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Cited by 393D Printing in Biomedical ResearchTissue Engineering and Regenerative MedicineDrug-Induced Hepatotoxicity and Protection

3D microperfusion of mesoscale human microphysiological liver models improves functionality and recapitulates hepatic zonation

Open access
Wesseler, Milan Finn, Taebnia, Nayere, Harrison, Sean, Youhanna, Sonia, Preiss, Lena C., Kemas, Aurino M., Vegvari, Akos, Mokry, Jaroslav +3 more·2023·Acta Biomaterialia
DOI 10.1016/j.actbio.2023.09.022
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Abstract

Hepatic in vitro models that accurately replicate phenotypes and functionality of the human liver are needed for applications in toxicology, pharmacology and biomedicine. Notably, it has become clear that liver function can only be sustained in 3D culture systems at physiologically relevant cell densities. Additionally, drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus, calling for models that capture this zonation. We demonstrate the manufacture of accurate liver microphysiological systems (MPS) via engineering of 3D stereolithography printed hydrogel chips with arrays of diffusion open synthetic vasculature channels at spacings approaching in vivo capillary distances. Chip designs are compatible with seeding of cell suspensions or preformed liver cell spheroids. Importantly, primary human hepatocytes (PHH) and hiPSC-derived hepatocyte-like cells remain viable, exhibit improved molecular phenotypes compared to isogenic monolayer and static spheroid cultures and form interconnected tissue structures over the course of multiple weeks in perfused culture. 3D optical oxygen mapping of embedded sensor beads shows that the liver MPS recapitulates oxygen gradients found in the acini, which translates into zone-specific acet-ami-no-phen toxicity patterns. Zonation, here naturally generated by high cell densities and associated oxygen and nutrient utilization along the flow path, is also documented by spatial proteomics showing increased concentration of periportal- versus perivenous-associated proteins at the inlet region and vice versa at the outlet region. The presented microperfused liver MPS provides a promising platform for the mesoscale culture of human liver cells at phenotypically relevant densities and oxygen exposures. STATEMENT OF SIGNIFICANCE: A full 3D tissue culture platform is presented, enabled by massively parallel arrays of high-resolution 3D printed microperfusion hydrogel channels that functionally mimics tissue vasculature. The platform supports long-term culture of liver models with dimensions of several millimeters at physiologically relevant cell densities, which is difficult to achieve with other methods. Human liver models are generated from seeded primary human hepatocytes (PHHs) cultured for two weeks, and from seeded spheroids of hiPSC-derived human liver-like cells cultured for two months. Both model types show improved functionality over state-of-the-art 3D spheroid suspensions cultured in parallel. The platform can generate physiologically relevant oxygen gradients driven by consumption rather than supply, which was validated by visualization of embedded oxygen-sensitive microbeads, which is exploited to demonstrate zonation-specific toxicity in PHH liver models.

AI snapshot

Written from the paper’s full text.

TL;DR

A 3D-printed hydrogel liver microphysiological system with diffusion-open synthetic vasculature supports mesoscale human liver cultures at >100 million cells/mL and establishes oxygen gradients resembling hepatic zonation. It improves primary human hepatocyte and hiPSC-derived hepatocyte-like cell phenotypes over 2D and static spheroid cultures and recapitulates zone-specific acetaminophen toxicity.

Key findings

  • The liver MPS contains three parallel cavities, each with 45 synthetic vasculature channels; the design uses 160 µm inter-channel spacing, a maximum diffusion distance of 190 µm, and supports cell densities >100 million cells mL⁻¹.
  • Oxygen mapping showed that a flow velocity of 0.87 mm s⁻¹ generated an oxygen gradient from ≈10% to ≈4%, close to the in vivo liver acinus; 2.19 mm s⁻¹ gave ≈14% to ≈9%, 0.22 mm s⁻¹ gave ≈5% to ≈0%, and stopping flow caused rapid hypoxia near 0%.
  • Primary human hepatocyte MPS formed dense interconnected tissue after 5 days, maintained albumin secretion over 14 days, and showed CYP3A4 induction by rifampicin; control metabolic rates were 8.3 pmol/min/10⁶ cells for 1-OH-midazolam and 1.4 pmol/min/10⁶ cells for N-desethylamodiaquine.
  • Non-targeted proteomics separated inlet and outlet regions: periportal markers ARG1, BAAT, PYGL, FBP1, and ASL were increased near the inlet, while perivenous markers were elevated near the outlet; at 5 mM acetaminophen, toxicity was low on the periportal side and increased toward the perivenous side.

