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Nano and microplastics are defined as particles smaller than 100 nm and 5 mm respectively. The widespread production and use of plastics in everyday life has resulted in significant accumulation of plastic debris in the environment. Over the last two decades there are increased concerns regarding the potential entry and accumulation of plastics in the human body with ingestion being one of the most important routes of exposure. However, the magnitude and nature of potential toxic effects of plastic exposure to human health is not yet fully understood. The liver is the body's principal detoxification organ and critically to this study recognised as the main accumulation site for particulates. In this study as the first of its kind the health impacts of long term low repeated polystyrene microplastics (1 and 5 µm) exposure was investigated in a functionally active 3D liver microtissue model, composed of primary human hepatocytes, Kupffer cells, sinusoidal endothelial cells and hepatic stellate cells. The highlight from the data includes microplastic-induced dose (3.125-25 µg/ml) and time dependent (up to 504 hr) increase in cell death and inflammation manifested by enhanced release of IL6, IL8 and TNF-α. The exposure to repeated dosing of the plastics also resulted in notable pathology manifested as aberrant tissue architecture, such as dilated bile canaliculi and large lipid droplets inside the hepatic cells. This toxicity matched extremely well to the accumulation of the materials with the cells of microtissue predominately in the organ macrophages. This study highlights the real issue and danger of microplastic exposure with potential for long-term accumulation and adverse effects of non-biodegradable plastics within the liver.
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Drug-Induced Liver Injury (DILI) constitutes hepatic damage attributed to drug exposure. DILI may be categorized as hepatocellular, cholestatic or mixed and might also involve immune responses. When DILI occurs in dose-dependent manner, it is referred to as intrinsic, while if the injury occurs spontaneously, it is termed as idiosyncratic. This review predominately focused on idiosyncratic liver injury. The established molecular mechanisms for DILI include (1) mitochondria dysfunction, (2) increased reactive oxygen species levels, (3) presence of elevated apoptosis and necrosis, (4) and bile duct injuries associated with immune mediated pathways. However, it should be emphasized that the underlying mechanisms responsible for DILI are still unknown. Prevention strategies are critical as incidences occur frequently, and treatment options are limited once the injury has developed. The aim of this review was to utilize retrospective cohort studies from across the globe to gain insight into epidemiological patterns. This review considers (1) what is currently known regarding the mechanisms underlying DILI, (2) discusses potential risk factors and (3) implications of the coronavirus pandemic on DILI presentation and research. Future perspectives are also considered and discussed and include potential new biomarkers, causality assessment and reporting methods.
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Doença Hepática Induzida por Substâncias e Drogas , Hepatopatias , Humanos , Estudos Retrospectivos , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatopatias/epidemiologia , Fatores de RiscoRESUMO
To reduce, replace, and refine in vivo testing, there is increasing emphasis on the development of more physiologically relevant in vitro test systems to improve the reliability of non-animal-based methods for hazard assessment. When developing new approach methodologies, it is important to standardize the protocols and demonstrate the methods can be reproduced by multiple laboratories. The aim of this study was to assess the transferability and reproducibility of two advanced in vitro liver models, the Primary Human multicellular microtissue liver model (PHH) and the 3D HepG2 Spheroid Model, for nanomaterial (NM) and chemical hazard assessment purposes. The PHH model inter-laboratory trial showed strong consistency across the testing sites. All laboratories evaluated cytokine release and cytotoxicity following exposure to titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles. No significant difference was observed in cytotoxicity or IL-8 release for the test materials. The data were reproducible with all three laboratories with control readouts within a similar range. The PHH model ZnO induced the greatest cytotoxicity response at 50.0 µg/mL and a dose-dependent increase in IL-8 release. For the 3D HepG2 spheroid model, all test sites were able to construct the model and demonstrated good concordance in IL-8 cytokine release and genotoxicity data. This trial demonstrates the successful transfer of new approach methodologies across multiple laboratories, with good reproducibility for several hazard endpoints.
