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Cited by 3Liver physiology and pathologyTissue Engineering and Regenerative Medicine3D Printing in Biomedical Research

Optimization of extracellular matrix for primary human hepatocyte cultures using mixed collagen-matrigel matrices

Open access
Seidemann, Lena, Prinz, Sarah, Scherbel, Jan-Constantin, Götz, Christina, Seehofer, Daniel, Damm, Georg·2023·IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund
DOI 10.17179/excli2022-5459PMC PMC9837384
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Abstract

Loss of differentiation of primary human hepatocytes (PHHs) ex vivo is a known problem of in vitro liver models. Culture optimizations using collagen type I and Matrigel reduce the dedifferentiation process but are not able to prevent it. While neither of these extracellular matrices (ECMs) on their own correspond to the authentic hepatic ECM, a combination of them could more closely resemble the in vivo situation. Our study aimed to systematically analyze the influence of mixed matrices composed of collagen type I and Matrigel on the maintenance and reestablishment of hepatic functions. Therefore, PHHs were cultured on mixed collagen-Matrigel matrices in monolayer and sandwich cultures and viability, metabolic capacity, differentiation markers, cellular arrangement and the cells’ ability to repolarize and form functional bile canaliculi were assessed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), functional assays and immunofluorescence microscopy. Our results show that mixed matrices were superior to pure matrices in maintaining metabolic capacity and hepatic differentiation. In contrast, Matrigel supplementation can impair the development of a proper hepatocytic polarization. Our systematic study helps to compose an optimized ECM to maintain and reestablish hepatic differentiation on cellular and multicellular levels in human liver models.

AI snapshot

Written from the paper’s full text.

TL;DR

Mixed collagen-Matrigel matrices better maintained metabolic capacity and hepatic differentiation of primary human hepatocytes in monolayer culture than pure collagen type I or Matrigel, but increasing Matrigel impaired hepatocytic polarization in sandwich culture. The authors selected 90C/10M for sandwich-culture follow-up because it stabilized E-cadherin, while pure Matrigel supported longer bile canaliculi but poor basolateral organization.

Key findings

  • In monolayer cultures over 6 days, mixed matrices generally sustained mitochondrial activity and protein concentrations better than pure matrices; XTT activity declined significantly on day 6 in 100C and in coatings containing ≥20% Matrigel, and 100C showed lower activity than almost any other coating.
  • Differentiation markers were better preserved on mixed matrices: E-Cad increased on day 2 but decreased by day 6 in 100C and 100M, while ALB and FGA decreased on days 4 and 6 in 100C and 100M; HNF4A declined under all conditions but was higher on day 6 in mixed matrices than 100C and partly than 100M, and CYP2C9 declined only on pure collagen I or Matrigel.
  • In sandwich cultures, dead cells increased over 6 days on 100M and were significantly higher than in collagen-containing cultures on days 4 and 6; 100M showed diffuse MRP2 and E-cadherin and OATP1B1 tracing whole membrane areas, indicating impaired polarization, whereas 100C formed defined polarized domains and 90C/10M largely resembled 100C but began losing the basolateral domain.
  • The CDF assay after 6 days showed functional bile canaliculi under all three sandwich conditions; 100M canaliculi were longer and more branched, with some clusters forming canaliculi networks.

Methods

  • Primary human hepatocytes were isolated from macroscopically tumor-free liver surgery samples by two-step EGTA/collagenase perfusion. Monolayer cultures used collagen type I/Matrigel ratios including 100C, 95C/5M, 90C/10M, 80C/20M, 70C/30M, and 100M for up to 6 days; sandwich cultures used 100C, 100M, or 90C/10M.
  • Monolayer assays from 5 donors included XTT, BCA protein, LDH/ALT/AST/g-GT enzyme activities, and glucose/lactate/urea metabolites; RT-qPCR from 3 donors measured CK-18, E-Cad, ALB, FGA/FGB/FGG, HNF4A, CYP3A4, and CYP2C9. Sandwich-culture assays from 3 donors included dead/alive staining, immunofluorescence for MRP2, ZO-1, E-cadherin, and OATP1B1, and the CDF bile canaliculi assay.
  • Data are mean + SEM; repeated measures two-way ANOVA with Bonferroni correction was used, with significance thresholds *p ≤ 0.0332, p ≤ 0.0021, *p ≤ 0.0002, p < 0.0001. PCR data were normalized to day 0 and the geometric mean of EEF2, 18S_rRNA, and GUSB.

