2024 Volume 49 Issue 10 Pages 425-434
The application of organoids derived from animal tissues and human-induced pluripotent stem cells to safety assessments of environmental chemicals has been introduced over the last decade. One of the objectives of this approach is to develop an alternative method for animal toxicological studies, while another is to focus on the local reactions of chemicals in each organ/tissue. One of the most important goals is bridging the toxicological properties of chemicals between animals and humans, which may be compared on a level playing field using healthy organoids derived from both animals and humans in vitro, excluding species difference in the absorption, distribution, metabolism, and excretion properties of chemicals in vivo. An overview of the application of organoid systems to safety assessments of environmental chemicals, including general toxicology, developmental toxicology, carcinogenicity, and mutagenicity, was provided herein, and bridging strategies using both animal and human organoids are proposed as a future perspective.
Several 2-dimensionally (2D) cultured cell systems, such as the Chinese hamster ovary (CHO) cell line, Chinese hamster lung/Ishidate Uta (CHL/IU) cell line, the human lymphoblast line TK6, Chinese hamster embryo (CHE) cell strains, the mouse fibroblast cell line BALB/c-3T3, and immortalized non-cancerous cell lines infected with a SV40/adenovirus, have been extensively applied to in vitro toxicological research. The CHO, CHL/IU, and TK6 cell lines are widely used for in vitro mammalian cell micronucleus/chromosome aberration tests (OECD, 1997, 2023). CHE cells and the BALB/c-3T3 cell line are used in cell transformation assays (Mascolo et al., 2010; OECD, 2015), while SV40-infected immortalized cells are applied to the mechanistic analysis of chemical toxicities (Bae et al., 2001; He et al., 2011). These cell lines/systems generally consist of uniform and immortalized cells, and bridging the gap between in vivo and in vitro systems for the extrapolation of risk assessments of chemicals to humans is challenging; however, the utilization of artifactual immortalized cells has been limited to mechanistic and hazardous evaluations and has yet to be expanded to human health risk assessments.
Sato et al. introduced a three-dimensional (3D) culture method using combined growth factors and laminin-rich Matrigel to support epithelial growth for murine intestinal epithelia in 2009, and crypt-like structures called organoids were shown to consist not only of tissue stem cells, but also various types of tissue-specific differentiated cells (Sato et al., 2009). Since then, organoid culturing from various organs/tissues, such as the lungs (Naruse et al., 2020; Rabata et al., 2020; Sachs et al., 2019), liver (Broutier et al., 2016; Huch et al., 2015), and colon (Sato et al., 2011a; Sato et al., 2011b), of animals and humans has been performed using organ-/tissue-specific media. A procedure to culture organoids from organs/tissues is shown in Fig. 1. Briefly, organs/tissues excised from healthy mice are physically minced with scissors, followed by enzymatic dissociation, and are then typically resuspended in liquid Matrigel to form a dome-like structure on a culture dish (Sato et al., 2009). As another method, dissociated cells are seeded with optimized medium onto a thin layer of Matrigel and incubated at 37°C overnight. On the next day, the supernatant including dead cells is removed, and viable cells attached to Matrigel are covered with additional Matrigel and overlaid with media to maintain the 3D culture (the Matrigel Bilayer Organoid Culture method) (Maru et al., 2019; Ishigamori et al., 2022). This organoid-culturing method, in contrast to the conventional Matrigel dome method (Jiang et al., 2022), is considered to be suitable for achieving a significantly high infection efficiency of virus vectors (Maru et al., 2019) as well as for the direct and secure exposure of chemicals. Passaging is conducted every 5-10 days, and organoids may be maintained throughout >10 passages. The development of these organoid culturing technologies has enabled us to utilize them in basic biology and medical science research, including tissue engineering and drug development.
A procedure to culture organoids from organs/tissues. EGF, epidermal growth factor.