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Óxido de Zinco , Humanos , Óxido de Zinco/toxicidade , Reprodutibilidade dos Testes , Interleucina-8 , Fígado , Linhagem Celular , Esferoides CelularesRESUMO
Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.
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Colágeno Tipo I , Neoplasias , Biomimética , Linhagem Celular Tumoral , Colágeno Tipo I/metabolismo , Matriz Extracelular , Colágenos Fibrilares/metabolismoRESUMO
In an effort to replace, reduce and refine animal experimentation, there is an unmet need to advance current in vitro models that offer features with physiological relevance and enhanced predictivity of in vivo toxicological output. Hepatic toxicology is key following chemical, drug and nanomaterials (NMs) exposure, as the liver is vital in metabolic detoxification of chemicals as well as being a major site of xenobiotic accumulation (i.e., low solubility particulates). With the ever-increasing production of NMs, there is a necessity to evaluate the probability of consequential adverse effects, not only in health but also in clinically asymptomatic liver, as part of risk stratification strategies. In this study, two unique disease initiation and maintenance protocols were developed and utilised to mimic steatosis and pre-fibrotic NASH in scaffold-free 3D liver microtissues (MT) composed of primary human hepatocytes, hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. The characterized diseased MT were utilized for the toxicological assessment of a panel of xenobiotics. Highlights from the study included: 1. Clear experimental evidence for the pre-existing liver disease is important in the augmentation of xenobiotic-induced hepatotoxicity and 2. NMs are able to activate stellate cells. The data demonstrated that pre-existing disease is vital in the intensification of xenobiotic-induced liver damage. Therefore, it is imperative that all stages of the wide spectrum of liver disease are incorporated in risk assessment strategies. This is of significant consequence, as a substantial number of the general population suffer from sub-clinical liver injury without any apparent or diagnosed manifestations.
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Hepatopatia Gordurosa não Alcoólica , Animais , Células Endoteliais/metabolismo , Hepatócitos , Humanos , Células de Kupffer , Fígado , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
We present a generic workflow combining physiology-based computational modeling and in vitro data to assess the clinical cholestatic risk of different drugs systematically. Changes in expression levels of genes involved in the enterohepatic circulation of bile acids were obtained from an in vitro assay mimicking 14 days of repeated drug administration for 10 marketed drugs. These changes in gene expression over time were contextualized in a physiology-based bile acid model of glycochenodeoxycholic acid. The simulated drug-induced response in bile acid concentrations was then scaled with the applied drug doses to calculate the cholestatic potential for each compound. A ranking of the cholestatic potential correlated very well with the clinical cholestasis risk obtained from medical literature. The proposed workflow allows benchmarking the cholestatic risk of novel drug candidates. We expect the application of our workflow to significantly contribute to the stratification of the cholestatic potential of new drugs and to support animal-free testing in future drug development.
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Benchmarking/métodos , Colestase/induzido quimicamente , Colestase/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Modelos Biológicos , Fluxo de Trabalho , Adulto , Animais , Colestase/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Preparações Farmacêuticas , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Adulto JovemRESUMO
BACKGROUND: Extensive DNA sequencing has led to an unprecedented view of the diversity of individual genomes and their evolution among patients with clear cell renal cell carcinoma (ccRCC). OBJECTIVE: To understand subclonal architecture and dynamics of patient-derived two-dimensional (2D) and three-dimensional (3D) ccRCC models in vitro, in order to determine whether they mirror ccRCC inter- and intratumor heterogeneity. DESIGN, SETTING, AND PARTICIPANTS: We have established a comprehensive platform of living renal cancer cell models from ccRCC surgical specimens. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: We confirmed the concordance of 2D and 3D patient-derived cell (PDC) models with the original tumor tissue in terms of histology, biomarker expression, cancer driver mutations, and copy number alterations. We addressed inter- and intrapatient heterogeneity by analyzing clonal dynamics during serial passaging. RESULTS AND LIMITATIONS: In-depth genetic characterization verified the presence of heterogeneous cell populations, and revealed a high degree of similarity between subclonal compositions of monolayer and organoid cell cultures and the corresponding parental ccRCCs. Clonal dynamics were evident during serial passaging of cells in vitro, suggesting that PDC cultures can offer insights into evolutionary potential and treatment susceptibility of ccRCC subclones in vivo. Proof-of-concept drug profiling using selected ccRCC-targeted therapy agents highlighted patient-specific vulnerabilities in PDC models that could not be anticipated by interrogating commercially available cell lines. CONCLUSIONS: We demonstrate that PDC models mirror inter- and intratumor heterogeneity of ccRCC in vitro. Based on our findings, we envision that the use of these models will advance our understanding of the trajectories that cause genetic diversity and their consequences for treatment on an individual level. PATIENT SUMMARY: In this study, we developed two- and three-dimensional patient-derived models from clear cell renal cell carcinoma (ccRCC) as "mini-tumors in a dish." We show that these cell models retain important features of the human ccRCCs such as the profound tumor heterogeneity, thus highlighting their importance for cancer research and precision medicine.