Methods

  • The MPS was fabricated by custom stereolithographic 3D printing of PEGDA hydrogel chips, sterilized, mounted in a custom holder and microscope-compatible incubator, and perfused at 120 µL min⁻¹ unless otherwise indicated.
  • Cell models included Hep G2 cells for oxygen mapping, primary human hepatocytes seeded at approximately 1.0 × 10⁶ cells per cavity and cultured for 14 days, and hiPSC-derived hepatocyte-like spheroids seeded on day 7 of differentiation and cultured for 54 days (up to 2 months).
  • Analyses included confocal FLIM/PLIM oxygen mapping with embedded sensor beads, qRT-PCR, LC-MS/MS metabolic assays, TMTpro non-targeted proteomics of inlet and outlet regions, histology/immunostaining, albumin ELISA, live/dead staining for APAP toxicity, and unpaired two-tailed heteroscedastic t-tests with significance at p ≤ 0.05.

Limitations

  • Stated: future work is needed for benchmarking with other cell sources, screening larger drug libraries, further confirmation of zonation, and establishment of disease models.
  • Stated: oxygen mapping was limited to the lower-most 0.5 mm of the culture volume because of optical scattering through dense cells, and imaging was performed only intermittently to minimize analysis-induced effects.
  • Evident from the text: zonation validation relies on inlet-versus-outlet bulk proteomics rather than single-cell spatial resolution, and the APAP toxicity pattern at 5 mM was reported from 3 independent experiments, with the highest APAP concentration showing reduced red signal likely due to prior cell disintegration.

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

Integrity signals

Registry

Crossref · 10.1016/j.actbio.2023.09.022

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 32 cited sources checked · 6 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Advanced 3D human hepatocyte culture models improve maintenance of the organotypic microenvironment, including multicellular spheroids and microphysiological systems.

    “Advanced 3D human hepatocyte culture models presented in recent years improve the maintenance of the organotypic microenvironment, including multicellular spheroids and microphysiological systems (MPS) [4][5][6][7][8][9] .”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [4] Lauschke, 2016, Novel 3D culture systems for studies of human liver function and assessments of the hepatotoxicity of drugs and drug candidates, Chem. Res. Toxicol. source
    • cites [5] Lin, 2016, Advances in engineered liver models for investigating drug-induced liver injury, Biomed Res. Int source
    • cites [6] Lauschke, 2019, 3D primary hepatocyte culture systems for analyses of liver diseases, drug metabolism, and toxicity: emerging culture paradigms and applications, Biotechnol. J. source
  • Premisecitation

    In vivo liver tissue oxygen gradients range from approximately 50 mmHg in periportal tissue to approximately 20 mmHg in perivenous tissue of the liver acinus.

    “which range from ≈50 mmHg in periportal tissue to ≈20 mmHg in perivenous tissue of the liver acinus [11] .”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Kietzmann, 2017, Metabolic zonation of the liver: the oxygen gradient revisited, Redox Biol. source
  • Findingown data

    Expression of perivenous markers was significantly elevated close to the outlet where oxygenation is lower.

    “In contrast, expression of perivenous markers was significantly elevated close to the outlet where oxygenation is lower.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [33] Ben-Moshe, 2019, Spatial sorting enables comprehensive characterization of liver zonation, Nat. Metab. source

      The Results section ('Spatial sorting enables isolating bulk hepatocyte populations') identifies CD73 as a pericentral marker, and 'Zonation patterns of the hepatocyte proteome' reports significant protein zonation, including pericentrally enriched pathways. The Introduction describes blood flowing from portal nodes toward draining central veins and generating an oxygen gradient, but our copy does not explicitly establish that oxygenation is lower near the outlet or report a significance test for the perivenous markers as a group. Page numbers were not available in our copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [34] Droin, 2021, Space-time logic of liver gene expression at sub-lobular scale, Nat. Metab. source

      The Introduction (page unavailable) describes blood flowing from the portal side to the central vein, an accompanying oxygen gradient, and genes expressed more strongly near the central vein. The Discussion (page unavailable) also describes the central region as subject to hypoxic stress. These passages support the direction of the claim, but the cited text does not establish a statistically significant elevation of perivenous markers close to an outlet. (LLM, conf=0.81)

