Cryopreservation of Hepatocyte Organoid-Derived Cells Generated from Human iPS Cells
Open accessAbstract
Hepatic organoids (iHOs) generated from human-induced pluripotent stem cell-derived hepatocyte-like cells (HLCs) have attracted attention as a new source of hepatocytes to replace primary human hepatocytes (PHHs) in drug discovery research. Recently, we established iHOs from HLCs, which proliferated for more than 10 passages, and developed a two-dimensional (2D) culture protocol for iHOs, by which the cells showed much higher hepatic functions. In this study, we examined the cryopreservation of iHOs for the purpose of improving their versatility. Cryopreserved iHOs had comparable cell proliferative capacity to fresh iHOs. Hepatocyte marker gene expression levels in the cryopreserved iHOs were comparable to those without cryopreservation. When cryopreserved iHOs were directly matured by our 2D culture protocol, they showed higher hepatocyte marker gene expression levels and higher CYP3A4 activity than HLCs. Some of the hepatic functions in 2D-cultured cryopreserved iHOs were at levels comparable to those in PHHs. These results suggest that cryopreserved iHOs also differentiated into mature hepatocytes by 2D culture. We expect cryopreserved iHOs to be used as a user-friendly cell source for drug discovery research in many laboratories.
AI snapshot
Written from the paper’s full text.
TL;DR
Cryopreserved human iPS cell-derived hepatic organoids (iHOs) retained viability, proliferative capacity, and hepatocyte marker expression comparable to fresh iHOs. When directly matured under a 2D protocol, cryopreserved iHOs generated hepatocyte-like cells with hepatic functions similar to fresh iHO-derived cells and some functions comparable to primary human hepatocytes.
Key findings
- Post-thaw viability of cryopreserved iHOs was approximately 70%; they showed morphology similar to fresh iHOs and grew to 10^4-fold in 18 d, though with slightly slower growth than fresh iHOs.
- Hepatocyte marker genes (ALB, HNF4α, CYP3A4, CYP2C9, CYP2C19, NTCP, UGT1A1) and proliferation/stem cell markers (Ki67, LGR5, EpCAM, CK19) in cryopreserved iHOs were comparable to fresh iHOs, with no significant differences reported.
- Cryopreserved iHOs directly differentiated into Cryo-iHO-Heps under 2D culture; hepatocyte marker gene expression was not significantly different from Fresh-iHO-Heps, and all genes except ALB were higher than in original HLCs.
- CYP3A4 activity in Cryo-iHO-Heps was similar to Fresh-iHO-Heps, much higher than HLCs, and comparable to PHHs; CYP2C9 and CYP2D6 activities were not as high as PHHs but were higher than HLCs and similar or higher than Fresh-iHO-Heps. Urea and ALB secretion were the same as Fresh-iHO-Heps, though ALB secretion was lower than in HLCs and PHHs.
Methods
- Human iPS cell lines Tic and YOW-iPS were differentiated into HLCs, then iHOs were established and passaged 1–5 times; iHOs were dissociated, suspended in STEM-CELLBANKER DMSO-free GMP grade, cooled to −80°C overnight, then stored at −150°C for at least 2 weeks before thawing.
- Cryopreserved iHOs were either cultured as organoids or directly seeded for 2D differentiation into iHO-Heps using a stepwise medium protocol; assays included CellTiter-Glo 3D viability, Countess viability, real-time RT-PCR, LC-MS/MS CYP activity with midazolam/diclofenac/bufuralol, P450-Glo CYP3A4 assay, immunofluorescence, ALB ELISA, and urea measurement.
- PHH comparison used one cryopreserved lot (HC4-24, XENOTECH); all results are based on three biological replicates, analyzed by unpaired two-tailed Student’s t-test with p < 0.05 considered significant.
Limitations
- The paper states that ALB secretion in Cryo-iHO-Heps and Fresh-iHO-Heps was lower than in HLCs and PHHs and “warrants further investigation in a future study.”
