2.3. Cells and culture media
HepG2/C3A, a clone of the HepG2 line derived from human hepatocellular carcinoma (ATCC CRL-10741), were used as the hepatocyte model. They were cultured in Minimal Essential Medium (MEM) with phenol red (Pan Biotech), 10% fetal bovine serum (FBS, Gibco), 1 mM hydroxy-ethylpiperazine-N-2-ethanesulfonic acid (HEPES, Gibco), 2 mM L-glutamine (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), and 100 U/mL penicillin, 100 μg/mL streptomycin (Pan Biotech).
The SK-HEP-1 cell line (ATCC, HTB-52) derived from an adenocarcinoma of the liver was used as the LSECs model. For maintenance, SK-HEP-1 cells were cultured in a mixture of 75% EGM-2 medium (Lonza) and 25% MEM (complemented as mentioned above).
All cells were cultured in 75 cm² flasks at 37°C in a humidified atmosphere with 5% of CO2. The culture medium was renewed every 2 days and the cells were passaged weekly at a confluence of 80-90%. To decrease the potential variability in the results, the cells were used between passages 10 and 20.
2.4. Experimental procedure of the cell cultures
2.4.1. Optimisation of common culture medium for HepG2/C3A and SK-HEP-1 cells
Culture medium optimisation was performed in static conditions, and different MEM/EGM-2 ratios were tested. The SK-HEP-1 cells were seeded in cell culture inserts (6-well format, polyethylene terephthalate membrane, 0.4 µm pore, THINCERT Greiner) at a density of 0.35 105 cell/cm2. The culture medium was renewed every 2 days in the apical (1 mL) and basal (2 mL) compartments, and the culture was maintained until confluence was attained (6-8 days). The HepG2/C3A were seeded in the wells of a 6-well plate at a density of of 105 cell/cm2. The culture was maintained for 4 days, and the medium (2 mL) was changed every 2 days. The cultures were continuously maintained at 37°C in a 5% CO2 supplied incubator and the assays were performed at the end of the experiments.
2.4.2. Dynamic monoculture and coculture in the IIDMP device
Each experiment lasted two days (Figure 1). The SK-HEP-1 inserts were maintained for 8 days for the formation of a confluent barrier, before performing the dynamic experiments, as mentioned in section 2.4.1. In parallel, 24 h before the dynamic experiments, HepG2/C3a cells were seeded in the biochips containing the hydroscaffold (4 105 cell/biochip), and the biochips were incubated overnight at 37°C in a humidified atmosphere with 5% of CO2.
On Day 0 of the experiment, the SK-HEP-1 previously grown for 8 days on inserts were transferred into the first well of the IIDMP device and the HepG2/C3a biochips were connected to the bottom of the device. As shown in Figure 1, three conditions were established: SK-HEP-1 monoculture (IIDMP with insert alone), HepG2/C3a monoculture (IIDMP with biochip alone) and coculture (IIDMP containing insert and biochip). Culture medium was added (1 mL in the apical insert side, 5 mL in the basal side and 4 mL in the reservoir well), the IIDMP was closed and connected to the pump. The entire setup was placed in the incubator and perfusion started at 10 µL/min for 48 h in a closed loop. For exposure to drugs, acetaminophen (APAP, Sigma-Aldrich) was loaded into the apical compartment of the insert at 1 mM before perfusion started (an insert without cells was used for HepG2/C3a monoculture experiments). After dilution in the total medium in the circuit (10 mL), the systemic concentration of APAP was 100 µM.
For each experiment, three IIDMP devices were used simultaneously to achieve 9 replicates/condition per experiment. At the end of the experiment, the pump was stopped, and the cover removed. The culture media were sampled, and the SK-HEP-1 inserts and HepG2/C3a biochips were detached from the device to perform the assays.