      Experimental — accuracy not yet measured
    • cites [35] Yu, 2022, Spatial transcriptome profiling of normal human liver, Sci. Data. source

      Technical Validation identifies GLUL, CYP3A4, and CYP1A1 as peri-central genes near the central veins, supporting the location of perivenous marker expression. The passage does not report that their expression was significantly elevated, and Background & Summary describes differing environments along the lobule without stating that oxygenation is lower near the central veins. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Interpretationown data

    In contrast to previous studies, zonation in this device is naturally generated by high cell densities and the corresponding oxygen and nutrient utilization along the flow path.

    “Importantly, in contrast to previous studies [12] , zonation in this device is naturally generated by high cell densities and the corresponding oxygen and nutrient utilization along the flow path.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [12] Tomlinson, 2019, In vitro liver zonation of primary rat hepatocytes, Front. Bioeng. Biotechnol. source
  • Premisecitation

    Hep G2 cells' oxygen consumption rate per tissue volume, 4 × 10-17 mol cell-1 s-1 at an estimated compact cell density of 1.1 × 109 cm-3 yielding 4 × 10-8 mol cm-3 s-1, is a good approximation of human liver tissue.

    “Hep G2 cells were used in this part of the study, since their oxygen consumption rate per tissue volume, 4 × 10 -17 mol cell -1 s -1 [29] at an estimated compact cell density of 1.1 × 10 9 cm -3 (cell volume of 850 μm 3 [30] ) yielding 4 × 10 -8 mol cm -3 s -1 , is a good approximation of human liver tissue.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [29] Weise, 2013, Analysis and comparison of oxygen consumption of HepG2 cells in a monolayer and three-dimensional high density cell culture by use of a matrigrid®, Biotechnol. Bioeng. source
    • cites [30] Wiśniewski, 2016, In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes, J. Proteomics. source
  • May combine two claims. 32
  • Classified as probably not a substantial claim. 11
  • The quote only partly states this claim. 1

  • May combine two claims.

    Interpretationown data

    The CYP3A4 induction by rifampicin suggests that nuclear hormone receptor signaling and cellular xenobiotic sensing remains intact in the perfused liver MPS.

    “After 72 h of rifampicin exposure, the perfused liver MPS system showed induction of CYP3A4 expression similar to spheroids, the gold standard for preclinical CYP induction measurements [32] , suggesting that nuclear hormone receptor signaling and cellular xenobiotic sensing remains intact.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] Hendriks, 2020, Clinically relevant cytochrome P450 3A4 induction mechanisms and drug screening in three-dimensional spheroid cultures of primary human hepatocytes, Clin. Pharmacol. Ther. source

      The Results (page unavailable) report CYP3A4 induction after 72 hours of rifampicin exposure in primary human hepatocyte spheroids, supporting their use as a comparator. But the paper calls rifampicin—not spheroids—the gold standard for CYP3A4 induction. The cited study does not test the perfused liver MPS; the inference about that system follows the citation in the citing sentence. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • Rests on an assumption: Rifampicin is a bona fide nuclear hormone receptor ligand.

  • May combine two claims.

    Findingown data

    The presented mesoscale culture system supports 3D interconnected liver tissue at physiological cell densities and oxygen gradients resembling those in the liver acinus in vivo.

    “Here, we present a mesoscale culture system of 3D interconnected liver tissue at physiological cell densities supporting oxygen gradients resembling those in the liver acinus in vivo ( Fig. 1 ).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: No model with physiological oxygen gradients along the perfusion axis of a continuous and densely perfused 3D tissue has been reported.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Cross-sectional analysis of printed devices shows that slight overcuring in the channel lumen results in equal channel width and wall thickness of 120 μm.

    “Cross-sectional analysis of printed devices shows that slight overcuring in the channel lumen results in equal channel width and wall thickness of 120 μm.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    The synthetic vasculature arrays are 3D printed in a mechanically stable yet diffusion-open hydrogel that can be directly connected to external perfusion tubing.

    “The synthetic vasculature arrays are 3D printed in a mechanically stable yet diffusion-open hydrogel that can be directly connected to external perfusion tubing.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    hiPSC-derived liver MPS cultures showed clear fusion of spheroids into dense tissues with little visible cell death in long-term culture (up to 2 months).