- The paper notes cryopreserved iHOs showed “a little slower growth than fresh iHOs” and that CYP2C9 and CYP2D6 activities were not as high as in PHHs.
- Evident from the text: PHH comparison relied on a single lot (HC4-24), and all results are based on three biological replicates; no in vivo or broader drug-toxicity validation is reported.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
4 of 12 cited sources checked · 2 supported · 0 flagged · accuracy not yet measured
- Premisecitation
Highly functional hepatocyte-like cells from iHOs were developed by a 2D culture protocol using a stepwise optimal culture medium.
“We also developed highly functional hepatocyte-like cells from iHOs by the two-dimensional (2D) culture protocol using a stepwise optimal culture medium.15)”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [15] 15) Inui J, Ueyama-Toba Y, Imamura C, Nagai W, Asano R, Mizuguchi H. Two-dimensionally cultured functional hepatocytes generated from human induced pluripotent stem cell-derived hepatic organoids for pharmaceutical research. <i>Biomaterials</i>, <b>318</b>, 123148 (2025). source
- Premisecitation
The predictive accuracy of hepatotoxicity in animal studies is only 50%.
“However, the predictive accuracy of hepatotoxicity in animal studies is only 50%.1,5)”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [1] 1) Ruoß M, Vosough M, Königsrainer A, Nadalin S, Wagner S, Sajadian S, Huber D, Heydari Z, Ehnert S, Hengstler JG, Nussler AK. Towards improved hepatocyte cultures: progress and limitations. <i>Food Chem. Toxicol.</i>, <b>138</b>, 111188 (2020). source
- cites [5] 5) Ware BR, Khetani SR. Engineered liver platforms for different phases of drug development. <i>Trends Biotechnol.</i>, <b>35</b>, 172–183 (2017). source
In “The Drug Development Pipeline” (page not provided), the paper says animal models are not always good predictors of human drug-induced liver injury. It does not give animal studies a 50% predictive accuracy; its <50% figure refers to the sensitivity of primary human hepatocytes in conventional culture. (LLM, conf=0.88)
Experimental — accuracy not yet measured
- Findingown data
Cryopreserved iHOs grew to 10^4-fold in 18 days.
“Cryopreserved iHOs grew to 10 4 -fold in 18 d”
Quote in the paperSubstantial claim - Findingown data
Gene expression in other drug-metabolizing enzymes and transporters was maintained at levels equivalent to or higher than those in HLCs.
“Gene expression in other drug-metabolizing enzymes and transporters was maintained at levels equivalent to or higher than those in HLCs (Supplementary Figs.S3A, S3B).”
Quote in the paperSubstantial claim - Findingown data
CYP3A4 activity in Cryo-iHO-Heps was much higher than in the original HLCs.
“The CYP3A4 activity in Cryo-iHO-Heps was similar to that in Fresh-iHO-Heps, but much higher than that in the original HLCs and comparable to that in PHHs (Fig. 3A).”
Quote in the paperSubstantial claim
- Findingown data
Some hepatic functions in 2D-cultured cryopreserved iHOs were at levels comparable to those in PHHs.
“Some of the hepatic functions in 2D-cultured cryopreserved iHOs were at levels comparable to those in PHHs.”
Quote in the paperSubstantial claim - Findingown data
The cell viability after cryopreservation was approximately 70%.
“The cell viability after cryopreservation was approximately 70% (Fig. 1C).”
Quote in the paperSubstantial claim - Findingown data
ALB secretion in Cryo-iHO-Heps and Fresh-iHO-Heps was lower than that in HLCs and PHHs.
“The finding that ALB secretion in Cryo-iHO-Heps and Fresh-iHO-Heps was lower than that in HLCs and PHHs, which was also the case in our previous report, 15) warrants further investigation in a future study.”
