Availability of hepatic tissue for the investigation of metabolic processes is severely limited. While primary hepatocytes or animal models are widely used in pharmacological applications, a change in methodology towards more sustainable and ethical assays is highly desirable. Stem cell derived hepatic cells are generally regarded as a viable alternative for the above model systems, if current limitations in functionality and maturation can be overcome. By combining microfluidic organ-on-a-chip technology with individually differentiated, multicellular hepatic tissue fractions, we aim to improve overall functionality of hepatocyte-like cells, as well as evaluate cellular composition and interactions with non-parenchymal cell populations towards the formation of mature liver tissue. Utilizing a multi-omic approach, we show the improved maturation profiles of hepatocyte-like cells maintained in a dynamic microenvironment compared to standard tissue culture setups without continuous perfusion. In order to evaluate the resulting tissue, we employ single cell sequencing to distinguish formed subpopulations and spatial localization. While cellular input was strictly defined based on established differentiation protocols of parenchyma, endothelial and stellate cell fractions, resulting hepatic tissue was shown to comprise a complex mixture of epithelial and non-parenchymal fractions with specific local enrichment of phenotypes along the microchannel. Following this approach, we show the importance of passive, paracrine developmental processes in tissue formation. Using such complex tissue models is a crucial first step to develop stem cell-derived in vitro systems that can compare functionally with currently used pharmacological and toxicological applications.

Hepatic physiology depends on the liver's complex structural composition which among others, provides high oxygen supply rates, locally differential oxygen tension, endothelial paracrine signaling, as well as residual hemodynamic shear stress to resident hepatocytes. While functional improvements were shown by implementing these factors into hepatic culture systems, direct cause-effect relationships are often not well characterized-obfuscating their individual contribution in more complex microphysiological systems. By comparing increasingly complex hepatic in vitro culture systems that gradually implement these parameters, we investigate the influence of the cellular microenvironment to overall hepatic functionality in pharmacological applications. Here, hepatocytes were modulated in terms of oxygen tension and supplementation, endothelial coculture, and exposure to fluid shear stress delineated from oxygen influx. Results from transcriptomic and metabolomic evaluation indicate that particularly oxygen supply rates are critical to enhance cellular functionality-with cellular drug metabolism remaining comparable to physiological conditions after prolonged static culture. Endothelial signaling was found to be a major contributor to differential phenotype formation known as metabolic zonation, indicated by WNT pathway activity. Lastly, oxygen-delineated shear stress was identified to direct cellular fate towards increased hepatic plasticity and regenerative phenotypes at the cost of drug metabolic functionality - in line with regenerative effects observed in vivo. With these results, we provide a systematic evaluation of critical parameters and their impact in hepatic systems. Given their adherence to physiological effects in vivo, this highlights the importance of their implementation in biomimetic devices, such as organ-on-a-chip systems. Considering recent advances in basic liver biology, direct translation of physiological structures into in vitro models is a promising strategy to expand the capabilities of pharmacological models.

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.

In vitro culture of primary hepatocytes for drug screening purposes remains a challenge as cells rapidly lose their function in conventional culture conditions. Thin oxygen permeable membranes have shown, through direct oxygenation, beneficial effects for long-term culture and cellular function with freshly isolated hepatocytes. However, culture of cryopreserved hepatocytes, a standard for the industry, has shown limits due to high cellular damage, leading to low cellular function. In addition, high sorption of drug screening compounds on PDMS oxygen permeable membranes has rendered evaluation of different molecules, aimed at the improvement of the culture of those cells difficult. Here, culture of cryopreserved hepatocytes was performed on PMP membranes, known to exhibit exceptionally low sorption characteristics. A mixture of anti-apoptotic and anti-inflammatory compounds to improve cell viability during adhesion was tested and evaluation in terms of cellular damage and drug metabolism was performed after 24 hr and 72 hr. Components of the mixture were shown to have beneficial effect on Reactive Oxygen Species production after 6 hr of adhesion as well as on mitochondrial activity and LDH release after 24 hr. Effects in improving recovery of albumin and drug metabolism, could be efficiently measured after 72 hr as a result of the use of PMP. The presented results demonstrate the compatibility of PMP oxygen-permeable membrane-based culture with cryopreserved hepatocytes for efficient drug screening.

