Laminin and hyaluronan supplementation of collagen hydrogels enhances endothelial function and tight junction expression on three-dimensional cylindrical microvessel-on-a-chip.

Vasculature-on-chip (VoC) models have become a prominent tool in the study of microvasculature functions because of their cost-effective and ethical production process. These models typically use a hydrogel in which the three-dimensional (3D) microvascular structure is embedded. Thus, VoCs are directly impacted by the physical and chemical cues of the supporting hydrogel. Endothelial cell (EC) response in VoCs is critical, especially in organ-specific vasculature models, in which ECs exhibit specific traits and behaviors that vary between organs. Many studies customize the stimuli ECs perceive in different ways; however, customizing the hydrogel composition accordingly to the target organ's extracellular matrix (ECM), which we believe has great potential, has been rarely investigated. We explored this approach to organ-specific VoCs by fabricating microvessels (MVs) with either human umbilical vein ECs or human brain microvascular ECs in a 3D cylindrical VoC using a collagen hydrogel alone or one supplemented with laminin and hyaluronan, components found in the brain ECM. We characterized the physical properties of these hydrogels and analyzed the barrier properties of the MVs. Barrier function and tight junction (ZO-1) expression improved with the addition of laminin and hyaluronan in the composite hydrogel.

A novel agonist for the HGF receptor MET promotes differentiation of human pluripotent stem cells into hepatocyte-like cells.

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.

Comparison of the transcriptomic profile of hepatic human induced pluripotent stem like cells cultured in plates and in a 3D microscale dynamic environment.

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.

Production of Cisplatin-Incorporating Hyaluronan Nanogels via Chelating Ligand-Metal Coordination.

Hyaluronan (HA) is a promising drug carrier for cancer therapy because of its CD44 targeting ability, good biocompatibility, and biodegradability. In this study, cisplatin (CDDP)-incorporating HA nanogels were fabricated through a chelating ligand-metal coordination cross-linking reaction. We conjugated chelating ligands, iminodiacetic acid or malonic acid, to HA and used them as a precursor polymer. By mixing the ligand-conjugated HA with CDDP, cross-linking occurred via coordination of the ligands with the platinum in CDDP, resulting in the spontaneous formation of CDDP-loaded HA nanogels. The nanogels showed pH-responsive release of CDDP, because the stability of the ligand-platinum complex decreases in an acidic environment. Cell viability assays for MKN45P human gastric cancer cells and Met-5A human mesothelial cells revealed that the HA nanogels selectively inhibited the growth of gastric cancer cells. In vivo experiments using a mouse model of peritoneal dissemination of gastric cancer demonstrated that HA nanogels specifically localized in peritoneal nodules after the intraperitoneal administration. Moreover, penetration assays using multicellular tumor spheroids indicated that HA nanogels had a significantly higher ability to penetrate tumors than conventional, linear HA. These results suggest that chelating-ligand conjugated HA nanogels will be useful for targeted cancer therapy.

Transplantation of pancreatic beta-cell spheroids in mice via non-swellable hydrogel microwells composed of poly(HEMA-co-GelMA).

Pancreatic islet transplantation is an effective treatment for type I diabetes mellitus. However, many problems associated with pancreatic islet engraftment remain unresolved. In this study, we developed a hydrogel microwell device for islet implantation, fabricated by crosslinking gelatin-methacryloyl (GelMA) and 2-hydroxyethyl methacrylate (HEMA) in appropriate proportions. The fabricated hydrogel microwell device could be freeze-dried and restored by immersion in the culture medium at any time, allowing long-term storage and transport of the device for ready-to-use applications. In addition, due to its non-swelling properties, the shape of the wells of the device was maintained. Thus, the device allowed pancreatic ß cell lines to form spheroids and increase insulin secretion. Intraperitoneal implantation of the ß cell line-seeded GelMA/HEMA hydrogel microwell device reduced blood glucose levels in diabetic mice. In addition, they were easy to handle during transplantation and were removed from the transplant site without peritoneal adhesions or infiltration by inflammatory cells. These results suggest that the GelMA/HEMA hydrogel microwell device can go from spheroid and/or organoid fabrication to transplantation in a single step.

Inflammatory liver tissue formation using oxygen permeable membrane based culture platform.