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Carcinoma de Células Renais , Neoplasias Renais , Biomarcadores Tumorais , Carcinoma de Células Renais/genética , Evolução Molecular , Heterogeneidade Genética , Humanos , Neoplasias Renais/genética , Medicina de PrecisãoRESUMO
The liver plays an important role in xenobiotic metabolism and represents a primary target for toxic substances. Many different in vitro cell models have been developed in the past decades. In this study, we used RNA-sequencing (RNA-Seq) to analyze the following human in vitro liver cell models in comparison to human liver tissue: cancer-derived cell lines (HepG2, HepaRG 3D), induced pluripotent stem cell-derived hepatocyte-like cells (iPSC-HLCs), cancerous human liver-derived assays (hPCLiS, human precision cut liver slices), non-cancerous human liver-derived assays (PHH, primary human hepatocytes) and 3D liver microtissues. First, using CellNet, we analyzed whether these liver in vitro cell models were indeed classified as liver, based on their baseline expression profile and gene regulatory networks (GRN). More comprehensive analyses using non-differentially expressed genes (non-DEGs) and differential transcript usage (DTU) were applied to assess the coverage for important liver pathways. Through different analyses, we noticed that 3D liver microtissues exhibited a high similarity with in vivo liver, in terms of CellNet (C/T score: 0.98), non-DEGs (10,363) and pathway coverage (highest for 19 out of 20 liver specific pathways shown) at the beginning of the incubation period (0 h) followed by a decrease during long-term incubation for 168 and 336 h. PHH also showed a high degree of similarity with human liver tissue and allowed stable conditions for a short-term cultivation period of 24 h. Using the same metrics, HepG2 cells illustrated the lowest similarity (C/T: 0.51, non-DEGs: 5623, and pathways coverage: least for 7 out of 20) with human liver tissue. The HepG2 are widely used in hepatotoxicity studies, however, due to their lower similarity, they should be used with caution. HepaRG models, iPSC-HLCs, and hPCLiS ranged clearly behind microtissues and PHH but showed higher similarity to human liver tissue than HepG2 cells. In conclusion, this study offers a resource of RNA-Seq data of several biological replicates of human liver cell models in vitro compared to human liver tissue.
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Biologia Computacional/métodos , Hepatócitos/metabolismo , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Transcriptoma , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Técnicas In Vitro , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , RNA-SeqRESUMO
Performing chemical analysis at the nanoliter (nL) scale is of paramount importance for medicine, drug development, toxicology, and research. Despite the numerous methodologies available, a tool for obtaining chemical information non-invasively is still missing at this scale. Observer effects, sample destruction and complex preparatory procedures remain a necessary compromise. Among non-invasive spectroscopic techniques, one able to provide holistic and highly resolved chemical information in-vivo is nuclear magnetic resonance (NMR). For its renowned informative power and ability to foster discoveries and life-saving applications, efficient NMR at microscopic scales is highly sought after, but so far technical limitations could not match the stringent necessities of microbiology, such as biocompatible handling, ease of use, and high throughput. Here we introduce a novel microsystem, which combines CMOS technology with 3D microfabrication, enabling nL NMR as a platform tool for non-invasive spectroscopy of organoids, 3D cell cultures, and early stage embryos. In this study we show its application to microlivers models simulating non-alcoholic fatty liver disease, demonstrating detection of lipid metabolism dynamics in a time frame of 14 days based on 117 measurements of single 3D human liver microtissues.
