Albumin Secretion Research Articles

Convenient rapid prototyping microphysiological niche for mimicking liver native basement membrane: Liver sinusoid on a chip

Liver is responsible for the metabolization processes of up to 90 % of compounds and toxins in the body. Therefore liver-on-a-chip systems, as an in vitro promising cell culture platform, have great importance for fundamental science and drug development. In most of the liver-on-a-chip studies, seeding cells on both sides of a porous membrane, which represents the basement membrane, fail to resemble the native characteristics of biochemical, biophysical, and mechanical properties. In this study, polycarbonate (PC) and polyethylene terephthalate (PET) membranes were coated with gelatin to address this issue by accurately mimicking the native basement membrane present in the space of Disse. Various coating methods were used, including doctor blade, gel micro-injection, electrospinning, and spin coating. Spin coating was demonstrated to be the most effective technique owing to the ability to produce thin gel thickness with desirable surface roughness for cell interactions on both sides of the membrane. HepG2 and EA.HY926 cells were seeded on the upper and bottom sides of the gelatin-coated PET membrane and cultured on-chip for 7 days. Cell viability increased from 90 % to 95 %, while apoptotic index decreased. Albumin secretion notably rose between days 1–7 and 4–7, while GST-α secretion decreased from day 1 to day 7. In conclusion, the optimized spin coating process reported here can effectively modify the membranes to better mimic the native basement membrane niche characteristics.

Geraniol intake improves age-related malnutrition in mice.

Geraniol is an acyclic monoterpenoid that is abundant in many plants, including rose, lemongrass, and lavender. As geraniol has various beneficial functions, rose oil rich in geraniol is not only used for aromatherapy but also as a supplement to promote health benefits. However, the beneficial effects of geraniol on age-related pathologies are unknown. In this study, we aimed to clarify the effects of geraniol intake on age-related pathologies. We orally administered geraniol to aged mice (age: 24-29 months) five times a week for 4 weeks and sampled their blood and various organs. We investigated age-related changes in the blood and organ samples. Furthermore, we treated HepG2 cells with geraniol and examined the expression level of the ALB gene and the amount of secreted albumin in vitro. Geraniol significantly increased blood albumin, total cholesterol, and red blood cell counts, indicating an improvement in nutritional markers in aged mice. Geraniol also transcriptionally increased the Alb gene expression in the liver of aged mice. Furthermore, treatment with geraniol significantly upregulated the ALB gene expression and the secretion of albumin in the conditioned medium of HepG2 cells. Geraniol increases serum albumin levels at the transcriptional level. Geraniol intake can be an effective strategy for age-related malnutrition. Geriatr Gerontol Int 2024; ••: ••-••.

Determining Rates of Molecular Secretion from Supernatant Concentration Measurements in a 3D-Bioprinted Human Liver Tissue Model.

The secretion rate of albumin is a key indicator of function in liver tissue models used for hepatotoxicity and pharmacokinetic testing. However, it is not generally clear how to determine molecular secretion rates from measurements of the molecular concentration in supernatant media. Here, we develop computational and analytical models of molecular transport in an experimental system that enable determination of albumin secretion rates based on measurements of albumin concentration in supernatant media. The experimental system is a 3D-bioprinted human liver tissue construct embedded in a 3D culture environment made from packed microgel particles swollen in liquid growth media. The mathematical models reveal that the range of albumin synthesis rates necessary to match experimentally measured albumin concentrations corresponds to reaction-limited conditions, where a steady state of albumin spatial distribution is rapidly reached between media exchanges. Our results show that temporally resolved synthesis rates can be inferred from serial concentration measurements of collected supernatant media. This link is critical to confidently assessing in vitro tissue performance in applications where critical quality attributes must be quantified, like in drug development and screening.

Enhanced Maturity and Functionality of Vascularized Human Liver Organoids through 3D Bioprinting and Pillar Plate Culture.

