RESUMO
Each year, millions to trillions of data points are generated to evaluate the response of chemicals and biologicals to human cells in vitro and in vivo using various technologies and endpoints. Despite the vast amount of data available, the development process has not become significantly more efficient in recent years. Given the increasing use of more complex physiological models, which are time-consuming and significantly more expensive, it is crucial to maximize the value of these valuable data through improved standardization.
Assuntos
Descoberta de Drogas , Descoberta de Drogas/normasRESUMO
Three-dimensional (3D) cell culture models are thought to mimic the physiological and pharmacological properties of tissues in vivo more accurately than two-dimensional cultures on plastic dishes. For the development of cancer therapies, 3D spheroid models are being created to reflect the complex histology and physiology of primary tumors with the hopes that drug responses will be more similar to and as predictive as those obtained in vivo. The effect of additional cell types in tumors, such as stromal cells, and the resulting heterotypic cell-cell crosstalk can be investigated in these heterotypic 3D cell cultures. Here, a high-throughput screening-compatible drug testing platform based on 3D multicellular spheroid models is described that enables the parallel assessment of toxicity on stromal cells and efficacy on cancer cells by drug candidates. These heterotypic microtissue tumor models incorporate NIH3T3 fibroblasts as stromal cells that are engineered with a reporter gene encoding secreted NanoLUC luciferase. By tracking the NanoLUC signal in the media over time, a time-related measurement of the cytotoxic effects of drugs on stromal cells over the cancer cells was possible, thus enabling the identification of a therapeutic window. An in vitro therapeutic index parameter is proposed to help distinguish and classify those compounds with broad cytotoxic effects versus those that are more selective at targeting cancer cells.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Esferoides Celulares/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Camundongos , Células NIH 3T3/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Esferoides Celulares/patologia , Células Estromais/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
As we witness steady progress towards the development of robust, scalable, and reproducible 3D tissue models for preclinical drug testing, there is a need for systematic physiological and pharmacological validation and benchmarking. Ongoing and future studies should generate evidence as to whether 3D tissue models are more predictive, help reduce the risk of failure rate, and can be used for decision making in the drug discovery and development pipeline. Here, we discuss the importance of harmonizing the validation of these models based on throughput capacity and physiological complexity as a requirement to establish their true translational capacity. We also outline our strategy for a novel 3D-tailored holistic drug discovery concept rather than piecemeal integration of 3D models into the current process.
Assuntos
Desenvolvimento de Medicamentos , Descoberta de Drogas , Pesquisa Translacional Biomédica , Animais , Humanos , Modelos BiológicosRESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Técnicas de Cultura de Órgãos/métodos , Testes de Toxicidade Aguda/métodos , Animais , Relação Dose-Resposta a Droga , Coração/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Preparações Farmacêuticas/administração & dosagem , Preparações Farmacêuticas/sangue , FarmacocinéticaRESUMO
Drug-induced liver injury (DILI) continues to be a major source of clinical attrition, precautionary warnings, and post-market withdrawal of drugs. Accordingly, there is a need for more predictive tools to assess hepatotoxicity risk in drug discovery. Three-dimensional (3D) spheroid hepatic cultures have emerged as promising tools to assess mechanisms of hepatotoxicity, as they demonstrate enhanced liver phenotype, metabolic activity, and stability in culture not attainable with conventional two-dimensional hepatic models. Increased sensitivity of these models to drug-induced cytotoxicity has been demonstrated with relatively small panels of hepatotoxicants. However, a comprehensive evaluation of these models is lacking. Here, the predictive value of 3D human liver microtissues (hLiMT) to identify known hepatotoxicants using a panel of 110 drugs with and without clinical DILI has been assessed in comparison to plated two-dimensional primary human hepatocytes (PHH). Compounds were treated long-term (14 days) in hLiMT and acutely (2 days) in PHH to assess drug-induced cytotoxicity over an 8-point concentration range to generate IC50 values. Regardless of comparing IC50 values or exposure-corrected margin of safety values, hLiMT demonstrated increased sensitivity in identifying known hepatotoxicants than PHH, while specificity was consistent across both assays. In addition, hLiMT out performed PHH in correctly classifying hepatotoxicants from different pharmacological classes of molecules. The hLiMT demonstrated sufficient capability to warrant exploratory liver injury biomarker investigation (miR-122, HMGB1, α-GST) in the cell-culture media. Taken together, this study represents the most comprehensive evaluation of 3D spheroid hepatic cultures up to now and supports their utility for hepatotoxicity risk assessment in drug discovery.