Organoid culturing from induced pluripotent stem cells (iPSCs) has been developed based on developmental biology independently from the methods described for organs/tissues. Not only epithelial tissue-derived organoids, but also non-epithelial tissues, such as cardiac and brain organoids, may be cultured from iPSCs (Sloan et al., 2018; Ergir et al., 2022). The heart has been considered to have limited capacity for self-repair and regeneration; however, Beltrami et al. (2003) found that the adult heart contains a heterogenous c-kit-positive stem cell population that may be grown in differentiation media for endothelial, smooth muscle, and cardiomyocyte lineages (Scalise et al., 2019; Scalise et al., 2021). A three-dimensional culturing technology in which cardiac microtissues are generated under scaffold-free conditions has been introduced, and they contain multiple cell types, e.g., epicardial cells, fibroblasts, several types of cardiomyocytes, and endocardial cells, which beat without external stimuli for more than 100 days (Ergir et al., 2022). Brain organoids are typically generated through the addition of region-specific extrinsic signaling molecules to culture media so that they contain structures resembling a specific brain region, and are referred to as ‘forebrain organoids’ or ‘midbrain organoids’, otherwise they may partly differentiate into non-ectoderm tissues (Di Lullo and Kriegstein, 2017). The generation of region-specific brain organoids resembling the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, midbrain, and cerebellum was recently reported (Kelley and Pașca, 2022). Organoids with an epithelial cell origin, such as pulmonary organoids (Yamamoto et al., 2017), hepatic organoids (Thompson and Takebe, 2020), and colon organoids (Crespo et al., 2017), are also generated from iPSCs. The application of iPSCs to the establishment of human healthy organoids enables us to eliminate the need for surgical specimens. Furthermore, iPSC-derived organoids may be suitable for application to developmental toxicology research because they recapitulate the developmental process of each organ/tissue in their stepwise differentiation protocols.
Many researchers have indicated the potential of human iPSC-derived organoids as a tool for disease modeling and drug discovery because iPSCs are derived from patients and healthy individuals to model the cellular phenotypes involved in disease progression (Vandana et al., 2023). The protocols for culturing iPSC-derived organoids involve the organ-specific developmental induction of 3D aggregates of iPSCs, followed by cultures in the presence of various small molecules to derive a specific cell fate, and they are based on the accumulated knowledge of developmental biology. The advantages of iPSC-derived organoids are their wide applicability to not only epithelial organs, but also those of mesenchymal and nervous origins, and the complete differentiation of stem cells to component cells in each organ, e.g., alveolar type 2 and type 1 cells in lung organoids (Ohnishi et al., 2024) and astrocytes and oligodendrocytes in addition to neurons in brain organoids (Di Lullo and Kriegstein, 2017). In contrast, completely differentiated cells lose their proliferative activities, resulting in incapability of passaging. On the other hand, the protocols for culturing tissue-derived organoids, which allow for robust and long-term cultures of primary epithelial cells, involve stem cell maintenance and cell proliferation/organ structural expansion processes using a combination of growth factors in culture media and small molecular inhibitors of kinases for long-term cultures in laminin-rich Matrigel to support epithelial growth (Sato et al., 2011a). In contrast, a number of technical difficulties are associated with obtaining completely differentiated cells in tissue-derived organoids; e.g., several types of stromal support cells were required to generate alveolar type 2 and type 1 cells in lung organoids (Barkauskas et al., 2017), and a long-term three-dimensional liver expansion system that mirrors the in vivo oval cell response was generated; however, these cells failed to express the markers of mature hepatocytes (Huch et al., 2015). Technological advances have been achieved in the induction of differentiation in tissue-derived organoids; e.g., fibroblast growth factor (FGF) ligands play distinct role in differentiation into alveolar type 2 and type 1 cells in lung organoids (Rabata et al., 2020), and hepatocyte organoids have been established using various small molecules and biologicals, including Wnt agonists, such as R-spondin1 and CHIR99021, epidermal growth factor, hepatocyte growth factor, FGF7, FGF10, and the TGF-β inhibitor A83-01 (Hu et al., 2018). One of the most important characteristics of both organoid types is the presence of not only pluripotent/tissue stem cells, but also different types of differentiated cells to varying degrees. Based on this background, organoids are defined as a three-dimensional structure derived from (pluripotent) stem cells, progenitor, and/or differentiated cells that self-organize through cell-cell and cell-matrix interactions to recapitulate aspects of the native tissue architecture and function in vitro (Marsee et al., 2021). Therefore, organoids derived from human iPSCs as well as those from animal/human tissues need to be applied to safety assessments of environmental radiation/chemicals, which have been evaluated in traditional test protocols using healthy animals/volunteers, particularly for evaluations of systemic effects and/or the identification of target organs/tissues. An overview of the application of iPSC-derived and animal/human tissue-derived organoids to toxicological evaluations targeting general (organ) toxicity, developmental toxicity, carcinogenicity, and genotoxicity is hereafter provided. The former two appear to mainly be conducted using human iPSC-derived organoids, while the latter two use animal tissue-derived organoids for the following reasons: (i) To detect various organ-specific as well as differentiated cell-specific toxicities in general in vitro toxicity evaluations, iPSC-derived organoids containing various types of completely differentiated cells in each organ are selected. (ii) Developmental toxicities are accurately characterized by the application of iPSC-derived organoids through an exposure to test chemicals in the developing stages from pluripotent cells through to organogenesis. (iii) To confirm the carcinogenicity of chemicals, repeated exposure to test substances is required, and physiological cell proliferation is needed for the accumulation of genetic and epigenetic alterations; therefore, tissue-derived organoids that allow for long-term maintenance through constant passages are suitable for evaluations of carcinogenicity. (iv) The metabolic activation of test chemicals in parallel with active cell proliferation is critical in genotoxicity studies, and tissue-derived organoids moderately harboring these characteristics are suitable for genotoxic evaluations of test chemicals.
