Each step in the cell and organoid cultures strictly adheres to aseptic techniques. Culture conditions are maintained at a temperature of 37℃ and in a 5% CO₂ environment. Please refer to Supplementary Table S1 for comprehensive details on each reagent.

Liver organoid generation from hASCs: The isolation of primary hepatocytes typically follows established protocols (16). Following the isolation from patient liver samples, primary hepatocytes are initially washed with advanced Dulbecco’s modified Eagle’s medium/Ham’s F-12 (DMEM/F12) and centrifuged at 300∼400 ×g for 5 minutes to pellet the cells. These cell pellets are then combined with Matrigel^TM (Corning) and seeded at a density of 3,000∼10,000 cells/well in a 48-well plate. Once the cells are seeded, the Matrigel is allowed to solidify, at which point starting media (SM) (18) is added. This SM is a blend of expansion media (20) supplemented with 25 ng/mL Noggin, 10 μM Y27632, and 100 ng/mL Wnt-3a. The composition of the expansion media (20) includes advanced DMEM/F12 supplemented with 1% penicillin-streptomycin (PS), 1x N-2, 1X B-27, 50 ng/mL epidermal growth factor (EGF), 25 ng/mL hepatocyte growth factor (HGF), 5 μM A83-01, 10 μM forskolin, 1.25 mM N-acetyl L-cysteine, 10 mM nicotinamide, 10 nM Leu15-gastrin I human, 10 ng/mL fibroblast growth factor 10 (FGF10), and 1 mg/mL R-spondin 1. The cells are maintained in the SM for three days. On the third day of culture in SM, the medium is replaced with expansion media, and after a further 10∼14 days, the organoids are resuspended in freshly mixed Matrigel. For long-term maintenance, the resuspension can be repeated every 7∼10 days for at least six months, with a dilution ratio ranging from 1：4 to 1：8 as appropriate.

Liver organoid differentiation from hPSCs: hPSCs, including both iPSCs and ESCs, undergo differentiation that mirrors the stages of liver development. Detailed steps for this process are provided in a previous study (8). Liver organoids derived from hPSCs are maintained and cultured using growth medium (GM), which consists of advanced DMEM/F12 supplemented with 1% PS, 1 mM GlutaMax, 1 mM HEPES, 1X N-2, 1X B-27 without vitamin A, 1X ITS, 50 ng/mL EGF, 25 ng/mL HGF, 10 ng/mL FGF-basic, 10 ng/mL Oncostatin M, 5 μM A83-01, 10 μM forskolin, 1 mM N-acetyl L-cysteine, 10 nM Leu15-gastrin I human, 10 mM nicotinamide, and 100 nM dexamethasone. For expansion, organoids cultured in a Matrigel dome and sustained with GM. The GM medium is replenished every three days. Regarding splitting, the organoids are isolated from the Matrigel matrix and can be divided either physically or chemically. Splitting is typically performed weekly, and the splitting ratio is adjusted between 1：4 and 1：10 according to the growth status. For differentiation, the cell medium is replaced with fresh EM a day after splitting. The composition of EM for the PSC-driven organoids differs slightly from the medium used for the ACS-driven organoid protocols. The EM consists of advanced DMEM/F12 supplemented with 1% PS, 1 mM GlutaMax, 1 mM HEPES, 1x N-2, 1X B-27 without vitamin A, 50 ng/mL EGF, 25 ng/mL HGF, 100 ng/mL FGF10, 1 mg/mL R-spondin 1, 25 ng/mL BMP7, 5 μM A83-01, 10 μM forskolin, 1 mM N-acetyl L-cysteine, 10 mM nicotinamide, and 10 nM Leu15-gastrin I human. Subsequently, the organoids are maintained in the EM for three days. Following this, the medium is exchanged with differentiation medium (DM) (8) and the cultures are maintained in DM for six days. Throughout this period, the DM medium is refreshed with fresh DM every two days. Upon completion of the differentiation process, the organoids are characterized and utilized for the subsequent toxicity assessments.

