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Cited by 329Drug-Induced Hepatotoxicity and ProtectionAnimal testing and alternativesLiver physiology and pathology

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Comparison of Hepatic 2D Sandwich Cultures and 3D Spheroids for Long-term Toxicity Applications: A Multicenter Study

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Bell, Catherine C, Dankers, Anita C A, Lauschke, Volker M, Sison-Young, Rowena, Jenkins, Roz, Rowe, Cliff, Goldring, Chris E, Park, Kevin, Regan, Sophie L, Walker, Tracy, Schofield, Chris, Baze, Audrey, Foster, Alison J, Williams, Dominic P, van de Ven, Amy W M, Jacobs, Frank, Houdt, Jos van, Lähteenmäki, Tuula, Snoeys, Jan, Juhila, Satu, Richert, Lysiane, Ingelman-Sundberg, Magnus·2018·Toxicological Sciences
DOI 10.1093/toxsci/kfx289PMC PMC5888952
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Abstract

Primary human hepatocytes (PHHs) are commonly used for in vitro studies of drug-induced liver injury. However, when cultured as 2D monolayers, PHH lose crucial hepatic functions within hours. This dedifferentiation can be ameliorated when PHHs are cultured in sandwich configuration (2Dsw), particularly when cultures are regularly re-overlaid with extracellular matrix, or as 3D spheroids. In this study, the 6 participating laboratories evaluated the robustness of these 2 model systems made from cryopreserved PHH from the same donors considering both inter-donor and inter-laboratory variability and compared their suitability for use in repeated-dose toxicity studies using 5 different hepatotoxins with different toxicity mechanisms. We found that expression levels of proteins involved in drug absorption, distribution, metabolism, and excretion, as well as catalytic activities of 5 different CYPs, were significantly higher in 3D spheroid cultures, potentially affecting the exposure of the cells to drugs and their metabolites. Furthermore, global proteomic analyses revealed that PHH in 3D spheroid configuration were temporally stable whereas proteomes from the same donors in 2Dsw cultures showed substantial alterations in protein expression patterns over the 14 days in culture. Overall, spheroid cultures were more sensitive to the hepatotoxic compounds investigated, particularly upon long-term exposures, across testing sites with little inter-laboratory or inter-donor variability. The data presented here suggest that repeated-dosing regimens improve the predictivity of in vitro toxicity assays, and that PHH spheroids provide a sensitive and robust system for long-term mechanistic studies of drug-induced hepatotoxicity, whereas the 2Dsw system has a more dedifferentiated phenotype and lower sensitivity to detect hepatotoxicity.

