Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

AIM: Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. METHODS: An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. RESULTS: Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/ß-catenin, transforming growth factor-ß, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. CONCLUSIONS: The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

Characterization of the proteome and metabolome of human liver sinusoidal endothelial-like cells derived from induced pluripotent stem cells.

The liver is a complex organ composed of several cell types organized hierarchically. Among these, liver sinusoidal endothelial cells (LSECs) are specialized vascular cells known to interact with hepatocytes and hepatic stellate cells (HSCs), and to be involved in the regulation of important hepatic processes in healthy and pathological situations. Protocols for the differentiation of LSECs from human induced pluripotent stem cells, hiPSCs, have been proposed and in-depth analysis by transcriptomic profiling of those cells has been performed. In the present work, an extended analysis of those cells in terms of proteome and metabolome has been implemented. The proteomic analysis confirmed the expression of important endothelial markers and pathways. Among them, the expression of patterns typical of LSECs such as PECAM1, VWF, LYVE1, STAB1 (endothelial markers), CDH13, CDH5, CLDN5, ICAM1, MCAM-CD146, ICAM2, ESAM (endothelial cytoskeleton), NOSTRIN, NOS3 (Nitric Oxide endothelial ROS), ESM1, ENG, MMRN2, THBS1, ANGPT2 (angiogenesis), CD93, MRC1 (mannose receptor), CLEC14A (C-type lectin), CD40 (antigen), and ERG (transcription factor) was highlighted. Besides, the pathway analysis revealed the enrichment of the endocytosis, Toll-like receptor, Nod-like receptor, Wnt, Apelin, VEGF, cGMP-PCK, and PPAR related signaling pathways. Other important pathways such as vasopressin regulated water reabsorption, fluid shear stress, relaxin signaling, and renin secretion were also highlighted. At confluence, the metabolome profile appeared consistent with quiescent endothelial cell patterns. The integration of both proteome and metabolome datasets revealed a switch from fatty acid synthesis in undifferentiated hiPSCs to a fatty oxidation in LSECs and activation of the pentose phosphate pathway and polyamine metabolism in hiPSCs-derived LSECs. In conclusion, the comparison between the signature of LSECs differentiated following the protocol described in this work, and data found in the literature confirmed the particular relevance of these cells for future in vitro applications.

Multi-omics analysis of hiPSCs-derived HLCs matured on-chip revealed patterns typical of liver regeneration.

Maturation of human-induced pluripotent stem cells (hiPSCs)-derived hepatocytes-like cells (HLCs) toward a complete hepatocyte phenotype remains a challenge as primitiveness patterns are still commonly observed. In this study, we propose a modified differentiation protocol for those cells which includes a prematuration in Petri dishes and a maturation in microfluidic biochip. For the first time, a large range of biomolecular families has been extracted from the same sample to combine transcriptomic, proteomic, and metabolomic analysis. After integration, these datasets revealed specific molecular patterns and highlighted the hepatic regeneration profile in biochips. Overall, biochips exhibited processes of cell proliferation and inflammation (via TGFB1) coupled with anti-fibrotic signaling (via angiotensin 1-7, ATR-2, and MASR). Moreover, cultures in this condition displayed physiological lipid-carbohydrate homeostasis (notably via PPAR, cholesterol metabolism, and bile synthesis) coupled with cell respiration through advanced oxidative phosphorylation (through the overexpression of proteins from the third and fourth complex). The results presented provide an original overview of the complex mechanisms involved in liver regeneration using an advanced in vitro organ-on-chip technology.

Metabolomic profiling during the differentiation of human induced pluripotent stem cells into hepatocyte-like cells.

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 characterized by fatty acid beta oxidation, sorbitol metabolism and pentose phosphate pathway, and glutamine and glucose metabolisms as various potential energy sources. The complex lipid metabolism switch was monitored via the reduction of lipid production from DE to HLCs step, whereas high glycerol production occurred mainly in HLCs. The nitrogen cycle, via urea production, was also a typical mechanism revealed in HLCs step. Our analysis may contribute to better understanding of differentiation and suggest new targets for improving iPSC maturation into functional hepatocytes.