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Hazard assessment, based on new approach methods (NAM), requires the use of batteries of assays, where individual tests may be contributed by different laboratories. A unified strategy for such collaborative testing is presented. It details all procedures required to allow test information to be usable for integrated hazard assessment, strategic project decisions and/or for regulatory purposes. The EU-ToxRisk project developed a strategy to provide regulatorily valid data, and exemplified this using a panel of > 20 assays (with > 50 individual endpoints), each exposed to 19 well-known test compounds (e.g. rotenone, colchicine, mercury, paracetamol, rifampicine, paraquat, taxol). Examples of strategy implementation are provided for all aspects required to ensure data validity: (i) documentation of test methods in a publicly accessible database; (ii) deposition of standard operating procedures (SOP) at the European Union DB-ALM repository; (iii) test readiness scoring accoding to defined criteria; (iv) disclosure of the pipeline for data processing; (v) link of uncertainty measures and metadata to the data; (vi) definition of test chemicals, their handling and their behavior in test media; (vii) specification of the test purpose and overall evaluation plans. Moreover, data generation was exemplified by providing results from 25 reporter assays. A complete evaluation of the entire test battery will be described elsewhere. A major learning from the retrospective analysis of this large testing project was the need for thorough definitions of the above strategy aspects, ideally in form of a study pre-registration, to allow adequate interpretation of the data and to ensure overall scientific/toxicological validity.
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Documentação , Processamento Eletrônico de Dados/legislação & jurisprudência , Regulamentação Governamental , Testes de Toxicidade , Toxicologia/legislação & jurisprudência , Animais , Células Cultivadas , Europa (Continente) , Humanos , Formulação de Políticas , Reprodutibilidade dos Testes , Estudos Retrospectivos , Medição de Risco , Terminologia como Assunto , Peixe-Zebra/embriologiaRESUMO
The importance of adsorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis is expected to grow substantially due to recent failures in detecting severe toxicity issues of new chemical entities during preclinical/clinical development. Traditionally, safety risk assessment studies for humans have been conducted in animals during advanced preclinical or clinical phase of drug development. However, potential drug toxicity in humans now needs to be detected in the drug discovery process as soon as possible without reliance on animal studies. The "omics", such as genomics, proteomics, and metabolomics, have recently entered pharmaceutical research in both drug discovery and drug development, but to the best of our knowledge, no applications in high-throughput safety risk assessment have been attempted so far. This paper reports an innovative method to anticipate adverse drug effects in an early discovery phase based on lipid fingerprints using human three-dimensional microtissues. The risk of clinical hepatotoxicity potential was evaluated for a data set of 22 drugs belonging to five different therapeutic chemical classes and with various drug-induced liver injury effect. The treatment of microtissues with repeated doses of each drug allowed collecting lipid fingerprints for five time points (2, 4, 7, 9, and 11 days), and multivariate statistical analysis was applied to search for correlations with the hepatotoxic effect. The method allowed clustering of the drugs based on their hepatotoxic effect, and the observed lipid impairments for a number of drugs was confirmed by literature sources. Compared to traditional screening methods, here multiple interconnected variables (lipids) are measured simultaneously, providing a snapshot of the cellular status from the lipid perspective at a molecular level. Applied here to hepatotoxicity, the proposed workflow can be applied to several tissues, being tridimensional microtissues from various origins.