Limitations

  • The authors state the study was explorative with a small set of donors due to economic reasons, and that parametric testing was chosen despite the small sample size.
  • The authors state they did not use growth factor-reduced Matrigel for economic reasons, so growth-factor confounding is possible; they argue this is unlikely but recommend future validation with growth factor-reduced Matrigel. They also state further functional investigations, such as drug uptake and excretion activity in sandwich-cultured PHHs on mixed matrices, are required.
  • Evident from the text: the study is in vitro and uses limited donor numbers (3 donors for RT-qPCR and sandwich-culture assays; 5 donors for monolayer assays), and the authors note donor-dependent actin expression patterns.

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

Integrity signals

Registry

DataCite · 10.17179/excli2022-5459

Cites

50 of 50 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

20 of 47 cited sources checked · 15 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    In the first few days of PHH cultivation, hepatic functions such as plasma protein production, drug metabolizing enzyme activities, glucose metabolism and urea synthesis are reduced.

    “Already in the first few days of cultivation, a reduction of hepatic functions can be observed, e.g. production of plasma proteins, drug metabolizing enzyme activities, glucose metabolism and urea synthesis (Godoy et al., 2013[15]).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Godoy et al., 2013[15] source

      The 3D liver models section (page unavailable) says hepatocyte dedifferentiation in 2D culture reduces plasma-protein production and detoxification associated with phase I and II enzymes. But the Isolated human hepatocytes section says enzyme activities, glucose metabolism and ammonia detoxification can remain functional for at least 48–72 hours; it does not establish that glucose metabolism or urea synthesis decline in the first few days. The citing sentence therefore overstates which functions the review identifies as declining at that time. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Cell-cell and cell-matrix interactions are important for improving hepatic functions in liver models.

    “The development of hepatocyte sandwich cultures (SCs), co-cultures with non-parenchymal cells and even more advanced 3-dimensional culturing techniques has revealed the importance of cell-cell and cell-matrix interactions for improving hepatic functions in liver models (Bachmann et al., 2015[2]; Duval et al., 2017[9]; Wei et al., 2018[45]).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites Bachmann et al., 2015[2] source
    • cites Duval et al., 2017[9] source
    • cites Wei et al., 2018[45] source
  • Premisecitation

    The expression of OATP1B1, selectively found in the sinusoidal hepatocyte membrane, declines rapidly in hepatocyte cultures.

    “The expression of the drug membrane transporter OATP1B1 that is selectively found in the sinusoidal hepatocyte membrane, declines rapidly in hepatocyte cultures (Ulvestad et al., 2011[41]).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Ulvestad et al., 2011[41] source

      The Summary of Papers reports an extensive decrease in OATP1B1 expression over time in plated primary human hepatocytes, and §4.1.1 places OATP1B1 at the basolateral membrane in the human liver. The claim is broader than the evidence: the decline was observed in plated cultures, whereas the Abstract reports preserved OATP1B1 expression for at least nine days in 3D bioreactor cultures. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Findingown data

    Mixed matrices were superior to pure matrices in maintaining metabolic capacity and hepatic differentiation.

    “Our results show that mixed matrices were superior to pure matrices in maintaining metabolic capacity and hepatic differentiation.”
    • Also stated in: § Introduction · plain
      “Our data reveal that supplementing collagen type I-based ECMs with Matrigel supports the maintenance of hepatocyte differentiation and their metabolic capacity in MCs.”
    Quote in the paperSubstantial claim
  • Findingown data

    Matrigel supplementation can impair the development of a proper hepatocytic polarization.