Multiscale-Engineered Muscle Constructs: PEG Hydrogel Micro-Patterning on an Electrospun PCL Mat Functionalized with Gold Nanoparticles.

The development of new, viable, and functional engineered tissue is a complex and challenging task. Skeletal muscle constructs have specific requirements as cells are sensitive to the stiffness, geometry of the materials, and biological micro-environment. The aim of this study was thus to design and characterize a multi-scale scaffold and to evaluate it regarding the differentiation process of C2C12 skeletal myoblasts. The significance of the work lies in the microfabrication of lines of polyethylene glycol, on poly(ε-caprolactone) nanofiber sheets obtained using the electrospinning process, coated or not with gold nanoparticles to act as a potential substrate for electrical stimulation. The differentiation of C2C12 cells was studied over a period of seven days and quantified through both expression of specific genes, and analysis of the myotubes' alignment and length using confocal microscopy. We demonstrated that our multiscale bio-construct presented tunable mechanical properties and supported the different stages skeletal muscle, as well as improving the parallel orientation of the myotubes with a variation of less than 15°. These scaffolds showed the ability of sustained myogenic differentiation by enhancing the organization of reconstructed skeletal muscle. Moreover, they may be suitable for applications in mechanical and electrical stimulation to mimic the muscle's physiological functions.

Metabolomics-on-a-chip approach to study hepatotoxicity of DDT, permethrin and their mixtures.

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 oxidative stress and cell mortality. Metabolomics profiling revealed that hepatocytes exposure to DDT150, PMT150 and DDT150 and PMT150 cause important modulation in intermediates of glutathione pathway and tricarboxylic acid cycle, amino acids and metabolites associated to hepatic necrosis and inflammation (α-ketoglutarate, arginine and 2-hydroxybutyrate). These changes were more striking in the combined group. Finally, DDT150 led to a significant increase of benzoate, decanoate, octanoate, palmitate, stearate and tetradecanoate, which illustrates the estrogen modulation. This study demonstrates the potential of metabolomics-on-a-chip approach to improve knowledge on the mode of action of pesticides.

Online monitoring of hepatic rat metabolism by coupling a liver biochip and a mass spectrometer.

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, hydroxytolbutamide, 3-methoxymorphinan and midazolam glucuronide, hydroxy repaglinide were not detected. Based on a pharmacokinetic model, we calculated in vitro intrinsic clearances in which adsorption onto the perfusion circuit was taken into account. Then, using a liver organ model, we extrapolated the in vitro intrinsic clearances to the in vivo clearances. The estimated in vivo clearances were in agreement with the literature data on rats for midazolam, dextromethorphan, phenacetin, tolbutamide and caffeine. Rosuvastatin, omeprazole and repaglinide prediction underestimated the in vivo data.

Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.

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. CYP1A2 phenacetin metabolism was found to be higher in biochips after 5, 9 and 13 days of culture. Thus, a 100-fold higher metabolic ratio of APAP in biochips was measured after 13 days of perfusion. These results demonstrated functional primary human hepatocyte culture in the bioreactor in a long-term culture. Copyright © 2016 John Wiley & Sons, Ltd.

Investigation of acetaminophen toxicity in HepG2/C3a microscale cultures using a system biology model of glutathione depletion.

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-treated experiments, and (iii) additional GSH literature data. The PK model was parameterized (i) using the in vitro kinetic data (at 160 µM, 1 mM, 10 mM), (ii) using the parameters resulting from a physiologically based pharmacokinetic (PBPK) literature model for APAP, and (iii) by literature data describing NAPQI formation. The PK-SB model predicted a ROS increase and GSH depletion due to the NAPQI formation. The transition from a detoxification phase and NAPQI and ROS accumulation was predicted for a NAPQI concentration ranging between 0.025 and 0.25 µM in the cytosol. In parallel, we performed a dose response analysis in biochips that shows a reduction of the final hepatic cell number appeared in agreement with the time and doses associated with the switch of the NAPQI detoxification/accumulation. As a result, we were able to correlate in vitro extracellular APAP exposures with an intracellular in silico ROS accumulation using an integration of a coupled mathematical and experimental liver on chip approach.