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Doença Hepática Induzida por Substâncias e Drogas , Lipidômica , Humanos , Fígado , Modelos Estatísticos , Medição de Risco/métodos , Esferoides Celulares , Fluxo de TrabalhoRESUMO
BACKGROUND: With ever-increasing exposure to engineered nanomaterials (NMs), there is an urgent need to evaluate the probability of consequential adverse effects. The potential for NM translocation to distal organs is a realistic prospect, with the liver being one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting (i.e. short life-span, reduced metabolic activity, lacking important cell populations, etc.). In this study, we scrutinize a 3D human liver microtissue (MT) model (composed of primary hepatocytes and non-parenchymal cells). This unique experiment benefits from long-term (3 weeks) repeated very low exposure concentrations, as well as incorporation of recovery periods (up to 2 weeks), in an attempt to account for the liver's recovery capacity in vivo. As a means of assessing the toxicological potential of NMs, cell cytotoxicity (cell membrane integrity and aspartate aminotransferase (AST) activity), pro/anti-inflammatory response and hepatic function were investigated. RESULTS: The data showed that 2 weeks of cell culture might be close to limits before subtle ageing effects start to overshadow low sub-lethal NM-induced cellular responses in this test system (adenylate kinase (AK) cytotoxicity assay). We showed that in vitro AST measurement are not suitable in a nanotoxicological context. Moreover, the cytokine analysis (IL6, IL8, IL10 and TNF-α) proved useful in highlighting recovery periods as being sufficient for allowing a reduction in the pro-inflammatory response. Next, low soluble NM-treated MT showed a concentration-dependent penetration of materials deep into the tissue. CONCLUSION: In this study the advantages and pitfalls of the multi-cellular primary liver MT are discussed. Furthermore, we explore a number of important considerations for allowing more meaningful in vitro vs. in vivo comparisons in the field of hepatic nanotoxicology.
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Doença Hepática Induzida por Substâncias e Drogas/etiologia , Hepatócitos/efeitos dos fármacos , Células de Kupffer/efeitos dos fármacos , Fígado/efeitos dos fármacos , Nanoestruturas/toxicidade , Técnicas de Cultura de Tecidos/métodos , Albuminas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Citocinas/metabolismo , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Células de Kupffer/metabolismo , Células de Kupffer/patologia , Fígado/metabolismo , Fígado/patologia , Testes de Função HepáticaRESUMO
Drug-induced liver injury (DILI) cannot be accurately predicted by animal models. In addition, currently available in vitro methods do not allow for the estimation of hepatotoxic doses or the determination of an acceptable daily intake (ADI). To overcome this limitation, an in vitro/in silico method was established that predicts the risk of human DILI in relation to oral doses and blood concentrations. This method can be used to estimate DILI risk if the maximal blood concentration (Cmax) of the test compound is known. Moreover, an ADI can be estimated even for compounds without information on blood concentrations. To systematically optimize the in vitro system, two novel test performance metrics were introduced, the toxicity separation index (TSI) which quantifies how well a test differentiates between hepatotoxic and non-hepatotoxic compounds, and the toxicity estimation index (TEI) which measures how well hepatotoxic blood concentrations in vivo can be estimated. In vitro test performance was optimized for a training set of 28 compounds, based on TSI and TEI, demonstrating that (1) concentrations where cytotoxicity first becomes evident in vitro (EC10) yielded better metrics than higher toxicity thresholds (EC50); (2) compound incubation for 48 h was better than 24 h, with no further improvement of TSI after 7 days incubation; (3) metrics were moderately improved by adding gene expression to the test battery; (4) evaluation of pharmacokinetic parameters demonstrated that total blood compound concentrations and the 95%-population-based percentile of Cmax were best suited to estimate human toxicity. With a support vector machine-based classifier, using EC10 and Cmax as variables, the cross-validated sensitivity, specificity and accuracy for hepatotoxicity prediction were 100, 88 and 93%, respectively. Concentrations in the culture medium allowed extrapolation to blood concentrations in vivo that are associated with a specific probability of hepatotoxicity and the corresponding oral doses were obtained by reverse modeling. Application of this in vitro/in silico method to the rat hepatotoxicant pulegone resulted in an ADI that was similar to values previously established based on animal experiments. In conclusion, the proposed method links oral doses and blood concentrations of test compounds to the probability of hepatotoxicity.