Gene expression: Total RNA is firstly extracted from the sample using RNA extraction reagents (easy-BLUE; iNtRON). The isolated RNA is subsequently converted into complementary DNA through reverse transcription polymerase chain reaction (RT-PCR) using TOPscript^TM RT DryMIX (Enzynomics). Specific primers (Supplementary Table S2) that are designed for the gene-specific markers are utilized to quantify the expression levels through real-time PCR using Fast SYBR^Ⓡ Green Master Mix (Applied Biosystems).

Flow cytometry: The organoids are dissociated into single cells using enzymes such as TrypLE^TM Express Enzyme (Gibco) for 10 minutes at 37℃. Subsequently, the single cells are then fixed, permeabilized, and sequentially blocked according to standard immunostaining protocols. Staining with an albumin (ALB)-specific antibody is then conducted, followed by flow cytometry analysis to assess the composition of ALB-positive cells within the liver organoids.

ALB, α1-antitrypsin, and urea secretion: To quantify the ALB, α1-antitrypsin (AAT), and urea levels, samples of the medium are collected 48-hour post-medium change and analyzed using a Human Albumin enzyme-linked immunosorbent assay (ELISA) Kit (Bethyl Laboratories), Human Alpha-1-Antitrypsin ELISA Quantitation Kit (Genway Biotech), or Urea Assay Kit (Cell Biolabs) according to the manufacturer’s protocols. The absorbance readings are measured using a microplate reader and normalized by the cell count.

Activation of drug-metabolizing enzyme: To assess cytochrome P450 (CYP) activity, the organoids are induced with specific inducers (20 μM rifampicin for CYP3A4) for 48 hours. Following this induction, the CYP enzyme activities are assessed using subtype-specific substrates and a P450-Glo Assay Kit (Promega) in accordance with the manufacturer’s protocols. Luciferase activity can be measured with a luminometer and normalized by the cell count.

Immunostaining: The organoids are initially fixed using 4% paraformaldehyde (Biosesang), followed by permeabilization of the cells using Triton X-100 at 21℃∼23℃, and subsequent blocking. Liver cell-specific antibodies (Supplemenary Table S3) are applied for immunostaining, following the protocol that are used in the flow cytometry for ALB detection. Fluorescent microscopy is used to capture images of the stained organoids.

Periodic acid-Schiff staining: To investigate the storage of glycogen, we performed a series of procedures on the organoids. Frozen sections are cut to a thickness of 10 μm at 20℃ using a cryostat microtome and treated with a periodic acid solution and then with Schiff’s reagent using a periodic acid-Schiff (PAS) staining kit (IHC WORLD). Subsequently, the nuclei are stained using hematoxylin solution according to the provided instructions. The images are captured by optical microscopy.

Indocyanine green staining: For the analysis of indocyanine green (ICG) uptake and release, organoids are washed with cold phosphate-buffered saline (PBS) (Welgene) to remove the Matrigel and then incubated with ICG (Sigma-Aldrich) for 15 minutes at 37℃ in a 5% CO₂ atmosphere. Microscopic images are captured to observe ICG uptake. Subsequently, the organoids undergo three gentle washes with PBS and fresh medium is added. After incubation for an hour at 37℃ in 5% CO₂, images are taken to observe ICG release.

Carboxyfluorescein diacetate staining: For the functional polarization assay, the organoids are detached from the Matrigel and incubated with culture media supplemented with carboxyfluorescein diacetate (CDFDA; Sigma-Aldrich) and Hoechst 33342 (Thermo Scientific) for 30 minutes at 37℃ in a 5% CO₂ atmosphere. After two gentle washes with cold PBS containing calcium and magnesium (Gibco), fluorescence images are captured using a confocal microscope.