Investigation of omeprazole and phenacetin first-pass metabolism in humans using a microscale bioreactor and pharmacokinetic models.

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 weight, respectively. Then the calculated hepatic and intestinal clearances led to predicting an oral bioavailability of 0.85 and 0.77 for phenacetin and omeprazole versus 0.92 and 0.78 using separate data from the simple monoculture of Caco-2 TC7 cells and hepatocytes in Petri dishes. When compared with the in vivo data, the results of oral bioavailability were overestimated (0.37 and 0.71, respectively). The feasibility of co-culture in a device allowing the integration of intestinal absorption, intestinal metabolism and hepatic metabolism in a single model was demonstrated. Nevertheless, further experiments with other drugs are needed to extend knowledge of the device to predict oral bioavailability and intestinal first-pass metabolism.

Coculture model of a liver sinusoidal endothelial cell barrier and HepG2/C3a spheroids-on-chip in an advanced fluidic platform.

The liver is one of the main organs involved in the metabolism of xenobiotics and a key organ in toxicity studies. Prior to accessing the hepatocytes, xenobiotics pass through the hepatic sinusoid formed by liver sinusoidal endothelial cells (LSECs). The LSECs barrier regulates the kinetics and concentrations of the xenobiotics before their metabolic processing by the hepatocytes. To mimic this physiological situation, we developed an in vitro model reproducing an LSECs barrier in coculture with a hepatocyte biochip, using a fluidic platform. This technology made dynamic coculture and tissue crosstalk possible. SK-HEP-1 and HepG2/C3a cells were used as LSECs and as hepatocyte models, respectively. We confirmed the LSECs phenotype by measuring PECAM-1 and stabilin-2 expression levels and the barrier's permeability/transport properties with various molecules. The tightness of the SK-HEP-1 barrier was enhanced in the dynamic coculture. The morphology, albumin secretion, and gene expression levels of markers of HepG2/C3a were not modified by coculture with the LSECs barrier. Using acetaminophen, a well-known hepatotoxic drug, to study tissue crosstalk, there was a reduction in the expression levels of the LSECs markers stabilin-2 and PECAM-1, and a modification of those of CLEC4M and KDR. No HepG2/C3a toxicity was observed. The metabolisation of acetaminophen by HepG2/C3a monocultures and cocultures was confirmed. Although primary cells are required to propose a fully relevant model, the present approach highlights the potential of our system for investigating xenobiotic metabolism and toxicity.

Transcriptomic profiling analysis of the effect of palmitic acid on 3D spheroids of ß-like cells derived from induced pluripotent stem cells.

Type 2 diabetes (T2D) is posing a serious public health concern with a considerable impact on human life and health expenditures worldwide. The disease develops when insulin plasma level is insufficient for coping insulin resistance, caused by the decline of pancreatic ß-cell function and mass. In ß-cells, the lipotoxicity exerted by saturated free fatty acids in particular palmitate (PA), which is chronically elevated in T2D, plays a major role in ß-cell dysfunction and mass. However, there is a lack of human relevant in vitro model to identify the underlying mechanism through which palmitate induces ß-cell failure. In this frame, we have previously developed a cutting-edge 3D spheroid model of ß-like cells derived from human induced pluripotent stem cells. In the present work, we investigated the signaling pathways modified by palmitate in ß-like cells derived spheroids. When compared to the 2D monolayer cultures, the transcriptome analysis (FDR set at  0.1) revealed that the 3D spheroids upregulated the pancreatic markers (such as GCG, IAPP genes), lipids metabolism and transporters (CD36, HMGSC2 genes), glucose transporter (SLC2A6). Then, the 3D spheroids are exposed to PA 0.5 mM for 72 h. The differential analysis demonstrated that 32 transcription factors and 135 target genes were mainly modulated (FDR set at  0.1) including the upregulation of lipid and carbohydrates metabolism (HMGSC2, LDHA, GLUT3), fibrin metabolism (FGG, FGB), apoptosis (CASP7). The pathway analysis using the 135 selected targets extracted the fibrin related biological process and wound healing in 3D PA treated conditions. An overall pathway gene set enrichment analysis, performed on the overall gene set (with pathway significance cutoff at 0.2), highlighted that PA perturbs the citrate cycle, FOXO signaling and Hippo signaling as observed in human islets studies. Additional RT-PCR confirmed induction of inflammatory (IGFBP1, IGFBP3) and cell growth (CCND1, Ki67) pathways by PA. All these changes were associated with unaffected glucose-stimulated insulin secretion (GSIS), suggesting that they precede the defect of insulin secretion and death induced by PA. Overall, we believe that our data demonstrate the potential of our spheroid 3D islet-like cells to investigate the pancreatic-like response to diabetogenic environment.

