TABLE 1.

Organoid-based toxicity assessment.

Organoid type	Organoid sources	Fabrication methods	Characteristics	Test substances	Toxic endpoints	References
Liver organoid	ESCs	Differentiation of ESCs into foregut endoderm progenitor populations that are further maturated into hepatic progenitor cells by the WNT signaling pathway, then cultured under 3D suspension culture conditions	By selecting the size of the organoids, it is possible to produce a more complex structure, including cholangiocytes and stromal cells, in case of large organoids	Acetaminophen	Viability (WST-1)	Sgodda et al. (2017)
	Primary human hepatocytes, hepatic stellate cells, and Kupffer cells	Produced with a mixture of 80% hepatocytes, 10% hepatic stellate cells, and 10% Kupffer cells and seeded onto non-adherent, round-bottomed plates	Composed of a representative population of cells in vivo, capable of maintaining high viability over a long period of time, and is metabolically active, able to respond to and metabolize drugs accurately	Environmental toxins (glyphosate, lead, mercury, thallium)	Viability (ATP and live cell staining)	Forsythe et al. (2018)
	Human liver tissue	Isolation of duct cells from human liver tissue by chopping and enzyme digestion, and embedding of the isolated duct cells in Matrigel followed by the addition of differentiation factors	Shows the characteristics of hepatocytes rather than HepG2 cells such as positive nuclear staining of HNF4A, clear positive glycogen accumulation, and a more stable albumin secretion	PL-inducing drugs (amikacin, amiodarone, sertraline, acetaminophen)	Viability (ATP), morphological changes, albumin secretion, expression of genes related to phospholipidosis	Lee et al. (2020)
	iPSCs	Differentiation of iPSCs into foregut, dissociation into single cells, and culture in a specialized medium to enhance organoid formation, followed by harvesting and resuspension in Matrigel on the Ultra-Low Attachment Multiwell Plate	Exhibits properties that include bile transport functions, allows assessment of susceptibility based on polymorphism at organoid resolution, and is amenable to high-throughput toxicity screening	238 marketed drugs including 206 DILI compounds and 32 non-DILI compounds	Biliary excretion capacity, cell viability (ATP), and mitochondrial membrane potential	Shinozawa et al. (2021)
Cardiac organoid	iPSCs and fibroblasts	Cell suspension consisted of 90% iPSC-derived CMs; 10% cardiac fibroblasts were suspended in a hydrogel, seeded in a specialized culture platform microwell (Biowire™ II), and subjected to a 10-weeks electrical field stimulation to promote maturation	Shows positive force frequency and post-rest potentiation and improved sarcomeric organization and is enriched for gene expression patterns of the corresponding adult human heart cardiac regions, enables polygenic cardiac disease modeling	6 cardioactive drugs (Zhao et al.), 12 cardioactive drugs (Feric et al.) and 8 reported positive inotropes (Qu et al.)	Contractility, Calcium transients, and gene expression related to cardiotoxicity pathway	Zhao et al. (2019), Feric et al. (2019),Qu et al. (2020)
	ESCs and fibroblasts	Cell suspension consisted of 90% ESC-derived CMs, and 10% dermal fibroblasts were re-suspended in aqueous scaffold materials (Matrigel and collagen I solution) in a dedicated bioreactor, followed by gelation and self-assembly during incubation to fabricate a miniature cardiac organoid that mimics a human ventricle	Shows organized sarcomeres with myofibrillar microstructures, upregulation of key genes related to calcium-handling, ion channel, and cardiac-specific proteins and allows direct measurement of stroke volume, EF, cardiac output, pressure volume loop, and so on	6 cardioactive drugs (Li et al.) and 25 cardioactive drugs (Keung et al.)	Contractility	Li et al., 2018, Keung et al., 2019
Cardiac organoid	iPSCs, hCMECs, and hCFs	Cell suspensions of iPSC-derived CMs, hCMECs, and hCFs in a ratio of 4:2:1 were seeded in ultra-low attachment spheroid microplates	Exhibits typical morphological and cellular compositional characteristics similar to those present in myocardium and are amenable to high-throughput toxicity screening	15 FDA approved structural cardiotoxins and 14 FDA approved nonstructural cardiotoxins	Viability (ATP), endoplasmic reticulum integrity, and mitochondrial membrane potential	Archer et al. (2018)
	iPSCs-CMs, hCFs, HUVECs, and hADSCs	A cell suspension composed of 50% iPSC-derived CMs and 50% non-myocytes (at a 4:2:1 ratio of FBs: HUVECs: hADSCs) was seeded into agarose hydrogel molds and submerged with culture medium, followed by incubation under hypoxic conditions and treatment with noradrenaline	Mimics the characteristics of myocardial infarction, including pathological metabolic changes, fibrosis and altered calcium handling, at transcriptomic, structural, and functional levels	doxorubicin	Contractility and apoptosis (TUNEL staining)	Richards et al. (2020)
