3D Liver Spheroid Research Articles

Mesenchymal Stromal Cell-Derived Extracellular Vesicles for Reversing Hepatic Fibrosis in 3D Liver Spheroids.

Hepatic fibrosis, arising from prolonged liver injury, entails the activation of hepatic stellate cells (HSCs) into myofibroblast-like cells expressing alpha-smooth muscle actin (α-SMA), thereby driving extracellular matrix deposition and fibrosis progression. Strategies targeting activated HSC reversal and hepatocyte regeneration show promise for fibrosis management. Previous studies suggest that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) can suppress HSC activation, but ensuring EV purity is essential for clinical use. This study investigated the effects of MSC-derived EVs cultured in chemically defined conditions on liver spheroids and activated HSCs. Umbilical cord- and bone marrow-derived MSCs were expanded in chemically defined media, and EVs were isolated using filtration and differential ultracentrifugation. The impact of MSC-EVs was evaluated on liver spheroids generated in Sphericalplate 5D™ and on human HSCs, both activated by transforming growth factor beta 1 (TGF-β1). MSC-EVs effectively reduced the expression of profibrotic markers in liver spheroids and activated HSCs induced by TGF-β1 stimulation. These results highlight the potential of MSC-EVs collected under chemically defined conditions to mitigate the activated phenotype of HSCs and liver spheroids, suggesting MSC-EVs as a promising treatment for hepatic fibrosis.

Mechanistic, functional and clinical aspects of pro-inflammatory cytokine mediated regulation of ADME gene expression in 3D human liver spheroids.

During systemic inflammation, pro-inflammatory cytokines alter metabolism and transport of drugs affecting the clincal outcome. We used an in vivo like human 3D liver spheroid model to study the effects and mechanisms of pro-inflammatory cytokines on the expression of nine different genes encoding enzymes responsible for the metabolism of > 90% of clinically used drugs. Treatment of spheroids with pathophysiologically relevant concentrations of IL-1β, IL-6 or TNFα resulted in a pronounced decrease in mRNA expression of CYP3A4 and UGT2B10 within 5 h. The reduction of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 mRNA expression was less pronounced, whereas the pro-inflammatory cytokines caused increased CYP2E1 and UGT1A3 mRNA expression. The cytokines did not influence expression of key nuclear proteins, nor the activities of specific kinases involved in the regulation of genes encoding drug metabolizing enzymes. However, ruxolitinib, a JAK1/2 inhibitor, inhibited the IL-6 dependent increase in CYP2E1 and the decrease in CYP3A4 and UGT2B10 mRNA expression. We evaluated the effect of TNFα in hepatocytes in 2D plates and found a rapid decrease in DME mRNA both in the absence or presence of the cytokines. Taken together, these data suggest that pro-inflammatory cytokines regulate multiple gene- and cytokine-specific events seen in in vivo and in 3D but not in 2D liver models. We propose that the 3D spheroid system is suitable for the prediction of drug metabolism under conditions of inflammation and constitutes a versatile system for short- and long-term pre-clinical and mechanistic studies of cytokine-induced changes in drug metabolism.

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

The Role of CTGF in Liver Fibrosis Induced in 3D Human Liver Spheroids.

Connective tissue growth factor (CTGF) is involved in the regulation of extracellular matrix (ECM) production. Elevated levels of CTGF can be found in plasma from patients with liver fibrosis and in experimental animal models of liver fibrosis, but the exact role of CTGF in, e.g., diet-induced human liver fibrosis is not entirely known. To address this question, we utilized a 3D human liver co-culture spheroid model composed of hepatocytes and non-parenchymal cells, in which fibrosis is induced by TGF-β1, CTGF or free fatty acids (FFA). Treatment of the spheroids with TGF-β1 or FFA increased COL1A1 deposition as well as the expression of TGF-β1 and CTGF. Recombinant CTGF, as well as angiotensin II, caused increased expression and/or production of CTGF, TGF-β1, COL1A1, LOX, and IL-6. In addition, silencing of CTGF reduced both TGF-β1- and FFA-induced COL1A1 deposition. Furthermore, we found that IL-6 induced CTGF, COL1A1 and TGF-β1 production, suggesting that IL-6 is a mediator in the pathway of CTGF-induced fibrosis. Taken together, our data indicate a specific role for CTGF and CTGF downstream signaling pathways for the development of liver inflammation and fibrosis in the human 3D liver spheroid model.