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.

In vitro culture of primary hepatocytes for drug screening purposes remains a challenge as cells rapidly lose their function in conventional culture conditions. Thin oxygen permeable membranes have shown, through direct oxygenation, beneficial effects for long-term culture and cellular function with freshly isolated hepatocytes. However, culture of cryopreserved hepatocytes, a standard for the industry, has shown limits due to high cellular damage, leading to low cellular function. In addition, high sorption of drug screening compounds on PDMS oxygen permeable membranes has rendered evaluation of different molecules, aimed at the improvement of the culture of those cells difficult. Here, culture of cryopreserved hepatocytes was performed on PMP membranes, known to exhibit exceptionally low sorption characteristics. A mixture of anti-apoptotic and anti-inflammatory compounds to improve cell viability during adhesion was tested and evaluation in terms of cellular damage and drug metabolism was performed after 24 hr and 72 hr. Components of the mixture were shown to have beneficial effect on Reactive Oxygen Species production after 6 hr of adhesion as well as on mitochondrial activity and LDH release after 24 hr. Effects in improving recovery of albumin and drug metabolism, could be efficiently measured after 72 hr as a result of the use of PMP. The presented results demonstrate the compatibility of PMP oxygen-permeable membrane-based culture with cryopreserved hepatocytes for efficient drug screening.

In the pharmaceutical industry, primary cultured hepatocytes is a standard tool used to assess hepatic metabolisms and toxicity in vitro. Drawbacks, however, include their functional deterioration upon isolation, mostly due to the lack of a physiological environment. Polydimethylsiloxane (PDMS) has been reported to improve the function of isolated hepatocytes by its high oxygen permeability when used as a material of microphysiological systems (MPS). However, its high chemical sorption property has impeded its practical use in drug development. In this study, we evaluated a new culture material, 4-polymethyl-1-pentene polymer (PMP), in comparison with PDMS and conventional tissue culture polystyrene (TCPS). First, we confirmed the high oxygen permeability and low sorption of PMP, and these properties were comparable with PDMS and TCPS, respectively. Moreover, using primary rat hepatocytes, we demonstrated maintained high levels of liver function at least for 1 week on PMP, with its low chemical sorption and high oxygen permeability being key factors in both revealing the potential of primary cultured hepatocytes and in performing an accurate evaluation of hepatic metabolisms. Taken together, we conclude that PMP is a superior alternative to both PDMS and TCPS, and a promising material for a variety of drug testing systems.

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.

The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA positive staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation density, flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static experiments carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon positive staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.

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.

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.

Purpose Implementation of Process Analytical Technologies (PAT) is an important consideration to increase process knowledge during pharmaceutical manufacturing. Currently used batch sampling methods, however, are limited in terms of temporal resolution when compared with continuous process sampling. Here, we show the importance of fluid dynamic characterization when repurposing available batch sampling sites comprising needle insertion mechanisms to generate continuous sample streams. Methods Rheological flow profiles in a needle-based sampling site were characterized by computational fluid dynamics. Observed characteristics were confirmed with a NovaSeptum sample system in reenacted sampling situations, and correlation of sample and system flow was achieved by simultaneous measurement of an acetone gradient. Results As a result of the sampling mechanism causing distinct flow patterns around the needle body, velocity fields are shown to generate orientation-dependent sample flow. Due to the inherent relationship between flow detachment and turbulent flows, extracted sample flow depends on system flow profiles, affecting sample correlation. A combination of active sample extraction, check valve addition, and geometrical changes in sampling site design is shown to reduce sample efflux variability, allowing for system parameter-independent sample generation. The resulting sample flow has been correlated to a simulated process flow, enabling a continuous representation of at-scale systems. Conclusions Repurposing existing sampling sites requires fluid dynamic characterization in order to generate representative sample streams, which allows the exchangeable inclusion of process analytics otherwise not compatible with at-scale systems. Resulting time-dependent, discrete samples can then be utilized for on- and off-line analysis to provide additional process knowledge according to the PAT initiative.

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.