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International audience; 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.

Hepatocyte growth factor (HGF) is the natural ligand of the MET receptor tyrosine kinase. This ligand-receptor couple is essential for the maturation process of hepatocytes. Previously, the rational design of a synthetic protein based on the assembly of two K1 domains from HGF led to the production of a potent and stable MET receptor agonist. In this study, we compared the effects of K1K1 with HGF during the differentiation of hepatocyte progenitors derived from human induced pluripotent stem cells (hiPSCs). In vitro, K1K1, in the range of 20 to 200 nM, successfully substituted for HGF and efficiently activated ERK downstream signaling. Analysis of the levels of hepatocyte markers showed typical liver mRNA and protein expression (HNF4α, albumin, alpha-fetoprotein, CYP3A4) and phenotypes. Although full maturation was not achieved, the results suggest that K1K1 is an attractive candidate MET agonist suitable for replacing complex and expensive HGF treatments to induce hepatic differentiation of hiPSCs.

In order to address the remaining issues of fragile structure and insufficient mass transfer faced in modular assembly-based liver tissue engineering, a Raschig ring-like hollowed micro-scaffold was proposed and fabricated using poly-e-caprolactone with 60% porosity and 11.4 mm2 effective surface area for cell immobilization. The method of cell inoculation, the types of cells for co-culture and the scalability of the proposed hollowed micro-scaffold in perfusion were all investigated to obtain an optimized organoid made of tissue modules. Extracellular matrix was found necessary to establish a hierarchical co-culture, and the triple co-culture of Human Hepatoma Hep G2 cells, liver sinusoid cell line TMNK-1 cells and fibroblasts (Swiss 3T3 cells) was recognized to be the most efficient to obtain higher cell attachment, proliferation and hepatic function. The equipped intersecting hollow channels provided in the micro-scaffold functioned as flow paths to promote mass transfer to the immobilized cells after the modules have been randomly packed into a bioreactor for perfusion culture, and resulted in enhanced albumin production and high cellular viability. Cell density comparable to those found in vivo were obtained in the perfused construct, which also maintained its rigid structure. Those results suggest that modular tissues made with hollowed micro-scaffold-based organoids hold great potential for scaling up tissue engineered constructs towards implantation.

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 upregulation of the APLN, LYVE1, VWF, ESAM and ANGPT2 genes while VEGFA appeared to be downregulated. Analysis of promoter motif activities highlighted several transcription factors (TFs) of interest in LSECs (IRF2, ERG, MEIS2, SPI1, IRF7, WRNIP1, HIC2, NFIX_NFIB, BATF, and PATZ1). Based on this investigation, we compiled the regulatory network involving the relevant TFs, their target genes as well as their related signaling pathways. The proposed hiPSC-derived LSEC model and its regulatory network were then confirmed by comparing the experimental data to primary human LSEC reference datasets. Thus, the presented model appears as a promising tool to generate more complex in vitro liver multi-cellular tissues.

For high-throughput anti-cancer drug screening, microwell arrays may serve as an effective tool to generate uniform and scalable tumor spheroids. However, microwell arrays are commonly anchored in non-oxygen-permeable culture plates, leading to limited oxygen supply for avascular spheroids. Herein, a polydimethylsiloxane (PDMS)-based oxygen-permeable microwell device is introduced for generating highly viable and functional hepatocellular carcinoma (HCC) spheroids. The PDMS sheets at the bottom of the microwell device provide a high flux of oxygen like in vivo neighboring hepatic sinusoids. Owing to the better oxygen supply, the generated HepG2 spheroids are larger in size and exhibit higher viability and proliferation with less cell apoptosis and necrosis. These spheroids also exhibit lower levels of anaerobic cellular respiration and express higher levels of liver-related functions. In anti-cancer drug testing, spheroids cultured in PDMS plates show a significantly stronger resistance against doxorubicin because of the stronger stem-cell and multidrug resistance phenotype. Moreover, higher expression of vascular endothelial growth factor-A produces a stronger angiogenesis capability of the spheroids. Overall, compared to the spheroids cultured in conventional non-oxygen-permeable plates, these spheroids can be used as a more favorable model for early-stage HCCs and be applied in high-throughput anti-cancer drug screening.

International audience; 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.