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Figure 1. Scheme of experimental procedure. Primary human hepatocytes (PHHs) were seeded in either 2Dsw or 3D spheroid configuration. Due to the time taken for spheroids to form (7–10 days) day 0 for 3D experiments is 7–10 days after seeding. Medium changes (± test compound) were performed every 2–3 days (grey boxes) and for 2Dsw cultures matrigel was re-overlaid as shown (black boxes). Sampling points for proteomics (triangles) and ATP measurements (circles) are indicated. Toxicity assays were done with cells from 3 different donors. The morphologies of cells from donor 1 in each of the systems at days 3, 7, and 14 are illustrated. These images were generated at Janssen.
Figure 1. Scheme of experimental procedure. Primary human hepatocytes (PHHs) were seeded in either 2Dsw or 3D spheroid configuration. Due to the time taken for spheroids to form (7–10 days) day 0 for 3D experiments is 7–10 days after seeding. Medium changes (± test compound) were performed every 2–3 days (grey boxes) and for 2Dsw cultures matrigel was re-overlaid as shown (black boxes). Sampling points for proteomics (triangles) and ATP measurements (circles) are indicated. Toxicity assays were done with cells from 3 different donors. The morphologies of cells from donor 1 in each of the systems at days 3, 7, and 14 are illustrated. These images were generated at Janssen.CC BY-NC · PubMed Central
Figure 2. Proteomic signatures of primary human hepatocytes are strongly influenced by the choice of in vitro culture paradigm. Heatmap visualization of mean-centered, sigma-normalized hierarchically clustered whole proteome data from freshly thawed PHH as well as from cultures from the same 3 donors in 2Dsw and 3D spheroid culture is shown (FDR = 5%). Enrichment analyses of the identified gene clusters revealed significantly affected pathways. In addition, ADME genes within each cluster are shown separately.
Figure 2. Proteomic signatures of primary human hepatocytes are strongly influenced by the choice of in vitro culture paradigm. Heatmap visualization of mean-centered, sigma-normalized hierarchically clustered whole proteome data from freshly thawed PHH as well as from cultures from the same 3 donors in 2Dsw and 3D spheroid culture is shown (FDR = 5%). Enrichment analyses of the identified gene clusters revealed significantly affected pathways. In addition, ADME genes within each cluster are shown separately.CC BY-NC · PubMed Central
Figure 3. PHH undergo distinct molecular changes in 2Dsw and 3D spheroid culture that substantially influence their phenotype and functionality. Semi-log volcano plots showing the distribution of up- and downregulated proteins in 2D sandwich or 3D spheroid culture at day 0 (A) and after 14 days in culture (B). The dashed lines indicate the significance threshold of p = .05. C, Venn diagram visualizing the overlap of dysregulated proteins at day 0 and day 14 in 2Dsw and 3D spheroid cultures compared with freshly thawed cells. Note that the vast majority of proteins are either affected in 2Dsw or in 3D culture but only few in both. 2Dsw = 2D sandwich culture. D, Principal component analysis of differentially expressed genes reveals that samples cluster by in vitro culture paradigm. Furthermore, the plot indicates that the molecular phenotypes of PHH in 3D spheroid cultures remain stable over time, whereas substantial changes occur in 2Dsw configuration. Thawed cells are also depicted. E, Targeted analysis of ADME proteins and other factors with importance for hepatic functionality indicates that their levels are overall increased in 3D spheroids compared with 2Dsw cultures from the same donor (ie, positive fold-change) throughout the culture time. Values on the ordinate indicate the fold change (FC), defined as protein abundance in 3D spheroids divided by protein abundance in 2Dsw cultures if FC > 1 and the negative inverse if FC < 1. All samples for proteomic analysis were generated at KaLyCell (2Dsw) and Karolinska Institutet (3D) and processed and analyzed at the University of Liverpool.
Figure 3. PHH undergo distinct molecular changes in 2Dsw and 3D spheroid culture that substantially influence their phenotype and functionality. Semi-log volcano plots showing the distribution of up- and downregulated proteins in 2D sandwich or 3D spheroid culture at day 0 (A) and after 14 days in culture (B). The dashed lines indicate the significance threshold of p = .05. C, Venn diagram visualizing the overlap of dysregulated proteins at day 0 and day 14 in 2Dsw and 3D spheroid cultures compared with freshly thawed cells. Note that the vast majority of proteins are either affected in 2Dsw or in 3D culture but only few in both. 2Dsw = 2D sandwich culture. D, Principal component analysis of differentially expressed genes reveals that samples cluster by in vitro culture paradigm. Furthermore, the plot indicates that the molecular phenotypes of PHH in 3D spheroid cultures remain stable over time, whereas substantial changes occur in 2Dsw configuration. Thawed cells are also depicted. E, Targeted analysis of ADME proteins and other factors with importance for hepatic functionality indicates that their levels are overall increased in 3D spheroids compared with 2Dsw cultures from the same donor (ie, positive fold-change) throughout the culture time. Values on the ordinate indicate the fold change (FC), defined as protein abundance in 3D spheroids divided by protein abundance in 2Dsw cultures if FC > 1 and the negative inverse if FC < 1. All samples for proteomic analysis were generated at KaLyCell (2Dsw) and Karolinska Institutet (3D) and processed and analyzed at the University of Liverpool.CC BY-NC · PubMed Central
