The liver plays an important role in metabolic homeostasis and numerous metabolic functions, including protein synthesis, hormone and bile production, and cholesterol and glucose homeostasis regulation (Builes Montaño et al., 2022; Chu et al., 2022; Liao et al., 2022). Liver cells consist of two major types: parenchymal and non-parenchymal cells. Parenchymal cells primarily comprise hepatocytes (∼80%) and bile-duct epithelial cells (BECs, also known as cholangiocytes; ∼20%), whereas non-parenchymal cells include Kupffer cells (macrophages), stellate cells, and sinusoidal endothelial cells (Jungermann and Kietzmann, 1996). BECs are responsible for bile acid collection and transportation, whereas hepatocytes are responsible for nutrient metabolism, xenobiotics, detoxification, and protein production (Miyajima et al., 2014; Rizki Safitri et al., 2018). Primary hepatocytes have proven to be useful tools in various biomedical studies, and have demonstrated their utility as ex vivo models for elucidating cellular and molecular events in liver physiology (Radko et al., 2019; Charni Natan and Goldstein, 2020). However, primary hepatocytes cannot be cultured for extended periods because this depletes liver function and viability under monolayer culture conditions (Sendi et al., 2018). Therefore, primary hepatocytes are unsuitable for long-term assay studies that require intact liver biology and specific functions (Bell et al., 2016). Ultimately, long-term culture methods for primary hepatocytes that can maintain liver metabolism and specific functions are necessary.

Health functional food ingredients must be evaluated for safety and functionality (Lee, 2013). The Organization for Economic Co-operation and Development (OECD) guidelines provide a standardized framework for conducting these evaluations (Kim et al., 2020b). The short-term dose toxicity evaluation results of single or multiple administration to animals within 24 h, according to OECD Test Guidelines (TG) 420, 423, and 425, and repeated dose toxicity evaluation results of administering health functional food ingredients to animals for 28 days or 90 days by long-term, according to OECD TG 408, 407, and 409, are to be submitted for safety and functionality for recognition (OECD, 2001; EFSA Scientific Committee, 2011; Jonsson et al., 2013). Additionally, the genotoxicity, reproductive toxicity, and chronic/carcinogenic toxicity results of animal tests must be submitted to confirm the safety and functionality of a health functional food. To comply with these OECD guidelines, a significant number of animals (at least 10 per group) must be used for each test, incurring significant costs and time constraints. Moreover, ethical concerns regarding animal testing have increased (MFDS, 2021a, 2021b). Accordingly, the requirement for experimental methods that replace animal experiments is increasing.

Cell lines and animal models have traditionally been used in several studies on the cellular signaling pathway, molecular development mechanism, and liver pathogenesis (Kim et al., 2020a). However, the two-dimensional (2D) culture of these cell lines is limited in terms of long-term and stable gene expression, while animal models are time consuming and expensive to develop (Milani Nejad and Janssen, 2014; Akbari et al., 2019). Organoids, which are three-dimensional (3D) cell culture systems, offer the structural and functional characteristics of an organ with self-renewal and maintained function (Kratochvil et al., 2019; Gómez Mariano et al., 2020). Organoids can mimic tissue structure and various cell types (Lancaster and Knoblich, 2014; Harrison et al., 2021). Several researchers have recently established organoids from adult tissues, such as the gut, brain, kidney, lung, retina, and liver (Qu et al., 2021). Because of their ability to replicate organ properties, organoids have been widely used in disease modeling, drug screening, and host-microbial interaction studies (Huch and Koo, 2015; Nakamura and Sato, 2017).

This study investigated the potential of liver organoids derived from primary mouse hepatocytes (OrgPH) to undergo long-term culture, mimic the functions of in vivo or in vitro environments, and serve as viable alternatives to animal models to ensure the safety and hepatic functional testing of health functional foods.

Reagents for hepatocyte isolation

1X Hank’s Balanced Salt solution (HBSS): 10X HBSS (Gibco), 1 M Hydroxyethyl piperazine ethane sulfonic acid (HEPES) buffer solution (Sigma-Aldrich Co.), and 7.5% NaHCO₃ (Daejung Chemical Co.); Solution 1: 1X HBSS, 34.2 mg/mL ethylene glycol-bis (2-aminoethyl ether) (Sigma-Aldrich Co.), and bovine serum albumin (BSA, RD tech); Solution 2: 1X HBSS, 0.4 M CaCl₂−2H₂O (Junsei Chemical Co., Ltd.), and BSA; Collagenase solution: 1X HBSS, 1 mg/mL collagenase 1A (Sigma-Aldrich Co., C9891), and 0.4 M CaCl₂−2H₂O. All solutions were filtered using Steritop™ Filter Units (Millipore).