Investigation of reversible histone acetylation and dynamics in gene expression regulation using 3D liver spheroid model

BackgroundThree-dimensional (3D) cell culture has emerged as an alternative approach to 2D flat culture to model more accurately the phenotype of solid tissue in laboratories. Culturing cells in 3D more precisely recapitulates physiological conditions of tissues, as these cells reduce activities related to proliferation, focusing their energy consumption toward metabolism and homeostasis.ResultsHere, we demonstrate that 3D liver spheroids are a suitable system to model chromatin dynamics and response to epigenetics inhibitors. To delay necrotic tissue formation despite proliferation arrest, we utilize rotating bioreactors that apply active media diffusion and low shearing forces. We demonstrate that the proteome and the metabolome of our model resemble typical liver functions. We prove that spheroids respond to sodium butyrate (NaBut) treatment, an inhibitor of histone deacetylases (HDACi), by upregulating histone acetylation and transcriptional activation. As expected, NaBut treatment impaired specific cellular functions, including the energy metabolism. More importantly, we demonstrate that spheroids reestablish their original proteome and transcriptome, including pre-treatment levels of histone acetylation, metabolism, and protein expression once the standard culture condition is restored after treatment. Given the slow replication rate (> 40 days) of cells in 3D spheroids, our model enables to monitor the recovery of approximately the same cells that underwent treatment, demonstrating that NaBut does not have long-lasting effects on histone acetylation and gene expression. These results suggest that our model system can be used to quantify molecular memory on chromatin.ConclusionTogether, we established an innovative cell culture system that can be used to model anomalously decondensing chromatin in physiological cell growth and rule out epigenetics inheritance if cells recover the original phenotype after treatment. The transient epigenetics effects demonstrated here highlight the relevance of using a 3D culture model system that could be very useful in studies requiring long-term drug treatment conditions that would not be possible using a 2D cell monolayer system.

Determination of the nanoparticle- and cell-specific toxicological mechanisms in 3D liver spheroids using scRNAseq analysis

Engineered nanomaterials (ENMs) are commonly used in consumer products, allowing exposure to target organs such as the lung, liver, and skin that could lead to adverse health effects in humans. To better reflect on toxicological effects in liver cells, it is important to consider the contribution of hepatocyte morphology, function, and intercellular interactions in a dynamic 3D microenvironment. Herein, we used a 3D liver spheroid model containing hepatocyte and Kupffer cells (KCs) to study the effects of three different material compositions, namely vanadium pentoxide (V2O5), titanium dioxide (TiO2), or graphene oxide (GO). Additionally, we used single-cell RNA sequencing (scRNAseq) to determine the nanoparticle (NP) and cell-specific toxicological responses. A general finding was that hepatocytes exhibit more variation in gene expression and adaptation of signaling pathways than KCs. TNF-α production tied to the NF-κB pathway was a commonly affected pathway by all NPs while impacts on the metabolic function of hepatocytes were unique to V2O5. V2O5 NPs also showed the largest number of differentially expressed genes in both cell types, many of which are related to pro-inflammatory and apoptotic response pathways. There was also evidence of mitochondrial ROS generation and caspase-1 activation after GO and V2O5 treatment, in association with cytokine production. All considered, this study provides insight into the impact of nanoparticles on gene responses in key liver cell types, providing us with a scRNAseq platform that can be used for high-content screening of nanomaterial impact on the liver, for use in biosafety and biomedical applications.

Hepatocyte Thorns, A Novel Drug-Induced Stress Response in Human and Mouse Liver Spheroids.

