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Cited by 0Liver physiology and pathologyDrug-Induced Hepatotoxicity and ProtectionTissue Engineering and Regenerative Medicine

The Hepatocyte Expansion Paradox: A Review of In Vitro Challenges and Advances

Open access
Kolahdouzmohammadi, Mina, Tjandra, Nicholas, Wu, Kevan, Nikoumaram, Raha, Oldani, Graziano·2026·Cells
DOI 10.3390/cells15161465PMC PMC13510449
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Abstract

The adult liver exhibits remarkable regenerative capacity in vivo; however, primary hepatocytes (PHs), the principal functional cells of the liver, swiftly forfeit their proliferative potential and specialized hepatic functions when isolated from their native microenvironment and cultured in vitro. While PHs are the benchmark for studying hepatic physiology, xenobiotic metabolism, and toxicological responses, their rapid dedifferentiation resulting in the loss of hepatic functions significantly limits their further application. Recent studies suggest three converging strategies to address the challenge of long-term maintenance and expansion of PHs. First, defined chemical and growth factor-based protocols can temporarily induce hepatocytes into a proliferative, progenitor-like state, followed by a maturation phase that restores differentiated hepatic functions. In addition, the inhibition of signaling pathways linked to cellular stress responses and identity loss can postpone dedifferentiation and preserve drug-metabolizing activity for prolonged durations, thereby enhancing disease modeling and toxicology studies. Finally, three-dimensional (3D) culture platforms generally improve hepatocyte maturation and functional stability, but these are often not scalable due to matrix dependence, technical complexity, handling requirements, and cost. This review thoroughly examines innovative methodological advancements designed to facilitate the proliferation and prolonged viability of healthy PHs, focusing on their translational relevance and clinical applicability.

AI snapshot

Written from the paper’s full text.

TL;DR

Primary hepatocytes rapidly lose proliferation and specialized hepatic functions in vitro, but defined growth-factor/chemical protocols can reversibly induce a proliferative progenitor-like state, while pathway inhibition and 3D systems can extend functional maintenance. The review recommends integrating reprogramming, pathway modulation, and 3D-culture strategies, while cautioning that scalability, donor variability, and mouse-to-human translation remain unresolved.

Key findings

Primary hepatocytes deteriorate within 24–48 h in 2D monolayer culture, with loss of polygonal shape, tight junctions, bile canaliculi, and downregulation of liver-specific genes such as albumin and HNF4A.

  • Defined protocols can reversibly expand hepatocytes: one protocol maintained mature identity for several months with expansion approaching 10^6-fold over ~4 weeks, classified as true expansion (TE); IL-6/EGF/HGF-derived induced hepatic progenitor cells proliferated for approximately 30 passages before redifferentiating and restoring albumin secretion, CYP450 activity, glycogen storage, and LDL uptake.
  • Inhibiting stress/identity-loss pathways or mechanical tension can postpone dedifferentiation; inhibiting YAP activation preserved polarity and metabolic activity without re-entering the cell cycle, sustaining mature function for around two weeks in certain matrix-independent platforms.
  • 3D culture, spheroids, and organoid-like aggregates improve hepatocyte maturation and functional stability, but no single 3D method fully replicates primary hepatocyte function; the most effective systems combine 3D architecture, customized ECM, biomechanical signals, inflammatory pathways, and multicellular interactions.

Methods

Narrative review of methodological breakthroughs from the past decade aimed at long-term culture and functional maintenance of healthy primary hepatocytes; no primary experimental data are reported.

  • Synthesizes studies using chemical modulation of culture media, co-culture and 3D systems, transcriptional reinforcement, and optimized isolation, distinguishing true expansion (TE) from functional maintenance (FM).
  • Main analysis is qualitative synthesis, with tables summarizing pharmacological and 3D/co-culture studies.

Limitations

Stated: 3D platforms often are not scalable due to matrix dependence, technical complexity, handling requirements, and cost; Matrigel dependence and multi-factor cytokine compositions constrain scalability and cost-effectiveness.