Quote in the paperNot a substantial claim - Findingown data
ALB secretion levels in Cryo-iHO-Heps were the same as in Fresh-iHO-Heps.
“The levels of urea and ALB secretion in Cryo-iHO-Heps were the same as in Fresh-iHO-Heps (Figs. 3B, 3C).”
Quote in the paperSubstantial claim - Findingown data
Hepatocyte marker gene expression levels in cryopreserved iHOs were comparable to those without cryopreservation.
“Hepatocyte marker gene expression levels in the cryopreserved iHOs were comparable to those without cryopreservation.”
Quote in the paperSubstantial claim - Findingown data
CYP2C9 and CYP2D6 activities in Cryo-iHO-Heps were not as high as those in PHHs.
“the CYP2C9 and CYP2D6 activities in Cryo-iHO-Heps were not as high as those in PHHs, although they were higher than those in the original HLCs and similar or higher than those in Fresh-iHO-Heps (Fig. 3A).”
Quote in the paperSubstantial claim - Findingown data
When cryopreserved iHOs were directly matured by the 2D culture protocol, they showed higher CYP3A4 activity than HLCs.
“When cryopreserved iHOs were directly matured by our 2D culture protocol, they showed higher hepatocyte marker gene expression levels and higher CYP3A4 activity than HLCs.”
Quote in the paperSubstantial claim - Findingown data
Cryopreserved iHOs had comparable cell proliferative capacity to fresh iHOs.
“Cryopreserved iHOs had comparable cell proliferative capacity to fresh iHOs.”
Quote in the paperSubstantial claim - Findingown data
CYP3A4 activity in Cryo-iHO-Heps was similar to that in Fresh-iHO-Heps.
“The CYP3A4 activity in Cryo-iHO-Heps was similar to that in Fresh-iHO-Heps, but much higher than that in the original HLCs and comparable to that in PHHs (Fig. 3A).”
Quote in the paperSubstantial claim - Findingown data
Cryopreserved iHOs showed hepatocyte marker gene expressions comparable to fresh iHOs.
“Cryopreserved iHOs showed hepatocyte marker gene expressions comparable to fresh iHOs (Fig. 1E).”
Quote in the paperSubstantial claim - Findingown data
Cryo-iHO-Heps had higher CYP3A4 activity than PHHs in a fluorescent assay.
“A fluorescent assay for CYP3A4 activity showed that Cryo-iHO-Heps had higher CYP3A4 activity than PHHs (Supplementary Fig. S4A).”
Quote in the paperNot a substantial claim - Findingown data
CYP3A4 activity in Cryo-iHO-Heps was comparable to that in PHHs.
“The CYP3A4 activity in Cryo-iHO-Heps was similar to that in Fresh-iHO-Heps, but much higher than that in the original HLCs and comparable to that in PHHs (Fig. 3A).”
Quote in the paperSubstantial claim - Findingown data
Expression levels of other drug-metabolizing enzymes and transporters in cryopreserved iHOs were similar to those in fresh iHOs.
“Although expression varied depending on the molecular species, in general, expression levels in cryopreserved iHOs were similar to those in fresh iHOs (Supplementary Figs.S1A, S1B).”
Quote in the paperSubstantial claim - Findingown data
Hepatocyte marker gene expressions were not significantly different between Cryo-iHO-Heps and Fresh-iHO-Heps.
“Hepatocyte marker gene expressions were not significantly different between Cryo-iHO-Heps and Fresh-iHO-Heps, although some of the gene expression levels differed depending on the types of genes.”
Quote in the paperSubstantial claim - Findingown data
No significant differences in proliferation/stem cell marker gene expressions were observed between cryopreserved and fresh iHOs.
“no significant differences in proliferation/stem cell marker gene expressions were observed between cryopreserved and fresh iHOs (Fig. 1F).”
Quote in the paperSubstantial claim - Findingown data
All genes except ALB showed higher expression in Cryo-iHO-Heps and Fresh-iHO-Heps than in the original HLCs.