International audience; Advances in organ-on-chip technology allowed the recapitulation of two or more organs interaction thanks to dedicated microbioreactors interconnected by microfluidic network. Here, we developed pancreas-liver coculture model in a microfluidic biochip, using rat Langerhans islets and hepatocytes. The behavior and functionality of the model were compared to islets and hepatocytes (with/without insulin) monocultures. We confirmed the insulin, glucagon and C-peptide secretions by islets monoculture and coculture. Furthermore, C-peptide and insulin secretions were higher in coculture after 5 days of culture. The islets coculture presented downregulation of Pdx1, Glut2, Gcg, App, Ins1, Neurod, Neurog3 and Gcgr genes, compared to monocultures. In hepatic compartment, the monocultures without insulin were negative to CK18 staining and displayed a weaker production of albumin when compared to monocultures with insulin. They also presented a moderate protein expression of CYP3A2, GLUT2, INSR and modulation of several hepatic genes. In coculture model, hepatocytes displayed albumin productions similar to those in monoculture with insulin. The hepatocytes cocultures were highly positive to INSR, GLUT2, CK18 and CYP3A2 immunostaining and allowed to recover mRNA levels similar to monoculture with insulin (Gcgr, Insr, Hnf4a, Igfbp1 and Alb). The result illustrated that islets can produce insulin to supplement the culture medium and recover hepatic functionality. We believe that our model illustrated the potential of organ-on-chip technology to reproduce cross-talk between liver and pancreas.

International audience; Liver modeling in disease via advanced in vitro physiological tissues remains a challenging issue, yet crucial for the future development of relevant tools for drug screening. In that regard, advanced technics such as human induced Pluripotent Stem Cells (hiPSCs) and organ-on-chip are promising technologies for the relevant reproduction of the in vivo micro physiology in in vitro culture conditions. In the present work, the maturation of carboxypeptidase M positive (CPM+) Hepatocyte-Like-Cells (HLCs) in microfluidic culture conditions was investigated. hiPSCs were differentiated in culture dishes until sorting and further amplified until seeding in biochips. After 2 weeks of maturation in biochips, evaluation of the cell metabolism was performed, and samples were collected for characterization via RNA sequencing, proteomics and metabolomics. The omics profile of biochips loaded with CPM+ HLCs was then compared with the one of previously cultured biochips loaded with unsorted HLCs. CPM+ HLCs presented advanced liver characteristics in terms of a higher activity of important Transcriptions Factors (TFs) such as HNF1, HNF4A, and CEBP/A, of the upregulation of steroids/corticoids-related nuclear receptors (FXR, NR3C2, NR4A1 genes), of phase I, and phase II metabolism genes (CYP1A1, CYP1B1, CYP2C18, CYP27A1, several UGT, SULT and GST genes). Further differences in the cellular reorganization, in the lipids and steroids metabolisms, in the OXPHOS respiration and in the expression of TGFβ signaling were also observed. The present study introduces a novel protocol using advances hiPSCs differentiation and sorting methods as well as the organ-on-chip technology to highlight important in vitro liver regeneration and hepatic maturation processes.

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 markers like PPARA. The performed analysis has allowed for the extraction of potential targets and pathways which would allow enhanced hepatic maturation in-vitro. From this investigation, NRF1 and SP3 appeared as transcription factors of importance. Complex epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) patterns were also observed during the differentiation process. Moreover, whole transcriptome analysis highlighted a response typical of the one observed in liver regeneration and hepatocyte proliferation. While a complete maturation of hepatocytes was yet to be obtained, the results presented in this work provide new insights into the process of liver development and highlight potential targets aimed to improve in-vitro liver regeneration.

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 biochip 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 the pancreatic islets to glucagon-like peptide 1 (GLP-1) stimulation in the biochips was reflected by the upregulation of mRNA levels of Gcgr, Reg3a, Neurog3, Ins1, Ins2, Stt and Glp-1r and by increased insulin secretion. The results obtained highlighted the functionality of the islets in the biochips and illustrated the potential of our pancreas-on-chip model for future pancreatic disease modeling and anti-diabetic drugs screening.