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Desenho de Fármacos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/diagnóstico , Hepatócitos/efeitos dos fármacos , Biomarcadores/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Descoberta de Drogas/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Hepatócitos/patologia , Humanos , Concentração Inibidora 50 , Fígado/efeitos dos fármacos , Fígado/patologia , Valor Preditivo dos Testes , Medição de Risco/métodos , Fatores de TempoRESUMO
The tumour microenvironment and tumour angiogenesis play a critical role in the development and therapy of many cancers, but in vitro models reflecting these circumstances are rare. In this study, we describe the development of a novel tri-culture model, using non-small cell lung cancer (NSCLC) cell lines (A549 and Colo699) in combination with a fibroblast cell line (SV 80) and two different endothelial cell lines in a hanging drop technology. Endothelial cells aggregated either in small colonies in Colo699 containing microtissues or in tube like structures mainly in the stromal compartment of microtissues containing A549. An up-regulation of hypoxia and vimentin, ASMA and a downregulation of E-cadherin were observed in co- and tri-cultures compared to monocultures. Furthermore, a morphological alteration of A549 tumour cells resembling "signet ring cells" was observed in tri-cultures. The secretion of proangiogenic growth factors like vascular endothelial growth factor (VEGF) was measured in supernatants. Inhibition of these proangiogenic factors by using antiangiogenic drugs (bevacizumab and nindetanib) led to a significant decrease in migration of endothelial cells into microtissues. We demonstrate that our method is a promising tool for the generation of multicellular tumour microtissues and reflects in vivo conditions closer than 2D cell culture.
Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Modelos Anatômicos , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Microambiente Tumoral , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Transformação Celular Neoplásica , Técnicas de Cocultura , Citocinas/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Imuno-Histoquímica , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Oxigênio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Células Estromais/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.
Assuntos
Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/terapia , Microambiente Tumoral/imunologia , Animais , Humanos , Esferoides CelularesRESUMO
This work evaluated gene expression differences between a hanging-drop 3D NSCLC model and 2D cell cultures and their in-vivo relevance by comparison to patient-derived data from The Cancer Genome Atlas. Gene expression of 2D and 3D cultures for Colo699 and A549 were assessed using Affymetrix HuGene 1.0 ST gene chips. Biostatistical analyses tested for reproducibility, comparability and significant differences in gene expression profiles between cell lines, experiments and culture methods. The analyses revealed a high interassay correlation within specific culture systems proving a high validity. 979 genes were altered in A549 and 1106 in Colo699 cells due to 3D cultivation. The overlap of changed genes between the cell lines was small (149), but the involved pathways in the reactome and GO- analyses showed a high overlap with DNA methylation, cell cycle, SIRT1, PKN1 pathway, DNA repair and oxidative stress as well known cancer-associated representatives. Additional specific GSEA-analyses revealed changes in immunologic and endothelial cell proliferation pathways, whereas hypoxic, EMT and angiogenic pathways were downregulated. Gene enrichment analyses showed 3D-induced gene up-regulations in the cell lines 38 to be represented in in-vivo samples of NSCLC patients using data of The Cancer Genome Atlas. Thus, our 3D NSCLC model might provide a tool for early drug development and investigation of microenvironment-associated mechanisms. However, this work also highlights the need for further individualization and model adaption to address remaining challenges.