The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel mechanisms of chronic drug-induced liver toxicity. Using this system, we present a novel drug-induced stress response in human and murine hepatocyte spheroids, wherein long slender filaments form after chronic treatment with four different drugs, of which three are PPARα antagonists. The morphology of the thorns varies between donors and the compounds used. They are mainly composed of diverse protein fibres, which are glycosylated. Their formation is inhibited by treatment with fatty acids or antioxidants. Treatment of mice with GW6471 revealed changes in gene and protein expression, such as those in the spheroids. In addition, similar changes in keratin expression were seen following the treatment of hepatotoxic drugs, including aflatoxin B1, paracetamol, chlorpromazine, cyclosporine, and ketoconazole. We suggest that thorn formation may be indicative of hepatocyte metaplasia in response to toxicity and that more focus should be placed on alterations of ECM-derived protein expression as biomarkers of liver disease and chronic drug-induced hepatotoxicity, changes that can be studied in stable in vivo-like hepatic cell systems, such as the spheroids.

The Polymorphic Nuclear Factor NFIB Regulates Hepatic CYP2D6 Expression and Influences Risperidone Metabolism in Psychiatric Patients.

The genetic background for interindividual variability of the polymorphic CYP2D6 enzyme activity remains incompletely understood and the role of NFIB genetic polymorphism for this variability was evaluated in this translational study. We investigated the effect of NFIB expression in vitro using 3D liver spheroids, Huh7 cells, and the influence of the NFIB polymorphism on metabolism of risperidone in patients in vivo. We found that NFIB regulates several important pharmacogenes, including CYP2D6. NFIB inhibited CYP2D6 gene expression in Huh7 cells and NFIB expression in livers was predominantly nuclear and reduced at the mRNA and protein level in carriers of the NFIB rs28379954 T>C allele. Based on 604 risperidone treated patients genotyped for CYP2D6 and NFIB, we found that the rate of risperidone hydroxylation was elevated in NFIB rs28379954 T>C carriers among CYP2D6 normal metabolizers, resulting in a similar rate of drug metabolism to what is observed in CYP2D6 ultrarapid metabolizers, with no such effect observed in CYP2D6 poor metabolizers lacking functional enzyme. The results indicate that NFIB constitutes a novel nuclear factor in the regulation of cytochrome P450 genes, and that its polymorphism is a predictor for the rate of CYP2D6 dependent drug metabolism in vivo.

Determination of the Nanoparticle- and Cell-Specific Toxicological Mechanisms in 3d Liver Spheroids Using Scrnaseq Analysis

Determination of the Nanoparticle- and Cell-Specific Toxicological Mechanisms in 3d Liver Spheroids Using Scrnaseq Analysis

Erratum: Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure.

An erratum was issued for:Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure. The Representative Results sectionwas updated. Figure 6 in the Representative Results section was updated from: to: The fourth paragraph in the Representative Results section was updated from: With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain >70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the ≥14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be ≥0.06 mg/mL whilst urea production should be ≥0.4 ng/µL before conducting an in vitro toxicological assessment with this model. to: With the inevitable development of a necrotic core, a known limitation of 3D liver spheroid cultures, the viability of this HepG2 based model had to be established to demonstrate it was able to sustain long-term (5-10 day) exposure regimes whilst maintaining the proliferative capability required to support the micronucleus assay5. Indeed, this 3D liver spheroid model has been shown to retain >70% viability over 10 days in culture. Based on this and in conjunction with the sustained liver-like functionality observed over the ≥14 day culture period, this 3D liver spheroid model can thus support long-term, repeated ENM exposure regimes up to 10 days long (i.e., before viability of the spheroids drop below 70%). For reference, it is advised that albumin levels for HepG2 spheroids seeded at 4000 cells/spheroid should be ≥50.0ng/μL whilst urea production should be ≥0.25ng/µL before conducting an in vitro toxicological assessment with this model.

Chloroquine diphosphate suppresses liver cancer via inducing apoptosis in Wistar rats using interventional therapy.