Liver tissues, composed of hepatocytes, cholangiocytes, stellate cells, Kupffer cells, and sinusoidal endothelial cells, are differentiated from endodermal and mesodermal germ layers. By mimicking the developmental process of the liver, various differentiation protocols have been published to generate human liver organoids (HLOs) in vitro using induced pluripotent stem cells (iPSCs). However, HLOs derived solely from the endodermal germ layer often encounter technical hurdles, such as insufficient maturity and functionality, limiting their utility for disease modeling and hepatotoxicity assays. To overcome this, we separately differentiated EpCAM+ endodermal progenitor cells (EPCs) and mesoderm-derived vascular progenitor cells (VPCs) from the same human iPSC line. These cells were then mixed in BME-2 matrix and concurrently differentiated into vascular human liver organoids (vHLOs). Remarkably, vHLOs exhibited significantly higher maturity than vasculature-free HLOs, as demonstrated by increased coagulation factor secretion, albumin secretion, drug-metabolizing enzyme (DME) expression, and bile acid transportation. To enhance assay throughput and miniaturize vHLO culture, we 3D bioprinted expandable HLOs (eHLOs) in BME-2 matrix on a pillar plate platform derived from EPCs and VPCs and compared with HLOs derived from endoderm alone. Compared to HLOs cultured in a 50 μL BME-2 matrix dome in a 24-well plate, vHLOs cultured on the pillar plate exhibited superior maturity, likely due to enhanced nutrient and signaling molecule diffusion. The integration of physiologically relevant patterned liver organoids with the unique pillar plate platform enhanced the capabilities for high-throughput screening and disease modeling.

Homogeneously complexed alginate-chitosan hydrogel microspheres for the viability enhancement of entrapped hepatocytes

The future deployment of biomedicine fields will require a new generation of biodegradable, biocompatible, and non-toxic hydrogels. Alginate and chitosan, naturally occurring polymers, have gained significant interest for hydrogel applications. However, integrating chitosan within alginate-based hydrogels to form microspheres with homogeneous distribution and a tailored surface charge remains challenging. Herein, we report the design and fabrication of homogeneously complexed alginate-chitosan hydrogel microspheres, demonstrating their ability to enhance the viability and liver-specific functionalities of entrapped hepatocytes. By exploring and optimizing the pH and ratio of alginate and chitosan solutions, we achieved well-controlled physicochemical properties, including the degree of sphericity, hydrophilicity, charge property, and surface roughness. Unlike traditional alginate-based hydrogel microspheres, hepatocytes entrapped in homogeneous alginate-chitosan microspheres displayed enhanced viability and liver-specific functions, including albumin secretion, urea synthesis, and cytochrome P-450 enzymatic activity. This work illustrates a potential pathway for manufacturing functionalized microspheres with tunable mechanical properties and functionalities based on biocompatible alginate and chitosan for hepatocyte applications.

Fasudil and viscosity of gelatin promote hepatic differentiation by regulating organelles in human umbilical cord matrix-mesenchymal stem cells