Pre-clinical drug screening is an important step in assessing the metabolic effects and hepatic toxicity of new pharmaceutical compounds. However, due to the complexity of the liver microarchitecture, simplified in vitro models do not adequately reflect in vivo situations. Especially spatial heterogeneity, known as metabolic zonation, is often lost due to limitations introduced by typical culture conditions. By culturing primary rat hepatocytes in varied ambient oxygen levels on either gas-permeable or non-permeable culture plates, we highlight the importance of biomimetic oxygen supply for the targeted induction of zonation-like phenotypes. Resulting cellular profiles illustrate the effect of pericellular oxygen concentration and consumption rates on hepatic functionality in terms of zone-specific metabolism and β-catenin signaling. We show that modulation of ambient oxygen tension can partially induce metabolic zonation in vitro when considering high supply rates, leading to in vivo-like drug metabolism. However, when oxygen supply is limited, similar modulation instead triggers an ischemic reprogramming, resembling metabolic profiles of hepatocellular carcinoma and increasing susceptibility toward drug-induced injury. Application of this knowledge will allow for the development of more accurate drug screening models to better identify adverse effects in hepatic drug metabolism.

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 processes (CYP450), PPAR signaling or arginine biosynthesis. A subset of 16 transcription factors (TFs) was extracted and identified as upstream regulators to HNF1A and PPARA. These TFs are also known as regulators to target genes engaged in the Wnt/βcatenin pathway, in the TGFβ/BMP/SMAD signaling, in the transition between epithelial mesenchymal transition (EMT) and mesenchymal epithelial transition (MET), in the homeostasis of lipids, bile acids and carbohydrates homeostasis, in drug metabolism, in the estrogen processing and in the oxidative stress response. Overall, the analysis allowed to confirm a partial zonation-like pattern in hiPSCs-derived HLCs in the microfluidic biochip environment. These results provide important insights into the reproduction of liver zonation in-vitro for a better understanding of the phenomenon.

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.

International audience; Interactions between the different liver cell types are critical to the maintenance or induction of their function in vitro. In this work, human-induced Pluripotent Stem Cells (hiPSCs)-derived Liver Sinusoidal Endothelial Cells (LSECs) and Hepatocytes-Like Cells (HLCs) were cultured and matured in a microfluidic environment. Both cell populations were differentiated in Petri dishes, detached, and inoculated in microfluidic biochips. In cocultures of both cell types, the tissue has exhibited a higher production of albumin (3.19 vs 5.31 μg/mL/106 cells in monocultures and cocultures) as well as a higher inducibility CYP450 over monocultures of HLCs. Tubular-like structures composed of LSECs and positive for the endothelial marker PECAM1, as well as a tissue more largely expressing Stabilin-2 were detected in cocultures only. In contrast, monocultures exhibited no network and less specific endothelial markers. The transcriptomic analysis did not reveal a marked difference between the profiles of both culture conditions. Nevertheless, the analysis allowed us to highlight different upstream regulators in cocultures (SP1, EBF1, and GATA3) and monocultures (PML, MECP2, and NRF1). In cocultures, the multi-omics dataset after 14 days of maturation in biochips has shown the activation of signaling related to hepatic maturation, angiogenesis, and tissue repair. In this condition, inflammatory signaling was also found to be reduced when compared to monocultures as illustrated by the activation of NFKB and by the detection of several cytokines involved in tissue injury in the latter. Finally, the extracted biological processes were discussed regarding the future development of a new generation of human in vitro hepatic models.

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 differentiation toward liver could be oriented.

International audience; 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 by the activation of upstream regulators such as HNF1A, MEIS2, NFIX, WRNIP1, SP4, TAL1. Their regulatory networks highlighted HNF4a as a bridge and linked them to important processes such as EMT–MET transitions, ECM remodeling and liver development pathways (HNF3, PPARA signaling, iron metabolism) along the different steps of differentiation.

International audience; 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 metabolomic data with transcriptomic contributed to explaining the kinetics of carbohydrate, lipid and nitrogen metabolism throughout differentiation. The information extracted during differentiation showed that the cells moved from an aerobic-like respiration pattern to a mitochondrial oxidative respiration pattern in both protocols. Reducing culture medium renewal led to reduced glucose consumption, followed by fructose production and significant extracellular lipogenesis throughout differentiation. We believe that the overall dataset can provide information on the sequence of process.

In the present study, we evaluated the performance of different protocols for the hepatic differentiation of human-induced pluripotent stem cells (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 with 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 the production rate for albumin in biochips (about 2,000 ng per day per 106 cells). Besides this, 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 a higher degree of hepatic differentiation in biochips, together with increased promoter motif activity for typical liver transcription factors such as estrogen related receptor alpha ( ESRRA), hepatic nuclear factor 1 ( HNF1A), hepatic nuclear factor 4 ( HNF4A), transcription factor 4 ( TCF4), and CCAAT enhancer binding protein alpha ( CEBPA). Gene set enrichment analysis identified several pathways related to the extracellular matrix, tissue reorganization, hypoxia-inducible transcription factor, and glycolysis that were differentially modulated in biochip cultures. However, the presence of CK19/ALB-positive cells and the ɑ-fetoprotein levels measured in the cultures still reflect primitive differentiation patterns. Overall, we identified key parameters for improved hepatic differentiation on-chip, including the maturation stage of hepatic progenitors, inoculation density, adhesion time, and perfusion flow rate. Optimization of these parameters further led to establish a protocol for reproducible differentiation of hiPSCs into hepatocyte-like cells in microfluidic biochips with significant improvements over Petri dish cultures.