Liquid chromatography-tandem mass spectrometry: Coupling of liquid chromatography-tandem mass spectrometry (LC-MS/MS) with chemical isotope labeling or isotope-labelled standard enables the quantitative examination of changes in the expression levels of targeted proteins and metabolites in liver organoids. The prerequisite sample preparations for the LC-MS experiments can be sequentially conducted–protein extraction, proteolytic digestion and chemical isotope labeling. If specific proteins are defined for quality control of liver organoids, add isotope-labeled counterpart peptides to each sample of liver organoids and their respective controls to be in suit with the mole quantities of the targeted proteins. In this paper, there are no restrictions on the types of instruments for LC-MS/MS experiments, excepting data analysis of MS/MS spectra obtained by LC-MS/MS. To enhance reliability of quantitative determination, there must be minimum requirements of search criteria as follows: (i) both software and proteome database must be validated, and (ii) protein searching against protein database are carried out with strict criteria as two missed cleavages per peptides, mass tolerance for precursors and fragment ions, fixed/variable modifications on amino acid residues of protein, the false discovery rate of <0.01 at both peptide and protein levels. The isotopic ratios of targeted proteins to controls are transformed into log2 scale and normalized by median subtraction. This allows for the quantitative comparison of changes in the expression levels of targeted proteins derived from liver organoids compared to the control groups (such as primary human hepatocytes, PHHs). There are no restrictions on the protein extraction methods, enzyme for proteolytic digestion, stable isotope labeling techniques, or LC-MS/MS configuration.

Freezing process: For long-term storage, liver organoids can be preserved by freezing. After separating the organoids from the Matrigel dome during cultivation, organoid pieces are obtained via physical or chemical dissociation. The remaining Matrigel and debris are thoroughly removed using basal medium (BM). The organoid pieces are suspended in a freezing solution such as mFreSR (STEMCELL Technologies) and transferred to cryotubes. They are placed in a cryotank and stored in a deep freezer for 24 hours before being transferred to a liquid nitrogen (LN₂) tank for long-term storage.

Thawing process: Organoid stock vials that were frozen in LN₂ are removed and rapidly thawed to approximately 50% in a 37℃ water bath. BM＋Y-27632 (Tocris) is added, and the stock solution is dissolved via pipetting. After sedimenting the organoid pieces using a centrifuge, they are washed 1∼2 times with BM. The washed organoid pieces are mixed with Matrigel and domed, and cultured with GM＋Y-27632. The following day, the medium is replaced with fresh GM＋Y-27632, and cultured for an additional 2 days. On the third day, the medium is replaced with fresh GM and cultured.

General requirements for the cell source: Liver organoids can be generated from human liver tissue-derived ASCs, or PSCs, including ESCs and iPSCs. When sourcing cells for organoid generation from human donors, it is essential to comply with the ethical guidelines and obtain approval from the institutional review board (IRB) to ensure compliance with ethical considerations.

Quality management: Before starting the production of organoids, a thorough assessment of the original cell sources is required. This involves examining whether the cells are capable of surviving and proliferating under the intended culture conditions. In addition, it is crucial to conduct rigorous checks for any contamination that may compromise the integrity of the cell culture, thus ensuring that the resulting organoids are of high quality and free from external pollutants. Furthermore, purification processes must be performed on hASCs to isolate and enrich the desired cell populations. This purification step enhances the consistency and reliability of the organoid culture by minimizing the presence of unwanted cell types. Another essential step of the preproduction process is validating the characteristics of the origin cells. This includes confirming that the cells exhibit the desired traits and functions relevant to their intended use in organoid development. These validations may include assessing specific molecular markers, determining cellular morphology, and performing functional assays to ensure that the cells possess the necessary properties for organoid formation and function. Furthermore, ensuring genetic stability is crucial for preventing any undesirable genetic alterations that may arise during cell culture and manipulation. Comprehensive genetic analyses should be conducted to verify the genomic integrity of the cells, thereby safeguarding against potential genetic abnormalities that could compromise the authenticity and functionality of the resulting organoids.

Cell characteristics and quality requirements of hASCs: For the generation of hASC-derived liver organoids, cells are sourced from PHHs obtained via biopsy of human clinical specimens (Fig. 1). The source cells for the generation of hASC-derived liver organoids exhibit stem cell and liver progenitor markers, such as LGR5, CK19, and SOX9. In particular, EpCAM＋ ductal cells are sorted, and organoids are generated in three-dimensional (3D) through the addition of extracellular matrix and liver-specific growth factors. EpCAM＋ cells possess bipotent characteristics, which enable them to differentiate into mature parenchymal cells in the liver, including cholangiocytes and hepatocytes (16, 21). Additionally, they must possess a normal karyotype, and remain uncontaminated by mycoplasma.