Figure 4. CYP catalyzed drug metabolism in 3D spheroids and 2Dsw cultures from the same donor. PHHs from donor 1 were cultured as either 3D spheroids or 2Dsw cultures and the activities of CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A4 were quantified by LC-MS/MS using phenacetin, amodiaquine, tolbutamide, dextromethorphan, and midazolam as probe substrates, respectively. As cells were incubated with probe substrates for different times (2h for spheroids and 30min for 2Dsw), activity data are shown as normalized per time and number of cells. Data represent the average of three 2Dsw incubations or 6 spheroids. Error bars indicate SD.
Figure 4. CYP catalyzed drug metabolism in 3D spheroids and 2Dsw cultures from the same donor. PHHs from donor 1 were cultured as either 3D spheroids or 2Dsw cultures and the activities of CYP1A2, CYP2C8, CYP2C9, CYP2D6, and CYP3A4 were quantified by LC-MS/MS using phenacetin, amodiaquine, tolbutamide, dextromethorphan, and midazolam as probe substrates, respectively. As cells were incubated with probe substrates for different times (2h for spheroids and 30min for 2Dsw), activity data are shown as normalized per time and number of cells. Data represent the average of three 2Dsw incubations or 6 spheroids. Error bars indicate SD.CC BY-NC · PubMed Central
Figure 5. The choice of culture format influences the sensitivity to selected hepatotoxins in a compound-specific manner. Hepatocytes from 3 different donors were cultured in 2Dsw and 3D spheroid configuration at 6 different laboratories. A, Hepatocyte viability remains stable in both culture methods for up to 2 weeks in culture as quantified by ATP measurements in untreated cells. Viability is represented as normalized to ATP levels from the same donor at day 3. For 2Dsw incubations, n = 6, for 3D spheroids n = 10–12. PHH from 3 donors cultured in either 3D spheroid or 2Dsw configuration were exposed to the hepatotoxic compounds acetaminophen (APAP, B), bosentan (C), diclofenac (D), fialuridine (E), or troglitazone (F) for up to 14 days. Furthermore, we determined the toxicity of troglitazone’s less-toxic structural analog pioglitazone (G). EC50 values were determined by quantification of intracellular ATP levels. Dashed lines indicate 10× therapeutic Cmax concentrations for the respective compound (human Cmax values: APAP = 139 µM [Sevilla-Tirado et al., 2003], bosentan = 7.4 µM [Gutierrez et al., 2013], diclofenac = 8 µM [G.D. Searle LLC Division of Pfizer Inc., FDA professional drug information—Arthrotec (2015) available at: http://www.drugs.com/pro/arthrotec.html (accessed: 4 September 2017)], fialuridine = 1 µM [Bowsher et al., 1994], pioglitazone = 2.9 µM [Wong et al., 2004], troglitazone = 3.6 µM corresponding to 400 mg recommended daily dose [Loi et al., 1999]). Dotted line indicates EC50 values above the highest tested concentration. All 3D spheroid experiments were performed at a minimum of 3 different laboratories (as indicated in Table 1), whereas for 2Dsw experiments, donor 1 was compared across 3 sites, while incubations for donors 2 and 3 were performed only at a single site. Error bars indicate SEM.
Figure 5. The choice of culture format influences the sensitivity to selected hepatotoxins in a compound-specific manner. Hepatocytes from 3 different donors were cultured in 2Dsw and 3D spheroid configuration at 6 different laboratories. A, Hepatocyte viability remains stable in both culture methods for up to 2 weeks in culture as quantified by ATP measurements in untreated cells. Viability is represented as normalized to ATP levels from the same donor at day 3. For 2Dsw incubations, n = 6, for 3D spheroids n = 10–12. PHH from 3 donors cultured in either 3D spheroid or 2Dsw configuration were exposed to the hepatotoxic compounds acetaminophen (APAP, B), bosentan (C), diclofenac (D), fialuridine (E), or troglitazone (F) for up to 14 days. Furthermore, we determined the toxicity of troglitazone’s less-toxic structural analog pioglitazone (G). EC50 values were determined by quantification of intracellular ATP levels. Dashed lines indicate 10× therapeutic Cmax concentrations for the respective compound (human Cmax values: APAP = 139 µM [Sevilla-Tirado et al., 2003], bosentan = 7.4 µM [Gutierrez et al., 2013], diclofenac = 8 µM [G.D. Searle LLC Division of Pfizer Inc., FDA professional drug information—Arthrotec (2015) available at: http://www.drugs.com/pro/arthrotec.html (accessed: 4 September 2017)], fialuridine = 1 µM [Bowsher et al., 1994], pioglitazone = 2.9 µM [Wong et al., 2004], troglitazone = 3.6 µM corresponding to 400 mg recommended daily dose [Loi et al., 1999]). Dotted line indicates EC50 values above the highest tested concentration. All 3D spheroid experiments were performed at a minimum of 3 different laboratories (as indicated in Table 1), whereas for 2Dsw experiments, donor 1 was compared across 3 sites, while incubations for donors 2 and 3 were performed only at a single site. Error bars indicate SEM.CC BY-NC · PubMed Central