Isolation of primary mouse hepatocytes

Primary mouse hepatocytes were isolated from 6- to 10-weeks-old male C57BL/6J mice using two-step collagenase perfusion. Briefly, mice were anesthetized with isoflurane. Then, catheters were inserted the inferior vena cava, and the portal vein was immediately severed. Each mouse’s liver was subsequently perfused with 50 mL of solution 1 (1X Hank’s buffer) for 5 min. Next, the liver was digested with 50 mL of collagenase 1A solution (Sigma-Aldrich Co., C9891). The digested liver was subsequently extracted from the body and disrupted in a Petri dish until the liver lobes were dissociated into cells. After dissociation, the cells were filtered four times using a 70 mm filter (Corning) with solution 2. Subsequently, the filtered cells were centrifuged at 600 g for 3 min at 20°C. The Institutional Animal Care and Use Committee of Ewha Womans University approved all experimental animals (approval number: 21-018t).

Construction of organoids from primary mouse hepatocytes

Isolated primary mouse hepatocytes were enumerated and seeded at a concentration of 200 cells/μL in Matrigel (Corning) in a 24-well plate. The plate was subsequently incubated at 37°C for 40 min to allow Matrigel solidification. After Matrigel solidification, organoid media was added to the plate without disturbing the Matrigel dome. The organoid media comprised HepatiCultTM Organoid Growth Medium (STEMCELL Technologies), 1% (v/v) penicillin/streptomycin (Gibco), and 10 μM Y-27632 (Abmole BioScince) according to the manufacturer’s instructions. The medium was replaced every 3∼4 days, excluding the addition of Y-27632. OrgPH were passaged every 7∼10 days.

AML12 cells and primary mouse hepatocyte cell culture

AML12 cells, which constitute a normal mouse hepatocyte cell line, were purchased from the American Type Culture Collection. AML12 cells were cultured in Dulbecco’s Modified Eagle Medium/F-12 (DMEN:F12; 1:1) medium (Hyclone™) supplemented with 10% fetal bovine serum (FBS) (Corning), 0.14 M sodium bicarbonate (Daejung), 1% penicillin and streptomycin (Gibco), 0.5 mM sodium pyruvate (Sigma-Aldrich Co.), and 40 ng/mL dexamethasone (Sigma-Aldrich Co.). The cells were incubated at 37°C in a 5% CO₂ humidified incubator.

The primary mouse hepatocyte isolation procedure was the same as that described above. The isolated primary hepatocytes were cultured in DMEM:F12 (1:1) medium, FBS, 1% penicillin and streptomycin, 1 mM L-methionine (Sigma-Aldrich Co.), 7.5% sodium bicarbonate (Daejung), 1 mg/mL insulin, 0.1 mM hydrocortisone (Sigma-Aldrich Co.), and 1 M HEPES buffer solution (Sigma-Aldrich Co.). The cells were incubated at 37°C in a 5% CO₂ humidified incubator.

Determination of liver function in organoids from primary mouse hepatocytes

AML12 cells were seeded in a six-well plate at a density of 0.2×10⁶ cells/well. After 4 h, AML12 cells were treated with 100 mM ethanol (Sigma-Aldrich Co.), 50 mM glucose (Sigma-Aldrich Co.), and 0.2 mg/mL cholesterol (Sigma-Aldrich Co.) for overnight.

OrgPH were embedded with Matrigel in a 48-well plate at a density of 200 cells/μL and the treatment concentrations of ethanol, glucose, and cholesterol were the same as those described above.