  • Stated: primary human hepatocytes show substantial donor-to-donor variability, and findings from mouse expansion systems should be validated independently in human systems before cross-species conclusions; genomic stability and tumorigenic risk require attention during long-term expansion, especially with sustained Wnt/YAP activation, serial passaging, epigenetic modulation, or lentiviral Foxa3/Hnf4a overexpression.
  • Evident from the text: as a narrative review, it does not report a systematic search strategy, inclusion criteria, or quantitative meta-analysis.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.3390/cells15161465

Cites

42 of 42 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

43 of 64 cited sources checked · 10 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Primary hepatocytes rapidly lose proliferative potential and specialized hepatic functions when isolated from their native microenvironment and cultured in vitro.

    “primary hepatocytes (PHs), the principal functional cells of the liver, swiftly forfeit their proliferative potential and specialized hepatic functions when isolated from their native microenvironment and cultured in vitro.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [2] Kaur, I., Vasudevan, A., Rawal, P., Tripathi, D.M., Ramakrishna, S., Kaur, S., and Sarin, S.K. (2023). Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements. Bioengineering, 10. source

      The Introduction (no page given) says that proliferation of functional adult hepatocytes is difficult to maintain in vitro and that isolated cells tend to change phenotype after a few days. Section 4.3 (no page given) says conventional cultures do not provide optimal hepatocyte function. These passages support a rapid decline, but not the categorical claim that primary hepatocytes forfeit both proliferative potential and specialized functions. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    Synchronized stimulation of IL-6, EGF, and HGF stimulates STAT3, ERK, and PI3K/AKT signaling pathways, facilitating transformation of adult hepatocytes into induced hepatic progenitor cells.

    “The synchronized stimulation of IL-6, EGF, and HGF stimulates STAT3, ERK, and PI3K/AKT signaling pathways, facilitating the transformation of adult hepatocytes into induced hepatic progenitor cells (iHPCs) [18].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Guo, 2022, IL6 supports long-term expansion of hepatocytes in vitro, Nat. Commun. source
  • Premisecitation

    Primary hepatocytes undergo severe morphological and functional deterioration within 24-48 h in two-dimensional monolayer culture, characterized by loss of polygonal shape, disruption of tight junctions, collapse of bile canaliculi, and gradual downregulation of liver-specific genes.

    “PHs undergo severe morphological and functional deterioration within 24–48 h in two-dimensional (2D) monolayer culture, characterized by a loss of polygonal shape, disruption of tight junctions, collapse of bile canaliculi, and gradual downregulation of liver-specific genes [13].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [13] Feng, 2019, The breakthrough in primary human hepatocytes in vitro expansion, Cancer Biol. Med. source

      The Introduction says primary human hepatocytes are difficult to culture and expand in vitro, but it does not describe deterioration within 24–48 hours in 2D monolayers. Searches for that timeframe and for the claimed changes in shape, tight junctions, bile canaliculi, and liver-specific genes found no supporting passage in our copy. (LLM, conf=0.72)

      Experimental — accuracy not yet measured
  • Premisecitation

    Wnt/β-catenin stimulation together with conventional mitogens such as HGF and EGF initiates a regeneration-like phase in which quiescent hepatocytes re-enter the cell cycle and expand while maintaining the ability to regain a mature hepatocyte phenotype upon cessation of mitogenic stimuli.

    “These signals initiate a regeneration-like phase: quiescent hepatocytes re-enter the cell cycle and experience significant expansion while maintaining the ability to regain a mature hepatocyte phenotype upon the cessation of mitogenic stimuli [10].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [10] Fu, 2018, Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens, Cell Res. source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
  • Premisecitation

    Primary hepatocytes exhibit considerable inter-donor variation in proliferative capacity, drug metabolism and stress tolerance, influenced by donor age, genetic background, clinical status and drug exposure history.

    “PHs exhibit considerable inter-donor variation in proliferative capacity, drug metabolism and stress tolerance, which are influenced by donor age, genetic background, clinical status and drug exposure history [15].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Xie, 2024, A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities, Stem Cell Res. Ther. source

      The Results section, “Hydrocortisone promotes PHHs expansion in vitro” (page unavailable), explicitly considers donor-related variability in hepatocyte expansion; the Discussion also refers to variability between PHH donor batches. The paper does not establish inter-donor variation in drug metabolism or stress tolerance, or attribute such variation to donor age, genetics, clinical status, or drug exposure history. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • May combine two claims. 26

  • May combine two claims.