Liver cancer ranks as the second leading cause of cancer-associated mortality worldwide. To date, neither current ablation therapy nor chemotherapy are considered ideal in improving the outcome of liver cancer. Therefore, more effective therapies for treating this devastating disease are urgently required. Interventional therapy has been used for numerous years in the treatment of different types of cancer, and is characterized by the direct delivery of anticancer drugs into the tumor. It has been reported that antimalarial chloroquine diphosphate (CQ) exerts effective anticancer activity against several types of cancer. However, its effect on liver cancer remains unclear. Therefore, in the present study, 2D monolayer cell culture and 3D spheroid in vitro models, and a rat model, were utilized to investigate the effect of CQ on liver cancer. CQ demonstrated an effective anticancer effect on HepG2 cells and 3D liver spheroids. Furthermore, the drug significantly inhibited cell growth and viability in the 2D and 3D in vitro models. The CQ-based intervention treatment effectively attenuated tumor size and weight, increased food intake and consumption of drinking water, and improved body weight and survival rate of rats in the in vivo model. In addition, treatment with CQ potently increased the expression levels of the apoptosis-related genes. Taken together, the findings of the present study may provide a novel insight into the development of safe and effective treatments for liver cancer.

Simulating Nanomaterial Transformation in Cascaded Biological Compartments to Enhance the Physiological Relevance of In Vitro Dosing Regimes: Optional or Required?

Would an engineered nanomaterial (ENM) still have the same identity once it reaches a secondary target tissue after a journey through several physiological compartments? Probably not. Does it matter? ENM pre-treatments may enhance the physiological relevance of in vitro testing via controlled transformation of the ENM identity. The implications of material transformation upon reactivity, cytotoxicity, inflammatory, and genotoxic potential of Ag and SiO2 ENM on advanced gastro-intestinal tract cell cultures and 3D liver spheroids are demonstrated. Pre-treatments are recommended for certain ENM only.

HepG2 (C3A) spheroids show higher sensitivity compared to HepaRG spheroids for drug-induced liver injury (DILI)

High-throughput, automation-friendly and therapeutically-predictive assays are needed in early drug discovery in order to prioritise compounds and reduce the risk of new drugs causing Drug-Induced Liver Injury (DILI). We evaluated the suitability of high-throughput 3D liver spheroid models of HepG2 (C3A clone) and HepaRG cell lines to predict DILI in early drug development. Spheroids were formed in 384-well ultra-low-attachment plates and dosed via direct acoustic droplet ejection at nine half-log spaced concentrations per compound. Spheroid viability was quantified with an ATP endpoint after a 4-day incubation with 150 drugs with known DILI liability. We derived a margin of safety for each cell line defined as the ratio between the IC50 values generated for each compound to their maximum plasma concentration Cmax which resulted in optimal classification accuracy. The margin of safety can be used to estimate a maximum safe Cmax for compounds in early drug discovery for which Cmax is not yet known. Both cell lines had similar level of accuracy in predicting DILI, with HepG2 spheroids being more sensitive. HepG2 spheroids had a sensitivity of 58% and a specificity of 83%, while HepaRG spheroids had a sensitivity of 47% and specificity of 86%. Ninety-nine of the 150 compounds were used to compare the relative sensitivities of HepG2 and HepaRG spheroids. HepaRG spheroids were more sensitive to 7 compounds and HepG2 spheroids were more sensitive to 34 compounds. In conclusion, across a diverse group of drugs HepG2 spheroids were more predictive of DILI compared to HepaRG spheroids.

P161 - Quantitative metabolic profiling of slowly cleared chemicals in in vitro models including 3D liver spheroid and hepatopac and hepatic clearance predictions from in vitro data using gastroplus PBPK modelling following topical application

P161 - Quantitative metabolic profiling of slowly cleared chemicals in in vitro models including 3D liver spheroid and hepatopac and hepatic clearance predictions from in vitro data using gastroplus PBPK modelling following topical application

P136 - Optimization of 3D liver spheroid culture conditions of individual hepatocyte donors and pooled hepatocyte donors

P136 - Optimization of 3D liver spheroid culture conditions of individual hepatocyte donors and pooled hepatocyte donors

Generation of liver organoids by 3D co-culturing of hepatocytes, hepatic stellate cells, and liver sinusoidal endothelial cells