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Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Administração Oral , Algoritmos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Simulação por Computador , Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Técnicas In Vitro , Dose Máxima Tolerável , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , Farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Máquina de Vetores de SuporteRESUMO
The potential for nanomaterial (NM) translocation to secondary organs is a realistic prospect, with the liver one of the most important target organs. Traditional in vitro or ex vivo hepatic toxicology models are often limiting and/or troublesome (i.e. short life-span reduced metabolic activity, lacking important cell populations, high inter-individual variability, etc.). Building on previous work, this study utilises a 3D human liver microtissue (MT) model (MT composed of mono-culture of hepatocytes or two different co-culture MT systems with non-parenchymal cell (NPC) fraction sourced from different donors) to investigate the importance of inter-donor variability of the non-parenchymal cell population in the overall governance of toxicological response following exposure to a panel of NMs. To the best of our knowledge, this is the first study of its kind to investigate inter-donor variability in hepatic NPC population. The data showed that the Kupffer cells were crucial in dictating the overall hepatic toxicity following exposure to the materials. Furthermore, a statistically significant difference was noted between the two co-culture MT models. However, the trend for particle-induced biological responses was similar between the co-cultures (cytotoxicity, cytokine production and caspase activity). Therefore, despite the recognition of some discrepancies in the absolute values between the co-culture models, the fact that the trends and patterns of biological responses were comparable between the multi-cellular models we propose the 3D liver MT to be a valuable tool in particle toxicology.
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Variação Biológica da População , Células de Kupffer/fisiologia , Fígado/patologia , Teste de Materiais/métodos , Nanoestruturas/toxicidade , Adulto , Células Cultivadas , Técnicas de Cocultura/métodos , Feminino , Hepatócitos/fisiologia , Humanos , Fígado/citologia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células/métodos , Testes de Toxicidade/métodosRESUMO
Primary human hepatocytes (PHHs) remain the gold standard for in vitro testing in the field of pharmacology and toxicology. One crucial parameter influencing the results of in vitro tests is the incubation period with test compounds. It has been suggested that longer incubation periods may be critical for the prediction of repeated dose toxicity. However, a study that systematically analyzes the relationship between incubation period and cytotoxicity in PHHs is not available. To close this gap, 30 compounds were tested in a concentration-dependent manner for cytotoxicity in cultivated cryopreserved PHHs (three donors per compound) for 1, 2 and 7 days. The median of the EC50 values of all compounds decreased 1.78-fold on day 2 compared to day 1, and 1.89-fold on day 7 compared to day 1. Median values of EC50 ratios of all compounds at day 2 and day 7 were close to one but for individual compounds the ratio increased up to almost six. Strong correlations were obtained for EC50 on day 1 and day 7 (R = 0.985; 95% CI 0.960-0.994), day 1 and day 2 (R = 0.964; 95% CI 0.910-0.986), as well as day 2 and day 7 (R = 0.981; 95% CI 0.955-0.992). However, compound specific differences also occurred. Whereas, for example, busulfan showed a relatively strong increase on day 7 compared to day 1, cytotoxicity of acetaminophen did not increase during longer incubation periods. To validate the observed correlations, a publicly available data set, containing data on the cytotoxicity of human hepatocytes cultivated as spheroids for incubation periods of 5 and 14 days, was analyzed. A high correlation coefficient of EC50 values at day 5 and day 14 was obtained (R = 0.894; 95% CI 0.798-0.945). In conclusion, the median cytotoxicity of the test compounds increased between 1 and 2 days of incubation, with no or only a minimal further increase until day 7. It remains to be studied whether the different results obtained for some individual compounds after longer exposure periods would correspond better to human-repeated dose toxicity.