Cell characteristics and quality requirements of hPSCs: For the generation of hPSC-derived liver organoids, iPSC cells are primarily sourced, while ESC-derived liver organoids are also required the same quality assessment standards (Fig. 1). iPSCs are readily available for procurement through commercial channels, such as stem cell banking systems, at either the national administration or industry levels. Additionally, iPSCs can be generated in-house through the process of reprogramming somatic cells, including skin fibroblasts, blood cells, bone marrow cells, and urine-derived somatic cells. This reprogramming entails the introduction of Yamanaka factors (22), a set of specific transcription factors, into the somatic cell types that induce them to revert to a pluripotent state resembling that of ESCs. iPSCs that are used in the production of liver organoids should express pluripotency markers including NANOG, SOX2, and OCT4. They should also demonstrate the capacity for differentiation into all three germ layers, possess a normal karyotype, and be confirmed free from contamination by mycoplasma.

In this section, we explore the essential factors involved in the generation of liver organoids derived from hASCs or hPSCs, considering the quality standards regarding their size, morphology, and cellular composition. In addition, we offer guidelines for quality assessment to validate the organ-specific functions of the liver organoids. These guidelines evaluate both in qualitative and quantitative criteria.

Size, morphology, and cellular composition quality guidelines for liver organoids: hASC-derived liver organoids exhibit a spherical shape (Fig. 2A). While there are no strict size requirements, researchers should aim for a minimum size that ensures functional relevance. The ASC-derived liver organoids primarily consist of two key cell types: hepatocytes and cholangiocytes.

hPSC-derived liver organoids offer greater flexibility in terms of size and complexity because of their pluripotent nature. Researchers should define both the minimum and maximum size requirements for consistency. The morphology of iPSC-derived organoids under growth conditions typically appears spherical (Fig. 2B, left), while under differentiation conditions, they may include specific structures such as bile ducts or vessel-like networks (Fig. 2B, right), which enhances their physiological relevance (18, 20). Their cellular compositions may include hepatocytes, cholangiocytes, hepatic stellate cells, sinusoidal endothelial cells, and immune cells (23-25).

Quality requirements for liver-specific functions of the organoids: The capacity to replicate major liver functions is one of the primary requirements for the quality assessment of liver organoids. The fundamental functions to be validated include the secretion of the essential proteins ALB and AAT. In addition, liver organoids exhibit the ability to produce urea, a waste product of protein metabolism, and the ability to store glycogen. The absorption and clearance of ICG validate the critical liver functions in the detoxification process of liver organoids. Moreover, the dynamic bile production and excretion, activation of drug-metabolizing enzymes, detoxification (or metabolism) of xenobiotics, including pharmaceutical compounds, should be assessed to validate the liver-specific functions of the organoids.

Genomic integrity and identity: Liver organoids produced for toxicity testing should exhibit a normal chromosomal karyotype. The source cells (liver tissue or iPSCs) should display the same short tandem repeat. For the long-term maintenance of the liver organoids, the chromosomal karyotype is recommended to be analyzed every 20∼30 passages.

For the practical utilization of liver organoids as a hepatotoxicity testing platform, it is required to have a set of minimum standards for their quality, including their size, targeted cell numbers per size, cell compositions, gene expression, and functional assay. Based on our previous experiences in producing liver organoids for use as a toxicity testing platform, we propose the following quality standards for each endpoint.

Liver organoid size standards: For hepatotoxicity testing, it is recommended to utilize hASC-derived liver organoids with a minimum size of 100 μm and an average cell count exceeding 1,000 cells/organoid. hPSC-derived liver organoids should fall within the diameter range of 100∼500 μm, with a prerequisite of more than 1,000 cells/organoid. The maximum size limit for hPSC-derived liver organoids is suggested because of potential limitations in nutrient and oxygen perfusion, which may lead to necrosis in the central regions. Conversely, hASC-derived liver organoids, with their inherent cystic morphology, do not exhibit such issues.