Antifouling action of polyisoprene-based coatings by inhibition of photosynthesis in microalgae.

Previous studies have demonstrated that ionic and non-ionic natural rubber-based coatings inhibit adhesion and growth of marine bacteria, fungi, microalgae, and spores of macroalgae. Nevertheless, the mechanism of action of these coatings on the different micro-organisms is not known. In the current study, antifouling activity of a series of these rubber-based coatings (one ionic and two non-ionic) was studied with respect to impacts on marine microalgal photosynthesis using pulse-amplitude-modulation (PAM) fluorescence. When grown in contact with the three different coatings, an inhibition of photosynthetic rate (relative electron transport rate, rETR) was observed in all of the four species of pennate diatoms involved in microfouling, Cocconeis scutellum, Amphora coffeaeformis, Cylindrotheca closterium, and Navicula jeffreyi. The percentage of inhibition ranged from 44% to 100% of the controls, depending on the species and the coating. The ionic coating was the most efficient antifouling (AF) treatment, and C. scutellum and A. coffeaeformis are the most sensitive and tolerant diatoms tested, respectively. Photosynthetic inhibition was reversible, as almost complete recovery of rETR was observed 48 h post exposure, after detachment of cells from the coatings. Thus, the antifouling activity seemed mostly due to an effect of contact with materials. It is hypothesized that photosynthetic activity was suppressed by coatings due to interference in calcium availability to the microalgal cells; Ca(2+) has been shown to be an essential micro/macro nutrient for photosynthesis, as well as being involved in cell adhesion and motility in pennate diatoms.

Development of a lung-liver in vitro coculture model for inhalation-like toxicity assessment.

Animal models are considered prime study models for inhalation-like toxicity assessment. However, in light of animal experimentation reduction (3Rs), we developed and investigated an alternative in vitro method to study systemic-like responses to inhalation-like exposures. A coculture platform was established to emulate inter-organ crosstalks between a pulmonary barrier, which constitutes the route of entry of inhaled compounds, and the liver, which plays a major role in xenobiotic metabolism. Both compartments (Calu-3 insert and HepG2/C3A biochip) were jointly cultured in a dynamically-stimulated environment for 72 h. The present model was characterized using acetaminophen (APAP), a well-documented hepatotoxicant, to visibly assess the passage and circulation of a xenobiotic through the device. Based on viability and functionality parameters the coculture model showed that the bronchial barrier and the liver biochip can successfully be maintained viable and function in a dynamic coculture setting for 3 days. In a stress-induced environment, present results reported that the coculture model emulated active and functional in vitro crosstalk that seemingly was responsive to xenobiotic exposure doses. The hepatic and bronchial cellular responses to xenobiotic exposure were modified in the coculture setting as they displayed earlier and stronger detoxification processes, highlighting active and functional organ crosstalk between both compartments.

Generation of ß-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids.