RESUMO
BACKGROUND: The focus of the outlined work is the establishment of a three-dimensional lung model for various drug-screening applications. METHODS: The non-small cell lung cancer (NSCLC) cell line Colo699 was cultivated as monolayer (2D) on plates for 5 days or as microtissues (3D) using a hanging-drop system for 5 and 10 days. Cells and microtissues were treated with afatinib (10-80 µM), cisplatin (100-800 µM) or vinorelbine (25-200 µM) for 24 or 48 hours (h). Cell proliferation and viability were analysed by intra-cellular adenosine triphosphate (ATP) and lactate dehydrogenase release (LDH) assays, annexin V/propidium iodide (PI) staining, and cell cycle determination. Microtissue morphology and size, as well as cell death were evaluated via phase contrast microscopy. RESULTS: Our results demonstrate the valid determination of viability and cell death using established assays in the 3D system for drug testing. The comparison of ATP, LDH and cytometry data showed moderate (0.40) to very strong (0.99) correlations. Thereby, we observed partially significant differences in drug efficacy between microtissues and 2D cultures dependent from the applied treatment and read-out method. Altogether, microtissues developed resistance to cisplatin and vinorelbine; but remained more vulnerable to afatinib. These findings were confirmed with microscopy. CONCLUSION: In summary, we established an NSCLC 3D test system with multiple assays compatible for drug-testing applications of substances with different mechanisms of action. In addition, our data support the usage of microtissues as more accurate tools for drug-efficacy testing with the possibility of long-term cultivation and treatment.
Assuntos
Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Pulmonares/patologia , Pulmão/citologia , Esferoides Celulares/citologia , Técnicas de Cultura de Tecidos/métodos , Afatinib , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Humanos , Pulmão/patologia , Quinazolinas/farmacologia , Células Tumorais Cultivadas , Vimblastina/análogos & derivados , Vimblastina/farmacologia , VinorelbinaRESUMO
INTRODUCTION: The interaction between the immune system and malignant diseases is a proven key target for cancer therapy. We describe an innovative 3D cell culture system comprising both immune and cancer cells to evaluate their interaction and immune cell infiltration to provide an innovative in vitro screening of immunomodulatory agents and biomarkers. METHODS: 3D tumor microtissues were cultivated using a hanging drops system. Human non-small-cell lung cancer cell lines were incubated for 7 days to form microtissues. On day 5, peripheral blood mononuclear cells (PBMC) were added with or without interleukin-2 (IL-2) for 24 or 48h. Viability of cancer cells and the infiltrating PBMC subpopulations were investigated by flow cytometry. Aggregation of tumor cells and PBMC and the infiltration of the PBMC into the tumor microtissues were analyzed by immunohistochemistry. Quantification of infiltration was measured by applying the TissueFAXS system. RESULTS: Immunohistochemistry revealed PBMC infiltration in all cell lines which increased under IL-2 stimulation. Analysis of infiltrating populations showed both lymphocyte subpopulations and monocytes within the tumor microtissues. In all three co-cultures, CD3+CD8+ and CD3+CD8+CD45R0+CD28+ lymphocytes were increased with IL-2, whereas CD3+CD8+CD45R0-CD28+ PBMCs were decreased with and without IL-2 stimulation. CONCLUSION: In summary, we present a novel cell culture system to study the interaction between cancer cells and immune cells in 3-dimensional microtissues. In addition, we report for the first time an in vitro infiltration assay based on 3D microtissues. This model has the potential to provide a tool for ex-vivo drug testing and biomarker screening of immunomodulatory agents.
