH) depletion. The SB model was parameterized using (i) transcriptomic data, (ii) qualitative results of time lapses ROS fluorescent curves for both control and 1-mM APAP …