Real-time quantitative polymerase chain reaction

Total RNA was purified from OrgPH and primary hepatocytes using the AccuPrep^Ⓡ Universal RNA Extraction Kit (Bioneer) following the manufacturer’s instructions. Total RNA from AML12 cells and liver tissue was purified using Trizol™ (Invtrogen™) following the manufacturer’s instructions. cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Quantitative PCR (qPCR) was conducted based on the guidelines accompanying the Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher Scientific). The relative expression levels of target mRNAs were normalized to glyceraldehyde 3-phosphate dehydrogenase (Gapdh), an internal control, using the 2^−ΔΔCT method (Livak and Schmittgen, 2001). All data are expressed as a relative quantity to each control value. The following primer sequences were used: albumin (Alb) forward and reverse primers: 5’-CCCTGATTACTCTGTATCCCT-3’ and 5’-CCAAGTTCTTAGGCTCTTCTAC-3’; arginase 1 (Arg1) forward and reverse primers: 5’-CCCTAATGACAGCTCCTTTC-3’ and 5’-CCACACTGACTCTTCCATTC-3’; glucose-6-phosphate (G6pc) forward and reverse primers: 5’-ATGACTTTGGGATCCAGTCG-3’ and 5’-TGGAACCAGATGGGAAAGAG-3’; cytochrome P450 family 1 subfamily A (Cyp1a1) forward and reverse primers: 5’-GTCCAGCTGTCAGATGATAAGG-3’ and 5’-TACATGAGGCTCCACGAGATA-3’; alcohol dehydrogenase (Adh) forward and reverse primers: 5’-GGGTGGACTTTTCGTTTGAA-3’ and 5’-CTACGACGACGCTTACACCA-3’; acetaldehyde dehydrogenase (Aldh) forward and reverse primers: 5’-CTCGTACCTGGTGGATTTG-3’ and 5’-CGTCGATGGGA ATGGTTT-3’; cytochrome P450 2E1 (Cyp2e1) forward and reverse primers: 5’-AGTCTCTGGTTGACCCTAAG-3’ and 5’-GGTCTCATGAACGAGGAATG-3’; glucose transporter 2 (Glut2) forward and reverse primers: 5’-CCCTTGTCACAGGCATTCTTA-3’ and 5’-CACAGCAGATAGGCCAAGTAG-3’; glucokinase (Gck) forward and reverse primers: 5’-CATCAGGAGGCCAGTGTAAAG-3’ and 5’-TCCCAGGTCTAAGGAGAGAAAG-3’; pyruvate kinase (Pkm) forward and reverse primers: 5’-TCCGGACTGGACTCATCAA-3’ and 5’-GGATGTTCTCGTCACACTTCTC-3’; 3-hydroxy-3-methyl-glutaryl-CoA reductase (Hmgcr) forward and reverse primers: 5’-GACGAAGATGTGTCCAAGAG-3’ and 5’-GAAGGCTAAACTCAGGGTAATC-3’; 3-hydroxy-3-methyl-glutaryl-CoA synthase (Hmgcs) forward and reverse primers: 5’-GATGGTGTAGATGCTGGAAAG-3’ and 5’-CAGTCAGGCAAAGAGAGTTG-3’; sterol regulatory element‐binding proteins 2 (Srebp2) forward and reverse primers: 5’-GGCGGACAACACACAATA-3’ and 5’-GCC AGACTTGTGCATCTT-3’; Gapdh forward and reverse primers: 5’-CATCACTGCCACCCAGAAGACTG-3’ and 5’-ATGCCAGTGAGCTTCCCGTTCAG-3’.

Statistical analysis

Data were analyzed using GraphPad PRISM 10 (GraphPad Software). Statistically significant mean differences in experimental liver-specific markers were analyzed using one-way analysis of variance (ANOVA) and post-hoc Tukey’s and Dunnett’s tests. Differences in liver functional enzyme activity between the two-groups were analyzed using the t-test. Statistical significance (P-value) was verified within 5%.

Morphological features of organoids from primary mouse hepatocytes

When hepatocytes were isolated from mouse liver and embedded in Matrigel, a single cell shape was observed. However, after 2∼3 days, this shape changed to a cluster-like or hollow shape and eventually grew in size. Organoids were embedded in Matrigel and cultured for a week, followed by passaging one week later. Even after the culture, the same shape was observed during the first week of culture, and the same shape was observed even after repeated cultivation (Fig. 1).

Figure 1. Characterization of organoids from primary mouse hepatocytes. Organoids from primary mouse hepatocytes altered their morphology with time on day 0 at passage 1 (A), day 3 at passage 2 (B), day 7 at passage 3 (C), and day 4 at passage 4 (D and E) in Matrigel 3D culture after isolating primary hepatocytes from 7-week-old mice. Microscopic image of organoids with an original magnification of ×40 (A–D) and ×200 (E). 