Since the identification of four different pancreatic ß-cell subtypes and bi-hormomal cells playing a role in the diabetes pathogenesis, the search for in vitro models that mimics such cells heterogeneity became a key priority in experimental and clinical diabetology. We investigated the potential of human induced pluripotent stem cells to lead to the development of the different ß-cells subtypes in honeycomb microwell-based 3D spheroids. The glucose-stimulated insulin secretion confirmed the spheroids functionality. Then, we performed a single cell RNA sequencing of the spheroids. Using a knowledge-based analysis with a stringency on the pancreatic markers, we extracted the ß-cells INS+/UCN3+ subtype (11%; ß1-like cells), the INS+/ST8SIA1+/CD9- subtype (3%, ß3-like cells) and INS+/CD9+/ST8SIA1-subtype (1%; ß2-like cells) consistently with literature findings. We did not detect the INS+/ST8SIA1+/CD9+ cells (ß4-like cells). Then, we also identified four bi-hormonal cells subpopulations including δ-like cells (INS+/SST+, 6%), γ-like cells (INS+/PPY+, 3%), α-like-cells (INS+/GCG+, 6%) and ε-like-cells (INS+/GHRL+, 2%). Using data-driven clustering, we extracted four progenitors' subpopulations (with the lower level of INS gene) that included one population highly expressing inhibin genes (INHBA+/INHBB+), one population highly expressing KCNJ3+/TPH1+, one population expressing hepatocyte-like lineage markers (HNF1A+/AFP+), and one population expressing stem-like cell pancreatic progenitor markers (SOX2+/NEUROG3+). Furthermore, among the cycling population we found a large number of REST+ cells and CD9+ cells (CD9+/SPARC+/REST+). Our data confirm that our differentiation leads to large ß-cell heterogeneity, which can be used for investigating ß-cells plasticity under physiological and pathophysiological conditions.

Investigation of the metabolomic crosstalk between liver sinusoidal endothelial cells and hepatocytes exposed to paracetamol using organ-on-chip technology.

Organ-on-chip technology is a promising in vitro approach recapitulating human physiology for the study of responses to drug exposure. Organ-on-chip cell cultures have paved new grounds for testing and understanding metabolic dose-responses when evaluating pharmaceutical and environmental toxicity. Here, we present a metabolomic investigation of a coculture of liver sinusoidal endothelial cells (LSECs, SK-HEP-1) with hepatocytes (HepG2/C3a) using advanced organ-on-chip technology. To reproduce the physiology of the sinusoidal barrier, LSECs were separated from hepatocytes by a membrane (culture insert integrated organ-on-chip platform). The tissues were exposed to acetaminophen (APAP), an analgesic drug widely used as a xenobiotic model in liver and HepG2/C3a studies. The differences between the SK-HEP-1, HepG2/C3a monocultures and SK-HEP-1/HepG2/C3a cocultures, treated or not with APAP, were identified from metabolomic profiles using supervised multivariate analysis. The pathway enrichment coupled with metabolite analysis of the corresponding metabolic fingerprints contributed to extracting the specificity of each type of culture and condition. In addition, we analysed the responses to APAP treatment by mapping the signatures with significant modulation of the biological processes of the SK-HEP-1 APAP, HepG2/C3a APAP and SK-HEP-1/HepG2/C3a APAP conditions. Furthermore, our model shows how the presence of the LSECs barrier and APAP first pass can modify the metabolism of HepG2/C3a. Altogether, this study demonstrates the potential of a "metabolomic-on-chip" strategy for pharmaco-metabolomic applications predicting individual response to drugs.

Correction: Generation of ß-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids.

Correction for 'Generation of ß-like cell subtypes from differentiated human induced pluripotent stem cells in 3D spheroids' by Lisa Morisseau et al., Mol. Omics, 2023, https://doi.org/10.1039/d3mo00050h.

Liver organ-on-chip models for toxicity studies and risk assessment.

