The Growing Importance of Three-Dimensional Models and Microphysiological Systems in the Assessment of Mycotoxin Toxicity

Current investigations in the field of toxicology mostly rely on 2D cell cultures and animal models. Although well-accepted, the traditional 2D cell-culture approach has evident drawbacks and is distant from the in vivo microenvironment. To overcome these limitations, increasing efforts have been made in the development of alternative models that can better recapitulate the in vivo architecture of tissues and organs. Even though the use of 3D cultures is gaining popularity, there are still open questions on their robustness and standardization. In this review, we discuss the current spheroid culture and organ-on-a-chip techniques as well as the main conceptual and technical considerations for the correct establishment of such models. For each system, the toxicological functional assays are then discussed, highlighting their major advantages, disadvantages, and limitations. Finally, a focus on the applications of 3D cell culture for mycotoxin toxicity assessments is provided. Given the known difficulties in defining the safety ranges of exposure for regulatory agency policies, we are confident that the application of alternative methods may greatly improve the overall risk assessment.

[1]  H. Wolinski,et al.  Dose and route dependent effects of the mycotoxin deoxynivalenol in a 3D gut-on-a-chip model with flow. , 2023, Toxicology in vitro : an international journal published in association with BIBRA.

[2]  E. Cimetta,et al.  A Porous Gelatin Methacrylate-Based Material for 3D Cell-Laden Constructs. , 2022, Macromolecular bioscience.

[3]  L. Griscom,et al.  Three-dimensional in vitro culture models in oncology research , 2022, Cell & bioscience.

[4]  C. Altamirano,et al.  Dynamic Culture of Mesenchymal Stromal/Stem Cell Spheroids and Secretion of Paracrine Factors , 2022, Frontiers in Bioengineering and Biotechnology.

[5]  H. S. Rho,et al.  A guide to the organ-on-a-chip , 2022, Nature Reviews Methods Primers.

[6]  Florian Petry,et al.  Large-Scale Production of Size-Adjusted β-Cell Spheroids in a Fully Controlled Stirred-Tank Reactor , 2022, Processes.

[7]  Jiadi Sun,et al.  Coexposure of Cyclopiazonic Acid with Aflatoxin B1 Involved in Disrupting Amino Acid Metabolism and Redox Homeostasis Causing Synergistic Toxic Effects in Hepatocyte Spheroids. , 2022, Journal of agricultural and food chemistry.

[8]  N. Mei,et al.  Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform. , 2022, ALTEX.

[9]  Pin-Chuan Chen,et al.  Engineering an integrated system with a high pressure polymeric microfluidic chip coupled to liquid chromatography-mass spectrometry (LC-MS) for the analysis of abused drugs , 2022, Sensors and Actuators B: Chemical.

[10]  Kitti Garai,et al.  The individual and combined effects of ochratoxin A with citrinin and their metabolites (ochratoxin B, ochratoxin C, and dihydrocitrinone) on 2D/3D cell cultures, and zebrafish embryo models. , 2021, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[11]  H. F. Carvalho,et al.  A modular, reversible sealing, and reusable microfluidic device for drug screening. , 2021, Analytica chimica acta.

[12]  Tae-Hyung Kim,et al.  Recent Advances in Multicellular Tumor Spheroid Generation for Drug Screening , 2021, Biosensors.

[13]  L. Delort,et al.  3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages , 2021, International Journal of Molecular Sciences.

[14]  E. Cimetta,et al.  Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing. , 2021, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[15]  Jiadi Sun,et al.  Application of triple co-cultured cell spheroid model for exploring hepatotoxicity and metabolic pathway of AFB1. , 2021, The Science of the total environment.

[16]  Aibo Wu,et al.  Fumonisin B1 triggers carcinogenesis via HDAC/PI3K/Akt signalling pathway in human esophageal epithelial cells. , 2021, The Science of the total environment.

[17]  Naresh Kumar,et al.  Cold Atmospheric Plasma Increases Temozolomide Sensitivity of Three-Dimensional Glioblastoma Spheroids via Oxidative Stress-Mediated DNA Damage , 2021, Cancers.

[18]  Matthew R. Lockett,et al.  Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells. , 2021, Toxicology in vitro : an international journal published in association with BIBRA.

[19]  I. Poliaček,et al.  Animal models of cough , 2021, Respiratory Physiology & Neurobiology.

[20]  K. Kim,et al.  Challenges of applying multicellular tumor spheroids in preclinical phase , 2021, Cancer cell international.

[21]  U. Marx,et al.  Demonstration of the first‐pass metabolism in the skin of the hair dye, 4‐amino‐2‐hydroxytoluene, using the Chip2 skin–liver microphysiological model , 2021, Journal of applied toxicology : JAT.