Background and Hypothesis: Recent progress with combination of chemicals in cell culture media prolongs hepatocyte (HC) function in vitro. However, without other cells of hepatic lineage that comprise natural liver, HC alone are not suitable for the study of complex liver diseases. Hepatic stellate cells (HSC) and liver sinusoidal endothelial cells (LSEC) play vital roles in physiological and pathological liver function in situ. We hypothesized that coculturing primary HC with HSC and LSEC as 3D liver organoids in the same chemical conditioned media would uphold HC function over time, creating a better physiological 3D liver microenvironment for liver disease research.
 Experimental Design: Freshly thawed primary human HCs were combined with immortalized human HSC alone, or together with immortalized human LSECs, to generate 3D liver spheroids. Spheroids were formed in HC maintenance media (HMM, Lonza) using low adhesion 96-well plates for 6 days before switching to the media with different combination of chemicals (SB31542, Forskolin, IWP2, DAPT, and LDN193189) for culturing another 14 days. Spheroids characterization, albumin secretion, mRNA transcription (CYP3A4), and histological analysis were performed for HC differentiation, maturation, and function. 
 Results: Both co-cultures of HC:HSC (2.5:1 ratio), and HC:HSC:LSEC (2.5:1:1) formed spheroids in HMM within 4 to 6 days (Fig.1A). The introduction of the different chemical-based media affected the roundness and diameter of spheroids differently (Fig.1B). In the presence of all 5 chemicals (5C), HC function was better maintained up to 21 days in HC:HSC:LSEC spheroids, measured by albumin, CYP3A4, and CK-19 secretion (Fig.1C). Surprisingly, 5C-based media significantly upregulated the expression of CK-19, which is one of the markers for cholangiocytes and liver precursor cells. 
 Conclusion and Potential Impact: 3D liver organoids composed of HC, HSC, and LSEC would create a niche environment mimicking the in vivo condition. Optimization of complex hepatic spheroids, as well as optimization of complex hepatic spheroid culturing media, would allow for the generation of a unique model for studying complex liver diseases.

Emerging Models of Drug Metabolism, Transporters, and Toxicity

This commentary summarizes expert mini-reviews and original research articles that have been assembled in a special issue on novel models of drug metabolism and disposition. The special issue consists of research articles or reviews on novel static or micro-flow based models of the intestine, liver, eye, and kidney. This issue reviews static intestinal systems like mucosal scrapings and cryopreserved intestinal enterocytes, as well as novel bioengineered or chemically engineered intestinal models derived from primary human tissue, iPSCs, enteroids, and crypts. Experts have reviewed hepatic systems like cryopermeabilized Metmax hepatocytes and longer term, hepatocyte coculture system from HµREL, yielding in vivo-like primary and secondary drug metabolite profiles. Additional liver models, including micropattern hepatocyte coculture, 3D liver spheroids, and microflow systems, applicable to the study of drug disposition and toxicology have also been reviewed. In this commentary, we have outlined expert opinions and current efforts on hepatic- and nephrotoxicity models. Ocular disposition models including corneal permeability models have been included within this special issue. This commentary provides a summary of in vivo mini-reviews of the issue, which have discussed the applications and drawbacks of pig and humanized mice models of P450, UGT, and rat organic anionic transporting polypeptide 1a4. While not extensively reviewed, novel positron emissions tomography imaging-based approaches to study the distribution of xenobiotics have been highlighted. This commentary also outlines in vitro and in vivo models of drug metabolism derived from breakthrough genetic, chromosomal, and tissue engineering techniques. The commentary concludes by providing a futuristic view of the novel models discussed in this issue.