2.5. Lucifer Yellow permeability assay
Lucifer Yellow (LY) is a fluorescent molecule used to test paracellular transport across the endothelial (SK-HEP-1) barrier. LY (LY CH dipotassium salt, Sigma-Aldrich) was diluted in Hanks' balanced salt solution (HBSS, with CaCl2 and MgCl2, Gibco) at 50 µM and loaded into the apical compartment of an empty insert and inserts with cells were cultured for 4-15 days. The basal compartment was filled with HBSS. The inserts were then incubated at 37°C and 5% of CO2. After 90 min, medium from the apical and basal compartments was collected. The fluorescence intensity was measured using a microplate reader (TECAN Spectafluor Plus) at excitation/emission wavelengths of 485/530 nm. The flow of LY was expressed by the calculation of the apparent permeability (Papp, m/s) as follows:
Papp = (dQ/dt) x (1/AxCa)
Where dQ/dt is the amount of LY transported during a given time (mol/s), Ca is the initial concentration of LY solution (mol/m3) and A is the surface of the insert (m2).
2.6. Permeability to dextrans
The SK-HEP-1 barrier’s permeability to molecules of different molecular weights was assessed using fluorescein isothiocyanate-dextrans (FITC-dextran 4, 70 and 150 kDa, Sigma-Aldrich). The assays were performed using confluent SK-HEP-1 barriers (8 days of culture) in static and dynamic (IIDMP device) conditions. The dextrans were diluted in the culture medium at a concentration of 100 µg/mL and deposited in the apical compartment of the culture inserts. Then, culture medium was sampled in the apical and basal compartments at different times. The FITC-dextran fluorescence intensity was measured using a microplate reader (TECAN Spectafluor Plus) at excitation/emission wavelengths of 490/525 nm.
2.7. Immunostaining assays
At the end of the experiments, the SK-HEP-1 inserts were washed with phosphate buffer saline solution (PBS, Gibco), fixed in PBS, 4% para-formaldehyde (PFA, MP biomedicals) for 30 min at room temperature, washed and stored at 4°C in PBS. Cells were permeabilised with 0.5% Triton X-100 in PBS solution for 30 min and blocked in PBS, 1% bovine serum albumin (BSA, Sigma Aldrich) for 30 min. The samples were incubated with primary antibodies overnight at 4°C in the dark. After washing with PBS, the secondary antibodies were added, and the samples incubated for a further 12 h at 4°C in the dark. Actin filaments were stained with Alexa Fluo 488 Phalloidin for 3h (Thermo Fisher, 1/50). Nuclei were stained with 10 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, D1306, Invitrogen) for 30 min at room temperature in the dark. Imaging was obtained with a laser scanning confocal microscope (LSM 710, Zeiss).
The primary antibodies used were mouse anti-PECAM-1/CD31 (ab24590, abcam, 1 µg/mL), rabbit anti-stabilin-2 (ab121893, abcam, 1 µg/mL), mouse anti-vimentin (ab8978, abcam, 1 µg/mL). Donkey anti-mouse Alexa Fluor 647 (ab150107, Abcam, 2 µg/mL) and goat anti-rabbit Alexa Fluor 488 (A11034, Invitrogen, 2 µg/mL) were used as secondary antibodies.
Total RNA was purified by phenol/chloroform extraction followed by alcohol precipitation, and RNA concentrations measured using a NanodropOne (Thermofisher Scientific). Reverse transcription reactions were performed using a High-capacity cDNA reverse transcription kit with RNase inhibitor (Applied Biosystems, ThermoFisher Scientific). Quantitative PCRs were performed using a StepOnePlus machine (Applied Biosystems, Thermofisher Scientific) in duplex reactions, mixing the cDNA with the TaqMan FAM-labelled probes of the selected gene (Applied Biosystems, ThermoFisher Scientific) and a β2-microglobulin-VIC-labeled probe (Table S1). The threshold cycle (CT) values were calculated at the upper linear range of the logarithm−2 amplification curve using the StepOne v2.3 software (Thermofisher scientific). The data were then expressed as 2−∆∆CT with ∆CT as the difference between the CT of the transcript of interest and the CT of the reference, and ∆∆CT is the difference between the mean ∆CT of the experimental samples and the mean ∆CT of the control samples (Livak and Schmittgen 2001). The relative quantity (RQ) corresponds to 2−∆∆CT which transforms the logarithmic−2 data into decimal values.
