In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System
Abstract
:1. Introduction
2. Results
2.1. General Metabolic Activity
2.2. Formation of Diclofenac Metabolites
2.3. Cell Morphology and Reorganization in the Bioreactor
2.4. Analysis of Transcriptomic Data upon Drug Application
3. Discussion
4. Materials and Methods
4.1. Bioreactor Technology
4.2. Isolation of Primary Human Liver Cells
4.3. 3D Culture of Primary Human Liver Cells
4.4. Analysis of Clinical Parameters
4.5. Analysis of Diclofenac and Its Metabolites
4.6. Histology and Immunohistochemistry
4.7. Microarray Analysis
4.8. Statistics
5. Conclusions
Supplementary Materials
Acknowledgments
Author Contributions
Conflicts of Interest
Abbreviations
2D | two-dimensional |
3D | three-dimensional |
300 µM BR | 300 µM bioreactors |
1000 µM BR | 1000 µM bioreactors |
A1AT | α-1-antitrypsin |
ABCB1 | ATP-binding cassette B1 |
ABCC2 | ATP-binding cassette C2 |
ABCG2 | ATP-binding cassette G2 |
ALB | albumin |
ALT | alanine transaminase |
ARG1 | arginase 1 |
APOC1 | apolipoprotein C-I |
ASL | argininosuccinate lyase |
AST | aspartate transaminase |
ATF3 | activating transcription factor 3 |
B2M | β -2-microglobulin |
BCRP | breast cancer resistance protein |
CK 18 | cytokeratin 18 |
control BR | control bioreactors |
CPS1 | mitochondrial carbamoyl-phosphate synthase 1 |
CYP | cytochrome P450 |
DILI | drug-induced liver injury |
DMSO | dimethyl sulfoxide |
FDR | False Discovery Rate |
GLDH | glutamate dehydrogenase |
GDF15 | growth differentiation factor 15 |
LDH | lactate dehydrogenase |
MS | mass spectrometry |
NRF2/KEAP1 | nuclear factor (erythroid-derived 2)-like 2/kelch-like ECH-associated protein 1 |
OTC | ornithine transcarbamoylase |
pHH | primary human hepatocytes |
SEM | standard errors of the mean |
TTR | transthyretin |
UDP | uridine diphosphate |
UGT2B7 | UDP glucuronosyltransferase 2B7 |
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Rank | GO ID | Size | p-Value | Description | Parent |
---|---|---|---|---|---|
Gene Ontology Biological Processes | |||||
1 | GO:0072376 | 67 (90) | 6.0 × 10−33 | Protein activation cascade | |
2 | GO:0006956 | 47 (69) | 1.8 × 10−31 | Complement activation | 1 |
3 | GO:0016064 | 94 (119) | 9.8 × 10−29 | Immunoglobulin mediated immune response | |
4 | GO:0002455 | 43 (67) | 1.0 × 10−27 | Humoral immune response mediated by circulating immunoglobulin | 3 |
5 | GO:0019724 | 97 (122) | 4.9 × 10−27 | B cell mediated immunity | 3 |
6 | GO:0002576 | 83 (87) | 1.4 × 10−26 | Platelet degranulation | |
7 | GO:0002687 | 89 (90) | 3.5 × 10−26 | Positive regulation of leukocyte migration | |
8 | GO:0006958 | 31 (52) | 2.0 × 10−22 | Complement activation, classical pathway | 1, 2, 3, 4, 5 |
9 | GO:0002688 | 79 (83) | 2.7 × 10−21 | Regulation of leukocyte chemotaxis | |
10 | GO:0002690 | 68 (69) | 2.8 × 10−18 | Positive regulation of leukocyte chemotaxis | 7, 9 |
Gene Ontology Cellular Component | |||||
1 | GO:0032994 | 37 (40) | 8.7 × 10−25 | Protein-lipid complex | |
2 | GO:0034358 | 35 (38) | 1.8 × 10−24 | Plasma lipoprotein particle | 1 |
3 | GO:0042613 | 12 (16) | 4.6 × 10−24 | MHC class II protein complex | |
4 | GO:0042611 | 22 (27) | 1.6 × 10−20 | MHC protein complex | 3 |
5 | GO:0031983 | 73 (82) | 1.1 × 10−19 | Vesicle lumen | |
6 | GO:0060205 | 72 (81) | 2.4 × 10−19 | Cytoplasmic membrane-bounded vesicle lumen | 5 |
7 | GO:0030134 | 48 (52) | 6.7 × 10−17 | ER to Golgi transport vesicle | |
8 | GO:0012507 | 38 (42) | 1.6 × 10−15 | ER to Golgi transport vesicle membrane | 7 |
9 | GO:0034364 | 23 (26) | 1.0 × 10−14 | High-density lipoprotein particle | 1, 2 |
10 | GO:0031091 | 60 (63) | 1.3 × 10−12 | Platelet α granule | |
Gene Ontology Molecular Functions | |||||
1 | GO:0042379 | 54 (58) | 2.8 × 10−9 | Chemokine receptor binding | |
2 | GO:0008009 | 43 (47) | 1.3 × 10−8 | Chemokine activity | 1 |
3 | GO:0030170 | 53 (56) | 5.3 × 10−7 | Pyridoxal phosphate binding | |
4 | GO:0003823 | 76 (107) | 2.0 × 10−6 | Antigen binding | |
5 | GO:0045236 | 17 (17) | 9.7 × 10−6 | CXCR chemokine receptor binding | 1, 2 |