Assuntos
Imunomodulação , Subpopulações de Linfócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias/imunologia , Antígenos de Superfície/metabolismo , Biomarcadores , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Cocultura , Citotoxicidade Imunológica , Humanos , Imunofenotipagem , Subpopulações de Linfócitos/metabolismo , Subpopulações de Linfócitos/patologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Monócitos/imunologia , Monócitos/metabolismo , Neoplasias/patologia , Esferoides Celulares , Células Tumorais CultivadasRESUMO
BACKGROUND: Radiation resistance presents a challenge to the effective treatment of cancer. If therapeutic compounds were capable of resensitizing resistant tumours then a concurrent chemo-radiation treatment could be used to overcome radiation resistance. METHODS: We have developed a phenotypic assay to investigate the response of radiation resistant breast cancer cells grown in 3D-microtissue spheroids to combinations of radiation and established chemotherapeutic drugs. The effects were quantified by real time high content imaging of GFP detection area over 14 days. Ten established chemotherapeutic drugs were tested for their ability to enhance the effects of radiation. RESULTS: Of ten analysed chemotherapeutics, vinblastine was the most effective compound, with docetaxel and doxorubicine being less effective in combination with radiation. To investigate the response in a model closer to the in vivo situation we investigated the response of heterotypic 3D microtissues containing both fibroblasts and breast cancer cells. Drug treatment of these heterotypic 3D cultures confirmed treatment with radiation plus vinblastine to be additive in causing breast cancer growth inhibition. We have validated the screen by comparing radiation sensitizing effects of known chemotherapeutic agents. In both monotypic and heterotypic models the concurrent treatment of vinblastine and radiation proved more effective inhibitors of mammary cancer cell growth. The effective concentration range of both vinblastine and radiation are within the range used in treatment, suggesting the 3D model will offer a highly relevant screen for novel compounds. CONCLUSIONS: For the first time comfortable 3D cell-based phenotypic assay is available, that allows high throughput screening of compounds with radiation therapy modulating capacity, opening the field to drug discovery.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/radioterapia , Técnicas de Cultura de Células/métodos , Tolerância a Radiação/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Docetaxel , Doxorrubicina/administração & dosagem , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Taxoides/administração & dosagem , Vimblastina/administração & dosagemRESUMO
Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and research applications. However, scientific and technical hurdles must be overcome before SCD in vitro methods undergo appropriate validation and become accepted in the regulatory arena.
Assuntos
Técnicas de Cultura/métodos , Células-Tronco/efeitos dos fármacos , Toxicologia/métodos , Animais , Ensaio de Unidades Formadoras de Colônias/métodos , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
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
Rational development of more physiologic in vitro models includes the design of robust and flexible 3D-microtissue-based multi-tissue devices, which allow for tissue-tissue interactions. The developed device consists of multiple microchambers interconnected by microchannels. Pre-formed spherical microtissues are loaded into the microchambers and cultured under continuous perfusion. Gravity-driven flow is generated from on-chip reservoirs through automated chip-tilting without any need for additional tubing and external pumps. This tilting concept allows for operating up to 48 devices in parallel in order to test various drug concentrations with a sufficient number of replicates. For a proof of concept, rat liver and colorectal tumor microtissues were interconnected on the chip and cultured during 8 days in the presence of the pro-drug cyclophosphamide. Cyclophosphamide has a significant impact on tumor growth but only after bio-activation by the liver. This effect was only observed in the perfused and interconnected co-cultures of different microtissue types on-chip, whereas the discontinuous transfer of supernatant via pipetting from static liver microtissues that have been treated with cyclophosphamide did not significantly affect tumor growth. The results indicate the utility and multi-tissue functionality of this platform. The importance of continuous medium circulation and tissue interaction is highlighted.
Assuntos
Técnicas de Cocultura/métodos , Fígado/citologia , Técnicas Analíticas Microfluídicas/instrumentação , Esferoides Celulares/citologia , Técnicas de Cultura de Tecidos/métodos , Animais , Antineoplásicos Alquilantes/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura/instrumentação , Ciclofosfamida/farmacologia , Células HCT116/citologia , Células HCT116/efeitos dos fármacos , Humanos , Técnicas Analíticas Microfluídicas/métodos , Ratos , Esferoides Celulares/efeitos dos fármacos , Técnicas de Cultura de Tecidos/instrumentaçãoRESUMO
A new generation of biomaterials focus on smart materials incorporating cells. Here, we describe a novel generation of synthetic nanofibrous implant functionalized with living microtissues for regenerative nanomedicine. The strategy designed here enhances the effectiveness of therapeutic implants compared to current approaches used in the clinic today based on single cells added to the implant.
RESUMO
In this article, we present a microfluidic platform, compatible with conventional 96-well formats, that enables facile and parallelized culturing and testing of spherical microtissues in a standard incubator. The platform can accommodate multiple microtissues (up to 66) of different cell types, formed externally by using the hanging-drop method, and enables microtissue interconnection through microfluidic channels for continuous media perfusion or dosage of substances. The platform contains 11 separate channels, and each channel has six tissue compartments. Primary rat liver tissues were cultured over 8 days, and multiple tumor tissues (HCT116) were exposed to various concentrations of 5-fluorouracil for platform characterization.