[22]  D. Kouroupis,et al.  Increased Mesenchymal Stem Cell Functionalization in Three-Dimensional Manufacturing Settings for Enhanced Therapeutic Applications , 2021, Frontiers in Bioengineering and Biotechnology.

[23]  Ana C. Henriques,et al.  Three-Dimensional Spheroids as In Vitro Preclinical Models for Cancer Research , 2020, Pharmaceutics.

[24]  Y. Matsuzaki,et al.  A Shaking-Culture Method for Generating Bone Marrow Derived Mesenchymal Stromal/Stem Cell-Spheroids With Enhanced Multipotency in vitro , 2020, Frontiers in Bioengineering and Biotechnology.

[25]  Keisuke Tanaka,et al.  Mycotoxin Deoxynivalenol Has Different Impacts on Intestinal Barrier and Stem Cells by Its Route of Exposure , 2020, Toxins.

[26]  A. Rettie,et al.  Microphysiological System Modeling of Ochratoxin A-Associated Nephrotoxicity. , 2020, Toxicology.

[27]  E. Brey,et al.  Optimization of Co-Culture Conditions for a Human Vascularized Adipose Tissue Model , 2020, Bioengineering.

[28]  S. Doak,et al.  Adaptation of the in vitro micronucleus assay for genotoxicity testing using 3D liver models supporting longer-term exposure durations , 2020, Mutagenesis.

[29]  P. Vulto,et al.  Direct On-Chip Differentiation of Intestinal Tubules from Induced Pluripotent Stem Cells , 2020, International journal of molecular sciences.

[30]  Bas J Blaauboer,et al.  New approach methodologies (NAMs) for human-relevant biokinetics predictions: Meeting the paradigm shift in toxicology towards an animal-free chemical risk assessment. , 2020, ALTEX.

[31]  Shayan S Nazari Generation of 3D Tumor Spheroids with Encapsulating Basement Membranes for Invasion Studies , 2020, Current protocols in cell biology.

[32]  U. Marx,et al.  Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies , 2020, Scientific Reports.

[33]  Yong Teng,et al.  Is It Time to Start Transitioning From 2D to 3D Cell Culture? , 2020, Frontiers in Molecular Biosciences.

[34]  R. Badhan,et al.  A dynamic perfusion based blood-brain barrier model for cytotoxicity testing and drug permeation , 2020, Scientific Reports.

[35]  Kristin M. Fabre,et al.  Drug-induced skin toxicity: gaps in preclinical testing cascade as opportunities for complex in vitro models and assays. , 2020, Lab on a chip.

[36]  Hansoo Park,et al.  Spheroid Culture System Methods and Applications for Mesenchymal Stem Cells , 2019, Cells.

[37]  Rossana E. Madrid,et al.  Microfluidics and hydrogel: A powerful combination , 2019 .

[38]  Abhishek Srivastava,et al.  Reproducing human and cross-species drug toxicities using a Liver-Chip , 2019, Science Translational Medicine.

[39]  F. Boccafoschi,et al.  Overview of natural hydrogels for regenerative medicine applications , 2019, Journal of Materials Science: Materials in Medicine.

[40]  A. Alshareeda,et al.  Scaffold-Free 3-D Cell Sheet Technique Bridges the Gap between 2-D Cell Culture and Animal Models , 2019, International journal of molecular sciences.

[41]  M. Filipič,et al.  Development of in vitro 3D cell model from hepatocellular carcinoma (HepG2) cell line and its application for genotoxicity testing , 2019, Archives of Toxicology.

[42]  P. Lelkes,et al.  An Air Bubble-Isolating Rotating Wall Vessel Bioreactor for Improved Spheroid/Organoid Formation. , 2019, Tissue engineering. Part C, Methods.

[43]  S. Ahadian,et al.  A Human Liver‐on‐a‐Chip Platform for Modeling Nonalcoholic Fatty Liver Disease , 2019, Advanced biosystems.

[44]  Noo Li Jeon,et al.  Engineering tumor vasculature on an injection-molded plastic array 3D culture (IMPACT) platform. , 2019, Lab on a chip.

[45]  L. Buzanska,et al.  Organoids are promising tools for species‐specific in vitro toxicological studies , 2019, Journal of applied toxicology : JAT.

[46]  Hui-chao Yan,et al.  Acute exposure to deoxynivalenol inhibits porcine enteroid activity via suppression of the Wnt/β-catenin pathway. , 2019, Toxicology letters.