6 | GO:0023026 | 15 (16) | 2.6 × 10−5 | MHC class II protein complex binding | 4 |
7 | GO:0060228 | 6 (6) | 3.1 × 10−5 | Phosphatidylcholine-sterol O-acyltransferaseactivator activity | |
8 | GO:0031210 | 15 (16) | 4.7 × 10−5 | Phosphatidylcholine binding | |
9 | GO:0019864 | 8 (12) | 1.1 × 10−4 | IgG binding | |
10 | GO:0023023 | 17 (19) | 1.6 × 10−4 | MHC protein complex binding | 4, 6 |
Rank | GO ID | Size | p-Value | Description | Parent |
---|---|---|---|---|---|
Gene Ontology Biological Processes | |||||
1 | GO:0042590 | 74 (79) | 1.2 × 10−46 | Antigen processing and presentation of exogenous peptide antigen via MHC class I | |
2 | GO:0002479 | 70 (75) | 2.1 × 10−36 | Antigen processing and presentation of exogenous peptide antigen via MHC class I, TAP-dependent | 1 |
3 | GO:0006521 | 59 (62) | 1.0 × 10−31 | Regulation of cellular amino acid metabolic process | |
4 | GO:0033238 | 79 (82) | 2.9 × 10−26 | Regulation of cellular amine metabolic process | |
5 | GO:0002576 | 83 (87) | 8.9 × 10−19 | Platelet degranulation | |
6 | GO:0044783 | 75 (77) | 3.9 × 10−17 | G1 DNA damage check point | |
7 | GO:0051437 | 68 (72) | 6.7 × 10−17 | Positive regulation of ubiquitin-protein ligase activity | |
8 | GO:0051351 | 86 (94) | 1.4 × 10−16 | Positive regulation of ligase activity | |
9 | GO:0051436 | 63 (67) | 2.1 × 10−16 | Negative regulation of ubiquitin-protein ligase activity | |
10 | GO:0051443 | 81 (89) | 3.0 × 10−16 | Positive regulation of ubiquitin-protein transferase activity | 8 |
Gene Ontology Cellular Component | |||||
1 | GO:0000502 | 63 (66) | 3.6 × 10−25 | Proteasome complex | |
2 | GO:1990204 | 78 (83) | 2.6 × 10−18 | Oxidoreductase complex | |
3 | GO:0043202 | 72 (73) | 2.5 × 10−16 | Lysosomal lumen | |
4 | GO:0005775 | 73 (77) | 1.4 × 10−15 | Vacuolar lumen | 3 |
5 | GO:0031091 | 60 (63) | 3.1 × 10−15 | Platelet α granule | |
6 | GO:0070469 | 65 (72) | 3.1 × 10−15 | Respiratory chain | |
7 | GO:0005746 | 59 (65) | 9.6 × 10−13 | Mitochondrial respiratory chain | 6 |
8 | GO:0022624 | 22 (24) | 3.9 × 10−12 | Proteasome accessory complex | 1 |
9 | GO:0031902 | 93 (99) | 3.1 × 10−11 | Late endosome membrane | |
10 | GO:0005839 | 20 (20) | 1.6 × 10−10 | Proteasome core complex | 1 |
Gene Ontology Molecular Functions | |||||
1 | GO:0009055 | 89 (99) | 8.2 × 10−21 | Electron carrier activity | |
2 | GO:0003743 | 48 (52) | 1.1 × 10−14 | Translation initiation factor activity | |
3 | GO:0016651 | 87 (95) | 9.7 × 10−14 | Oxidoreductase activity, acting on NAD(P)H | |
4 | GO:0016655 | 49 (53) | 1.6 × 10−12 | Oxidoreductase activity, acting on NAD(P)H, quinon or similar compound as acceptor | 3 |
5 | GO:0008135 | 75 (83) | 2.9 × 10−11 | Translation factor activity, nucleic acid binding | |
6 | GO:0004298 | 21 (21) | 4.3 × 10−8 | Threonine-type endopeptidase activity | |
7 | GO:0070003 | 21 (21) | 4.3 × 10−8 | Threonine-type peptidase activity | 6 |
8 | GO:0016830 | 45 (47) | 1.1 × 10−6 | Carbon-carbon lyase activity | |
9 | GO:0003954 | 36 (39) | 2.4 × 10−6 | NADH dehydrogenase activity | 3, 4 |
10 | GO:0008137 | 36 (39) | 2.4 × 10−6 | NADH dehydrogenase (ubiquinone) activity | 3, 4, 9 |
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Knöspel, F.; Jacobs, F.; Freyer, N.; Damm, G.; De Bondt, A.; Van den Wyngaert, I.; Snoeys, J.; Monshouwer, M.; Richter, M.; Strahl, N.; et al. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System. Int. J. Mol. Sci. 2016, 17, 584. https://doi.org/10.3390/ijms17040584
Knöspel F, Jacobs F, Freyer N, Damm G, De Bondt A, Van den Wyngaert I, Snoeys J, Monshouwer M, Richter M, Strahl N, et al. In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System. International Journal of Molecular Sciences. 2016; 17(4):584. https://doi.org/10.3390/ijms17040584
Chicago/Turabian StyleKnöspel, Fanny, Frank Jacobs, Nora Freyer, Georg Damm, An De Bondt, Ilse Van den Wyngaert, Jan Snoeys, Mario Monshouwer, Marco Richter, Nadja Strahl, and et al. 2016. "In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System" International Journal of Molecular Sciences 17, no. 4: 584. https://doi.org/10.3390/ijms17040584