    Premisecitation

    Hepatocytes isolated from a donor liver show transient viability and functionality in culture and usually decline within days due to loss of polarity, reduced expression of liver-specific genes, and cellular senescence.

    “Hepatocytes isolated from a donor liver show transient viability and functionality in culture, and usually decline within days due to loss of polarity, reduced expression of liver-specific genes, and cellular senescence [3,4].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [3] Cooreman, 2019, Primary hepatocytes and their cultures for the testing of drug-induced liver injury, Adv. Pharmacol.

      Not checked in the free run.

    • cites [4] Zhang, 2018, In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity, Cell Stem Cell source

      The Introduction describes difficulty expanding primary human hepatocytes in vitro, and the Results report senescence and cessation of proliferation during culture. The Discussion reports reduced maturation-related gene expression during passage. The paper does not establish that donor hepatocytes usually lose viability and function within days, or that loss of polarity causes that decline; no page numbers are available in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    SIRT1 activity is essential for transformation of primary hepatocytes into proliferative progenitor-like cells; inhibition of SIRT1 results in growth cessation and triggers apoptosis.

    “The activity of SIRT1 is essential for the transformation of PHs into proliferative progenitor-like cells; the inhibition of SIRT1 results in growth cessation and triggers apoptosis [10].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [10] Fu, 2018, Expansion and differentiation of human hepatocyte-derived liver progenitor-like cells and their use for the study of hepatotropic pathogens, Cell Res. source

  • May combine two claims.

    Premisecitation

    After partial hepatectomy or injury, hepatocytes can re-enter the cell cycle and proliferate extensively in vivo, but this regenerative response requires a tightly controlled interplay of mitogenic growth factors, cytokines, metabolic cues, and mechanical stimuli that are difficult to recapitulate ex vivo.

    “After partial hepatectomy or injury, hepatocytes can re-enter the cell cycle and proliferate extensively in vivo, but this regenerative response requires a tightly controlled interplay of mitogenic growth factors, cytokines, metabolic cues, and mechanical stimuli that are difficult to recapitulate ex vivo [12].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [12] Michalopoulos, 2017, Hepatostat: Liver regeneration and normal liver tissue maintenance, Hepatology source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Following withdrawal into hepatic maturation media, expression of albumin and HNF4A was restored, while levels of alpha-fetoprotein and Ki-67 diminished.

    “Following withdrawal into hepatic maturation media, the expression of albumin and HNF4A was restored, although levels of alpha-fetoprotein (AFP) and Ki-67 diminished.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Jiang, 2023, Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation, JHEP Rep. source

  • May combine two claims.

    Premisecitation

    Hepatocytes cultured in 2D systems swiftly impair liver-specific gene expression, CYP450 activity, and synthetic capability, leading to reduced viability and inadequate predictive accuracy for drug testing.

    “Across multiple studies, hepatocytes cultured in 2D systems swiftly impair liver-specific gene expression, CYP450 activity, and synthetic capability, ultimately leading to reduced viability and inadequate predictive accuracy for drug testing [24,25].”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [24] Vorrink, 2017, Endogenous and xenobiotic metabolic stability of primary human hepatocytes in long-term 3D spheroid cultures revealed by a combination of targeted and untargeted metabolomics, FASEB J. source

      The Results report rapid loss of liver-specific gene expression and hepatic functions in 2D culture, while the Discussion reports lower CYP activity than in 3D culture. The Abstract says 2D dedifferentiation impairs prediction of human drug metabolism. The cited paper does not establish the claimed reduction in cell viability or show that the listed changes cause it. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [25] Chang, 2017, Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity, Toxicol. Vitr. source

  • May combine two claims.

    Premisecitation

    Induced hepatic progenitor cells proliferate for approximately 30 passages and thereafter redifferentiate to restore albumin secretion, CYP450 activity, glycogen storage, and LDL uptake, while rescuing Fah−/− mice following transplantation, supporting classification as true expansion.