    “In contrast, Matrigel supplementation can impair the development of a proper hepatocytic polarization.”
    • Also stated in: § Introduction · plain
      “In SCs, however, Matrigel can impair the development of a proper hepatocytic polarization.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 27
  • May combine two claims. 43
  • The quote only partly states this claim. 1

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

    Premisecitation

    The apical membrane is equipped with transport proteins such as MRP2 that eliminate bile acids and xenobiotics from the cell.

    “The apical membrane is equipped with transport proteins that eliminate bile acids and xenobiotics from the cell, like multidrug resistance-associated protein 2 (MRP2) (Godoy et al., 2013[15]; Ito et al., 2005[20]).”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites Godoy et al., 2013[15] source
    • cites Ito et al., 2005[20] source

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

    Premisecitation

    Serna-Márquez et al. reported a positive influence of collagen on viability and aggregation of rat hepatocytes cultured on soft conditions.

    “Serna-Márquez et al. additionally examined the influence of different combinations of collagen type I and Matrigel conjugated to polyacrylamide hydrogels and reported a positive influence of collagen on viability and aggregation of rat hepatocytes cultured on soft conditions (Serna-Márquez et al., 2020[35]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Serna-Márquez et al., 2020[35] source

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

    Premisecitation

    The hepatocyte basal membrane faces the sinusoid and expresses transmembrane transporters that manage exchange of substances with the sinusoidal blood stream.

    “The hepatocyte's basal membrane faces the sinusoid. It expresses several transmembrane transporters that manage the exchange of substances with the sinusoidal blood stream (Godoy et al., 2013[15]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Godoy et al., 2013[15] source

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

    Premisecitation

    Hepatocytes are epithelial cells that feature a unique type of polarization.

    “Hepatocytes are epithelial cells that feature a unique type of polarization (Müsch, 2014[29]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Müsch, 2014[29] source

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

    Premisecitation

    Adjacent hepatocytes form small apical membrane domains that border the lumen of a bile canaliculus.

    “Adjacent hepatocytes form small apical membrane domains that border the lumen of a bile canaliculus (Slim et al., 2014[38]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Slim et al., 2014[38] source

  • Classified as probably not a substantial claim.

    Premisecitation

    In vivo, the distribution of ECM components varies between different parts of the liver acinus.

    “In vivo, the distribution of ECM components varies between the different parts of the liver acinus, the smallest organizational unit of the liver (Godoy et al., 2013[15]; Martinez-Hernandez and Amenta, 1993[26]).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites Godoy et al., 2013[15] source
    • cites Martinez-Hernandez and Amenta, 1993[26] source

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

    Premisecitation

    The hepatocyte cell membrane is compartmentalized into specific domains, separating blood from bile flow.

    “Their cell membrane is compartmentalized in specific domains, thus separating blood from bile flow (Pradhan-Sundd and Monga, 2019[32]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Pradhan-Sundd and Monga, 2019[32] source

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

    Premisecitation

    Tight junctions in the lateral membranes between neighboring cells flank the apical membrane domains and seal the bile canalicular lumen.

    “Tight junctions (TJs) in the lateral membranes between two neighboring cells flank the apical membrane domains and seal the bile canalicular lumen (Slim et al., 2014[38]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Slim et al., 2014[38] source

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

    Premisecitation

    Attempts to mimic an in vivo-like ECM using decellularized liver ECMs have produced diverging results.

    “Attempts to mimic an in vivo-like ECM have been made by utilizing decellularized liver ECMs with diverging results (Alevra Sarika et al., 2020[1]; Bual and Ijima, 2019[7]).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Alevra Sarika et al., 2020[1] source
    • cites Bual and Ijima, 2019[7] source

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

    Premisecitation

    The most commonly applied ECM components for hepatocyte cultures are collagen type I and Matrigel.