BackgroundHuman mesenchymal stem cells originating from umbilical cord matrix are a promising therapeutic resource, and their differentiated cells are spotlighted as a tissue regeneration treatment. However, there are limitations to the medical use of differentiated cells from human umbilical cord matrix-mesenchymal stem cells (hUCM-MSCs), such as efficient differentiation methods.MethodsTo effectively differentiate hUCM-MSCs into hepatocyte-like cells (HLCs), we used the ROCK inhibitor, fasudil, which is known to induce endoderm formation, and gelatin, which provides extracellular matrix to the differentiated cells. To estimate a differentiation efficiency of early stage according to combination of gelatin and fasudil, transcription analysis was conducted. Moreover, to demonstrate that organelle states affect differentiation, we performed transcription, tomographic, and mitochondrial function analysis at each stage of hepatic differentiation. Finally, we evaluated hepatocyte function based on the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4 in mature HLCs.ResultsFasudil induced endoderm-related genes (GATA4, SOX17, and FOXA2) in hUCM-MSCs, and it also induced lipid droplets (LDs) inside the differentiated cells. However, the excessive induction of LDs caused by fasudil inhibited mitochondrial function and prevented differentiation into hepatoblasts. To prevent the excessive LDs formation, we used gelatin as a coating material. When hUCM-MSCs were induced into hepatoblasts with fasudil on high-viscosity (1%) gelatin-coated dishes, hepatoblast-related genes (AFP and HNF4A) showed significant upregulation on high-viscosity gelatin-coated dishes compared to those treated with low-viscosity (0.1%) gelatin. Moreover, other germline cell fates, such as ectoderm and mesoderm, were repressed under these conditions. In addition, LDs abundance was also reduced, whereas mitochondrial function was increased. On the other hand, unlike early stage of the differentiation, low viscosity gelatin was more effective in generating mature HLCs. In this condition, the accumulation of LDs was inhibited in the cells, and mitochondria were activated. Consequently, HLCs originated from hUCM-MSCs were genetically and functionally more matured in low-viscosity gelatin.ConclusionsThis study demonstrated an effective method for differentiating hUCM-MSCs into hepatic cells using fasudil and gelatin of varying viscosities. Moreover, we suggest that efficient hepatic differentiation and the function of hepatic cells differentiated from hUCM-MSCs depend not only on genetic changes but also on the regulation of organelle states.

Examining the hepatotoxic potential of cannabidiol, cannabidiol-containing hemp extract, and cannabinol at consumer-relevant exposure concentrations in primary human hepatocytes.

Hemp extracts and consumer products containing cannabidiol (CBD) and/or other phytocannabinoids derived from hemp have entered the marketplace in recent years. CBD is an approved drug in the United States for the treatment of certain seizure disorders. While effects of CBD in the liver have been well characterized, data on the effects of other cannabinoids and hemp extracts in the liver and methods for studying these effects in vitro are limited. This study examined the hepatotoxic potential of CBD, CBD concentration-matched hemp extract, and cannabinol (CBN), at consumer-relevant concentrations determined by in silico modeling, in vitro using primary human hepatocytes. Primary human hepatocytes exposed to between 10-nM and 25-μM CBD, CBN, or hemp extract for 24 and 48 h were evaluated by measuring lactate dehydrogenase release, apoptosis, albumin secretion, urea secretion, and mitochondrial membrane potential. Cell viability was not significantly affected by CBD, CBN, or the hemp extract at any of the concentrations tested. Exposure to hemp extract induced a modest but statistically significant decrease in albumin secretion, urea secretion, and mitochondrial membrane potential at the highest concentration tested whereas CBD only induced a modest but statistically significant decrease in albumin secretion compared with vehicle control. Although this study addresses data gaps in the understanding of cannabinoid hepatoxicity in vitro, additional studies will be needed to determine how these results correlate with relevant consumer exposure and the biological effects of cannabinoids in human liver.

3D spheroid HepaRG and fluorescent biphasic tracer for CYP3A4-mediated antibiotic interaction monitoring in sepsis.

Cytochrome P450 3A4 (CYP3A4) is a crucial enzyme in the metabolism of xenobiotics, particularly in drug metabolism interactions (DDIs), making it a significant factor in clinical drug use. However, current assay techniques are both laborious and costly, making it difficult to construct a high-throughput monitoring method that can be used in conjunction with the clinic. This poses certain safety hazards for drug combination. Therefore, it is crucial to develop a synchronized monitoring method for the inhibition and induction of CYP3A4. In this study, we utilized 3D culture technology to develop a HepaRG cells spheroid model. The CYP450 and transporter expression, the albumin secretion, and urea synthesis capacity characteristics were analyzed. The NEN probe was utilized as a tracer molecule for CYP3A4. The fluorescence intensity of metabolites was characterized by laser confocal technique to determine the inhibition and expression of CYP3A4 in the HepaRG cell spheroid model by the antibiotics for sepsis. The results indicate that the HepaRG sphere model successfully possessed the physiological phenotype of the liver, which could be used for drug interaction monitoring. Through positive drug testing, NEN probe was able to achieve bidirectional characterization of CYP3A4 induction and inhibition. The monitoring method described in this paper was successfully applied to drug interaction monitoring of commonly used antibiotics in sepsis patients, which is a convenient and rapid monitoring method. The proposed method offers a new strategy for monitoring CYP3A4-mediated drug-drug interactions with a high-throughput assay, which will help to improve the safety of clinical drug combination.