During chronic liver injury, inflammation leads to liver fibrosis, particularly due to the activation of hepatic stellate cells (HSCs). The involvement of inflammatory cytokines in HSC activation and the interplay among different liver cells are elaborated. To examine their interactions in vitro, many cultured liver tissue models are performed in organoid or spheroid culture with random 3D structure. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with non-parenchymal cells such as the human-derived HSC line (LX-2) and liver sinusoidal endothelial cell line (TMNK-1). The cocultured tissue had high usability with simple operation of separating solid and liquid phases with improved liver functions such as albumin production and hepatic cytochrome P450 3A4 activity. We also studied the effects of stimulation by both oxygen tension and the key pro-fibrogenic cytokine, transforming growth factor beta (TGF-ß), on HSC activation. Gene expression of collagen type I and alpha-smooth muscle actin were enhanced in the hierarchical coculture under lower oxygen tension and TGF-ß1 stimulation. Therefore, this hierarchical in vitro cocultured liver tissue could provide a useful platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in future liver fibrogenesis studies.

Spheroid on-demand printing and drug screening of endothelialized hepatocellular carcinoma model at different stages.

Hepatocellular carcinoma (HCC) poses a significant threat to human health and medical care. Its dynamic microenvironment and stages of development will influence the treatment strategies in clinics. Reconstructing tumor-microvascular interactions in different stages of the microenvironment is an urgent need forin vitrotumor pathology research and drug screening. However, the absence of tumor aggregates with paracancerous microvascular and staged tumor-endothelium interactions leads to bias in the antitumor drug responses. Herein, a spheroid-on-demand manipulation strategy was developed to construct staged endothelialized HCC models for drug screening. Pre-assembled HepG2 spheroids were directly printed by alternating viscous and inertial force jetting with high cell viability and integrity. A semi-open microfluidic chip was also designed to form a microvascular connections with high density, narrow diameter, and curved morphologies. According to the single or multiple lesions in stages Ⅰ or Ⅰ HCC, endothelialized HCC models from micrometer to millimeter scale with dense tumor cell aggregation and paracancerous endothelial distribution were successively constructed. A migrating stage Ⅰ HCC model was further constructed under TGF-ßtreatment, where the spheroids exhibited a more mesenchymal phenotype with a loose cell connection and spheroid dispersion. Finally, the stage ⅠHCC model showed stronger drug resistance compared to the stage Ⅰ model, while the stage III showed a more rapid response. The corresponding work provides a widely applicable method for the reproduction of tumor-microvascular interactions at different stages and holds great promise for the study of tumor migration, tumor-stromal cell interactions, and the development of anti-tumor therapeutic strategies.

Enhanced podocyte differentiation and changing drug toxicity sensitivity through pressure-controlled mechanical filtration stress on a glomerulus-on-a-chip.

Podocytes, localized in the glomerulus, are a prognostic factor of proteinuria in kidney disease and are exposed to distinct physiological stimuli from basal to apical filtration flow. Research studies on drug discovery and disease modeling for glomerulopathy have developed a glomerulus-on-a-chip and studied podocyte mechanobiology to realize alternative methods to animal experiments. However, the effect of filtration stimulus on podocytes has remained unclear. Herein, we report the successful development of a user-friendly filtration culture device and system that can precisely control the filtration flow using air pressure control by incorporating a commercially available culture insert. It allows mouse podocytes to be cultured under filtration conditions for three days with a guarantee of maintaining the integrity of the podocyte layer. Using our system, this study demonstrated that podocyte damage caused by hyperfiltration resulting from glomerular hypertension, a common pathophysiology of many glomerulopathies, was successfully recapitulated and that filtration stimulus promotes the maturation of podocytes in terms of their morphology and gene expression. Furthermore, we demonstrated that filtration stimulus induced different drug responsiveness in podocytes than those seen under static conditions, and that the difference in drug responsiveness was dependent on the pharmacological mechanism. Overall, this study has revealed differentiating and pharmacodynamic properties of filtration stimulus and brings new insights into the research field of podocyte mechanobiology towards the realization of glomerulus-on-a-chip.

Efficient High-Density hiPSCs Expansion in Simple Dialysis Device.

iPSCs are potential cell types that can be used for regenerative medicine. Suspension culture is the main approach to produce a sufficient amount of required cells to realize the application of hiPSCs for the transplantation of the large organ. The dialysis culture system holds the potential to reduce the cost by utilizing endogenous growth factors and recycle the remaining exogenous growth factors at the same time. However, the current large scale dialysis culture system was not optimized for expanding the high-density culture. Several problems such as the requirement of the large volume and technical complexity make the optimization of this system remain challenging. Also, the interference of mechanical stress in the dynamic suspension culture may reduce cellular viability, pluripotency, and differentiation capacity. Here, we describe the simple miniaturized dialysis platform to evaluate the feasibility of high-density hiPSCs culture by combining the utilization of endogenous growth factors supported by a continuous exchange of nutrition and toxic metabolic product in a low mechanical stress culture environment in a viscoelastic medium.

Differentiation of Human Induced Pluripotent Stem Cells into Definitive Endoderm Using Simple Dialysis Culture Device.