    “These iHPCs proliferate for approximately 30 passages and thereafter redifferentiate to restore albumin secretion, CYP450 activity, glycogen storage, and low-density lipoprotein (LDL) uptake, while also rescuing Fah−/− mice following transplantation, supporting classification as TE.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Guo, 2022, IL6 supports long-term expansion of hepatocytes in vitro, Nat. Commun. source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Dose-response studies indicate that IL-6 initiates proliferation, but elevated IL-6 in conjunction with EGF/HGF maintains prolonged cycling.

    “Dose–response studies indicate that IL-6 initiates proliferation, but elevated IL-6 in conjunction with EGF/HGF maintains prolonged cycling.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [18] Guo, 2022, IL6 supports long-term expansion of hepatocytes in vitro, Nat. Commun. source

  • May combine two claims.

    Premisecitation

    ROCK inhibitor Y-27632 and GSK3β inhibitor CHIR99021 reduce actomyosin tension and stabilize β-catenin, creating a proliferative but reversible progenitor-like state.

    “Jiang et al. employed ROCK inhibitor Y-27632 and GSK3β inhibitor CHIR99021 to reduce actomyosin tension and stabilize β-catenin, therefore creating a proliferative but reversible progenitor-like state [19].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Jiang, 2023, Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation, JHEP Rep. source

      The Results section on reprogramming reports that Y27632 and CHIR99021 produce proliferating hepatic progenitor-like cells, and the Abstract Results says those cells can return to a mature state. The Discussion identifies ROCK inhibition and possible WNT/β-catenin activation, but does not establish that the drugs reduced actomyosin tension or stabilized β-catenin. Page numbers are unavailable in our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Hepatocyte-derived organoids undergo an initial 30-day quiescent phase post-transplantation, followed by rapid and extensive liver repopulation akin to that of primary hepatocytes.

    “Hu et al. demonstrated that hepatocyte-derived organoids undergo an initial 30-day quiescent phase post-transplantation, succeeded by rapid and extensive liver repopulation akin to that of PHs [16].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [16] Hu, 2018, Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids, Cell source

      The section “Human Hep-Orgs Engraft and Repopulate Damaged Mouse Liver” (p. 12) reports stable serum human albumin for the first 30 days in mice receiving fetal organoids, followed by graft proliferation at the same rate as primary-cell controls. It also reports an initial lag and subsequent proliferation for organoids derived from primary human hepatocytes (p. 13), but does not establish that their lag lasted 30 days or that either graft was strictly quiescent. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Fmoc-FF hydrogels alone failed to maintain hepatocyte viability or function, but addition of integrin-binding RGD dramatically increased cell survival and CYP450 activity, surpassing both 2D cultures and Matrigel for long-term cultures.

    “Self-assembling peptide hydrogels formed from Fmoc-FF alone failed to maintain hepatocyte viability or function, but the addition of integrin-binding motifs such as RGD (Arg-Gly-Asp) dramatically increased cell survival and CYP450 activity, surpassing both 2D cultures and Matrigel for long-term cultures [30].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [30] MacPherson, 2021, Peptide-based scaffolds for the culture and maintenance of primary human hepatocytes, Sci. Rep. source

      The Results section (page not provided) reports low viability in Fmoc-FF alone and significantly higher survival and CYP450 activity with Fmoc-FF/RGD. It does not support an unqualified claim that the RGD hydrogel surpassed both comparators in long-term cultures: at day 14, CYP450 function exceeded Matrigel but compared well with the 2D control, while survival was comparable to Matrigel. (LLM, conf=0.89)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    PSC-derived hepatocyte-like cells generally show lower functional recapitulation than primary hepatocytes, and most fail to self-renew post-differentiation, requiring repeated rounds of de novo differentiation.

    “PSC-derived hepatocyte-like cells illustrate this well; they generally show lower functional recapitulation than PHs, and most fail to self-renew post-differentiation, requiring repeated rounds of de novo differentiation that constrain their direct substitutability with adult hepatocyte systems [40].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [40] Nuciforo, 2021, Organoids to model liver disease, JHEP Rep. source

  • May combine two claims.