“Importantly, all of the genes, except for ALB, showed higher expression in Cryo-iHO-Heps and Fresh-iHO-Heps than in the original HLCs (Fig. 2C).”
Quote in the paperSubstantial claim - Findingown data
When cryopreserved iHOs were directly matured by the 2D culture protocol, they showed higher hepatocyte marker gene expression levels than HLCs.
“When cryopreserved iHOs were directly matured by our 2D culture protocol, they showed higher hepatocyte marker gene expression levels and higher CYP3A4 activity than HLCs.”
Quote in the paperSubstantial claim - Findingown data
Similar results were obtained in iHOs derived from another iPS cell line, YOW-iPS.
“Similar results were obtained in iHOs derived from another iPS cell line, YOW-iPS (Supplementary Fig. S2).”
Quote in the paperNot a substantial claim - Findingown data
Cryopreserved iHOs showed a little slower growth than fresh iHOs.
“although cryopreserved iHOs showed a little slower growth than fresh iHOs (Fig. 1D).”
Quote in the paperNot a substantial claim - Findingown data
Urea secretion levels in Cryo-iHO-Heps were the same as in Fresh-iHO-Heps.
“The levels of urea and ALB secretion in Cryo-iHO-Heps were the same as in Fresh-iHO-Heps (Figs. 3B, 3C).”
Quote in the paperSubstantial claim - Findingown data
Cryo-iHO-Heps showed a hepatocyte-like polygonal morphology similar to Fresh-iHO-Heps.
“The resulting iHO-Heps, Cryo-iHO-Heps, showed a hepatocyte-like polygonal morphology similar to Fresh-iHO-Heps without cryopreservation (Fig. 2B).”
Quote in the paperSubstantial claim - Findingown data
Cryopreserved iHOs had thick cell layers similar to fresh iHOs.
“Cryopreserved iHOs had thick cell layers similar to fresh iHOs (Fig. 1B).”
Quote in the paperSubstantial claim - Findingnone given
Cryopreserved iHOs maintained high hepatocyte marker gene expressions.
“In summary, cryopreserved iHOs maintained high hepatocyte marker gene expressions.”
Quote in the paperSubstantial claim - Findingown data
Some functions in Cryo-iHO-Heps were comparable to those of PHHs.
“Some of the functions in Cryo-iHO-Heps were comparable to those of PHHs.”
Quote in the paperSubstantial claim - Interpretationown data
The functions of iHOs were maintained after cryopreservation.
“These findings suggest that the functions of iHOs were maintained after cryopreservation.”
Quote in the paperSubstantial claim - Interpretationown data
Cryopreserved iHOs also differentiated into mature hepatocytes by 2D culture.
“These results suggest that cryopreserved iHOs also differentiated into mature hepatocytes by 2D culture.”
Quote in the paperSubstantial claim - Interpretationown data
Except for ALB secretion, iHOs could be matured into iHO-Heps even after cryopreservation.
“Except in regard to ALB secretion, iHOs could be matured into iHO-Heps even after cryopreservation and Cryo-iHO-Heps had the similar or higher levels of hepatic functions as compared with Fresh iHO-Heps and HLCs.”
Quote in the paperSubstantial claim - Premisecitation
The expressions and activities of drug-metabolizing enzymes are different in humans and animals.
“One of the main reasons is that the expressions and activities of drug-metabolizing enzymes are different in humans and animals.6)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [6] 6) Martignoni M, Groothuis GMM, de Kanter R. Species differences between mouse, rat, dog, monkey and human CYP-mediated drug metabolism, inhibition and induction. <i>Expert Opin. Drug Metab. Toxicol.</i>, <b>2</b>, 875–894 (2006). source
- Premisecitation
Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market.
“Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market.1)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [1] 1) Ruoß M, Vosough M, Königsrainer A, Nadalin S, Wagner S, Sajadian S, Huber D, Heydari Z, Ehnert S, Hengstler JG, Nussler AK. Towards improved hepatocyte cultures: progress and limitations. <i>Food Chem. Toxicol.</i>, <b>138</b>, 111188 (2020). source
- Interpretationown data
The cell proliferation rate of cryopreserved iHOs was sufficiently high.
“The cell proliferation rate of cryopreserved iHOs was also sufficiently high enough.”
Quote in the paperSubstantial claim - Interpretationown data
A large amount of cryopreserved iHOs can be prepared from a small amount of cryopreserved stock.
“Since the proliferation rate of cryopreserved iHOs was about 10 4 -fold in 18 d, we conclude that a large amount of cryopreserved iHOs can be prepared from a small amount of cryopreserved stock.”
Quote in the paperSubstantial claim - Interpretationown data
Cryo-iHO-Heps had similar or higher levels of hepatic functions compared with Fresh-iHO-Heps and HLCs.
“Except in regard to ALB secretion, iHOs could be matured into iHO-Heps even after cryopreservation and Cryo-iHO-Heps had the similar or higher levels of hepatic functions as compared with Fresh iHO-Heps and HLCs.”
Quote in the paperSubstantial claim
- May combine two claims. 6
- Classified as probably not a substantial claim. 4
May combine two claims.
PremisecitationiHOs from HLCs maintained high cell proliferative capacity and expression of hepatocyte marker genes for a long time.
“The hepatic organoids (iHOs) from HLCs maintained high cell proliferative capacity and expression of hepatocyte marker genes for a long time.”
Quote in the paperSubstantial claim1 of 1 sources checked- cites [15] 15) Inui J, Ueyama-Toba Y, Imamura C, Nagai W, Asano R, Mizuguchi H. Two-dimensionally cultured functional hepatocytes generated from human induced pluripotent stem cell-derived hepatic organoids for pharmaceutical research. <i>Biomaterials</i>, <b>318</b>, 123148 (2025). source
Classified as probably not a substantial claim.
PremisecitationPHHs are the best in vitro hepatocyte models in current drug discovery research.
“Primary (cryopreserved) human hepatocytes (PHHs) are the best in vitro hepatocyte models in current drug discovery research.7,8)”
Quote in the paperSubstantial claim1 of 2 sources checked- cites [7] 7) Gómez-Lechón MJ, Tolosa L, Conde I, Donato MT. Competency of different cell models to predict human hepatotoxic drugs. <i>Expert Opin. Drug Metab. Toxicol.</i>, <b>10</b>, 1553–1568 (2014). source
- cites [8] 8) Yokoyama Y, Sasaki Y, Terasaki N, Kawataki T, Takekawa K, Iwase Y, Shimizu T, Sanoh S, Ohta S. Comparison of drug metabolism and its related hepatotoxic effects in HepaRG, cryopreserved human hepatocytes, and HepG2 cell cultures. <i>Biol. Pharm. Bull.</i>, <b>41</b>, 722–732 (2018). source
The paper’s introductory passage (labelled Abstract in our copy; page unavailable) calls human hepatocytes the gold standard of human hepatic models for toxicological studies. It does not say cryopreserved hepatocytes are the best models across current drug discovery research: the Discussion notes their variability and says HepaRG cells may be more feasible for early drug-discovery screening. (LLM, conf=0.86)
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataCYP2C9 and CYP2D6 activities in Cryo-iHO-Heps were similar or higher than in Fresh-iHO-Heps.
“the CYP2C9 and CYP2D6 activities in Cryo-iHO-Heps were not as high as those in PHHs, although they were higher than those in the original HLCs and similar or higher than those in Fresh-iHO-Heps (Fig. 3A).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataCYP2C9 and CYP2D6 activities in Cryo-iHO-Heps were higher than in the original HLCs.