Polyethylene Terephthalate Fiber Modified with Type I Collagen as a 3D Scaffold Material for Bioartificial Liver

Acute and chronic liver failure are clinically significant conditions, and the artificial liver support system (ALSS) is emerging as a novel and effective approach for the clinical management of liver failure. Within this framework, scaffold materials occupy a pivotal position as integral components of the bioreactor. Elevating the performance capabilities of these scaffolds not only augments the therapeutic efficacy of the artificial liver but also lays the groundwork for refining and selecting large-scale hepatocyte culture models. In this study, we introduced a novel hepatocyte scaffold material designated as PET-COL, crafted by coating polyethylene terephthalate (PET) with collagen. This involved a sequence of modifications, including alkaline hydrolysis, EDC/NHS activation and crosslinking, as well as collagen conjugation. The physicochemical attributes of the scaffold were thoroughly characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), second harmonic generation (SHG), water contact angle measurements, and high-performance liquid chromatography–mass spectrometry (HPLC-MS). Furthermore, an investigation into the material’s biological properties was conducted that encompassed SEM (HepaRG growth), fluorescence staining (assessment of cell viability), staining by trypan blue (HepaRG counting), CCK-8 (proliferation of cells), biochemical testing, and immunosorbent assay. Our findings revealed that collagen was covalently bonded to the PET surface, leading to a substantial enhancement in the material’s hydrophilicity (p < 0.001). The quantity of collagen coating was determined to be precisely 33.30 μg per scaffold. Human liver progenitor HepaRG cells thrived on the PET-COL material. Compared with the untreated group, cell viability, albumin secretion, urea synthesis, and the expression levels of CYP3A4 and CPS1 increased significantly (p < 0.001), demonstrating remarkable biological vitality. The PET-COL scaffold, as developed in this study, holds immense potential for application in bioartificial livers.

In Situ Self-Assembling Liver Spheroids with Synthetic Nanoscaffolds for Preclinical Drug Screening Applications.

Drug-induced liver injury (DILI) is one of the most common reasons for acute liver failure and a major reason for the withdrawal of medications from the market. There is a growing need for advanced in vitro liver models that can effectively recapitulate hepatic function, offering a robust platform for preclinical drug screening applications. Here, we explore the potential of self-assembling liver spheroids in the presence of electrospun and cryomilled poly(caprolactone) (PCL) nanoscaffolds for use as a new preclinical drug screening tool. This study investigated the extent to which nanoscaffold concentration may have on spheroid size and viability and liver-specific biofunctionality. The efficacy of our model was further validated using a comprehensive dose-dependent acetaminophen toxicity protocol. Our findings show the strong potential of PCL-based nanoscaffolds to facilitate in situ self-assembly of liver spheroids with sizes under 350 μm. The presence of the PCL-based nanoscaffolds (0.005 and 0.01% w/v) improved spheroid viability and the secretion of critical liver-specific biomarkers, namely, albumin and urea. Liver spheroids with nanoscaffolds showed improved drug-metabolizing enzyme activity and greater sensitivity to acetaminophen compared to two-dimensional monolayer cultures and scaffold-free liver spheroids. These promising findings highlight the potential of our nanoscaffold-based liver spheroids as an in vitro liver model for drug-induced hepatotoxicity and drug screening.

