Comparison of hydroxyapatite and honeycomb micro-structure in bone tissue engineering using electrospun beads-on-string fibers.

Thick honeycomb-like electrospun scaffold with nanoparticles of hydroxyapatite (nHA) recently demonstrated its potential to promote proliferation and differentiation of a murine embryonic cell line (C3H10T1/2) to osteoblasts. In order to distinguish the respective effects of the structure and the composition on cell differentiation, beads-on-string fibers were used to manufacture thick honeycomb-like scaffolds without nHA. Mechanical and biological impacts of those beads-on string fibers were evaluated. Uniaxial tensile test showed that beads-on-string fibers decreased the Young Modulus and maximal stress but kept them appropriate for tissue engineering. C3H10T1/2 were seeded and cultured for 6 days on the scaffolds without any growth factors. Viability assays revealed the biocompatibility of the beads-on-string scaffolds, with adequate cells-materials interactions observed by confocal microscopy. Alkaline phosphatase staining was performed at day 6 in order to compare the early differentiation of cells to bone fate. The measure of stained area and intensity confirmed the beneficial effect of both honeycomb structure and nHA, independently. Finally, we showed that honeycomb-like electrospun scaffolds could be relevant candidates for promoting bone fate to cells in the absence of nHA. It offers an easier and faster manufacture process, in particular in bone-interface tissue engineering, permitting to avoid the dispersion of nHA and their interaction with the other cells.

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.

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.

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.

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.

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.

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.

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.

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.

Validation of HepG2/C3A Cell Cultures in Cyclic Olefin Copolymer Based Microfluidic Bioreactors.

Organ-on-chip (OoC) technology is one of the most promising in vitro tools to replace the traditional animal experiment-based paradigms of risk assessment. However, the use of OoC in drug discovery and toxicity studies remain still limited by the low capacity for high-throughput production and the incompatibility with standard laboratory equipment. Moreover, polydimethylsiloxanes, the material of choice for OoC, has several drawbacks, particularly the high absorption of drugs and chemicals. In this work, we report the development of a microfluidic device, using a process adapted for mass production, to culture liver cell line in dynamic conditions. The device, made of cyclic olefin copolymers, was manufactured by injection moulding and integrates Luer lock connectors compatible with standard medical and laboratory instruments. Then, the COC device was used for culturing HepG2/C3a cells. The functionality and behaviour of cultures were assessed by albumin secretion, cell proliferation, viability and actin cytoskeleton development. The cells in COC device proliferated well and remained functional for 9 days of culture. Furthermore, HepG2/C3a cells in the COC biochips showed similar behaviour to cells in PDMS biochips. The present study provides a proof-of-concept for the use of COC biochip in liver cells culture and illustrate their potential to develop OoC.

Development of Liver-on-Chip Integrating a Hydroscaffold Mimicking the Liver's Extracellular Matrix.

The 3Rs guidelines recommend replacing animal testing with alternative models. One of the solutions proposed is organ-on-chip technology in which liver-on-chip is one of the most promising alternatives for drug screening and toxicological assays. The main challenge is to achieve the relevant in vivo-like functionalities of the liver tissue in an optimized cellular microenvironment. Here, we investigated the development of hepatic cells under dynamic conditions inside a 3D hydroscaffold embedded in a microfluidic device. The hydroscaffold is made of hyaluronic acid and composed of liver extracellular matrix components (galactosamine, collagen I/IV) with RGDS (Arg-Gly-Asp-Ser) sites for cell adhesion. The HepG2/C3A cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregates and proliferated, forming 3D spheroids. The cell viability, functionality, and spheroid integrity were investigated and compared to static cultures. The results showed a 3D aggregate organization of the cells up to large spheroid formations, high viability and albumin production, and an enhancement of HepG2 cell functionalities. Overall, these results highlighted the role of the liver-on-chip model coupled with a hydroscaffold in the enhancement of cell functions and its potential for engineering a relevant liver model for drug screening and disease study.

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.

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.

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.

Investigation of the hepatic development in the coculture of hiPSCs-derived LSECs and HLCs in a fluidic microenvironment.

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.

Investigation of steatosis profiles induced by pesticides using liver organ-on-chip model and omics analysis.

Several studies have reported a correlation between pesticides exposure and metabolic disorders. Dichlorodiphenyltrichloroethane (DDT) and permethrin (PMT), two pesticides highly prevalent in the environment, have been associated to dysregulation of liver lipids and glucose metabolisms and non-alcoholic fatty liver disease (NAFLD). However, the effects of DDT/PMT mixtures and mechanisms mediating their action remain unclear. Here, we used multi-omic to investigate the liver damage induced by DDT, PMT and their mixture in rat liver organ-on-chip. Organ-on-chip allow the reproduction of in vivo-like micro-environment. Two concentrations, 15 and 150 µM, were used to expose the hepatocytes for 24 h under perfusion. The transcriptome and metabolome analysis suggested a dose-dependent effect for all conditions, with a profile close to control for pesticides low-doses. The comparison between control and high-doses detected 266/24, 256/24 and 1349/30 genes/metabolites differentially expressed for DDT150, PMT150 and Mix150 (DDT150/PMT150). Transcriptome modulation reflected liver inflammation, steatosis, necrosis, PPAR signaling and fatty acid metabolism. The metabolome analysis highlighted common signature of three treatments including lipid and carbohydrates production, and a decrease in amino acids and krebs cycle intermediates. Our study illustrates the potential of organ-on-chip coupled to multi-omics for toxicological studies and provides new tools for chemical risk assessment.

Analysis of the behavior of 2D monolayers and 3D spheroid human pancreatic beta cells derived from induced pluripotent stem cells in a microfluidic environment.

The limited availability of primary human ß-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 ß-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 ß-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 ß-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 ß-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.

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.

Cryogel-Integrated Biochip for Liver Tissue Engineering.

Microfluidic systems and polymer hydrogels have been widely developed for tissue engineering. Yet, only a few tools combining both approaches, especially for in vitro liver models, are being explored. In this study, an alginate-based cryogel-integrated biochip was engineered for dynamic hepatoma cell line culture in three dimensions (3D). The alginate cryogel was covalently cross-linked in the biochip at subzero temperatures (T < 0 °C) to create a scaffold with high mechanical stability and an interconnected macroporous network. By varying the alginate concentration and the cross-linker ratio, Young's modulus of the cryogel can be fine-tuned between 1.5 and 29 kPa, corresponding to the range of stiffness of the different physiological states of the liver. We demonstrated that HepG2/C3A cells can be cultured and maintained as viable under dynamic conditions in this device up to 6 days. Albumin synthesis and glucose consumption increased over the cell culture days. Moreover, a 3D cell structure was observed across the entire height of the biochip, which was preserved following alginate lyase treatment to remove the cryogel-based scaffold. In summary, these results represent a proof of concept of an interesting cell culture technology that should be further investigated to engineer healthy and cirrhotic liver models.

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.