General (organ) toxicityMatsui and Shinozawa comprehensively described drug-induced organ toxicities in the organoids of five areas or organs (the liver, heart, kidneys, gastrointestinal tract, and brain) (Shinozawa et al., 2021). 2D-cultured human primary hepatocytes, which express cytochrome P450 enzymes (CYPs) just after their establishment, did not proliferate in vitro, rapidly underwent dedifferentiation, and lost their enzyme activity (Elaut et al., 2006). In contrast, 3D-hepatic organoids established from the bile duct epithelia of liver biopsy samples expanded and differentiated to hepatocytes (Huch et al., 2015), and differentiated hepatic organoids were induced in liver maturation media from stably expandable foregut cells, which were generated from iPSCs, and may be cryopreserved over long periods (Shinozawa et al., 2021). In in vitro toxicological evaluations using hepatic organoids, acetaminophen reduced cell viability, indicating its advanced metabolic activation by CYPs (Sgodda et al., 2017); amine-containing cationic amphiphilic drugs, e.g., amiodarone, induced phospholipidosis in hepatic organoids (Lee et al., 2020). A high-throughput live imaging analysis using liver organoids was also introduced; 238 pharmaceuticals were evaluated for the induction of drug-induced liver injury by measuring cell viability and the transport properties of a fluorescent bile acid as a marker of cholestasis, demonstrating the ability of this system to harbor functional bile canaliculi-like structures, an essential component for modeling defective bile excretion (Shinozawa et al., 2021). A multi-omics data acquisition system using liver organoids was more recently reported, in which a biochemical analysis of albumin production, CYP expression, the release of ALT/AST, and microscopy-based morphological profiling as well as single-cell transcriptomics were conducted, revealing the morphological/molecular characteristics of steatosis and mitochondrial perturbation, in addition to biochemical changes induced by known hepatotoxic drugs, e.g., a combination of tenofovir and inarigivir (Zhang et al., 2023).
Toxicological evaluations using cardiac organoids were also described in the above review (Shinozawa et al., 2021). An in vitro system that allows for the direct measurement of cardiac performance as a pump by fabricating a 3D electromechanically coupled human ventricle-like cardiac organoid chamber was introduced (Li et al., 2018; Keung et al., 2019). On the other hand, cardiac organoids, which are frequently called cardiac microtissues, have self-organized structures that accurately retain the biological characteristics and functions of heart tissue because they contain the main cardiac cell types, such as cardiomyocytes, cardiac fibroblasts, and/or endothelial cells (Archer et al., 2018). Cardiac fibroblasts and endothelial cells contribute to contractile performance and electrical conduction (Saini et al., 2015; Brutsaert, 2003), suggesting their involvement in the development of and/or responses to structural cardiotoxicity. Nguyen et al. cultured human cardiac microtissues derived from human iPSC into cardiomyocytes and cardiac fibroblasts, conducted a proteomic analysis after treatment with anticancer anthracyclines, including doxorubicin, idarubicin, and epirubicin, and concluded that anthracycline toxic mechanisms may involve mitochondrial functions and the NF-ĸB signaling pathway (Nguyen et al., 2021). In other studies, doxorubicin was shown to induce the characteristics of cardiotoxic damage, including cell apoptosis, inflammation, fibrosis, and mitochondrial damage, as well as functional/structural changes, such as pathological metabolic shifts and fibrosis recapitulating hallmarks of myocardial infarction in cardiac organoids (Richards et al., 2020; Chen et al., 2023).
In the application of liver and cardiac organoids, biochemical parameters, such as organ-specific deviation enzymes, which are also used in in vivo studies, as well as proteomic and transcriptomics are evaluated for the detection of general (organ) toxicity. Other organ biomarkers, including kidney injury molecule-1 in kidney organoid cases (Morizane et al., 2015), have also been reported. In addition, organoid systems, in which diverse cells self-organize into microtissues with in vivo-like architectures, enable physiological function tests measuring cellular transport activity in liver cases, electrophysical tests for cardiac contraction activity, and the evaluation of transepithelial electrical resistance in 3D gastrointestinal microtissues as a marker for gut barrier function (Peters et al., 2019) (Fig. 2). The application of these physiological parameters in toxicology studies using organoid systems is considered to be more advantageous than 2D-cultured cell systems.