Liver organoid cell composition requirements: Given the primary objective of liver organoids as platform for hepatotoxicity evaluation, it is important that their cellular composition predominantly reflects that of hepatocytes. Quantitatively assessing the proportion of hepatocytes within the liver organoids is determined by measuring the expression level of ALB using flow cytometry. Because human liver tissue typically consists of >70% hepatocytes (26, 27), it is recommended to utilize organoids that express ALB at levels >70%.

Gene expression and functional assays: Liver organoids are required to express liver-specific functional markers, including ALB, HNF4a, MDRs, BSEP, MRPs, and major drug-metabolizing enzymes, including CYP3A4, CYP1A2, CYP2C9, and CYP2D6, at both the RNA and protein levels. The liver-specific gene expression is quantitatively assessed using quantitative RT-PCR, followed by the detection of the protein levels in the supernatant of the organoid culture, which potentially reflects the liver functionality of the organoids. Liver organoids synthesize and secrete serum proteins, such as ALB and AAT, which are detected using ELISA. Alternatively, relative quantitative analysis can be performed to assess hepatocyte function using LC-MS/MS. This method enables the quantitative assessment of ALB protein levels, which can be compared with controls, such as PHHs. Moreover, the activity of drug-metabolizing enzymes can be measured after induced by the drug. The ammonia removal process generated through amino acid metabolism, which is one of the important liver functions, is observed through the production of urea. Additionally, liver organoids accumulate glycogen, absorb and excrete ICG, and have the ability to excrete bile, all of which can be qualitatively assessed using PAS, ICG, and CDFDA staining, respectively.

The quality assessment criteria for the above functions are evaluated quantitatively or qualitatively, as described in Table 1 and 2. The quantitative criteria were determined by referring to previously reported PHH data (8). For the specific COU for hepatotoxicity testing, it is recommended to use liver organoids that fulfill the quantitative evaluation criteria for at least the following liver-specific functions: (i) the cellular composition for ALB expression; and (ii) CYP3A4 activity. These functions serve as fundamental and essential standard indicators of liver functionality for drug metabolism.

Among the quality quantification criteria (Table 1), it is recommended to perform batch-wise toxicity evaluations for ALB secretion and CYP3A4 activity. Other quantitative and qualitative quality evaluations should be performed for all parameters after the initial production of organoids, and thereafter, monitoring every 10∼20 passages is recommended.

Quality assurance in the storage and preservation of organoids depends on two key factors: post-thaw viability and the absence of microbial contamination. Organoids are cryopreserved for extended periods, which requires careful handling during both storage and subsequent post-thawing. It is crucial to ensure that organoids retain a viability threshold of >70% upon thawing; failure to meet this standard warrants the consideration of using an alternative batch from the frozen stocks. Furthermore, sustained, long-term maintenance requires rigorous measures to prevent microbial contamination. The detection of mycoplasma, a common contaminant, is achieved via PCR analysis using the supernatant medium from the organoid cultures following 48 hours of incubation. Negative PCR results, compared to the positive controls, indicate the absence of mycoplasma contamination. It is recommended to perform the contamination assessment every 10∼20 passages to maintain the integrity of the organoid cultures.

Liver organoids must be produced in accordance with ethical and medical guidelines. Here, we outline specific criteria for selecting liver samples to create organoids derived from tissue-derived ASCs. Human liver tissue sampling should only be conducted from donors who willingly consent to participate in the research, particularly among patients who require surgical treatment. Donors over the age of 80, experiencing bleeding in the surgical region, not requiring surgical treatment, or under the age of 5 years old should be excluded. Sampling of liver tissue from patients with liver cancer or other liver diseases should be performed on the removed liver tissue from their surgical treatment. For other donors, tissue sampling may involve a portion of the tissue biopsy obtained for diagnosis. The personal information of any patient is codified and managed using clinical trial numbers, which are encrypted for protection. Encrypted information can only be accessed by the research participants, and disclosure to third parties is prohibited. When recruiting participants, medical staff must thoroughly explain that participation in the study will not result in any disadvantages or discriminatory treatment for the patient, and efforts should be made to ensure the autonomy and protection of vulnerable subjects. Patient consent, sample acquisition, and all other aspects of organoid research must be conducted under IRB approval.