Therapeutic use of differentiated organ cells from human induced pluripotent stem cells (hiPSCs) is one of the promising strategies for regenerative medicine. Differentiation into definitive endoderm is an essential process in the preparation of metabolic organs. However, the manufacturability of differentiation is limited due to the high-cost cytokines required for the differentiation of endodermal lineage. Furthermore, the cytokines remaining in the used culture medium and possible endogenous factors are removed along with toxic metabolites by the medium replacement. To address these problems, the application of dialysis culture can retain and fully utilize their accumulation to create a better culture environment that contributes to differentiation cost reduction.

Scalable Formation of Highly Viable and Functional Hepatocellular Carcinoma Spheroids in an Oxygen-Permeable Microwell Device for Anti-Tumor Drug Evaluation.

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.

Differentiation of Human-Induced Pluripotent Stem Cell-Derived Endocrine Progenitors to Islet-like Cells Using a Dialysis Suspension Culture System.

The production of functional islet-like cells from human-induced pluripotent stem cells (hiPSCs) is a promising strategy for the therapeutic use and disease modeling for type 1 diabetes. However, the production cost of islet-like cells is extremely high due to the use of expensive growth factors for differentiation. In a conventional culture method, growth factors and beneficial autocrine factors remaining in the culture medium are removed along with toxic metabolites during the medium change, and it limits the efficient utilization of those factors. In this study, we demonstrated that the dialysis suspension culture system is possible to reduce the usage of growth factors to one-third in the differentiation of hiPSC-derived endocrine progenitor cells to islet-like cells by reducing the medium change frequency with the refinement of the culture medium. Furthermore, the expression levels of hormone-secretion-related genes and the efficiency of differentiation were improved with the dialysis suspension culture system, possibly due to the retaining of autocrine factors. In addition, we confirmed several improvements required for the further study of the dialysis culture system. These findings showed the promising possibility of the dialysis suspension culture system for the low-cost production of islet-like cells.

High density culture of pancreatic islet-like 3D tissue organized in oxygen-permeable porous scaffolds with external oxygen supply.

Transplantation of macroencapsulated pancreatic islets within semipermeable membranes is a promising approach for the treatment of type 1 diabetes. Encapsulation beneficially isolates the implants from the host immune system. Deleteriously however, it also limits oxygen supply to the cells. This creates challenges in loading islets at the amount and density required to meet the practical demands of clinical usage. To overcome this challenge, we investigated the feasibility of using macroporous scaffolds made of an oxygen-permeable polymer, poly(dimethylsiloxane) (PDMS) by culturing pancreatic islet-like three-dimensional tissue made of a rat pancreatic beta cell line on the scaffolds. With external oxygenation, the density and function of cells on the PDMS scaffold were more than three times and almost two times higher than those without oxygenation, respectively. This suggests that the oxygenation afforded by the PDMS scaffolds allows for high-density loading of islet tissue into the devices.

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.

Morphological Manipulation of DNA Gel Microbeads with Biomolecular Stimuli.

Hydrogels are essential in many fields ranging from tissue engineering and drug delivery to food sciences or cosmetics. Hydrogels that respond to specific biomolecular stimuli such as DNA, mRNA, miRNA and small molecules are highly desirable from the perspective of medical applications, however interfacing classical hydrogels with nucleic acids is still challenging. Here were demonstrate the generation of microbeads of DNA hydrogels with droplet microfluidic, and their morphological actuation with DNA strands. Using strand displacement and the specificity of DNA base pairing, we selectively dissolved gel beads, and reversibly changed their size on-the-fly with controlled swelling and shrinking. Lastly, we performed a complex computing primitive-A Winner-Takes-All competition between two populations of gel beads. Overall, these results show that strand responsive DNA gels have tantalizing potentials to enhance and expand traditional hydrogels, in particular for applications in sequencing and drug delivery.

Coculture with hiPS-derived intestinal cells enhanced human hepatocyte functions in a pneumatic-pressure-driven two-organ microphysiological system.

Examining intestine-liver interactions is important for achieving the desired physiological drug absorption and metabolism response in in vitro drug tests. Multi-organ microphysiological systems (MPSs) constitute promising tools for evaluating inter-organ interactions in vitro. For coculture on MPSs, normal cells are challenging to use because they require complex maintenance and careful handling. Herein, we demonstrated the potential of coculturing normal cells on MPSs in the evaluation of intestine-liver interactions. To this end, we cocultured human-induced pluripotent stem cell-derived intestinal cells and fresh human hepatocytes which were isolated from PXB mice with medium circulation in a pneumatic-pressure-driven MPS with pipette-friendly liquid-handling options. The cytochrome activity, albumin production, and liver-specific gene expressions in human hepatocytes freshly isolated from a PXB mouse were significantly upregulated via coculture with hiPS-intestinal cells. Our normal cell coculture shows the effects of the interactions between the intestine and liver that may occur in vivo. This study is the first to demonstrate the coculturing of hiPS-intestinal cells and fresh human hepatocytes on an MPS for examining pure inter-organ interactions. Normal-cell coculture using the multi-organ MPS could be pursued to explore unknown physiological mechanisms of inter-organ interactions in vitro and investigate the physiological response of new drugs.