Advantages and toxic markers in safety assessments of chemicals using iPSC-derived/tissue-derived organoids. iPSC, induced pluripotent stem cell.
Caporale et al. demonstrated that organoids have two key advantages that warrant their comparison with fetal progenitors. Organoids may be derived from iPSCs, which are highly standardized, self-renewing sources, and the application of organoids to developmental toxicity research overcomes the inherent limitations, both technical and ethical, of fetal progenitor procurement. Furthermore, particularly in brain organoid cases, organoids allow exposure in developing stages from pluripotency through to an intermediate of corticogenesis, a stage that features a relevant representation of progenitor cells, early neurons, and developmental trajectories that match at the transcriptomic level (Caporale et al., 2022). A mixture of common endocrine-disrupting chemicals induced abnormalities in the transcriptome patterns of exposed organoids at estimated concentration levels equivalent to what humans are the exposed to in the environment (Caporale et al., 2022). Li et al. summarized advances in the use of human iPSC-derived organoids for developmental toxicity and teratogenicity assessments of environmental chemicals, including heavy metals, persistent organic pollutants, nanomaterials, and ambient air pollutants (Li et al., 2022). With better understanding of the early stages of embryonic development, such as the blastula, gastrula, and neurula stages, recent studies in emerging fields have examined the use of stem cell cultures to generate a new type of organized embryo-like structure, called embryoids (Fu et al., 2021) (Fig. 2).
CarcinogenicityThe endpoint of traditional in vivo carcinogenicity studies on chemicals is a histopathological evaluation of organs/tissues (OECD, 2018). Genomic and/or proteomic analyses of tumor tissues enable tumor-specific genomic mutations/epigenomic alterations and protein modifications, such as the phosphorylation of kinases, to be detected. Organoids treated with factors that induce carcinogenesis have been introduced; for example, H. pylori infection in gastric organoids resulted in the phosphorylation of c-Met receptors or the activation of beta-catenin signaling (Zhang et al., 2022). However, parameters at the molecular level harbor limitations that cannot reflect the biological characteristics of tumors, such as their autonomous growth and invasiveness. In the study by Naruse et al., mouse lung, liver, and mammary tissue-derived organoids were exposed to several genotoxic carcinogens in vitro during their passages, and were then subcutaneously injected into nude mice. As the endpoint of this experiment, the carcinogenic properties of carcinogens were evaluated as the tumorigenicity or histopathological characteristics of tumors, such as multilayered epithelia with or without invasiveness and the expression of the tumor-specific phosphorylation of kinases (Naruse et al., 2020). Wang et al. recently proposed an organoid-based cell transformation assay for mouse intestinal carcinogenicity screening in which the concentration of each chemical for anchorage-independent growth was evaluated (Wang et al., 2024), and the development of a protocol in which the carcinogenicity of chemicals may be evaluated in vitro is expected for other organs (Fig. 2).
GenotoxicityResearch on mutagenesis has recently focused on analyses of the genome-scale mutational signatures of carcinogens using organoid systems. Experimental mutational signatures may explain etiological links to patterns that are found in human who have had the same exposures (Melki et al., 2020). The rationale for applying organoids to genotoxicity studies is similarities in the metabolism of chemicals to that in in vivo systems. Komiya et al. reported that acrylamide exhibited mutagenic activity in mouse lung organoids, but did not induce mutagenesis in other in vitro test systems, indicating the retention of the key metabolic activation enzyme CYP2E1 in organoids, but not in other in vitro mammalian cell systems (Komiya et al., 2021). Furthermore, based on CYP1A1, CYP1A2, CYP3A4, and NQO1 expression levels, Caipa Garcia et al. demonstrated that the metabolic activities of dietary carcinogens, e.g., aflatoxin B1, aristolochic acid I, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, in human liver, kidney, or colon organoids varied between tissues, reflecting the DNA adduct and tumor formation specificities of each carcinogen (Caipa Garcia et al., 2024). These metabolic characteristics of organoids, particularly those derived from humans, are one of the most distinct advantages for their application to not only genotoxicity studies, but all fields of safety assessments of environmental chemicals (Fig. 2).