Expression of liver-specific markers in organoids from primary mouse hepatocytes

Liver-specific markers, Alb, Arg1, G6pc, and Cyp1a1 were selected to confirm the gene expression in OrgPH, AML12 cells, and primary mouse hepatocytes. The expression of liver-specific marker genes, including Alb (P<0.0001), Arg1 (P<0.01), G6pc (P<0.001), and Cyp1a1 (P<0.001), in primary hepatocytes, was significantly higher than that in AML12 cells; nevertheless, the expression of these four markers eventually decreased in primary hepatocytes (P<0.05) (Fig. 2A).

Figure 2. mRNA expression levels of liver-specific markers in primary hepatocytes (A) and organoids from primary mouse hepatocytes compared with those in AML12 cells (B). Alb, Arg1, G6pc, and Cyp1a1 were selected as liver-specific markers. Primary hepatocytes (PH) were cultured for 10 days after being isolated from mouse livers, and the expression levels of liver-specific markers were measured at 0, 2, 4, and 10 days using qPCR. OrgPH was cultured in Matrigel for 28 days after being isolated from mouse livers, and the expression level of liver-specific markers was measured at 7, 14, and 28 days using qPCR. All data are expressed as the mean±SD (n=3). Error bars represent the mean±SEM. *P<0.05, **P<0.01, and ****P<0.0001. PH, primary hepatocyte; OrgPH, liver organoids from primary mouse hepatocytes; G6Pc, glucose-6-phosphatase; CYP1A1, cytochrome P450 family 1 subfamily a member 1. 

Alb expression in OrgPH was significantly higher than that in AML12 cells on day 7 (P<0.05) but significantly lower on days 14 (P<0.05) and 28 (P<0.01). In contrast, the mRNA expression level of Arg1 in OrgPH was confirmed to be elevated after 14 days, although no significant difference from the AML12 cells was noted on day 7. The expression level of G6pc in OrgPH was significantly higher than that in the AML 12 cells on day 7 (P<0.05), and it eventually increased. Finally, the expression level of Cyp1a1 in OrgPH was consistent with that in AML12 cells on day 7; nonetheless, it eventually increased significantly (P<0.05) (Fig. 2B).

Assessment of glucose metabolism in organoids from primary mouse hepatocytes

Glucose treatment was administered to evaluate glucose metabolism in OrgPH. The mRNA expression levels of Glut2 and Pkm increased significantly in the glucose-treated group compared with those in the control group (P<0.05, P<0.01) (AML12). However, the expression levels of Glut2, Gck, and Pkm genes in OrgPH were indifferent between the control and glucose-treated groups (Fig. 3).

Figure 3. mRNA expression levels of glucose metabolism genes in AML12 cells and organoids from primary mouse hepatocytes. AML12 cells and OrgPH were treated with 50-mM glucose for 24 h. The expression levels of Glut2 (A), Gck (B), and Pkm (C) were determined using qPCR. All data are expressed as the mean±SD (n=3). Error bars represent the mean±SEM. *P<0.05, and **P<0.01 vs. control group. OrgPH, liver organoids from primary mouse hepatocytes; Glut2, glucose transport 2; GcK, glucokinase; PKM, pyruvate kinase. 

Assessment of cholesterol metabolism in organoids from primary mouse hepatocytes

To evaluate cholesterol metabolism in OrgPH, both models were treated with cholesterol. The mRNA expression levels of the Hmgcs, Hmgcr, and Srebp2 cholesterol metabolism-related genes decreased significantly in cholesterol-treated AML12 cells (P<0.05). However, the expression levels of Hmgcs, Hmgcr, and Srebp2 genes in OrgPH increased significantly in the cholesterol-treated group compared with those in the control group (P<0.01, P<0.05). When treated with cholesterol, an opposite trend was observed in AML12 cells and OrgPH (Fig. 4).

Figure 4. mRNA expression levels of cholesterol metabolism-related genes in AML12 cells and organoids from primary mouse hepatocytes. AML12 cells and OrgPH were treated with 0.2 mg/mL cholesterol for 24 h. The expression levels of Hmgcs (A), Hmgcr (B), and Srebp2 (C) were determined using qPCR. All data are expressed as the mean±SD (n=3). Error bars represent the mean±SEM. *P<0.05, and **P<0.01 vs. control group. OrgPH, liver organoids from primary mouse hepatocytes; HMGCS, 3-hydroxy-3-methyl-glutaryl-CoA synthase; HMGCR, 3-hydroxy-3-methyl-glutaryl-CoA reductase; SREBP2, sterol regulatory element‐binding proteins 2. 