“the CYP2C9 and CYP2D6 activities in Cryo-iHO-Heps were not as high as those in PHHs, although they were higher than those in the original HLCs and similar or higher than those in Fresh-iHO-Heps (Fig. 3A).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataSimilar results were obtained in another set of experiments.
“Similar results were obtained in another set of experiments (Supplementary Fig. S4B).”
Quote in the paperNot a substantial claimMay combine two claims.
PremisecitationUp to 85% of cardiovascular and gastrointestinal adverse effects and more than 90% of adverse effects related to hematologic toxicity in drug development can be predicted in animal studies.
“Up to 85% of cardiovascular and gastrointestinal adverse effects and more than 90% of adverse effects related to hematologic toxicity in drug development can be predicted in animal studies, 2)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [2] 2) Olson H, Betton G, Robinson D, Thomas K, Monro A, Kolaja G, Lilly P, Sanders J, Sipes G, Bracken W, Dorato M, Van Deun K, Smith P, Berger B, Heller A. Concordance of the toxicity of pharmaceuticals in humans and in animals. <i>Regul. Toxicol. Pharmacol.</i>, <b>32</b>, 56–67 (2000). source
May combine two claims.
PremisecitationHLCs have lower hepatic function than PHHs and almost no proliferative capacity.
“However, HLCs have lower hepatic function than PHHs and almost no proliferative capacity. 13)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites [13] 13) Song Z, Cai J, Liu Y, Zhao D, Yong J, Duo S, Song X, Guo Y, Zhao Y, Qin H, Yin X, Wu C, Che J, Lu S, Ding M, Deng H. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. <i>Cell Res.</i>, <b>19</b>, 1233–1242 (2009). source
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAnimal studies remain the gold standard for preclinical study.
“and this remains the gold standard for preclinical study.3,4)”
Quote in the paperNot a substantial claim0 of 2 sources checked- cites [3] 3) Tralau T, Luch A. Drug-mediated toxicity: illuminating the “bad” in the test tube by means of cellular assays? <i>Trends Pharmacol. Sci.</i>, <b>33</b>, 353–364 (2012). source
- cites [4] 4) Hartung T, Daston G. Are <i>in vitro</i> tests suitable for regulatory use? <i>Toxicol. Sci.</i>, <b>111</b>, 233–237 (2009). source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenAll results are based on three biological replicates.
“All results are based on three biological replicates.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationreasoningCryopreserved iHOs can be directly seeded onto plates and matured with the 2D culture protocol, making it possible to prepare highly functional hepatocytes without complicated manipulations.
“In addition, cryopreserved iHOs can be directly seeded onto plates and matured with our 2D culture protocol, making it possible to prepare highly functional hepatocytes without complicated manipulations.”
Quote in the paperSubstantial claim- Rests on an assumption: Culturing organoids is more technically demanding than culturing conventional cells.
- Rests on an assumption: Organoids embedded in Matrigel are difficult to apply to pharmaceutical research due to sequestration of drugs in the gels, low throughput, and limited available model systems.
The paper relies on this without support.
PHHs rapidly lose hepatic functions after seeding and lack proliferative capacity.
“They rapidly lose hepatic functions after seeding and lack proliferative capacity.”
The paper relies on this without support.
Long-term culture of PHHs is difficult.
“In addition, long-term culture of PHHs has been considered difficult.”
The paper relies on this without support.
Culturing organoids is more technically demanding than culturing conventional cells.
“Culturing of organoids is more technically demanding than culturing conventional cells, and this drawback has hindered the widespread use of organoids and their derivatives.”
The paper relies on this without support.
Organoids embedded in Matrigel are difficult to apply to pharmaceutical research due to sequestration of drugs in the gels, low throughput, and limited available model systems.
“In addition, organoids embedded in Matrigel are difficult to apply to pharmaceutical research due to sequestration of the drugs in the gels, low throughput, and limited available model systems.”