In safety assessments of developing industrial chemicals, including pharmaceuticals, improvements in labor productivity, time saving, and accuracy of safety profiles to human health are important issues. The high-throughput screening of the safety of chemicals using an organoid system represents a promising tool for developing industrial chemicals; however, the prediction of toxicological target organs/tissues is needed before the utilization of efficient high-throughput in vitro systems. Therefore, information on the toxicokinetics and/or target organs of each chemical needs to be obtained before the application of organoid systems to safety assessments of chemicals. Chemical structure-based pharmacological or pharmacokinetic predictions may be helpful for the identification of candidate target organs/tissues before the exposure of chemicals to animals or humans (Fig. 3).
Bridging strategies using animal and human organoids in safety assessments of environmental chemicals. iPSC, induced pluripotent stem cell; GLP, good laboratory practice.
In safety assessments of environmental pollutants, the collection of epidemiological data on damage to the health of animals and/or humans is important. Aflatoxicosis was first found in the early 1960s in England where it killed 100,000 turkey poults, and the major target organ was thereafter identified as the liver in various animal species and humans (Newberne and Butler, 1969). Measurements of the levels of hemoglobin adducts from acrylamide are used for its exposure assessment. Acrylamide has been shown to cause tumors in various organs/tissues, e.g., thyroid follicles, the lungs, testicular tunica mesothelium, mammary glands, and skin mesenchymal tissue; furthermore, it is associated with neurotoxicity that may inhibit the development and reproduction in animals and/or humans, with levels ranging widely in non-occupationally exposed individuals and non-smokers, indicating that dietary exposure to acrylamide markedly varies (Pedersen et al., 2022). The origin of the incidental discovery of acrylamide exposure from processed food was severe neurotoxic symptoms in cows and fish death in a fish farm due to the leakage of water containing incomplete polymerized acrylamide from a tunnel construction site in September 1997 in southern Sweden (Hagmar et al., 2001). The most important goal of safety assessments of chemicals is human health; however, these findings had been obtained using not only laboratory animals, but also domestic and wild animals to identify target organs, leading to early risk management for human health. An advantage of the application of organoids to safety assessments of environmental chemicals is that organoids may be established not only from laboratory animals, but also other species, including dogs, horses, and chickens (Elbadawy et al., 2022; Thompson et al., 2022; Wang et al., 2022).
One of the objectives of the application of animal- and human-derived organoids to safety assessments of environmental chemicals is the development of an alternative method for animal toxicological studies that achieves the 3Rs (Replacement, Reduction, and Refinement of animals used in research) and the high-throughput assessment of chemicals. Another aim is to focus on the local reactions of chemicals in each organ/tissue for mechanistic/molecular-based analyses. Additionally, one of the most important goals of this approach is bridging the toxicological properties of chemicals between animals and humans, which will allow comparisons on a level playing field using healthy organoids derived from both animals and humans in vitro, excluding species differences in the absorption, distribution, metabolism, and excretion properties of chemicals in vivo. Regarding mechanistic/molecular-based analyses as well as screening for toxic biomarkers of chemicals, transcriptome and/or proteome data in organoids have recently demonstrated the signaling pathways related to each toxic reaction in general and developmental toxicities, as described above. The mechanisms underlying target organ specificities are expected to be elucidated by comparisons of carcinogenicity and genotoxicity in a number of organ-derived organoids. Comparisons of toxic properties in animals and humans may reveal differences in the cytotoxic concentrations of chemicals, as well as the expression of the same biomarkers, which are screened with proteomics and/or transcriptomics using animal-derived organoids followed by evaluations of species differences using human-derived organoids (Fig. 3). The toxic concentration of each chemical and toxic findings, including the expression properties of biomarkers, are expected to be similar or different between animal and human organoids. Therefore, animal and human organoid systems are applicable to risk assessments of chemicals, referring to blood/tissue concentration data in each species, in addition to hazard evaluations for human health. Findings in organoid systems may be confirmed in vivo at the individual level in animal models and in humans, and new information on target organs may be applied to organoid experiments.
Experience and knowledge of toxicological reactions caused by environmental chemicals in organoid systems have steadily accumulated over the last decade. Organoids may be established from both animals and humans, and the importance of bridging the toxicologic properties of chemicals between animals and humans was reconfirmed in this review. We have accumulated large amounts of in vivo toxicological data over the last 50 years, and new technologies, such as analyses of genome-scale mutational signatures in experimental systems and human tissues, will contribute further to this field. Organoid research that bridges the gap between animals and humans will continue to be developed and expand.
This study was supported by Research on Risk of Chemical Substances from the Ministry of Health, Labour and Welfare of Japan (22KD1001). We are grateful to Dr. Akihiro Hirata and Masami Komiya for their excellent collaborative work.
Conflict of interestThe authors declare that there is no conflict of interest.