Evaluation of ethanol metabolism in organoids from primary mouse hepatocytes

When AML12 cells were treated with ethanol, the mRNA expression levels of Adh, and Aldh increased significantly in the ethanol-treated group compared with those in the control group (P<0.05). However, Cyp2e1 expression was increased compared with that in the control, although no statistical difference was confirmed. When OrgPH were treated with ethanol, Adh, Aldh, and Cyp2e1 expression levels increased significantly in the ethanol-treated group compared with that in the control group (P<0.05) (Fig. 5).

Figure 5. mRNA expression levels of ethanol metabolism genes in AML12 cells and organoids from primary mouse hepatocytes. AML12 cells and OrgPH were treated with 100 mM ethanol for 24 h. The expression levels of Adh (A), Aldh (B), and Cyp2e1 (C) were determined using qPCR. All data are expressed as the mean±SD (n=3). Error bars represent the mean±SEM. *P<0.05 vs. control group. OrgPH, liver organoids from primary mouse hepatocytes; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase 2; CYP2E1, cytochrome P450 2E1.

This study aimed to examine the capacity of OrgPH to achieve normal liver function and long-term culture. In vivo model experiments can be time consuming, and expensive and may raise ethical concerns because of animal use. Furthermore, for the safety and functionality of healthy functional foods to be recognized, numerous animals should be used because the results of single, repeated, genetic and chronic experiments must be submitted following the OECD guidelines (MFDS, 2021b). Alternative methods are required to replace or reduce the reliance on animal experiments. Additionally, in vitro models do not easily explain the functional and physiological changes of in vivo models (Kratochvil et al., 2019; Rezakhani et al., 2021). Therefore, this study focused on producing OrgPH using a 3D culture system. This approach enabled more effective simulation of an in vivo environment, providing a more physiologically relevant setting for studying liver function and responses. On comparing the results of OrgPH with those of AML12 cells, which are commonly used in normal mouse liver models, similar trends were observed.

Several studies have suggested that OrgPH morphology potentially exhibits a grape-like or hollow shape, whereas others have also suggested that it forms a rosette shape (Hu et al., 2018; Brooks et al., 2021; Nuciforo and Heim, 2020; Peng et al., 2021). In this study, a uniform shape was confirmed 2∼3 days after embedding in Matrigel; the morphology assumed a grape-like shape after the first culture was initiated, and it gradually appearing hollow (Fig. 1).

To investigate the expression of common markers of liver metabolic function in OrgPH, we selected the major liver metabolic markers related to the urea cycle, glucose homeostasis, and detoxification metabolism, including Arg1, G6pc, and Cyp1a1. Their expression levels were compared among AML12 cells, primary hepatocytes, and OrgPH (Zhu et al., 2020). The liver synthesizes albumin, which plays several roles, including the maintenance of osmotic pressure, the binding of various substances, such as hormones, drugs, and long-chain fatty acids, antioxidant activity, and anti-inflammatory functions (Miller and Jedrzejczak, 2001; Carvalho and Verdelho Machado, 2018). In primary hepatocytes, expression level of Alb peaked on day 0 after isolation from the liver, gradually decreasing with time. Furthermore, the expression levels of other genes, including Arg1, G6pc, and Cyp1a1, were significantly higher than those in AML12 cells on day 0; nevertheless, they gradually decreased over time (Fig. 2A). This could be because when primary hepatocytes are isolated from mouse liver tissue and cultured on a 2D monolayer, they experience a short life span and lack of cell proliferation, resulting in decreased metabolic function with time. Furthermore, primary hepatocytes rapidly lose their morphological characteristics (Shulman and Nahmias, 2012; Rose et al., 2021).

In contrast, Arg1, G6pc, and Cyp1a1 expression levels in OrgPH gradually increased compared with those in AML12 cells (Fig. 2B). Thus, these results suggest that the OrgPH model is potentially more suitable for long-term experiments on the urea cycle, glucose homeostasis, and detoxification metabolism. However, the Alb expression level in OrgPH was lower than that in AML12 cells (Fig. 2B). Nevertheless, OrgPH maintained 50% of the Alb expression levels for 14 days. Therefore, these results suggest that OrgPH potentially performs liver metabolic functions and that the OrgPH model can mimic liver metabolic functions for a longer period than primary hepatocytes. Moreover, it can mitigate the difficulty of repeatedly isolating hepatocytes from mice for primary hepatocyte experimentation and circumvent ethical concerns.