Nondestructive Optical Toxicity Assays of 3D Liver Spheroids with Optical Coherence Tomography

AbstractDrug‐induced liver injury (DILI) is the leading cause of failure in preclinical drug development and withdrawals. Given the central role of the liver in drug metabolism and detoxification, strong incentives exist to create new physiologically relevant, human‐based, 3D in vitro liver models which will offer better prediction of DILI. However, most cell metabolic assays are designed for 2D culture and are not always suitable to assess 3D cultures; in addition, there is an emerging need to develop novel nondestructive assays to assess cell activity in a time‐lapse fashion. In this study, optical coherence tomography (OCT) is used to measure the viability on 3D liver spheroids after acetaminophen exposure used as a model of DILI. Using HepaRG cells, 3D liver spheroids are grown simultaneously to the conventional 2D DILI model for 9 d and are exposed to 5, 10, 20, and 40 × 10−3 m acetaminophen for 24 h. It is shown that OCT can image noninvasively, and label free, the cell metabolic activity, thus enabling the assessment of viability of the 3D spheroids. This correlates well with cellular metabolic activity measured by biochemical assays. This method is translatable to any 3D tissue model and can be performed using any commercial OCT system.

Design and fabrication of a liver-on-a-chip platform for convenient, highly efficient, and safe in situ perfusion culture of 3D hepatic spheroids.

Spheroid-based three-dimensional (3D) liver culture models, offering a desirable biomimetic microenvironment, are useful for recapitulating liver functions in vitro. However, a user-friendly, robust and specially optimized method has not been well developed for a convenient, highly efficient, and safe in situ perfusion culture of spheroid-based 3D liver models. Here, we have developed a biomimetic and reversibly assembled liver-on-a-chip (3D-LOC) platform and presented a proof of concept for long-term perfusion culture of 3D human HepG2/C3A spheroids for building a 3D liver spheroid model. On the basis of a fast and reversible seal of concave microwell-based PDMS-membrane-PDMS sandwich multilayer chips, it enables a high-throughput and parallel perfusion culture of 1080 cell spheroids in a high mass transfer and low fluid shear stress biomimetic microenvironment as well as allowing the convenient collection and analysis of the cell spheroids. In terms of reducing spheroid loss and maintaining cell morphology and viability in long-term perfusion culture, the cell spheroids in the 3D-LOC were more safe and efficient. Notably, the polarisation, liver-specific functions, and metabolic activity of the cell spheroids in 3D-LOC were also remarkably improved and exhibited better long-term maintenance over conventional perfusion methods. Additionally, a robust micromilling method that incorporates secondary PDMS coating techniques (SPCs) for fabricating V-shaped concave microwells was also developed. The V-shaped concave microwell arrays exhibited a higher distribution density and aperture ratio, making it easy to form large-scale and uniform-sized cell spheroids with minimum cell loss. In summary, the proposed 3D-LOC could provide a convenient and robust solution for the long-term safe perfusion culture of hepatic spheroids and be beneficial for a variety of potential applications including development of bio-artificial livers, disease modeling, and drug toxicity screening.

Phenotypic Characterization of Toxic Compound Effects on Liver Spheroids Derived from iPSC Using Confocal Imaging and Three-Dimensional Image Analysis.

Cell models are becoming more complex to better mimic the in vivo environment and provide greater predictivity for compound efficacy and toxicity. There is an increasing interest in exploring the use of three-dimensional (3D) spheroids for modeling developmental and tissue biology with the goal of accelerating translational research in these areas. Accordingly, the development of high-throughput quantitative assays using 3D cultures is an active area of investigation. In this study, we have developed and optimized methods for the formation of 3D liver spheroids derived from human iPS cells and used those for toxicity assessment. We used confocal imaging and 3D image analysis to characterize cellular information from a 3D matrix to enable a multi-parametric comparison of different spheroid phenotypes. The assay enables characterization of compound toxicities by spheroid size (volume) and shape, cell number and spatial distribution, nuclear characterization, number and distribution of cells expressing viability, apoptosis, mitochondrial potential, and viability marker intensities. In addition, changes in the content of live, dead, and apoptotic cells as a consequence of compound exposure were characterized. We tested 48 compounds and compared induced pluripotent stem cell (iPSC)-derived hepatocytes and HepG2 cells in both two-dimensional (2D) and 3D cultures. We observed significant differences in the pharmacological effects of compounds across the two cell types and between the different culture conditions. Our results indicate that a phenotypic assay using 3D model systems formed with human iPSC-derived hepatocytes is suitable for high-throughput screening and can be used for hepatotoxicity assessment in vitro.