Validating Well-Functioning Hepatic Organoids for Toxicity Evaluation.

"Organoids", three-dimensional self-organized organ-like miniature tissues, are proposed as intermediary models that bridge the gap between animal and human studies in drug development. Despite recent advancements in organoid model development, studies on toxicity using these models are limited. Therefore, in this study, we aimed to analyze the functionality and gene expression of pre- and post-differentiated human hepatic organoids derived from induced pluripotent stem cells and utilize them for toxicity assessment. First, we confirmed the functional similarity of this hepatic organoid model to the human liver through various functional assessments, such as glycogen storage, albumin and bile acid secretion, and cytochrome P450 (CYP) activity. Subsequently, utilizing these functionally validated hepatic organoids, we conducted toxicity evaluations with three hepatotoxic substances (ketoconazole, troglitazone, and tolcapone), which are well known for causing drug-induced liver injury, and three non-hepatotoxic substances (sucrose, ascorbic acid, and biotin). The organoids effectively distinguished between the toxicity levels of substances with and without hepatic toxicity. We demonstrated the potential of hepatic organoids with validated functionalities and genetic characteristics as promising models for toxicity evaluation by analyzing toxicological changes occurring in hepatoxic drug-treated organoids.

A 3D spheroid model of quadruple cell co-culture with improved liver functions for hepatotoxicity prediction

Drug-induced liver injury (DILI) is one of the major concerns during drug development. Wide acceptance of the 3 R principles and the innovation of in-vitro techniques have introduced various novel model options, among which the three-dimensional (3D) cell spheroid cultures have shown a promising prospect in DILI prediction. The present study developed a 3D quadruple cell co-culture liver spheroid model for DILI prediction via self-assembly. Induction by phorbol 12-myristate 13-acetate at the concentration of 15.42 ng/mL for 48 hours with a following 24-hour rest period was used for THP-1 cell differentiation, resulting in credible macrophagic phenotypes. HepG2 cells, PUMC-HUVEC-T1 cells, THP-1-originated macrophages, and human hepatic stellate cells were selected as the components, which exhibited adaptability in the designated spheroid culture conditions. Following establishment, the characterization demonstrated the competence of the model in long-term stability reflected by the maintenance of morphology, viability, cellular integration, and cell-cell junctions for at least six days, as well as the reliable liver-specific functions including superior albumin and urea secretion, improved drug metabolic enzyme expression and CYP3A4 activity, and the expression of MRP2, BSEP, and P-GP accompanied by the bile acid efflux transport function. In the comparative testing using 22 DILI-positive and 5 DILI-negative compounds among the novel 3D co-culture model, 3D HepG2 spheroids, and 2D HepG2 monolayers, the 3D culture method significantly enhanced the model sensitivity to compound cytotoxicity compared to the 2D form. The novel co-culture liver spheroid model exhibited higher overall predictive power with margin of safety as the classifying tool. In addition, the non-parenchymal cell components could amplify the toxicity of isoniazid in the 3D model, suggesting their potential mediating role in immune-mediated toxicity. The proof-of-concept experiments demonstrated the capability of the model in replicating drug-induced lipid dysregulation, bile acid efflux inhibition, and α-SMA upregulation, which are the key features of liver steatosis and phospholipidosis, cholestasis, and fibrosis, respectively. Overall, the novel 3D quadruple cell co-culture spheroid model is a reliable and readily available option for DILI prediction.