International audience; 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 reached 2500 ng/mL/day for both protocols. The transcription factor motif activity analysis via transcriptomic data obtained with the Cap Analysis Gene Expression (CAGE) technology confirmed a typical hepatic profile for both protocols (such as activation of HNF1 A and HNF1B promoters). However, specific patterns were also detected with higher mRNA levels of WT1, MTF1, HCFC1, and PGCα1 in protocol 2. The overall transcriptomic analysis also revealed a modulation of targets involved in glucose homeostasis, in cell remodeling, lipid pathways and chromatin processing. However, the alpha-fetoprotein (AFP) production remained very high and the function of CYP3A4 was weak in both protocols illustrating still an immature profile in both protocols.

Establishing a bile duct in vitro is valuable to obtain relevant hepatic tissue culture systems for cell-based assays in chemical and drug metabolism analyses. The cyst constitutes the initial morphogenesis for bile duct formation from biliary epithelial cells (BECs) and serves the main building block of bile duct network morphogenesis from the ductal plate during embryogenesis in rodents. Cysts have been commonly cultured via Matrigel-embedded culture, which does not allow structural organisation and restricts the productivity and homogeneity of cysts. In this study, we propose a new method utilising oxygen permeable honeycomb microwells for efficient cyst establishment. Primary mouse BECs were seeded on four sizes of honeycomb microwell (46, 76, 126, and 326 µm-size in diameter). Matrigel in various concentrations was added to assist in cyst formation. The dimension accommodated by microwells was shown to play an important role in effective cyst formation. Cytological morphology, bile acid transportation, and gene expression of the cysts confirmed the favourable basic bile duct function compared to that obtained using Matrigel-embedded culture. Our method is expected to contribute to engineered in vitro liver tissue formation for cell-based assays.

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 3 hr 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 dependent as far as it decreased with the time of culture. Perivenous and periportal liver patterns were characterized by the immunostaining of the hepatic markers. We put in evidence a spatio temporal hepatic organization. We observed the overexpression since 24 hr of perfusion of the APC and PCK1 proteins upstream in the oxygen-rich area of the device. The overexpression of GS, GCK, CYP1A, and HIFα proteins were observed downstream in the oxygen-poor area. Then, CYP3A2 and β-catenin spatial reorganization was achieved after 48 hr of culture. The results presented a partial zonation-like pattern that was superimposed with an oxygen gradient profile.

In vitro models of the liver microvasculature, especially with respect to cancer cell extravasation, should include not only endothelial and cancer cells but also surrounding cells to mimic the physiological situation. To this end, in the present study, we established a physiologically-relevant hierarchical co-culture model by stacking layers of primary rat hepatocytes (Hep), hepatic stellate cells embedded in collagen gel (LX-2) and endothelial cells (HUVECs) on a specially designed oxygen-permeable polydimethylsiloxane PDMS bottom plate. The model was used to investigate the role and contribution of each of the three cell types in pancreatic cancer and promyeloblast cell adhesion. In particular, we showed an increase in albumin production by the primary hepatocytes and in the consumption of the produced vascular endothelial growth factors (VEGFs). Furthermore, in co-culture, the HUVECs exhibited a mature vascular endothelial and non-inflamed phenotype, as evidenced by Stabilin-1, lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), intercellular adhesion molecule (ICAM-1), and vascular adhesion protein-1 (VAP-1) expression. The HUVECs were also successfully activated with an inflammatory cytokine and their ICAM-1 response was found to be higher in monoculture compared to co-culture. Additionally, the adhesion of MiaPaCa-2 pancreatic cancer cells and HL60 promyeloblasts was tested in both cases (i.e.: activation or not by an inflammatory cytokine). It has been found that their adhesion was always reduced in the co-culture model. These results highlight the importance of integrating hepatic stellate cells in the design of biomimetic models of the hepatic endothelial barrier.