    “The most commonly applied ECM components for hepatocyte cultures are collagen type I and Matrigel, a commercially available BM matrix extract from Engelbreth-Holm-Swarm (EHS) mouse sarcoma (Serna-Márquez et al., 2020[35]; Zeilinger et al., 2016[49]).”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites Serna-Márquez et al., 2020[35] source
    • cites Zeilinger et al., 2016[49] source

      The “2D and 3D culture models for PHH” section identifies rat-tail collagen and Matrigel as ECM components used in hepatocyte sandwich cultures. It does not establish that they are the most commonly applied components across hepatocyte cultures generally. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    Relative gene expression levels were calculated by normalization to the day 0 gene expression level and the geometric average of the three reference genes as described in Vandesompele et al. (2002).

    “Relative gene expression levels were calculated by normalization to the day 0 gene expression level and the geometric average of the three reference genes as described in Vandesompele et al. (2002[42]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Vandesompele et al. (2002[42]) source

      The Results section on normalization-factor calculation (page unavailable) recommends the geometric mean of at least three stable internal control genes, supporting that part of the method. The passages do not describe normalization to a day 0 gene-expression level, so the citation does not establish that the full procedure was described in this paper. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    E-cadherin is a key element of adherens junctions, mediating cell-cell contacts and outside-in signaling.

    “E-cadherin is a key element of adherens junctions, thereby mediating cell-cell contacts and outside-in signaling.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Treyer and Müsch, 2013[40] source

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

    Premisecitation

    E-cadherin participates in the organization of tight junctions, influencing polarization through the determination of apical and basolateral membrane areas.

    “It further participates in the organization of tight junctions, thus influencing polarization through the determination of apical and basolateral membrane areas (Treyer and Müsch, 2013[40]).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Treyer and Müsch, 2013[40] source

  • May combine two claims.

    Premisecitation

    EGF, FGF and PDGF play a major role in liver regeneration and are closely linked to dedifferentiation events during hepatocyte proliferation.

    “It is known, that EGF, FGF and PDGF play a major role in liver regeneration and therefore are closely linked to dedifferentiation events during hepatocyte proliferation (Hoffmann et al., 2020[18]).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Hoffmann et al., 2020[18] source

      The Results > Growth factors section links EGF and FGF signaling to hepatocyte proliferation and describes PDGF signaling as significant, though replaceable, in liver regeneration. The Results > Temporal sequence of regeneration section also places these factors in the regenerative response. Neither section links them to hepatocyte dedifferentiation; a search for that link found no supporting passage. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Cultures on 90C/10M and 80C/20M showed a decrease in protein concentration compared to the initial protein concentration, but stabilized their protein content over days 4 to 6.

    “Cultures on 90 % collagen type I / 10 % Matrigel (90C/10M) and 80 % collagen type I/20 % Matrigel (80C/20M) showed a decrease in comparison to the initial protein concentration, but stabilized their protein content over days 4 to 6.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PHHs cultured on mixed matrices presented a stable CYP2C9 expression, which was even increased by trend on day 2.

    “PHHs cultured on mixed matrices presented a stable expression, which was even increased by trend on day 2.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Actin expression correlated with formation of bile canaliculi and surrounded the canalicular network.

    “Additionally, the expression correlated with formation of bile canaliculi and surrounded the canalicular network.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In 100C cultures, MRP2 expression was scarce, but where detected it was either co-expressed with ZO-1 or in direct proximity to ZO-1-positive membrane areas.

    “MRP2 expression was scarce, but where it could be detected, it was either co-expressed with ZO-1 or in direct proximity to ZO-1-positive membrane areas.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    CYP3A4 expression already decreased on day 2 independently of the applied coatings, but remained stable thereafter.

    “The expression levels of CYP3A4 already decreased on day 2 independently of the applied coatings, but remained stable thereafter.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    PHHs cultured between two layers of 100% collagen adhered homogeneously and in high density forming hepatocyte-typical hexagonal shapes.

    “PHHs adhered homogeneously and in high density forming hepatocyte-typical hexagonal shapes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 100C cultures, E-Cad expression was differently distributed over the cell layer, with some cells showing intracellular E-Cad accumulations and weak membrane staining, and others being intracellularly clear with strong membrane expression.