To evaluate liver function in OrgPH, gene expression alterations confirmed upon treatment with ethanol, glucose, and cholesterol as well as the tendency of gene expression were compared with those in AML12 cells.

Ethanol is mandatorily eliminated from the body, and without its removal, it accumulates in the body, potentially causing various diseases and affecting metabolic pathways (Zakhari, 2006). Ethanol metabolism in the liver is initiated by ADH, ALDH, and CYP2E1 (Clemens, 2006). Eventually, ethanol is decomposed into water and carbon dioxide for easy elimination from the body (NIAAA, 2007). A previous study demonstrated decreased expression of Adh and Aldh, and increased expression of Cyp2e1 in the livers of chronic ethanol-treated mice (Liu et al., 2012; Matson et al., 2013). Another study demonstrated an increased expression of Aldh in the livers of acute ethanol-treated mice (Bak et al., 2016). In our study, the expression levels of Adh, Aldh, and Cyp2e1 increased in OrgPH with acute and high-concentration ethanol (Katz et al., 2001; Liu et al., 2010), consistent with previous studies (Fig. 5). In summary, these results suggest that OrgPH potentially provides a hepatic in vivo environment for detoxification metabolism.

Glucose metabolism in the liver is an extremely important function in glycemic control. When blood glucose levels increase, the liver absorbs it via GLUT2 and is regulated by the rate-limiting enzymes GCK, G6Pc, and PKM (Han et al., 2016). In this study, glucose metabolism in OrgPH was evaluated and compared with that in AML12 cells. When AML12 cells were treated with glucose, Glut2 and Pkm increased significantly; however, no significant difference was observed in OrgPH. However, the expression of corresponding genes was confirmed, suggesting that glucose metabolism can be evaluated using primary mouse hepatocyte-derived organoids.

Body cholesterol is synthesized in the liver via HMGCS, HMGCR, and SREBP2 gene regulation and potentially causes several health problems, including liver damage (Horton et al., 2002; Trapani et al., 2012; Liao et al., 2022). Therefore, we confirmed genetic changes based on cholesterol treatment. In AML 12 cells, the expression of the three genes decreased in the cholesterol-treated groups; nevertheless, opposite results were obtained in the cholesterol-treated groups of OrgPH (Fig. 4). In several studies on cholesterol diets in rodents, the expression of Hmgcr, Hmgcs, and Srebp2 increased, and dietary cholesterol reportedly increased due to lipid metabolism disorders (Beppu et al., 2012; Chen et al., 2017; Liu et al., 2018). Interestingly, our data coincide with the experimental results from cholesterol diet-based liver function assessments using animal models, and these results suggest that OrgPH maintains a hepatic environment in vivo, even in long-term culture.

In conclusion, we examined the potential of OrgPH to maintain liver function during long-term culture. Liver function in primary hepatocytes decreased within two days after their isolation from the liver. However, it was maintained in OrgPH even during long-term culture, and hepatic functional metabolism was confirmed through glucose, ethanol, and cholesterol treatment. Therefore, OrgPH can be used to explain hepatic in vivo conditions and metabolism. Consequently, organoids can address the challenges encountered when using animal models and can be used in high-throughput drug screening and toxicity evaluation of health functional foods cheaply and with minimal use of animals. While further research is required to assess the efficiency of hepatocyte organoids in the hepatotoxicity evaluation of health functional foods, the organoid model is a potentially useful alternative to animal models.

This research was supported by the BK21 FOUR (Fostering Outstanding Universities for Research) funded by the Ministry of Education (MOE, Korea) and National Research Foundation of Korea (NRF-5199990614253, Education Research Center for 4IR-Based Health Care).

This research was supported by grants provided by the National Research Foundation of Korea (NRF), funded by the Korean Government through the Ministry of Science and ICT (MSIT) (No. 2020R1A2C1102451 and 2023R1A2C1007243).

The authors declare no conflict of interest.

Concept and design: HMK, KSK. Analysis and interpretation: HMK, Yerin K, Yuri K, KSK. Data collection: HMK, KSK. Writing the article: HMK, KSK. Critical revision of the article: YJK, KSK. Final approval of the article: all authors. Statistical analysis: HMK. Obtained funding: KSK. Overall responsibility: KSK.