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Doença Hepática Induzida por Substâncias e Drogas/etiologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Hepatócitos/efeitos dos fármacos , Testes de Toxicidade/métodos , Acetaminofen/toxicidade , Técnicas de Cultura de Células/métodos , Células Cultivadas , Criopreservação , Relação Dose-Resposta a Droga , Humanos , Fatores de TempoRESUMO
Three-Dimensional (3D) liver microtissues, specifically prepared from primary human hepatocytes (PHH) in coculture with nonparenchymal cells (NPCs), have been shown to be a valuable tool for in vitro toxicology. However, a lack of thorough characterization on a functional, transcriptomic, and proteomic level of such models during long-term cultivation is evident. By integrating multiple omics technologies, we provide in this study an in-depth long-term characterization of 3D microtissues composed of PHH from three different donors cocultured with primary NPCs. The 3D human liver microtissues (hLiMTs) exhibited stable adenosine triphosphate (ATP) content and albumin secretion over 5 weeks. Histological analysis indicated a healthy liver tissue with polarized expression of bile salt export pump (BSEP) and multidrug resistance protein 2 (MRP2) in a structure reminiscent of bile canaliculi. The 3D microtissues exhibited stable basal and inducible cytochrome P450 activities up to 5 weeks in culture. Analysis of 40,716 transcripts using RNA arrays revealed distinct similarities to native human liver gene expression. Long-term culture showed a stable phenotype up to 5 weeks, with differences in liver gene expression primarily attributed to individual donors. Proteomic profiling of 2200 unique proteins by label-free LC-MS/MS revealed a relatively stable protein expression where only 7.3% were up- or downregulated more than twofold from day 7 to 35 in culture. Taken together, these results suggest that hLiMTs represent a responsive and physiologically relevant in vitro liver model that maintains stable function over 5 weeks and is therefore well suited for repeated-dose toxicity testing.
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The epithelial cells lining the proximal tubules of the kidney mediate complex transport processes and are particularly vulnerable to drug toxicity. Drug toxicity studies are classically based on two-dimensional cultures of immortalized proximal tubular cells. Such immortalized cells are dedifferentiated, and lose transport properties (including saturable endocytic uptake) encountered in vivo. Generating differentiated, organotypic human microtissues would potentially alleviate these limitations and facilitate drug toxicity studies. Here, we describe the generation and characterization of kidney microtissues from immortalized (HK-2) and primary (HRPTEpiC) human renal proximal tubular epithelial cells under well-defined conditions. Microtissue cultures were done in hanging drop GravityPLUS™ culture plates and were characterized for morphology, proliferation and differentiation markers, and by monitoring the endocytic uptake of albumin. Kidney microtissues were successfully obtained by co-culturing HK-2 or HRPTEpiC cells with fibroblasts. The HK-2 microtissues formed highly proliferative, but dedifferentiated microtissues within 10 days of culture, while co-culture with fibroblasts yielded spherical structures already after 2 days. Low passage HRPTEpiC microtissues (mono- and co-culture) were less proliferative and expressed tissue-specific differentiation markers. Electron microscopy evidenced epithelial differentiation markers including microvilli, tight junctions, endosomes, and lysosomes in the co-cultured HRPTEpiC microtissues. The co-cultured HRPTEpiC microtissues showed specific uptake of albumin that could be inhibited by cadmium and gentamycin. In conclusion, we established a reliable hanging drop protocol to obtain functional kidney microtissues with proximal tubular epithelial cell lines. These microtissues could be used for high-throughput drug and toxicology screenings, with endocytosis as a functional readout.
Assuntos
Células Epiteliais/citologia , Túbulos Renais Proximais/citologia , Cultura Primária de Células/métodos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura/métodos , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Fibroblastos/metabolismo , Humanos , Técnicas de Cultura de Tecidos/métodosRESUMO
Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.
Assuntos
Sinalização do Cálcio , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Cardiovasculares , Miócitos Cardíacos/metabolismo , Esferoides Celulares/metabolismo , Alicerces Teciduais/química , Adulto , Conexina 43/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Esferoides Celulares/citologiaRESUMO
The adsorption geometry of 1,3,5-tris(4-mercaptophenyl)benzene (TMB) on Cu(111) is determined with high precision using two independent methods, experimentally by quantitative low energy electron diffraction (LEED-I(V)) and theoretically by dispersion corrected density functional theory (DFT-vdW). Structural refinement using both methods consistently results in similar adsorption sites and geometries. Thereby a level of confidence is reached that allows deduction of subtle structural details such as molecular deformations or relaxations of copper substrate atoms.