Tubular bile duct structure mimicking bile duct morphogenesis for prospective in vitro liver metabolite recovery.

BACKGROUND: Liver metabolites are used to diagnose disease and examine drugs in clinical pharmacokinetics. Therefore, development of an in vitro assay system that reproduces liver metabolite recovery would provide important benefits to pharmaceutical research. However, liver models have proven challenging to develop because of the lack of an appropriate bile duct structure for the accumulation and transport of metabolites from the liver parenchyma. Currently available bile duct models, such as the bile duct cyst-embedded extracellular matrix (ECM), lack any morphological resemblance to the tubular morphology of the living bile duct. Moreover, these systems cannot overcome metabolite recovery issues because they are established in isolated culture systems. Here, we successfully established a non-continuous tubular bile duct structure model in an open-culture system, which closely resembled an in vivo structure. This system was utilized to effectively collect liver metabolites separately from liver parenchymal cells. RESULTS: Triple-cell co-culture of primary rat hepatoblasts, rat biliary epithelial cells, and mouse embryonic fibroblasts was grown to mimic the morphogenesis of the bile duct during liver development. Overlaying the cells with ECM containing a Matrigel and collagen type I gel mixture promoted the development of a tubular bile duct structure. In this culture system, the expression of specific markers and signaling molecules related to biliary epithelial cell differentiation was highly upregulated during the ductal formation process. This bile duct structure also enabled the separate accumulation of metabolite analogs from liver parenchymal cells. CONCLUSIONS: A morphogenesis-based culture system effectively establishes an advanced bile duct structure and improves the plasticity of liver models feasible for autologous in vitro metabolite-bile collection, which may enhance the performance of high-throughput liver models in cell-based assays.

Induction of in vitro Metabolic Zonation in Primary Hepatocytes Requires Both Near-Physiological Oxygen Concentration and Flux.

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 ß-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.

Modular assembly-based approach of loosely packing co-cultured hepatic tissue elements with endothelialization for liver tissue engineering.

BACKGROUND: In liver tissue engineering, co-culturing hepatocytes with typical non-parenchymal hepatic cells to form cell aggregates is available to mimic the in vivo microenvironment and promote cell biological functions. With a modular assembly approach, endothelialized hepatic cell aggregates can be packed for perfusion culture, which enables the construction of large-scale liver tissues. Since tightly packed aggregates tend to fuse with each other and block perfusion flows, a loosely packed mode was introduced in our study. METHODS: Using an oxygen-permeable polydimethylsiloxane (PDMS)-based microwell device, highly dense endothelialized hepatic cell aggregates were generated as hepatic tissue elements by co-culturing hepatocellular carcinoma (HepG2) cells, Swiss 3T3 cells, and human umbilical vein endothelial cells (HUVECs). The co-cultured aggregates were then harvested and applied in a PDMS-fabricated bioreactor for 10 days of perfusion culture. To maintain appropriate interstitial spaces for stable perfusion, biodegradable poly-L-lactic acid (PLLA) scaffold fibers were used and mixed with the aggregates, forming a loosely packed mode. RESULTS: In a microwell co-culture, Swiss 3T3 cells significantly contributed to the formation of hepatic cell aggregates. HUVECs developed a peripheral distribution in aggregates for endothelialization. In the perfusion culture, compared with pure HepG2 aggregates, HepG2/Swiss 3T3/HUVECs co-cultured aggregates exhibited a higher level of cell proliferation and liver-specific function expression (i.e., glucose consumption and albumin secretion). Under the loosely packed mode, co-cultured aggregates showed a characteristic histological morphology with cell migration and adhesion to fibers. The assembled hepatic tissue elements were obtained with 32% of in vivo cell density. CONCLUSIONS: In a co-culture of HepG2, Swiss 3T3, and HUVECs, Swiss 3T3 cells were observed to be beneficial for the formation of endothelialized hepatic cell aggregates. Loosely packed aggregates enabled long-term perfusion culture with high viability and biological function. This study will guide us in constructing large-scale liver tissue models by way of aggregate-based modular assembly.

Microwell-based pancreas-on-chip model enhances genes expression and functionality of rat islets of Langerhans.

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.