Assuntos
Técnicas de Cultura de Células , Dispositivos Lab-On-A-Chip/estatística & dados numéricos , Fígado/citologia , Microfluídica/métodos , Esferoides Celulares/citologia , Animais , Fluoruracila/farmacologia , Células HCT116 , Humanos , Fígado/efeitos dos fármacos , Cultura Primária de Células , Ratos , Esferoides Celulares/efeitos dos fármacosRESUMO
BACKGROUND: The liver has a crucial role in metabolic homeostasis as well as being the principal detoxification centre of the body, removing xenobiotics and waste products which could potentially include some nanomaterials (NM). With the ever increasing public and occupational exposure associated with accumulative production of nanomaterials, there is an urgent need to consider the possibility of detrimental health consequences of engineered NM exposure. It has been shown that exposure via inhalation, intratracheal instillation or ingestion can result in NM translocation to the liver. Traditional in vitro or ex vivo hepatic nanotoxicology models are often limiting and/or troublesome (i.e. reduced metabolism enzymes, lacking important cell populations, unstable with very high variability, etc.). METHODS: In order to rectify these issues and for the very first time we have utilised a 3D human liver microtissue model to investigate the toxicological effects associated with a single or multiple exposure of a panel of engineered NMs (Ag, ZnO, MWCNT and a positively charged TiO2). RESULTS: Here we demonstrate that the repeated exposure of the NMs is more damaging to the liver tissue as in comparison to a single exposure with the adverse effects more significant following treatment with the Ag and ZnO as compared with the TiO2 and MWCNT NMs (in terms of cytotoxicity, cytokine secretion, lipid peroxidation and genotoxicity). CONCLUSIONS: Overall, this study demonstrates that the human microtissue model utilised herein is an excellent candidate for replacement of traditional in vitro single cell hepatic models and further progression of liver nanotoxicology.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Dano ao DNA , Fígado/efeitos dos fármacos , Nanoestruturas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Albumina Sérica/biossíntese , Testes de Toxicidade Aguda/métodos , Células Cultivadas , Doença Hepática Induzida por Substâncias e Drogas/imunologia , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Técnicas de Cocultura , Citocinas/agonistas , Citocinas/metabolismo , Hepatócitos/citologia , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Nanotubos de Carbono/química , Nanotubos de Carbono/toxicidade , Nanotubos de Carbono/ultraestrutura , Albumina Sérica Humana , Prata/química , Prata/toxicidade , Células Estromais/citologia , Titânio/química , Titânio/toxicidade , Óxido de Zinco/química , Óxido de Zinco/toxicidadeRESUMO
Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Therapy today includes surgical removal of the tumour and neoadjuvant and adjuvant chemotherapy. The 5-year survival rates for patients with localised disease are between 50 and 70%, but in patients with metastases the prognosis remains poor (â¼ 20%). The aim of this study was the development of a biological relevant OS 3D microtissue model, which is suitable for drug development. Microtissues were formed by the hanging drop method with the established OS cell lines SaOS-2, HOS and MG-63, as well as with cells derived from osteoblastic and chondroblastic OS patient material. Histological characterisation of the microtissues with H/E- and Ki-67-(proliferation), as well as apoptosis staining (TUNEL) revealed the inherent histological heterogeneity of OS. Microtissues from SaOS-2 and HOS cell lines were exposed to doxorubicin, cisplatin, taurolidine, pemetrexed and taxol and the viability was assessed by the CellTiter-GLO(®) Luminescent Cell Viability Assay. The obtained IC50-values for 3D cultures were all higher (1.7 to >16,000-fold) when compared to corresponding cells grown in 2D monolayer culture, except for pemetrexed that was inactive in 2D and 3D cultures. Doxorubicin did not affect the viability of chondroblastic monolayer cultures whereas on 3D microtissues an IC50-value of 2.3 µM was obtained. The 3D microtissues reflect the tissue heterogeneity of OS and are potential suitable tools for drug development towards personalised medicine.