    Premisecitation

    Multiple comparative studies indicate that spheroid cultures sustain elevated liver-specific functions such as albumin secretion, urea production, and drug-metabolizing enzyme activity, while demonstrating enhanced sensitivity to hepatotoxic compounds and a longer lifespan.

    “Multiple comparative studies indicate that spheroid cultures sustain elevated liver-specific functions, such as albumin secretion, urea production, and drug-metabolizing enzyme activity, while simultaneously demonstrating enhanced sensitivity to hepatotoxic compounds and a longer lifespan [26,27].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [26] Bell, 2016, Characterization of primary human hepatocyte spheroids as a model system for drug-induced liver injury, liver function and disease, Sci. Rep. source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [27] Bell, 2018, Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study, Toxicol. Sci. source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    3D liver organoid systems better preserve cell identity and enable longer-term propagation compared with traditional 2D models while also supporting patient-specific disease modeling and drug testing.

    “Among the most impactful advances has been the rise of 3D liver organoid systems, which better preserve cell identity and enable longer-term propagation compared with traditional 2D models while also supporting patient-specific disease modeling and drug testing approaches [40,41].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [40] Nuciforo, 2021, Organoids to model liver disease, JHEP Rep. source

      The Introduction and Table 1 describe long-term expansion of some liver organoids, and the Future perspectives section supports patient-derived disease modeling and drug testing (page numbers unavailable). The comparison with traditional 2D models is too broad: Table 1 rates cancer cell lines as highly maintainable, while the Adult-derived liver organoids section says primary human hepatocytes retain a more mature phenotype than hepatocyte organoids and notes that adult-derived organoids have limited expansion. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [41] Afonso, 2023, Human liver organoids: From generation to applications, Hepatology source

      The Introduction says 3D cultures better resemble liver tissue than 2D cultures, and the Liver Disease Modeling and Drug Discovery sections describe patient-derived organoids that retain individual genetic backgrounds and support drug testing. The section on organoids derived from primary liver tissue also describes their expansion, but does not establish a general longer-term propagation advantage over 2D models; it reports that adult human hepatocyte organoids deteriorated after 2.5 months. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Under optimized conditions, PHH-derived SHPCs exhibited polarized morphology, functional bile canaliculi, persistent expression of hepatocyte-specific transcription factors and transporters, and maintained CYP450 activity, leading to drug metabolism capacity comparable to that of primary hepatocytes.

    “Under optimized conditions, PHH-derived SHPCs exhibited polarized morphology, functional bile canaliculi, persistent expression of hepatocyte-specific transcription factors, and transporters and maintained CYP450 activity, leading to drug metabolism capacity comparable to that of PHs.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] Sengupta, 2020, Co-culture with mouse embryonic fibroblasts improves maintenance of metabolic function of human small hepatocyte progenitor cells, Curr. Res. Toxicol. source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Human primary hepatocytes show substantial donor-to-donor variability not typically observed to the same degree in inbred mouse models, and this variability can persist even within engineered expansion or maturation protocols derived from the same donor.

    “Human primary hepatocytes also show substantial donor-to-donor variability that is not typically observed to the same degree in inbred mouse models, and this variability can persist even within engineered expansion or maturation protocols derived from the same donor [4,15].”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [4] Zhang, 2018, In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity, Cell Stem Cell source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [15] Xie, 2024, A two-step strategy to expand primary human hepatocytes in vitro with efficient metabolic and regenerative capacities, Stem Cell Res. Ther. source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Several dramatic expansion results, including near-unlimited theoretical fold-expansion and multi-month clonal proliferation, have been demonstrated almost exclusively in mouse hepatocytes, whereas human systems have generally achieved more modest expansion with measurable trade-offs in redifferentiation and repopulation capacity at higher expansion levels.