The paper relies on this without support.
Cryopreservation would facilitate transportation, making iHOs available to a larger number of facilities.
“Moreover, cryopreservation would facilitate transportation, making iHOs available to a larger number of facilities.”
Cites
Modeling Development and Disease with Organoids
Clevers, H. (2016) · Cell · doi:10.1016/j.cell.2016.05.082 · Cited by 3,196 · see citing works
Concordance of the toxicity of pharmaceuticals in humans and in animals
Olson, H., Betton, G., Robinson, D., Thomas, K., Monro, A., Kolaja, G., Lilly, P., Sanders, J., Sipes, G., Bracken, W., et al. (2000) · Regulatory Toxicology and Pharmacology · doi:10.1006/rtph.2000.1399 · Cited by 2,050 · see citing works
Martignoni, M., Groothuis, G. M. M., & de Kanter, R. (2006) · Expert Opinion on Drug Metabolism & Toxicology · doi:10.1517/17425255.2.6.875 · Cited by 1,478 · see citing works
Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells
Si-Tayeb, K., Noto, F. K., Nagaoka, M., Li, J., Battle, M. A., Duris, C., North, P. E., Dalton, S., & Duncan, S. A. (2010) · Hepatology · doi:10.1002/hep.23354 · Cited by 1,282 · see citing works
Progress and potential in organoid research
Rossi, G., Manfrin, A., & Lutolf, M. P. (2018) · Nature Reviews Genetics · doi:10.1038/s41576-018-0051-9 · Cited by 1,182 · see citing works
Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids
Hu, H., Gehart, H., Artegiani, B., LÖpez-Iglesias, C., Dekkers, F., Basak, O., van Es, J., Chuva de Sousa Lopes, S. M., Begthel, H., Korving, J., et al. (2018) · Cell · doi:10.1016/j.cell.2018.11.013 · Cited by 837 · see citing works
Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells
Song, Z., Cai, J., Liu, Y., Zhao, D., Yong, J., Duo, S., Song, X., Guo, Y., Zhao, Y., Qin, H., et al. (2009) · Cell Research · doi:10.1038/cr.2009.107 · Cited by 499 · see citing works
Are In Vitro Tests Suitable for Regulatory Use?
Hartung, T., & Daston, G. (2009) · Toxicological Sciences · doi:10.1093/toxsci/kfp149 · Cited by 202 · see citing works
Competency of different cell models to predict human hepatotoxic drugs
Gómez-Lechón, M. J., Tolosa, L., Conde, I., & Donato, M. T. (2014) · Expert Opinion on Drug Metabolism & Toxicology · doi:10.1517/17425255.2014.967680 · Cited by 199 · see citing works
Takayama, K., Morisaki, Y., Kuno, S., Nagamoto, Y., Harada, K., Furukawa, N., Ohtaka, M., Nishimura, K., Imagawa, K., Sakurai, F., et al. (2014) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1413481111 · Cited by 193 · see citing works
Self-citationYokoyama, Y., Sasaki, Y., Terasaki, N., Kawataki, T., Takekawa, K., Iwase, Y., Shimizu, T., Sanoh, S., & Ohta, S. (2018) · Biological and Pharmaceutical Bulletin · doi:10.1248/bpb.b17-00913 · Cited by 160 · see citing works
Liver Organoids: Recent Developments, Limitations and Potential
Harrison, S. P., Baumgarten, S., Verma, R., Lunov, O., Dejneka, A., & Sullivan, G. J. (2021) · Frontiers in Medicine · doi:10.3389/fmed.2021.574047 · Cited by 141 · see citing works
Liver organoid as a 3D in vitro model for drug validation and toxicity assessment
Brooks, A., Liang, X., Zhang, Y., Zhao, C., Roberts, M. S., Wang, H., Zhang, L., & Crawford, D. H. (2021) · Pharmacological Research · doi:10.1016/j.phrs.2021.105608 · Cited by 109 · see citing works