    “An MPS culture stained for live cell marker calcein AM and dead cell marker propidium iodide on day 40 of culture showed clear fusion of spheroids into dense tissues and with little visible cell death ( Fig. 4 c).”
    • Also stated in: § 4. Discussion · plain
      “The hiPSC-based liver-like spheroid based MPS cultures enabled the formation of dense, interconnected 3D tissues and exhibited high viability in long-term cultures (2 months), assessed by live/dead stained liver MPS.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    An intermediate flow speed of 0.87 mm s-1 generated an oxygen gradient from approximately 10% to approximately 4%, in close proximity of the measured gradient in liver acinus blood, and was consequently used in the rest of further experiments.

    “An intermediate flow speed of 0.87 mm s -1 generated an oxygen gradient from ≈10% to ≈4%, in close proximity of the measured gradient in liver acinus blood, and was consequently used in the rest of further experiments.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At low concentrations (1-3 mM APAP) no measurable cell death was observed, whereas complete cell death was observed at a very high concentration of 50 mM.

    “At low concentrations (1-3 mM APAP) we observed no measurable cell death, whereas complete cell death was observed at a very high concentration of 50 mM ( Fig. 7 ).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingcitation

    Immunohistochemical analyses of hiPSC-derived MPS tissue sections confirmed dense continuous tissue with clear protein expression of hepatocyte markers ALB and CK18.

    “Furthermore, immunohistochemical analyses of hiPSC-derived MPS tissue sections confirmed the formation of a dense continuous tissue with clear protein expression of the hepatocyte markers ALB and CK18 [31] ( Fig. 4 e).”
    • Also stated in: § 4. Discussion · boosted
      “The confirmation of the presence of liver typical proteins such as albumin in histological analysis of the liver tissues further supports this and the presence of cytokeratin 18, expressed on hepatocyte plasma membrane surface, confirms the presence of hepatocyte-like cells.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Kania, 2004, Generation of glycogen- and albumin-producing hepatocyte-like cells from embryonic stem cells, Biol. Chem. source

  • May combine two claims.

    Findingown data

    Morphologically, the perfused tissue remained intact and limited remodeling was visible after tissue formation throughout the culture period.

    “Morphologically, the perfused tissue remained intact and limited remodeling was visible after tissue formation throughout the culture period.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Simple pipetting of the spheroid suspension into the chip cavities resulted in a thick sediment of separate spheroids evenly distributed across the cavity area, in the absence or presence of suspended oxygen sensor beads.

    “Simple pipetting of the spheroid suspension into the chip cavities resulted in a thick sediment of separate spheroids evenly distributed across the cavity area, in the absence or presence of suspended oxygen sensor beads ( Fig. 4 ab).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Pathway analysis of the differentially expressed proteins revealed that biosynthesis of amino acids and gluconeogenesis were increased at the inlet region, whereas drug metabolism, amino acid degradation, steroid hormone biosynthesis and fatty acid metabolism were upregulated close to the outlet.

    “Notably, pathway analysis of the differentially expressed proteins revealed that, among other functions, biosynthesis of amino acids and gluconeogenesis were increased at the inlet region, whereas drug metabolism, amino acid degradation, steroid hormone biosynthesis and fatty acid metabolism were upregulated close to the outlet ( Table 1 ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Among the top proteins with the highest differences in zonal expression were CKL and INS-IGF2, which were upregulated on the inlet side, as well as LMNB1 and SDF2L1 on the outlet side.

    “Among the top proteins with the highest differences in zonal expression were the signal transducer CKL and the metabolic regulator INS-IGF2, which were upregulated on the inlet side, as well as structural components (LMNB1) and chaperones (SDF2L1) on the outlet side ( Fig. 6 c).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingnone given

    The looser initially formed tissue is observed to contract further during the first 5 days of culture and likely forms more dense tissues between the vasculature channels while forming small voids around it.

    “The looser initially formed tissue is observed to contract further during the first 5 days of culture and likely forms more dense tissues between the vasculature channels while forming small voids around it.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    hiPSC-derived liver MPS cultures showed improved maturation compared to spheroid suspension culture in long-term culture (up to 2 months), with increased expression of hepatic markers, CYP enzymes and albumin, and decreased AFP.