Cyanotoxin cylindrospermopsin disrupts lipid homeostasis and metabolism in a 3D in vitro model of the human liver

Cylindrospermopsin, a potent hepatotoxin produced by harmful cyanobacterial blooms, poses environmental and human health concerns. We used a 3D human liver in vitro model based on spheroids of HepG2 cells, in combination with molecular and biochemical assays, automated imaging, targeted LC-MS-based proteomics, and lipidomics, to explore cylindrospermopsin effects on lipid metabolism and the processes implicated in hepatic steatosis. Cylindrospermopsin (1 μM, 48 h) did not significantly affect cell viability but partially reduced albumin secretion. However, it increased neutral lipid accumulation in HepG2 spheroids while decreasing phospholipid levels. Simultaneously, cylindrospermopsin upregulated genes for lipogenesis regulation (SREBF1) and triacylglycerol synthesis (DGAT1/2) and downregulated genes for fatty acid synthesis (ACLY, ACCA, FASN, SCD1). Fatty acid uptake, oxidation, and lipid efflux genes were not significantly affected. Targeted proteomics revealed increased levels of perilipin 2 (adipophilin), a major hepatocyte lipid droplet-associated protein. Lipid profiling quantified 246 lipid species in the spheroids, with 28 significantly enriched and 15 downregulated by cylindrospermopsin. Upregulated species included neutral lipids, sphingolipids (e.g., ceramides and dihexosylceramides), and some glycerophospholipids (phosphatidylethanolamines, phosphatidylserines), while phosphatidylcholines and phosphatidylinositols were mostly reduced. It suggests that cylindrospermopsin exposures might contribute to developing and progressing towards hepatic steatosis or metabolic dysfunction-associated steatotic liver disease (MASLD).

High-Scale 3D-Bioprinting Platform for the Automated Production of Vascularized Organs-on-a-Chip.

3D bioprinting possesses the potential to revolutionize contemporary methodologies for fabricating tissue models employed in pharmaceutical research and experimental investigations. This is enhanced by combining bioprinting with advanced organs-on-a-chip (OOCs), which includes a complex arrangement of multiple cell types representing organ-specific cells, connective tissue, and vasculature. However, both OOCs and bioprinting so far demand a high degree of manual intervention, thereby impeding efficiency and inhibiting scalability to meet technological requirements. Through the combination of drop-on-demand bioprinting with robotic handling of microfluidic chips, a print procedure is achieved that is proficient in managing three distinct tissue models on a chip within only a minute, as well as capable of consecutively processing numerous OOCs without manual intervention. This process rests upon the development of a post-printing sealable microfluidic chip, that is compatible with different types of 3D-bioprinters and easily connected to a perfusion system. The capabilities of the automized bioprint process are showcased through the creation of a multicellular and vascularized liver carcinoma model on the chip. The process achieves full vascularization and stable microvascular network formation over 14 days of culture time, with pronounced spheroidal cell growth and albumin secretion of HepG2 serving as a representative cell model.

Effect of Donor Age and Liver Steatosis on Potential of Decellularized Liver Matrices to be used as a Platform for iPSC-Hepatocyte Culture.

Decellularization of discarded whole livers and their recellularization with patient-specific induced pluripotent stem cells (iPSCs) to develop a functional organ is a promising approach to increasing the donor pool. The effect of extracellular matrix (ECM) of marginal livers on iPSC-hepatocyte differentiation and function has not been shown. To test the effect of donor liver ECM age and steatosis, young and old, as well as no, low, and high steatosis livers, are decellularized. All livers are decellularized successfully. High steatosis livers have fat remaining on the ECM after decellularization. Old donor liver ECM induces lower marker expression in early differentiation stages, compared to young liver ECM, while this difference is closed at later stages and do not affect iPSC-hepatocyte function significantly. High steatosis levels of liver ECM lead to higher albumin mRNA expression and secretion while at later stages of differentiation expression of major cytochrome (CYP) 450enzymes is highest in low steatosis liver ECM. Both primary human hepatocytes and iPSC-hepatocytes show an increase in fat metabolism marker expression with increasing steatosis levels most likely induced by excess fat remaining on the ECM. Overall, removal of excess fat from liver ECM may be needed for inducing proper hepatic function after recellularization.

Gut-liver microphysiological systems revealed potential crosstalk mechanism modulating drug metabolism.