Olgasi, C., Cucci, A., & Follenzi, A. (2020) · International Journal of Molecular Sciences · doi:10.3390/ijms21176215 · Cited by 100 · see citing works
Engineered Liver Platforms for Different Phases of Drug Development
Ware, B. R., & Khetani, S. R. (2017) · Trends in Biotechnology · doi:10.1016/j.tibtech.2016.08.001 · Cited by 90 · see citing works
Towards improved hepatocyte cultures: Progress and limitations
Ruoß, M., Vosough, M., Königsrainer, A., Nadalin, S., Wagner, S., Sajadian, S., Huber, D., Heydari, Z., Ehnert, S., Hengstler, J. G., et al. (2020) · Food and Chemical Toxicology · doi:10.1016/j.fct.2020.111188 · Cited by 77 · see citing works
Tricot, T., Verfaillie, C. M., & Kumar, M. (2022) · Cells · doi:10.3390/cells11030442 · Cited by 35 · see citing works
Advancements in Disease Modeling and Drug Discovery Using iPSC-Derived Hepatocyte-like Cells
Blaszkiewicz, J., & Duncan, S. A. (2022) · Genes · doi:10.3390/genes13040573 · Cited by 28 · see citing works
Drug-mediated toxicity: illuminating the ‘bad’ in the test tube by means of cellular assays?
Tralau, T., & Luch, A. (2012) · Trends in Pharmacological Sciences · doi:10.1016/j.tips.2012.03.015 · Cited by 24 · see citing works
Inui, J., Ueyama-Toba, Y., Mitani, S., & Mizuguchi, H. (2023) · PLoS ONE · doi:10.1371/journal.pone.0285783 · Cited by 5 · see citing works
Self-citationInui, J., Ueyama-Toba, Y., Imamura, C., Nagai, W., Asano, R., & Mizuguchi, H. (2025) · Biomaterials · doi:10.1016/j.biomaterials.2025.123148 · Cited by 5 · see citing works
Self-citation
Flagged first, then by citations
Cited by
Sorted by citations
Walsh, E., Rocha, C., Lépine, P., Sirois, J., Durcan, T. M., & Ben, R. N. (2026) · Organoids · doi:10.3390/organoids5030032
- Authors
- Nagai, W., Inui, J., Ueyama-Toba, Y., Asano, R., & Mizuguchi, H.
- Affiliations
- The University of Osaka (JP)
- National Institute of Biomedical Innovation, Health and Nutrition (JP)
- Venue
- Biological and Pharmaceutical Bulletin
- Publisher
- Pharmaceutical Society of Japan
- Published
- 2025-09-17
- Volume
- 48
- Issue
- 9
- Pages
- 1391-1398
- ISSN
- 0918-6158 (ISSN-L)
- 1347-5215
- OpenAlex
- W4414224775
- Semantic Scholar
- 06ab88868e05c46a19b12889f4487bb7df62072e
- Cited by
- 1
- References
- 21
- Citations by year
- 2026: 1
- FWCI
- 0.26
- Type
- journal article
- Language
- en
- Open access
- diamond
- doi.org
- Licence
- cc-by-nc
- Locations
- Biological and Pharmaceutical Bulletin (publishedVersion, cc-by-nc)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Health Sciences › Medicine › Hepatology › Liver physiology and pathology
- Keywords
- cryopreservation, hepatocyte organoids, induced pluripotent stem cells, hepatocyte-like cells, monolayer culture, hepatocyte differentiation, CYP3A4 activity, primary human hepatocytes, drug discovery
- MeSH terms
- Cell Differentiation, Cells, Cultured, Humans, Organoids, Cryopreservation, Cell Culture Techniques, Hepatocytes, Cell Proliferation, Cytochrome P-450 CYP3A, Induced Pluripotent Stem Cells
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-23.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-23.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