    “Several of the most dramatic expansion results reported to date—including near-unlimited theoretical fold-expansion and multi-month clonal proliferation—have been demonstrated almost exclusively in mouse hepatocytes [18,31], whereas human systems have generally achieved more modest expansion with measurable trade-offs in redifferentiation and repopulation capacity at higher expansion levels [4,17]”
    Quote in the paperSubstantial claim3 of 4 sources checked
    • cites [18] Guo, 2022, IL6 supports long-term expansion of hepatocytes in vitro, Nat. Commun. source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [31] Peng, 2018, Inflammatory Cytokine TNFα Promotes the Long-Term Expansion of Primary Hepatocytes in 3D Culture, Cell source

      Split into 2 atoms — 0 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 1 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [4] Zhang, 2018, In Vitro Expansion of Primary Human Hepatocytes with Efficient Liver Repopulation Capacity, Cell Stem Cell source
    • cites [17] Igarashi, 2025, Generation of human adult hepatocyte organoids with metabolic functions, Nature source

  • May combine two claims.

    Premisecitation

    Self-assembled microtissues facilitate improved cell-cell interactions and have greater physiological relevance in cell polarity than 2D systems.

    “These self-assembled microtissues facilitate improved cell–cell interactions and have greater physiological relevance in cell polarity than 2D systems [19,20,25,26].”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [19] Jiang, 2023, Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation, JHEP Rep. source

      The Introduction and Results discuss hepatocyte dedifferentiation, proliferation, and culture with small-molecule drugs, but do not address self-assembled microtissues or compare them with 2D systems. Searches for microtissues, cell–cell interactions, and cell polarity found no passage supporting the attributed claim. (LLM, conf=0.74)

      Experimental — accuracy not yet measured
    • cites [20] Sun, 2019, Maintenance of Primary Hepatocyte Functions In Vitro by Inhibiting Mechanical Tension-Induced YAP Activation, Cell Rep. source
    • cites [25] Chang, 2017, Characterization of rat or human hepatocytes cultured in microphysiological systems (MPS) to identify hepatotoxicity, Toxicol. Vitr. source

  • May combine two claims.

    Premisecitation

    Spheroids significantly promote development of bile canaliculi-like networks and maintain stable metabolic zonation, characteristics infrequently preserved in 2D environments.

    “Spheroids significantly promote the development of bile canaliculi-like networks and maintain stable metabolic zonation, both of which are characteristics that are infrequently preserved in 2D environments [19].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [19] Jiang, 2023, Small molecule drugs promote repopulation of transplanted hepatocytes by stimulating cell dedifferentiation, JHEP Rep. source

      The Results section on reprogramming hepatocytes reports small-molecule-induced proliferation and maturation, not spheroid culture. Searches for spheroids, bile canaliculi, metabolic zonation, and 2D comparisons found no passage in our copy addressing the attributed claim; page numbers were unavailable. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationreasoning

    The morphological and functional deterioration of primary hepatocytes in 2D culture eventually results in cellular senescence and severely restricts long-term culture viability.

    “These alterations eventually result in cellular senescence and severely restrict long-term culture viability.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Collagen, laminin, Matrigel, and decellularized liver scaffolds enhance hepatic identity but reduce actomyosin tension, increasing the risk of dedifferentiation.

    “Collagen, laminin, Matrigel, and decellularized liver scaffolds enhance hepatic identity; however, they reduce actomyosin tension, which is achieved through ROCK inhibition, micropatterning, or tension-modulating cocktails, increasing the risk of dedifferentiation.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Sun, 2019, Maintenance of Primary Hepatocyte Functions In Vitro by Inhibiting Mechanical Tension-Induced YAP Activation, Cell Rep. source

  • May combine two claims.

    Premisecitation

    Three-dimensional culture platforms generally improve hepatocyte maturation and functional stability but are often not scalable due to matrix dependence, technical complexity, handling requirements, and cost.

    “Finally, three-dimensional (3D) culture platforms generally improve hepatocyte maturation and functional stability, but these are often not scalable due to matrix dependence, technical complexity, handling requirements, and cost.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Li, 2024, HDAC inhibitors support long-term expansion of porcine hepatocytes in vitro, Biomed. Pharmacother. source

  • May combine two claims.