    “Expression profiling of liver markers obtained from tissues cultured for 54 days showed increased differentiation in the MPS compared to spheroid suspension culture, as indicated by strongly elevated expression of hepatic markers, CYP enzymes and albumin, and with decreased expression of the progenitor cell marker AFP ( Fig. 4 d).”
    • Also stated in: § 4. Discussion · plain
      “Expression profiling showed a significant increase in liver typical markers in the liver MPS compared to spheroid suspension cultures at day 60 of differentiation.”
    • Also stated in: § 5. Conclusion · plain
      “hiPSC-based liver MPS allowed the use of hiPSC-derived hepatocyte-like spheroids in long-term cultures (up to 2 months) with improved maturation compared to spheroid suspension culture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Flow velocity in microfluidic channels traversing the liver acinus model at 2.19 mm s-1 resulted in hyperoxygenation with an oxygen gradient in the cell compartment from approximately 14% to approximately 9%.

    “Flow velocity in microfluidic channels traversing the liver acinus model at 2.19 mm s -1 resulted in hyperoxygenation with an oxygen gradient in the cell compartment from ≈14% to ≈9%”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression levels of secreted hepatic factors (ALB), drug metabolizing enzymes (CYP2E1, CYP3A4, CYP2C8, CYP2D6 and CYP4F12), and factors involved in fatty acid and carbohydrate metabolism (FASN, SREBF1, and PCK1) were orders of magnitude higher than in 2D culture and similar or even higher than in 3D spheroids.

    “Notably, expression levels were orders of magnitude higher than in 2D culture and similar or even higher than in 3D spheroids ( Fig. 5 b; Figure S4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    The setup enabled perfusion of the chips with pre-conditioned culture medium and efficiently prevented bubbles from being introduced and trapped in the synthetic vasculature networks during long-term culture.

    “This enabled perfusion of the chips with pre-conditioned culture medium and efficiently prevented bubbles from being introduced and trapped in the synthetic vasculature networks during longterm culture.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisenone given

    Flow-over and flow-through perfusion models provide close contact and highly efficient gas-and nutrient-exchange with cells in direct contact with the perfusion liquid.

    “Flow-over and flow-through perfusion models provide close contact and consequently highly efficient gas-and nutrient-exchange with cells in direct contact with the perfusion liquid.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Spheroid culture formats are reasonably easy to use, reproducible, allow medium to high throughput screening, and have shown vast improvements in retention of mature phenotypes compared to 2D adhesion cultures.

    “Spheroid culture formats are reasonably easy to use, reproducible, allow medium to high throughput screening, and have shown vast improvements in retention of mature phenotypes compared to 2D adhesion cultures [10] .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Ingelman-Sundberg, 2022, 3D human liver spheroids for translational pharmacology and toxicology, Basic Clin. Pharmacol. Toxicol. source

      Not checked in the free run.

  • May combine two claims.

    Study design and conductnone given

    The devices could withstand swelling and deswelling without damage during sterilization with 70% ethanol and 2.5% glutaraldehyde, and during cell seeding where the hydrogel chips are taken out of their immersion fluid for up to three hours.

    “The devices could withstand swelling and deswelling without damage during sterilization with 70% ethanol and 2.5% glutaraldehyde, and during cell seeding where the hydrogel chips are taken out of their immersion fluid for up to three hours.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The temperature was monitored to be 35.5-37.0°C during long-term culture.

    “The temperature was monitored to be 35.5-37.0°C during long-term culture.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Premisenone given

    The flowing liquid in flow-over and flow-through perfusion models exerts super-physiological levels of shear stress and may prevent the establishment of local tissue concentration gradients present in vivo.

    “However, the flowing liquid also exerts super-physiological levels of shear stress and may prevent the establishment of local tissue concentration gradients present in vivo .”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductnone given

    The developed post-printing sterilization protocol reliably prevented contamination stemming from imprinted bacteria while avoiding damage to the structure of the hydrogel chips.

    “The developed post-printing sterilization protocol reliably prevented contamination stemming from imprinted bacteria while avoiding damage to the structure of the hydrogel chips.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    The MPS design enables longitudinal imaging of the entire replicated liver acinus in situ and supports live oxygen mapping based on embedded microsensor beads to monitor establishment and maintenance of physiological oxygen gradients.