[47]  E. Danen,et al.  3D Cell-Based Assays for Drug Screens: Challenges in Imaging, Image Analysis, and High-Content Analysis , 2019, SLAS discovery : advancing life sciences R & D.

[48]  Joseph C. Wu,et al.  Progress, obstacles, and limitations in the use of stem cells in organ-on-a-chip models. , 2019, Advanced drug delivery reviews.

[49]  D. Ingber,et al.  Modelling cancer in microfluidic human organs-on-chips , 2019, Nature Reviews Cancer.

[50]  Jessica K. Chang,et al.  YAP-independent mechanotransduction drives breast cancer progression , 2018, bioRxiv.

[51]  Julia Hoeng,et al.  A lung/liver-on-a-chip platform for acute and chronic toxicity studies. , 2018, Lab on a chip.

[52]  Andreas Fischer,et al.  Human Endothelial Cell Spheroid-based Sprouting Angiogenesis Assay in Collagen. , 2018, Bio-protocol.

[53]  G. Jenkins,et al.  Reprint of: A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies. , 2018, Mutation research. Genetic toxicology and environmental mutagenesis.

[54]  L P Ferreira,et al.  Design of spherically structured 3D in vitro tumor models -Advances and prospects. , 2018, Acta biomaterialia.

[55]  L. Gribaldo,et al.  Human 3D Cultures as Models for Evaluating Magnetic Nanoparticle CNS Cytotoxicity after Short- and Repeated Long-Term Exposure , 2018, International journal of molecular sciences.

[56]  Woojung Shin,et al.  Microfluidic Organ-on-a-Chip Models of Human Intestine , 2018, Cellular and molecular gastroenterology and hepatology.

[57]  E. Kumacheva,et al.  Hydrogel microenvironments for cancer spheroid growth and drug screening , 2018, Science Advances.

[58]  Markus Schulz,et al.  Validation of the 3D Skin Comet assay using full thickness skin models: Transferability and reproducibility. , 2018, Mutation research. Genetic toxicology and environmental mutagenesis.

[59]  Sigrid A. Langhans Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning , 2018, Front. Pharmacol..

[60]  P. Peters,et al.  Humans in a Dish: The Potential of Organoids in Modeling Immunity and Infectious Diseases , 2017, Front. Microbiol..

[61]  T. Neumann,et al.  Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity. , 2017, JCI insight.

[62]  K. Ulgen,et al.  Advances in microfluidic devices made from thermoplastics used in cell biology and analyses. , 2017, Biomicrofluidics.

[63]  R. Hegde,et al.  Modeling tumor cell adaptations to hypoxia in multicellular tumor spheroids , 2017, Journal of experimental & clinical cancer research : CR.

[64]  Raffaella Corvi,et al.  In vitro genotoxicity testing-Can the performance be enhanced? , 2017, Food and chemical toxicology : an international journal published for the British Industrial Biological Research Association.

[65]  Michael J. Cronce,et al.  Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function. , 2017, Lab on a chip.

[66]  S. Ardestani,et al.  Monitoring ZEO apoptotic potential in 2D and 3D cell cultures and associated spectroscopic evidence on mode of interaction with DNA , 2017, Scientific Reports.

[67]  I. Rustenbeck,et al.  A 3D microfluidic perfusion system made from glass for multiparametric analysis of stimulus-secretioncoupling in pancreatic islets , 2017, Biomedical microdevices.

[68]  J. Sung,et al.  Microtechnology-Based Multi-Organ Models , 2017, Bioengineering.

[69]  Ye Fang,et al.  Three-Dimensional Cell Cultures in Drug Discovery and Development , 2017, SLAS discovery : advancing life sciences R & D.

[70]  Terrance J Kavanagh,et al.  Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity. , 2017, Toxicology in vitro : an international journal published in association with BIBRA.

[71]  Alireza Mashaghi,et al.  An end-user perspective on Organ-on-a-Chip : Assays and usability aspects , 2017 .

[72]  E. Leclerc,et al.  Hepatocytes cocultured with Sertoli cells in bioreactor favors Sertoli barrier tightness in rat , 2017, Journal of applied toxicology : JAT.

[73]  Shawn P. Carey,et al.  Three-dimensional collagen matrix induces a mechanosensitive invasive epithelial phenotype , 2017, Scientific Reports.

[74]  Jong Hwan Sung,et al.  A pumpless multi‐organ‐on‐a‐chip (MOC) combined with a pharmacokinetic–pharmacodynamic (PK–PD) model , 2017, Biotechnology and bioengineering.

[75]  David J Hughes,et al.  Three-dimensional perfused human in vitro model of non-alcoholic fatty liver disease , 2017, World journal of gastroenterology.