    “E-Cad expression was differently distributed over the cell layer. Some cells showed intracellular E-Cad accumulations and a weak membrane staining. In contrast, other cells were intracellularly clear and displayed a strong membrane expression.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    100M showed, in addition to a confluent arrangement, also a clustered arrangement forming strains of cells alternating with elliptical voids.

    “In contrast, 100M showed additionally to a confluent arrangement also a clustered arrangement forming strains of cells alternating with elliptical voids.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In mixed ECM cultures, FGA also decreased on day 6, but was still higher than in the 100C cultures.

    “In the mixed ECM cultures, FGA also decreased on day 6, but was still higher than in the 100C cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PHHs cultured on mixed matrix coatings showed a higher HNF4A expression on day 6 than on 100C and partly also than on 100M.

    “But PHHs cultured on mixed matrix coatings still showed a higher HNF4A expression on day 6 than on 100C and partly also than on 100M.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Glucose increased again on day 4 in all cell cultures.

    “Glucose increased again on day 4 in all cell cultures.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    E-Cad expression increased significantly on day 2 of all MCs, but decreased again on day 6 in the 100C- and 100M-cultured PHHs.

    “Expression of the epithelial cell-cell adhesion protein E-Cad increased significantly on day 2 of all MCs, but decreased again on day 6 in the 100C- and 100M-cultured PHHs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    While 100C and 90C/10M cultures showed rather similar pictures of CDF spots and short lines, bile canaliculi formed by 100M-cultured hepatocytes were longer and more branched.

    “But while the 100C and 90C/10M cultures showed rather similar pictures of CDF spots and short lines, the bile canaliculi formed by 100M-cultured hepatocytes were longer and more branched.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CDF is secreted into bile via the apical hepatocyte membrane transporter MRP2, allowing visualization of bile canaliculi by fluorescence microscopy.

  • May combine two claims.

    Findingown data

    A clear stabilizing effect of the mixed matrices was not seen for HNF4A, although its mRNA levels on day 6 were higher on most mixed matrix combinations than on pure collagen type I and in part also higher than on pure Matrigel.

    “Such a clear stabilizing effect of the mixed matrices was not seen for HNF4A, although its mRNA levels on day 6 were higher on most mixed matrix combinations than on pure collagen type I and in part also higher than on pure Matrigel.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 90C/10M cultures, OATP1B1 expression between cells was increased and more comparable to 100M cultures.

    “In contrast, OATP1B1 expression between cells was increased and more comparable to 100M cultures.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In 100M SCs, E-Cad expression was more diffuse than in 90C/10M and 100C cultures; accumulations at cell borders could hardly be detected.

    “E-Cad expression was more diffuse, than in the 90C/10M and 100C cultures; accumulations at cell borders could hardly be detected.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    High confluency was witnessed in collagen-containing SCs, while the conformation of PHHs cultured between two 100% Matrigel layers varied between small cell clusters, larger spheroid-like formations, and confluent layers.

    “We furthermore witnessed a high confluency in our collagen-containing SCs, while the conformation of PHHs cultured between two 100 % Matrigel layers varied between small cell clusters over larger spheroid-like formations to confluent layers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In 90C/10M cultures, MRP2 accumulated in spots or spotted lines, although MRP2-positive areas were rather few.

    “MRP2 accumulated in spots or spotted lines, although in general, MRP2-positive areas were rather few.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In 90C/10M cultures, E-Cad-positive cell-cell contacts were defined more precisely and intracellular accumulations appeared less than in 100C.

    “E-Cad-positive cell-cell contacts were defined more precisely and intracellular accumulations appeared less than in 100C.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Albumin mRNA levels were stabilized over the culture time on mixed matrices, in contrast to downregulated expressions on pure ECMs.

    “In contrast to the downregulated expressions on pure ECMs, albumin mRNA levels were stabilized over the culture time on mixed matrices.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    While general metabolic functions like glucose and lactate formation stabilized with ongoing culture time ECM-independently, critical hepatic functions like urea synthesis were stabilized only in Matrigel-supplemented cultures.