Kidney organoid	ESCs and iPSCs	Differentiation of 3D cultured PSC-derived cavitated spheroids into segmented, nephron-like kidney organoids by GSK3b inhibition to result in cell population with proximal tubular, podocyte, and endothelial characteristics	3D culture system that reconstitutes functional, structured epithelia modelling the epiblast, kidney tubular cells, and podocyte-like cells	cisplatin and gentamicin	Expression of kidney injury biomarker (Kim-1)	Freedman et al. (2015)
	ESCs and iPSCs	PSC-derived intermediate mesoderm was treated with GSK-3b inhibitor and FGF9 to differentiate into nephron progenitor cells of metanephric mesenchyme and to further induce pretubular aggregates and renal vesicles	Contains epithelial nephron-like structures expressing markers of podocytes, proximal tubules, loops of Henle, and distal tubules in an organized, continuous arrangement	gentamicin and cisplatin	Expression of kidney injury biomarker (Kim-1)	Morizane et al. (2015)
	iPSCs	Monolayer culture of PSC-derived posterior primitive streak to induce intermediate mesoderm, followed by a 3D culture to promote self-organizing nephrogenic events leading to organoid formation	Contains nephrons associated with a collecting duct network surrounded by renal interstitium and endothelial cells	cisplatin	Apoptosis (caspase-3)	Takasato et al. (2015)
	Renal fibroblasts, huvec, and RPTECs	Renal fibroblasts and huvec were combined in a 50:50 ratio and seeded into a microwell plate using a 3D bioprinter, followed by the addition of RPTECs	Shows formation of extensive microvascular networks and tight junctions and expression of renal uptake and efflux transporters	cisplatin	Viability (LDH release) and expression of genes related to fibrosis (TGF beta)	King et al., 2017
Intestinal organoid	Ileal small intestinal tissue	Digested human primary ileal tissue, isolated intestinal crypts by passing the minced tissue sections through filter mesh, and suspended in Matrigel and cultured in a specialized medium	Enteroids collected with a mean diameter of 600 μm indicate the presence of Paneth cells, enteroendocrine cells, goblet cells, and enterocytes	31 marketed diarrheagenic and non-diarrheagenic drugs	Viability (ATP)	Belair et al. (2020)
	fibroblasts and primary small intestinal epithelial cells	Generated by seeding small intestinal epithelial cells on a supportive layer of fibroblasts under air-liquid interface conditions in a transwell format	Composed of a monolayer of simple columnar epithelial cells with basally positioned nuclei, consistent with enterocytes	39 marketed drugs with different diarrhea-genic risk	Barrier function and Cell proliferation (MTT assay)	Peters et al. (2019)
Brain organoid	iPSCs	Cultured embryoid bodies generated from iPSC were embedded in Matrigel and seeded onto plates in organoid differentiation media to form cerebral organoids and then transferred to a spinner platform for long-term culture	Exhibits similar developmental. Patterns and contains multiple types of brain cells (NSCs, neurons, and astrocytes), and distinct multi-layered, cortical-like neuronal zone and choroid plexus	Ethanol	Caspase 3 activity and functional and morphological alterations of mitochondria	Arzua et al. (2020)
	ESCs	ESC-derived cerebral organoids generated by adding dual SMAD-signaling inhibitors to neural induction medium to promote neuroepithelial expansion and recapitulate fetal cerebral development in vitro	Displays the features of brain development, including the expression of neural stem cells, neurons, and astrocyte markers, indicating cerebral organoid stages up to early fetal stage	acrylamide	Apoptosis, expression of genes related to the NRF2 pathway, and tau phosphorylation	Bu et al. (2020)
	iPSCs	Embryoid bodies differentiated from iPSC were cultured in a selected growth factors and ECM culture conditions to generate neuroectoderm, followed by development into neural epithelial tissue, then plated and incubated on an orbital shaker to form cerebral organoids	Exhibits complex morphology and neuron-specific cell differentiation and expresses marker genes related to forebrain, hippocampus, hindbrain, and prefrontal cortex	vincristine	Caspase 3 activity	Liu et al. (2019)
	iPSCs	Neural progenitor cells differentiated from iPSC were differentiated into brainspheres by incubating in a dedicated differentiation medium under constant gyratory shaking for up to 8 weeks	Expresses mature neuronal markers (NF200, βIII-tubulin and MAP2), astrocytic marker GFAP, and oligodendrocyte marker O1, indicating that diverse neuronal and glial cell populations are present	rotenone	Reactive oxygen species (ROS) and mitochondrial function	Pamies et al. (2018)