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Metadata

Authors
Bell, C. C., Dankers, A. C. A., Lauschke, V. M., Sison-Young, R., Jenkins, R., Rowe, C., Goldring, C. E., Park, K., Regan, S. L., Walker, T., Schofield, C., Baze, A., Foster, A. J., Williams, D. P., van de Ven, A. W. M., Jacobs, F., Houdt, J. V., Lähteenmäki, T., Snoeys, J., Juhila, S., Richert, L., & Ingelman-Sundberg, M.
Affiliations
  • Karolinska Institutet (SE)
  • Janssen (Belgium) (BE)
  • University of Liverpool (GB)
  • MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, University of Liverpool, Liverpool, UK
  • AstraZeneca (United Kingdom) (GB)
  • GlaxoSmithKline (United Kingdom) (GB)
  • GlaxoSmithKline Research & Development Limited (United Kingdom) (GB)
  • KaLy Cell, Plobsheim, France
  • Orion Corporation (Finland) (FI)
  • Université Bourgogne Franche-Comté (FR)
  • Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden
ORCID
Venue
Toxicological Sciences
Publisher
Oxford University Press (OUP)
Published
2018-01-10
Volume
162
Issue
2
Pages
655-666
ISSN
  • 1096-0929 (ISSN-L)
  • 1096-6080
DOI
10.1093/toxsci/kfx289
PMC
PMC5888952
OpenAlex
W2782718389
Cited by
327
References
47
Citations by year
2026: 40 · 2025: 38 · 2024: 28 · 2023: 31 · 2022: 39 · 2021: 60 · 2020: 47 · 2019: 28 · 2018: 16
FWCI
41.55
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Toxicological Sciences (publishedVersion, cc-by-nc)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • AstraZeneca
  • Vetenskapsrådet (2015-02760, 2016-06222, 01154, 2016-01154, 2016-01153)
  • Medical Research Council (MR/L006758/1)
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
  • Health Sciences › Veterinary › Small Animals › Animal testing and alternatives
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
Keywords
primary human hepatocytes, multicellular spheroids, drug-induced liver injury, hepatotoxicity, repeated-dose toxicity, hepatocyte dedifferentiation, cryopreserved hepatocytes, inter-laboratory variability, donor variability, cytochrome P450 expression
MeSH terms
Cell Survival, Cells, Cultured, Cytochrome P-450 Enzyme System, Dose-Response Relationship, Drug, Pharmaceutical Preparations, Humans, Male, Middle Aged, Models, Biological, Predictive Value of Tests, Time Factors, Cryopreservation, Toxicity Tests, Spheroids, Cellular, Coculture Techniques, Hepatocytes, Chemical and Drug Induced Liver Injury, Primary Cell Culture

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