The liver is a key organ that plays a pivotal role in metabolism and ensures a variety of functions in the body, including homeostasis, synthesis of essential components, nutrient storage, and detoxification. As the centre of metabolism for exogenous molecules, the liver is continuously exposed to a wide range of compounds, such as drugs, pesticides, and environmental pollutants. Most of these compounds can cause hepatotoxicity and lead to severe and irreversible liver damage. To study the effects of chemicals and drugs on the liver, most commonly, animal models or in vitro 2D cell cultures are used. However, data obtained from animal models lose their relevance when extrapolated to the human metabolic situation and pose ethical concerns, while 2D static cultures are poorly predictive of human in vivo metabolism and toxicity. As a result, there is a widespread need to develop relevant in vitro liver models for toxicology studies. In recent years, progress in tissue engineering, biomaterials, microfabrication, and cell biology has created opportunities for more relevant in vitro models for toxicology studies. Of these models, the liver organ-on-chip (OoC) has shown promising results by reproducing the in vivo behaviour of the cell/organ or a group of organs, the controlled physiological micro-environment, and in vivo cellular metabolic responses. In this review, we discuss the development of liver organ-on-chip technology and its use in toxicity studies. First, we introduce the physiology of the liver and summarize the traditional experimental models for toxicity studies. We then present liver OoC technology, including the general concept, materials used, cell sources, and different approaches. We review the prominent liver OoC and multi-OoC integrating the liver for drug and chemical toxicity studies. Finally, we conclude with the future challenges and directions for developing or improving liver OoC models.

Investigation of the Exometabolomic Profiles of Rat Islets of Langerhans Cultured in Microfluidic Biochip.

Diabetes mellitus (DM) is a complex disease with high prevalence of comorbidity and mortality. DM is predicted to reach more than 700 million people by 2045. In recent years, several advanced in vitro models and analytical tools were developed to investigate the pancreatic tissue response to pathological situations and identify therapeutic solutions. Of all the in vitro promising models, cell culture in microfluidic biochip allows the reproduction of in-vivo-like micro-environments. Here, we cultured rat islets of Langerhans using dynamic cultures in microfluidic biochips. The dynamic cultures were compared to static islets cultures in Petri. The islets' exometabolomic signatures, with and without GLP1 and isradipine treatments, were characterized by GC-MS. Compared to Petri, biochip culture contributes to maintaining high secretions of insulin, C-peptide and glucagon. The exometabolomic profiling revealed 22 and 18 metabolites differentially expressed between Petri and biochip on Day 3 and 5. These metabolites illustrated the increase in lipid metabolism, the perturbation of the pentose phosphate pathway and the TCA cycle in biochip. After drug stimulations, the exometabolome of biochip culture appeared more perturbed than the Petri exometabolome. The GLP1 contributed to the increase in the levels of glycolysis, pentose phosphate and glutathione pathways intermediates, whereas isradipine led to reduced levels of lipids and carbohydrates.

Analysis of the transcriptome and metabolome of pancreatic spheroids derived from human induced pluripotent stem cells and matured in an organ-on-a-chip.

Functional differentiation of pancreatic like tissue from human induced pluripotent stem cells is one of the emerging strategies to achieve an in vitro pancreas model. Here, we propose a protocol to cultivate hiPSC-derived ß-like-cells coupling spheroids and microfluidic technologies to improve the pancreatic lineage maturation. The protocol led to the development of spheroids producing the C-peptide and containing cells positive to insulin and glucagon. In order to further characterize the cellular and molecular profiles, we performed full transcriptomics and metabolomics analysis. The omics analysis confirmed the activation of key transcription factors together with the upregulation of genes and the presence of metabolites involved in functional pancreatic tissue development, extracellular matrix remodeling, lipid and fatty acid metabolism, and endocrine hormone signaling. When compared to static 3D honeycomb cultures, dynamic 3D biochip cultures contributed to increase specifically the activity of the HIF transcription factor, to activate the calcium activated cation channels, to enrich the glucagon and insulin pathways and glycolysis/gluconeogenesis, and to increase the secretion of serotonin, glycerol and glycerol-3-phosphate at the metabolic levels.