The small intestine and liver play important role in determining oral drug's fate. Both organs are also interconnected through enterohepatic circulation, which imply there are crosstalk through circulating factors such as signaling molecules or metabolites that may affect drug metabolism. Coculture of hepatocytes and intestinal cells have shown to increase hepatic drug metabolism, yet its crosstalk mechanism is still unclear. In this study, we aim to elucidate such crosstalk by coculturing primary human hepatocytes harvested from chimeric mouse (PXB-cells) and iPSc-derived intestinal cells in a microphysiological systems (MPS). Perfusion and direct oxygenation from the MPS were chosen and confirmed to be suitable features that enhanced PXB-cells albumin secretion, cytochrome P450 (CYP) enzymes activity while also maintaining barrier integrity of iPSc-derived intestine cells. Results from RNA-sequencing showed significant upregulation in gene ontology terms related to fatty acids metabolism in PXB-cells. One of such fatty acids, arachidonic acid, enhanced several CYP enzyme activity in similar manner as coculture. From the current evidences, it is speculated that the release of bile acids from PXB-cells acted as stimuli for iPSc-derived intestine cells to release lipoprotein which was ultimately taken by PXB-cells and enhanced CYP activity.

A highly efficient cell culture method using oxygen-permeable PDMS-based honeycomb microwells produces functional liver organoids from human induced pluripotent stem cell-derived carboxypeptidase M liver progenitor cells.

Recent advancements in bioengineering have introduced potential alternatives to liver transplantation via the development of self-assembled liver organoids, derived from human-induced pluripotent stem cells (hiPSCs). However, the limited maturity of the tissue makes it challenging to implement this technology on a large scale in clinical settings. In this study, we developed a highly efficient method for generating functional liver organoids from hiPSC-derived carboxypeptidase M liver progenitor cells (CPM+ LPCs), using a microwell structure, and enhanced maturation through direct oxygenation in oxygen-permeable culture plates. We compared the morphology, gene expression profile, and function of the liver organoidwith those of cells cultured under conventional conditions using either monolayer or spheroid culture systems. Our results revealed that liver organoids generated using polydimethylsiloxane-based honeycomb microwells significantly exhibited enhanced albumin secretion, hepatic marker expression, and cytochrome P450-mediated metabolism. Additionally, the oxygenated organoids consisted of both hepatocytes and cholangiocytes, which showed increased expression of bile transporter-related genesas well as enhanced bile transport function. Oxygen-permeable polydimethylsiloxane membranes may offer an efficient approach to generating highly mature liver organoids consisting of diverse cell populations.

Reproducible generation of human liver organoids (HLOs) on a pillar plate platform via microarray 3D bioprinting.

Human liver organoids (HLOs) hold significant potential for recapitulating the architecture and function of liver tissues in vivo. However, conventional culture methods of HLOs, forming Matrigel domes in 6-/24-well plates, have technical limitations such as high cost and low throughput in organoid-based assays for predictive assessment of compounds in clinical and pharmacological lab settings. To address these issues, we have developed a unique microarray 3D bioprinting protocol of progenitor cells in biomimetic hydrogels on a pillar plate with sidewalls and slits, coupled with a clear bottom, 384-deep well plate for scale-up production of HLOs. Microarray 3D bioprinting, a droplet-based printing technology, was used to generate a large number of small organoids on the pillar plate for predictive hepatotoxicity assays. Foregut cells, differentiated from human iPSCs, were mixed with Matrigel and then printed on the pillar plate rapidly and uniformly, resulting in coefficient of variation (CV) values in the range of 15-18%, without any detrimental effect on cell viability. Despite utilizing 10-50-fold smaller cell culture volume compared to their counterparts in Matrigel domes in 6-/24-well plates, HLOs differentiated on the pillar plate exhibited similar morphology and superior function, potentially due to rapid diffusion of nutrients and oxygen at the small scale. Day 25 HLOs were robust and functional on the pillar plate in terms of their viability, albumin secretion, CYP3A4 activity, and drug toxicity testing, all with low CV values. From three independent trials of in situ assessment, the IC50 values calculated for sorafenib and tamoxifen were 6.2 ± 1.6 μM and 25.4 ± 8.3 μM, respectively. Therefore, our unique 3D bioprinting and miniature organoid culture on the pillar plate could be used for scale-up, reproducible generation of HLOs with minimal manual intervention for high-throughput assessment of compound hepatotoxicity.