[76]  Jong Hwan Sung,et al.  Microfluidic Gut-liver chip for reproducing the first pass metabolism , 2017, Biomedical Microdevices.

[77]  Jos Joore,et al.  High-throughput compound evaluation on 3D networks of neurons and glia in a microfluidic platform , 2016, Scientific Reports.

[78]  Minsung Kim,et al.  Monitoring the effects of doxorubicin on 3D-spheroid tumor cells in real-time , 2016, OncoTargets and therapy.

[79]  Albert van den Berg,et al.  Direct quantification of transendothelial electrical resistance in organs-on-chips. , 2016, Biosensors & bioelectronics.

[80]  Stephen J. Evans,et al.  Critical review of the current and future challenges associated with advanced in vitro systems towards the study of nanoparticle (secondary) genotoxicity , 2016, Mutagenesis.

[81]  Stephanie J Hachey,et al.  3D microtumors in vitro supported by perfused vascular networks , 2016, Scientific Reports.

[82]  Uwe Marx,et al.  Biology-inspired microphysiological system approaches to solve the prediction dilemma of substance testing. , 2016, ALTEX.

[83]  Paul Wilmes,et al.  A microfluidics-based in vitro model of the gastrointestinal human–microbe interface , 2016, Nature Communications.

[84]  Qasem Ramadan,et al.  In vitro micro-physiological immune-competent model of the human skin. , 2016, Lab on a chip.

[85]  C. Luo,et al.  Gel integration for microfluidic applications. , 2016, Lab on a chip.

[86]  M. Ingelman-Sundberg,et al.  Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease , 2016, Scientific Reports.

[87]  Catarina Brito,et al.  Adaptable stirred-tank culture strategies for large scale production of multicellular spheroid-based tumor cell models. , 2016, Journal of biotechnology.

[88]  Alessandro Bevilacqua,et al.  3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained , 2016, Scientific Reports.

[89]  S. Fey,et al.  From 2D to 3D--a New Dimension for Modelling the Effect of Natural Products on Human Tissue. , 2015, Current pharmaceutical design.

[90]  Uwe Marx,et al.  The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture , 2015, Journal of visualized experiments : JoVE.

[91]  Holger Weber,et al.  Endothelial cell spheroids as a versatile tool to study angiogenesis in vitro , 2015, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[92]  Zhilong Yu,et al.  A microfluidic live cell assay to study anthrax toxin induced cell lethality assisted by conditioned medium , 2015, Scientific Reports.

[93]  Michael G. Roper,et al.  Microfluidics-to-mass spectrometry: a review of coupling methods and applications. , 2015, Journal of chromatography. A.

[94]  L. weiswald,et al.  Spherical Cancer Models in Tumor Biology1 , 2015, Neoplasia.

[95]  Marissa Nichole Rylander,et al.  Review of collagen I hydrogels for bioengineered tissue microenvironments: characterization of mechanics, structure, and transport. , 2014, Tissue engineering. Part B, Reviews.

[96]  J. Kelm,et al.  3D cell culture systems modeling tumor growth determinants in cancer target discovery. , 2014, Advanced drug delivery reviews.

[97]  F. Bidard,et al.  A three dimensional thermoplastic microfluidic chip for robust cell capture and high resolution imaging. , 2014, Biomicrofluidics.

[98]  R. Tuan,et al.  Functional comparison of human-induced pluripotent stem cell-derived mesenchymal cells and bone marrow-derived mesenchymal stromal cells from the same donor. , 2014, Stem cells and development.

[99]  D. Beebe,et al.  The present and future role of microfluidics in biomedical research , 2014, Nature.

[100]  B. Herpers,et al.  A 3D in vitro model of differentiated HepG2 cell spheroids with improved liver-like properties for repeated dose high-throughput toxicity studies , 2014, Archives of Toxicology.

[101]  Nima Milani-Nejad,et al.  Small and large animal models in cardiac contraction research: advantages and disadvantages. , 2014, Pharmacology & therapeutics.

[102]  T. Bartosh,et al.  Preparation of anti-inflammatory mesenchymal stem/precursor cells (MSCs) through sphere formation using hanging-drop culture technique. , 2014, Current protocols in stem cell biology.

[103]  Mark Ungrin,et al.  Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates , 2013, Journal of visualized experiments : JoVE.

[104]  Ali Khademhosseini,et al.  Hydrogel-coated microfluidic channels for cardiomyocyte culture. , 2013, Lab on a chip.

[105]  K. Ren,et al.  Materials for microfluidic chip fabrication. , 2013, Accounts of chemical research.