    “The MPS design enables longitudinal imaging of the entire replicated liver acinus in situ and supports live oxygen mapping based on embedded microsensor beads to monitor the establishment and maintenance of physiological oxygen gradients, which translate into zone-specific functional differences.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    The MPS is mounted in a custom designed microscopy-compatible table-top incubator with temperature and gas control and heated glasses for condensation-free optical access, ensuring robust tubing connection in a controlled and aseptic environment that can be monitored during long-term culture.

    “The MPS is mounted in a custom designed microscopy-compatible table-top incubator with temperature and gas control as well as heated glasses for condensation-free optical access, ensuring robust tubing connection in a controlled and aseptic environment that can be monitored during long-term cul-ture.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Studies of oxygen gradients in the human liver most commonly report oxygen tension in periportal approximately 8% (60-65 mmHg) and perivenous approximately 4% (30-35 mmHg) blood.

    “Studies of oxygen gradients in the human liver most commonly report the oxygen tension in periportal ≈8% (60-65 mmHg) and perivenous ≈4% (30-35 mmHg) blood [26] .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Jungermann, 2000, Oxygen: modulator of metabolic zonation and disease of the liver, Hepatology source

  • May combine two claims.

    Interpretationnone given

    Freeform 3D printing of diffusion-open synthetic vasculature chips allowed for massive parallel microperfusion in three independent culture cavities per chip, enabling stable, long-term perfusion cultures with live imaging capabilities.

    “Freeform 3D printing of diffusion-open synthetic vasculature chips allowed for massive parallel microperfusion in three independent culture cavities per chip, enabling stable, long-term perfusion cultures with live imaging capabilities.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The increased CYP3A4 and CYP2E1 in the MPS indicate molecular zonation without a deterioration in overall phenotypes.

    “Particularly, the perivenous zonation markers CYP3A4 and CYP2E1 were significantly increased in the MPS, indicating molecular zonation without a deterioration in overall phenotypes.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CYP3A4 and CYP2E1 are perivenous zonation markers.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Tissue oxygen concentrations are more difficult to measure, with reported values of approximately 3% (20-25 mmHg) using microelectrodes.

    “Tissue oxygen concentrations are more difficult to measure, with reported values of ≈3% (20-25 mmHg) using microelectrodes [27] .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [27] Kessler, 1974, Oxygen supply to tissue in normoxia and in oxygen deficiency, Microvasc. Res. source

  • May combine two claims.

    Premisecitation

    The dose responses seen in this study are similar to doses previously used in 3D primary hepatocyte spheroids and close to in vivo levels for APAP overdosing resulting in drug induced liver injury or failure.

    “The dose responses seen in this study are similar to doses previously used in 3D primary hepatocyte spheroids [40] and close to in vivo levels for APAP overdosing resulting in drug induced liver injury or failure [41] .”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [40] Bell, 2017, Transcriptional, functional, and mechanistic comparisons of stem cell-derived hepatocytes, HepaRG cells, and three-dimensional human hepatocyte spheroids as predictive in vitro systems for drug-induced liver injury, Drug Metab. Dispos. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [41] Schulz, 2020, Revisited: therapeutic and toxic blood concentrations of more than 1100 drugs and other xenobiotics, Crit. Care. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    The oxygen consumption rate in human liver is reported to be 0.06 mL g-1 min-1, corresponding to 4 × 10-8 mol cm-3 s-1 for an approximate liver mass density of 1 g cm-3.

    “The oxygen consumption rate in human liver is reported to be 0.06 mL g -1 min -1 [28] , corresponding to 4 × 10 -8 mol cm -3 s -1 for an approximate liver mass density of 1 g cm -3 .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [28] Lutz, 1975, Oxygen supply and uptake in the liver and the intestine, Pflügers Arch, Eur. J. Physiol. source

  • May combine two claims.

    Interpretationown data

    The developed liver MPS is suitable for the organotypic long-term culture of human liver cells and replicates in vivo-like continuous oxygen gradients in connected mesoscale liver tissue.

    “We conclude that the developed liver MPS is suitable for the organotypic long-term culture of human liver cells and replicates in vivo -like continuous oxygen gradients in connected mesoscale liver tissue.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    Seeded tissue densities can be estimated from the volume of 1 million primary human hepatocytes with an average cellular volume of 2.3 × 10^3 μm^3 resulting in 2.3 μL total cell volume, corresponding to an upper limit on the cell density of 4.3 × 10^8 cells mL-1 located within the MPS acinus model volume of 20 μL (excluding the synthetic vasculature channels).