A High-Throughput Microphysiological Liver Chip System to Model Drug-Induced Liver Injury Using Human Liver Organoids

Drug-induced liver injury (DILI) is a major failure mode in pharmaceutical development. This study aims to address the limitations of existing preclinical models by assessing a high-throughput, microfluidic liver-on-a-chip system, termed "Curio Barrier Liver Chips," and its capacity to recapitulate the effects of chronic hepatotoxic drug treatment through metabolic and phenotypic characterization. Curio Barrier liver chips (Curiochips), fabricated in an 8× 2 well configuration, were utilized to establish three dimensional liver organoid cultures. Human-induced pluripotent stem cells were differentiated into human liver organoids, and their viability, liver-specific functions, and pharmacological responses were assessed over 28 days. The Curiochips successfully maintained liver physiology and function, showing strong albumin secretion and cytochrome (CYP) P450 activities for 28 days. Unlike traditional models requiring millimolar drug concentrations to detect hepatotoxicity, this platform showed increased sensitivity for acetaminophen and fialuridine at micromolar concentrations. In situ differentiation of foregut spheroids to liver organoids was also achieved, further simplifying the establishment of liver chips. Furthermore, the chips demonstrated viability, function, and DILI responsiveness for 28 days, making this an improved model for studying idiosyncratic DILI with prolonged drug exposure and high-throughput capabilities compared to other available systems or primary human hepatocytes. The Curiochips offer an advanced, miniaturized invitro model for early-stage drug development and a sensitive, responsive, and cost-effective means to detect direct hepatotoxicity. Induced pluripotent stem cell liver organoids, in conjunction with the Curiochip, deliver a high-throughput platform with robust functionality and pharmacological responsiveness that make it a promising tool for improving the prediction and understanding of DILI risk prediction, especially with prolonged drug exposure. The model also opens new avenues for research in other chronic liver diseases.

Microfluidic Fabrication of MicroRNA-Induced Hepatocyte-Like Cells/Human Umbilical Vein Endothelial Cells-Laden Microgels for Acute Liver Failure Treatment.

Acute liver failure (ALF) is a critical life-threatening disease that occurs due to a rapid loss in hepatocyte functions. Hepatocyte transplantation holds great potential for ALF treatment, as it rapidly supports liver biofunctions and enhances liver regeneration. However, hepatocyte transplantation is still limited by renewable and ongoing cell sources. In addition, intravenously injected hepatocytes are primarily trapped in the lungs and have limited efficacy because of the rapid clearance in vivo. Here, we designed a Y-shaped DNA nanostructure to deliver microRNA-122 (Y-miR122), which could induce the hepatic differentiation and maturation of human mesenchymal stem cells. mRNA sequencing analysis revealed that the Y-miR122 promoted important hepatic biofunctions of the induced hepatocyte-like cells including fat and lipid metabolism, drug metabolism, and liver development. To further improve hepatocyte transplantation efficiency and therapeutic effects in ALF treatment, we fabricated protective microgels for the delivery of Y-miR122-induced hepatocyte-like cells based on droplet microfluidic technology. When cocultured with human umbilical vein endothelial cells in microgels, the hepatocyte-like cells exhibited an increase in hepatocyte-associated functions, including albumin secretion and cytochrome P450 activity. Notably, upon transplantation into the ALF mouse model, the multiple cell-laden microgels effectively induced the restoration of liver function and enhanced liver regeneration. Overall, this study presents an efficient approach from the generation of hepatocyte-like cells to hepatocyte transplantation in ALF therapy.