    Premisecitation

    Valproic acid induces chromatin relaxation in primary hepatocytes, facilitating long-term, karyotypically stable development, effective gene modification, and therapeutic restoration following microencapsulated transplantation.

    “The use of valproic acid induces chromatin relaxation in PHs, facilitating long-term, karyotypically stable development, effective gene modification, and therapeutic restoration following microencapsulated transplantation [21].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [21] Li, 2024, HDAC inhibitors support long-term expansion of porcine hepatocytes in vitro, Biomed. Pharmacother. source

  • May combine two claims.

    Premisecitation

    Inhibiting cell spreading and reducing actin tension, Src kinase, tankyrase and PI3K all inhibit YAP activation, enabling hepatocytes to preserve polarity and metabolic activity without re-entering the cell cycle.

    “This inhibition enables hepatocytes to preserve polarity and metabolic activity without re-entering the cell cycle [20].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [20] Sun, 2019, Maintenance of Primary Hepatocyte Functions In Vitro by Inhibiting Mechanical Tension-Induced YAP Activation, Cell Rep. source

  • May combine two claims.

    Premisecitation

    Scaffold stiffness, degradability, and proteolytic stability are important factors affecting hepatocyte behavior.

    “In addition to structural support, scaffold stiffness, degradability, and proteolytic stability are all important factors affecting hepatocyte behavior, emphasizing the importance of closely mimicking the biomechanical properties of liver tissue in the design of in vitro models [29].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [29] Biswas, 2022, Chemically Modified Dipeptide Based Hydrogel Supports Three-Dimensional Growth and Functions of Primary Hepatocytes, ACS Appl. Bio Mater. source

  • May combine two claims.

    Premisecitation

    3D culture systems suffer poor scalability, non-specific adsorption of small molecules onto scaffold materials, technical complexity, and large batch-to-batch variability of ECM components, impairing reproducibility.

    “Often, the problems cited are poor scalability, non-specific adsorption of SMs onto scaffold materials, technical complexity, and the large batch-to-batch variability of ECM components, impairing reproducibility [21].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Li, 2024, HDAC inhibitors support long-term expansion of porcine hepatocytes in vitro, Biomed. Pharmacother. source

  • May combine two claims.

    Interpretationreasoning

    Many expanded hepatocyte systems still display immature or mixed hepatic phenotypes, variability across donors and protocols, and incomplete recovery of key adult functions such as robust CYP activity and long-term metabolic competence.

    “More broadly, many expanded systems still display immature or mixed hepatic phenotypes, variability across donors and protocols, and incomplete recovery of key adult functions such as robust CYP activity and long-term metabolic competence.”
    Quote in the paperSubstantial claim
26 of 26

  • The paper relies on this without support.

    Animal-derived hepatocytes have species-specific metabolic pathways and regulatory networks that significantly limit their translational value.

    “Hepatocytes derived from animals are more accessible; however, the presence of species-specific metabolic pathways and regulatory networks significantly limits their translational value.”
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Authors
Kolahdouzmohammadi, M., Tjandra, N., Wu, K., Nikoumaram, R., & Oldani, G.
Affiliations
  • University of British Columbia (CA)
  • Simon Fraser University (CA)
Corresponding author
Oldani, Graziano
ORCID
Venue
Cells
Publisher
MDPI AG
Published
2026-08-15
Volume
15
Issue
16
Pages
1465
ISSN
2073-4409 (ISSN-L)
DOI
10.3390/cells15161465
PMC
PMC13510449
OpenAlex
W7203654039
Semantic Scholar
ed7b109ef8679bfaba7c17b6129f85c9eed7a9f8
Cited by
0
References
41
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Cells (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • cIRcle (University of British Columbia) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • Open Collections
Funders
BC Children's Hospital
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Keywords
primary hepatocytes, hepatocyte expansion, hepatocyte dedifferentiation, hepatocyte proliferation, hepatocyte maturation, liver disease modeling, drug metabolism, hepatotoxicity testing, liver regeneration
MeSH terms
Animals, Cell Differentiation, Humans, Cell Culture Techniques, Hepatocytes, Cell Proliferation

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 ›

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