    “Seeded tissue densities can be estimated from the volume of 1 million primary human hepatocytes with an average cellular volume of 2.3 × 10 3 μm 3 [39] resulting in 2.3 μL total cell volume.This corresponds to an upper limit on the cell density of 4.3 × 10 8 cells mL -1 located within the MPS acinus model volume of 20 μL (excluding the synthetic vasculature channels).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Yoshikado, 2017, Comparison of methods for estimating unbound intracellular-to-medium concentration ratios in rat and human hepatocytes using statinss, Drug Metab. Dispos. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationown data

    When cultured without perfusion through the synthetic vasculature, cells remained round and did not aggregate, due to very limited or absent metabolic activity in their environment depleted of oxygen and nutrients, leading to tissue anoxia.

    “In contrast, when cultured without perfusion through the synthetic vasculature, cells remained round and did not aggregate due to very limited or absent metabolic activity in their environment depleted of oxygen and nutrients (Figure S3).”
    • Also stated in: § 4. Discussion · plain
      “This was contrasted by the failure of cells to fuse into dense, connected tissues in control liver MPS cultures with disabled perfusion leading to tissue anoxia.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Former results using a single layer of perfusion channels showed full anoxia in the cultured cell volume within 10 min of stopping perfusion of medium.

    “This is in agreement with our former results using a single layer of perfusion channels, where full anoxia in the cultured cell volume was observed within 10 min of stopping perfusion of medium [19] .”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [19] Wesseler, 2022, Optical 4D oxygen mapping of microperfused tissue models with tunable in vivo-like 3D oxygen microenvironments, Lab Chip source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Premisecitation

    The intracellular in vivo oxygen gradient across the different zones has been estimated to range from approximately 6% (45-50 mmHg) on the periportal side of the acinus to approximately 2% (15-20 mmHg) on the perivenous side.

    “The intracellular in vivo oxygen gradient across the different zones has been estimated to range from ≈6% (45-50 mmHg) on the periportal side of the acinus to ≈2% (15-20 mmHg) on the perivenous side [11] .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [11] Kietzmann, 2017, Metabolic zonation of the liver: the oxygen gradient revisited, Redox Biol. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Oxygen utilization in these mesoscale tissue constructs drove the formation of a physiologically relevant oxygen gradient, which propagated into functional specification.

    “Oxygen utilization in these mesoscale tissue constructs drove the formation of a physiologically relevant oxygen gradient, which propagated into functional specification.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Interpretationown data

    The primary hepatocyte MPS promotes primary hepatocyte survival and supports high maintenance of liver-typic expression profiles, with preserved liver function over 14 days, vastly improved over 2D cultures and also over 3D spheroid cultures.

    “The primary hepatocyte MPS results show that the system not only promotes primary hepatocyte survival but also supports high maintenance of liver-typic expression profiles, which vastly improved over 2D cultures and also over 3D spheroid cultures.”
    • Also stated in: § 5. Conclusion · plain
      “Primary human hepatocyte-based liver MPS showed formation of dense 3D tissues with high viability and preserved liver function over the course of 14 days compared to 2D culture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    These patterns of toxicity are in agreement with the increased expression of the perivenous markers CYP2E1 and CYP3A4 in the liver MPS, which drive APAP hepatotoxicity, and recapitulate in vivo APAP toxicity profiles.

    “Notably, these patterns of toxicity are in agreement with the increased expression of the perivenous markers CYP2E1 and CYP3A4 in the liver MPS, which drive APAP hepatotoxicity, and recapitulate in vivo APAP toxicity profiles [37] .”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [37] Heldring, 2022, Unraveling the effect of intra- and intercellular processes on acetaminophen-induced liver injury, Npj Syst. Biol. Appl. source

      Checked before — its earlier verdict is held back until a new check runs.

    • Rests on an assumption: APAP is mainly metabolized by perivenous CYP enzymes CYP2E1 and CYP3A4 into the reactive hepatotoxic metabolite NAPQI.
    • Rests on an assumption: CYP3A4 and CYP2E1 are perivenous zonation markers.
39 of 39

  • The paper relies on this without support.