    “While general metabolic functions like glucose and lactate formation stabilized with ongoing culture time ECM-independently, critical hepatic functions like urea synthesis were stabilized only in Matrigel-supplemented cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Although the polarization marker pattern in 100M SCs did not appear well organized, the accumulation of actin filaments around bile canaliculi and the CDF assay revealed the formation of functioning bile canaliculi in 100M SCs building even more complex formations than in collagen-containing SCs.

    “Although the polarization marker pattern in the 100M SCs did not appear well organized, the accumulation of actin filaments around bile canaliculi and the CDF assay revealed the formation of functioning bile canaliculi in the 100M SCs building even more complex formations than in the collagen-containing SCs.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: CDF is secreted into bile via the apical hepatocyte membrane transporter MRP2, allowing visualization of bile canaliculi by fluorescence microscopy.

  • May combine two claims.

    Findingown data

    Concerning mRNA expression levels of E-Cad, mixed matrices showed superiority over the pure ECMs and especially over the 100% Matrigel cultures.

    “Concerning mRNA expression levels of E-Cad, mixed matrices showed superiority over the pure ECMs and especially over the 100 % Matrigel cultures.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The ECM is an integral part of the complex liver architecture and restoration of cell-ECM contacts is crucial for preservation of the differentiated hepatic phenotype in cell culture.

    “Although scarcely distributed, the ECM is an integral part of the complex liver architecture and the restoration of cell-ECM contacts is crucial for the preservation of the differentiated hepatic phenotype in cell culture (Fraczek et al., 2013[12]; Ye et al., 2019[48]).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Fraczek et al., 2013[12] source
    • cites Ye et al., 2019[48]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Culturing hepatocytes in a sandwich configuration between two layers of extracellular matrix allows them to regain polarity and permits functional analysis of hepatocyte biliary excretion.

    “Culturing hepatocytes in a sandwich configuration between two layers of extracellular matrix (ECM) allows them to regain polarity and permits functional analysis of hepatocyte biliary excretion (Deharde et al., 2016[8]; Fardel et al., 2019[11]).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Deharde et al., 2016[8] source
    • cites Fardel et al., 2019[11]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Collagen type I is present all over the liver lobule, basement membrane formation is only observed in portal and central spaces, and collagen type IV can also be found in the space of Disse.

    “Collagen type I is present all over the liver lobule, while the formation of a basement membrane (BM) is only observed in the portal and central spaces. However, collagen type IV, a typical BM element, can also be found in the space of Disse (Martinez-Hernandez and Amenta, 1993[26]).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Martinez-Hernandez and Amenta, 1993[26] source

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

    Premisecitation

    OATP1B1 is a liver-specific transporter of bile acids, hormones and other substances that is expressed uniquely at the basolateral membrane.

    “Organic anion transporting polypeptide 1B1 (OATP1B1) is a liver-specific transporter of bile acids, hormones and other substances that is expressed uniquely at the basolateral membrane (Obaidat et al., 2012[30]).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Obaidat et al., 2012[30]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

    Premisecitation

    ZO-1 and -2 mediate the connection to the actin cytoskeleton and are crucial elements of the blood-bile barrier.

    “The TJ proteins zonula occludens (ZO)-1 and -2 mediate the connection to the actin cytoskeleton and are crucial elements of the blood-bile barrier (Itoh et al., 2021[21]).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Itoh et al., 2021[21]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

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

    Premisecitation

    Matrigel is a mixture of the basement membrane proteins collagen type IV, laminin, entactin and heparan sulphate proteoglycane.

    “Matrigel is a mixture of the BM proteins collagen type IV, laminin, entactin and heparan sulphate proteoglycane (Benton et al., 2014[4]; Hughes et al., 2010[19]).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Benton et al., 2014[4] source
    • cites Hughes et al., 2010[19] source

  • Classified as probably not a substantial claim.

    Premisecitation

    A previous study observed that application of Matrigel in combination with classic collagen can have a beneficial influence on hepatocyte morphology and polarization.

    “In a previous study, we observed that the application of Matrigel in combination with the classic collagen culture can have a beneficial influence on hepatocyte morphology and polarization (Deharde et al., 2016[8]).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Deharde et al., 2016[8] source

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

    Study design and conductnone given

    EEF2, 18S_rRNA and GUSB served as reference genes for RT-qPCR.