[106]  Elmar Heinzle,et al.  3D organotypic cultures of human HepaRG cells: a tool for in vitro toxicity studies. , 2013, Toxicological sciences : an official journal of the Society of Toxicology.

[107]  Jean-Pierre Gillet,et al.  The clinical relevance of cancer cell lines. , 2013, Journal of the National Cancer Institute.

[108]  D. Seliktar Designing Cell-Compatible Hydrogels for Biomedical Applications , 2012, Science.

[109]  S. Takayama,et al.  Quantitative Analysis of Molecular Absorption into PDMS Microfluidic Channels , 2012, Annals of Biomedical Engineering.

[110]  Maria Vinci,et al.  Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation , 2012, BMC Biology.

[111]  J. Miao,et al.  A practical guide for the fabrication of microfluidic devices using glass and silicon. , 2012, Biomicrofluidics.

[112]  Thomas Hartung,et al.  From alternative methods to a new toxicology. , 2011, European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V.

[113]  Shang-Tian Yang,et al.  High-throughput 3-D cell-based proliferation and cytotoxicity assays for drug screening and bioprocess development. , 2011, Journal of biotechnology.

[114]  Nikolay Bazhanov,et al.  Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties , 2010, Proceedings of the National Academy of Sciences.

[115]  Robert Kavlock,et al.  The U.S. Environmental Protection Agency Strategic Plan for Evaluating the Toxicity of Chemicals , 2010, Journal of toxicology and environmental health. Part B, Critical reviews.

[116]  Hanry Yu,et al.  Towards a human-on-chip: culturing multiple cell types on a chip with compartmentalized microenvironments. , 2009, Lab on a chip.

[117]  N. Kotov,et al.  In vitro toxicity testing of nanoparticles in 3D cell culture. , 2009, Small.

[118]  S. Pun,et al.  3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers. , 2008, Bioconjugate chemistry.

[119]  Aaron Sin,et al.  Development of a Microscale Cell Culture Analog To Probe Naphthalene Toxicity , 2008, Biotechnology progress.

[120]  M. Ashraf,et al.  Cytotoxicity of Fumonisin B1 in Spheroid and Monolayer Cultures of Rat Hepatocytes , 2008 .

[121]  D. Beebe,et al.  PDMS absorption of small molecules and consequences in microfluidic applications. , 2006, Lab on a chip.

[122]  Yoshito Ikada,et al.  Challenges in tissue engineering , 2006, Journal of The Royal Society Interface.

[123]  R. G. Allen,et al.  Replicative senescence: a critical review , 2004, Mechanisms of Ageing and Development.

[124]  A. Abbott Cell culture: Biology's new dimension , 2003, Nature.

[125]  Martin Fussenegger,et al.  Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. , 2003, Biotechnology and bioengineering.

[126]  J. Bennett,et al.  Mycotoxins , 2003, Clinical Microbiology Reviews.

[127]  G. Whitesides,et al.  Poly(dimethylsiloxane) as a material for fabricating microfluidic devices. , 2002, Accounts of chemical research.

[128]  Tony F. Chan,et al.  Active contours without edges , 2001, IEEE Trans. Image Process..

[129]  Natasha Loder,et al.  UK researchers call for limits on animal experiment ‘red tape’ , 2000, Nature.

[130]  P. Carmeliet Mechanisms of angiogenesis and arteriogenesis , 2000, Nature Medicine.

[131]  H. Augustin,et al.  Tensional forces in fibrillar extracellular matrices control directional capillary sprouting. , 1999, Journal of cell science.

[132]  L. Kunz-Schughart,et al.  Multicellular tumor spheroids: intermediates between monolayer culture and in vivo tumor , 1999, Cell biology international.

[133]  D. Eaton,et al.  Metabolism and toxicity of aflatoxins M1 and B1 in human-derived in vitro systems. , 1998, Toxicology and applied pharmacology.

[134]  C Haanen,et al.  A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. , 1995, Journal of immunological methods.

[135]  N. Kanopoulos,et al.  Design of an image edge detection filter using the Sobel operator , 1988, IEEE J. Solid State Circuits.

[136]  Regina Luttge,et al.  Electrical monitoring approaches in 3-dimensional cell culture systems: Toward label-free, high spatiotemporal resolution, and high-content data collection in vitro , 2021 .

[137]  Sangeeta Khare,et al.  Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology. , 2015, ALTEX.

[138]  Thomas Hartung,et al.  Food for thought...on alternative methods for chemical safety testing. , 2010, ALTEX.

[139]  Juergen Friedrich,et al.  Spheroid-based drug screen: considerations and practical approach , 2009, Nature Protocols.