    Liver function can only be sustained in 3D culture systems at physiologically relevant cell densities.

    “Notably, it has become clear that liver function can only be sustained in 3D culture systems at physiologically relevant cell densities.”

  • The paper relies on this without support.

    Drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus.

    “Additionally, drug metabolism and drug-induced cellular toxicity often follow distinct spatial micropatterns of the metabolic zones in the liver acinus, calling for models that capture this zonation.”

  • The paper relies on this without support.

    An increasing number of applications require liver cultures that are functionally stable for multiple days to weeks.

    “An increasing number of applications require liver cultures that are functionally stable for multiple days to weeks.”

  • The paper relies on this without support.

    No model with physiological oxygen gradients along the perfusion axis of a continuous and densely perfused 3D tissue has been reported.

    “While these different culture format approaches are promising and have modeled important expression and secretory characteristics of liver tissue, no model with physiological oxygen gradients along the perfusion axis of a continuous and densely perfused 3D tissue has been reported.”

  • The paper relies on this without support.

    APAP is mainly metabolized by perivenous CYP enzymes CYP2E1 and CYP3A4 into the reactive hepatotoxic metabolite NAPQI.

    “which is mainly metabolized by the perivenous CYP enzymes CYP2E1 and CYP3A4 into the reactive and hepatotoxic metabolite N -acetyl-p -benzoquinoneimine (NAPQI).”

  • The paper relies on this without support.

    CYP3A4 and CYP2E1 are perivenous zonation markers.

    “Particularly, the perivenous zonation markers CYP3A4 and CYP2E1 were significantly increased in the MPS”

  • The paper relies on this without support.

    Rifampicin is a bona fide nuclear hormone receptor ligand.

    “using the bona fide nuclear hormone receptor ligand rifampicin”
7 of 7

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conflicting rifampicin exposure times for CYP3A4 expression

    Minor

    The results describe the CYP3A4 expression measurement as following 72 hours of rifampicin exposure, but the caption for that expression measurement specifies 48 hours.

1 of 1

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Authors
Wesseler, M. F., Taebnia, N., Harrison, S., Youhanna, S., Preiss, L. C., Kemas, A. M., Vegvari, A., Mokry, J., Sullivan, G. J., Lauschke, V. M., & Larsen, N. B.
Affiliations
  • Technical University of Denmark (DK)
  • Oslo University Hospital (NO)
  • Karolinska Institutet (SE)
  • Merck KGaA, Darmstadt (Germany) (DE)
Corresponding author
  • Sullivan, Gareth J.
  • Lauschke, Volker M.
  • Larsen, Niels B.
ORCID
Venue
Acta Biomaterialia
Publisher
Elsevier BV
Published
2023-11
Volume
171
Pages
336-349
ISSN
1742-7061 (ISSN-L)
DOI
10.1016/j.actbio.2023.09.022
OpenAlex
W4386865650
Semantic Scholar
aa1ecdff25b0150399a08d9c1afcb79138d91c7c
Cited by
39
References
47
Citations by year
2026: 16 · 2025: 13 · 2024: 9 · 2023: 1
FWCI
2.99
Type
journal article
Language
en
Open access
Licence
Locations
  • Acta Biomaterialia (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Technical University of Denmark, DTU Orbit (Technical University of Denmark, DTU) (submittedVersion, cc-by)
  • St Andrews Research Repository (St Andrews Research Repository) (submittedVersion, cc-by)
Funders
  • Innovative Medicines Initiative
  • Research Council of Norway (262613, 247624)
  • Technical University of Denmark
  • Danmarks Frie Forskningsfond
  • McGill University
  • Robert-Bosch Foundation GmbH
  • Kungliga Tekniska Högskolan
  • Independent Research Fund Denmark (7017\u201300366B, 2035-00186B)
  • Vetenskapsrådet (2016\u201301153, 2019\u201301837, 01154)
  • Novo Nordisk Foundation (NNF16OC0022166)
Topics
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
Keywords
microphysiological systems, stereolithography, oxygen gradients, hepatic zonation, primary human hepatocytes, liver spheroids, acetaminophen toxicity, spatial proteomics, drug metabolism
MeSH terms
Humans, Liver, Oxygen, Hydrogels, Hepatocytes

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 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

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Statistics and arithmetic are recomputed, not judged.

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