    “EEF2, 18S_rRNA and GUSB served as reference genes.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    Long-term cultured PHHs showed higher expression levels of HNF4A and CYP2C9 when cultured on mixed matrices than on pure collagen type I and for HNF4A also than on pure Matrigel.

    “Taken together, long-term cultured PHHs showed higher expression levels of HNF4A and CYP2C9 when cultured on mixed matrices than on pure collagen type I and for HNF4A also than on pure Matrigel.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Ethical approval and informed consent were obtained for human liver tissue samples.

    “All patients gave their informed consent according to the ethical guidelines of the Medical Faculty of Leipzig University (Ethical vote: registration number 322/17-ek, date 2020/06/10 ratified on 2021/11/30 and registration number 006/17-ek, date 2017/03/21 ratified on 2019/02/12).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cells incubated without primary antibodies served as negative controls.

    “Cells incubated without primary antibodies served as negative controls.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Due to the explorative design of the study, parametric testing was used despite the small sample size.

    “Due to the explorative design of the study we decided for parametric testing despite the small sample size.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    For each time point and culture condition, dead and alive cells were counted manually in 3 randomly chosen microscopy images.

    “For each time point and culture condition, dead and alive cells were counted manually in 3 randomly chosen microscopy images.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    To strengthen conclusions, assays on multiple levels were performed to validate the results.

    “To strengthen conclusions, we perfomed assays on multiple levels to validate our results.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    A certain concentration of Matrigel components can dampen cell isolation stress, support cellular regeneration and growth as well as stabilize hepatic functions in the subsequent culture.

    “We conclude that a certain concentration of Matrigel components can dampen cell isolation stress, support cellular regeneration and growth as well as stabilize hepatic functions in the subsequent culture.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Adding Matrigel to collagen type I can boost the positive Matrigel influence while negative effects are minimized.

    “Our approach of adding Matrigel to collagen type I can boost the positive Matrigel influence while negative effects are minimized.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    Mixed collagen-Matrigel matrices combine the positive traits of both ECM types and allow for a better development of cell-cell as well as cell-matrix contacts.

    “We suggest that mixed collagen-Matrigel matrices combine the positive traits of both ECM types and allow for a better development of cell-cell as well as cell-matrix contacts.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Interpretationreasoning

    Hepatocytes on the brink of apoptosis have a better chance to survive when open binding sites for ECM components are saturated.

    “Hence, hepatocytes on the brink of apoptosis have a better chance to survive.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Interpretationnone given

    In addition to cell-cell contacts, cell-matrix interactions are crucial for the maintenance of a differentiated hepatocyte phenotype.

    “In addition to cell-cell contacts, cell-matrix interactions are crucial for the maintenance of a differentiated hepatocyte phenotype.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The ECM composition has a clear influence on the maintenance of hepatocyte differentiation in vitro, and mixed collagen-Matrigel matrices provide a beneficial environment in this regard.

    “Taken together with our functional data, the ECM composition has a clear influence on the maintenance of hepatocyte differentiation in vitro and mixed collagen-Matrigel matrices provide a beneficial environment in this regard.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Matrigel offers a framework for the formation of cell-cell contacts that enable hepatocyte polarization, although PHHs cultured on pure Matrigel showed several features of dedifferentiation and especially an altered E-cadherin expression.

    “We conclude, that although the PHHs cultured on pure Matrigel showed several features of dedifferentiation and especially an altered E-cadherin expression, Matrigel offers a framework for the formation of cell-cell contacts that enable hepatocyte polarization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The 90C/10M cultures majorly reflected the polarization of the 100C cultures but started losing the well-defined basolateral membrane domain.

    “Taken together, the 90C/10M cultures majorly reflected the polarization of the 100C cultures but started losing the well-defined basolateral membrane domain.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The study's results strongly hint at ECM-dependency of the maintenance of the differentiated hepatocytic phenotype, while polarization is tied to the ability to form cell-cell contacts.

    “Our results strongly hint at ECM-dependency of the maintenance of the differentiated hepatocytic phenotype, while polarization is tied to the ability to form cell-cell contacts.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    TGF-β activates epithelial to mesenchymal transition in hepatocytes, leading to dedifferentiation and loss of function.

    “In contrast, TGF-β activates the epithelial to mesenchymal transition (EMT) in hepatocytes leading also to dedifferentiation and loss of function (Xu et al., 2009[47]).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Xu et al., 2009[47]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Interpretationreasoning

    A successful establishment of an in vivo-like in vitro liver model relies on both traits (ECM-dependent metabolism and polarization tied to cell-cell contacts).

    “Notwithstanding, a successful establishment of an in vivo-like in vitro liver model relies on both traits.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    A dedifferentiation-associated downregulation of albumin, HNF4A and its downstream targets in the cytochrome P450 enzyme group in primary hepatocyte cultures is well described.

    “A dedifferentiation-associated downregulation of albumin, the liver-enriched transcription factor HNF4A and its downstream targets in the cytochrome P450 enzyme group in primary hepatocyte cultures is well described (Elaut et al., 2006[10]).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Elaut et al., 2006[10] source

  • May combine two claims.

    Premisecitation

    Collagen type I is present all over the liver lobule, while other ECM elements are differently distributed over the sinusoid.

    “While collagen type I is present all over the liver lobule, there is an abundance of other ECM elements, that are differently distributed over the sinusoid (Martinez-Hernandeza and Amenta, 1993[26]).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martinez-Hernandeza and Amenta, 1993[26]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Premisecitation

    Loss of cell-matrix interactions leads to senescence, triggering apoptotic pathways.

    “It is known that loss of cell-matrix interactions leads to senescence, triggering apoptotic pathways (Gómez-Lechón et al., 1998[16]).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Gómez-Lechón et al., 1998[16]

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

65 of 65

  • The authors state this as an assumption.

    Mixed collagen-Matrigel matrices have not been evaluated as ECM for primary hepatocyte cultures of human origin before.

    “To the best of our knowledge, mixed collagen-Matrigel matrices have not been evaluated as ECM for primary hepatocyte cultures of human origin before.”

  • The paper relies on this without support.

    CDF is secreted into bile via the apical hepatocyte membrane transporter MRP2, allowing visualization of bile canaliculi by fluorescence microscopy.

    “The fluorescent CDF is secreted into the bile via the apical hepatocyte membrane transporter MRP2 allowing the visualization of the bile canaliculi by fluorescence microscopy.”
2 of 2

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.

  • Results name a 100G culture that was not among the tested conditions

    Minor

    The morphology results report a 100G culture, but the methods specify only 100C, 100M, and 90C/10M sandwich cultures. The stated culture condition is therefore inconsistent with the study's own condition labels.

1 of 1

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Related

Authors
Seidemann, L., Prinz, S., Scherbel, J.-C., Götz, C., Seehofer, D., & Damm, G.
Affiliations
University Hospital Leipzig (DE)
ORCID
Venue
IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund
Publisher
IfADo - Leibniz Research Centre for Working Environment and Human Factors, Dortmund
Published
2023
Volume
22
Pages
12-34
ISSN
1611-2156 (ISSN-L)
DOI
10.17179/excli2022-5459
PMC
PMC9837384
OpenAlex
W4317567935
Semantic Scholar
19e48db0f4317e1aaec1594b93a7c7e9ec593b1e
Cited by
3
References
50
Citations by year
2024: 3
FWCI
0.39
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • EXCLI journal (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Leibniz-Institut für Arbeitsforschung an der TU Dortmund (IfADo)
Funders
  • Bundesministerium für Bildung und Forschung (031L0256C)
  • Universität Leipzig
Topics
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
  • Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
Keywords
extracellular matrix, primary human hepatocytes, type I collagen, Matrigel, hepatocyte culture, hepatic differentiation, hepatocyte polarization, bile canaliculi, sandwich culture, liver model

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.

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Statistics and arithmetic are recomputed, not judged.

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