Detection and quantitative evaluation of APAP and APAP metabolites was performed with LC-HRMS. The HPLC system (Infinity 1290, Agilent Technologies, France) with DAD, was connected to a Q-TOF micro hybrid quadrupole time of flight mass spectrometer (Agilent 6538, Agilent Technologies, France) with electrospray ionisation (ESI). HPLC was carried out on a Thermo Hypersyl Gold C18 (USP L1) column (150 × 4.6 mm ID, 5 µm, 175 A), connected to an Agilent Infinity 1290 HPLC at 40°C. The solvent system was A: 0.1% formic acid in H2O and B: acetonitrile. The gradient programme began with 5% B, held at 5% for 1 min and ramped up to 20% B at 5 min and to 95% at 7 min, held at 95% for 3 min, then decreased to the initial condition and held at 5 % for 1 min. The flow rate was set at 1 mL/min. All compound responses were measured in ESI+ and ESI- alternatively and were calibrated externally. The ESI Gas Temp was 350 °C, at electrospray voltage +3800 V or – 3500V. Drying gas was set at 10 L/min and the nebuliser was at 30 psi. Fragment voltage was set at 110 V. The HRMS spectrum was registered at 5 Hz in the mass range of 50 to 1200 m/z with internal calibration. Software MassHunter (Version B.07.00, Agilent Technologies, Santa Clara, CA 95051, United States) was used for data processing, quantification, and data acquisition. APAP and APAP metabolites were validated by the conjunction of exact mass and retention time from standards.
3. Results
The culture of cells of different origin in the same system requires an adapted coculture medium capable of maintaining both cell types in good conditions, without impairing their characteristics and functionalities. The routine culture medium used in our conditions for SK-HEP-1 is EGM-2/MEM (75%/25%) and the cells formed a well-structured cell monolayer at confluence, as needed for the barrier function (Figure 2A, Figure S3). On the other hand, when SK-HEP-1 cells were cultured in HepG2/C3a medium, which is based on MEM only, the endothelial cell morphology was greatly altered, and the cells failed to form a confluent monolayer (Figure S3). In an attempt to, first, create the endothelial barrier, and then to switch to a hepatocyte culture medium, SK-HEP-1 cells were cultured in their normal medium for 6 days, followed by culture in MEM for 3 days (as the coculture period). In these conditions again, the endothelial cells failed to maintain a confluent monolayer (Figure S3). Finally, when cells were maintained in EGM-2/MEM (25%/75%) medium for 7 days, the SK-HEP-1 cells formed a confluent monolayer (Figure 2A and Figure S3) and exhibited the characteristic morphology of SK-HEP-1, as when cultured in their original medium. The gene expression levels of several LSECs markers were investigated. No major differences were observed for most of the genes when cells were cultured in EGM-2/MEM (25%/75%) when compared to their original medium. A downregulation of CLEC4M and VCAM1 was observed when cells were maintained in EGM-2/MEM (25%/75%) in comparison with native medium, with fold changes (FC) of 0.25 and 0.48, respectively (Figure 2B). Based on the results obtained with SK-HEP-1 and HepG2/C3a cells, the mixture of EGM-2/MEM (25%/75%) was chosen for the dynamic coculture experiments. To facilitate the comparisons between monoculture and coculture, this medium was also used for SK-HEP-1 and HepG2/C3a maintenance in monocultures.
LSECs act as a physical barrier to molecules and play a significant role in transportation from circulating blood to the hepatocytes. Therefore, before using SK-HEP-1 to form a liver endothelial barrier in our coculture model, it was essential to characterise the formation, integrity, and permeability of the barrier. The SK-HEP-1 cells were seeded in static inserts using the selected coculture medium and followed over time. The cells proliferated continuously to reach full confluence and form homogenous and continuous monolayers from Days 7-8 and thereafter (Figure S4). Then, overgrowth could be observed, resulting in the formation of a second layer of cells on top of the first one (Day 10, Figure S4). Nevertheless, the formation of continuous layers of confluent cells was confirmed by nuclei, vimentin, and actin stainings. As shown in Figure 3A, the tissue was dense with contiguous cells and a well-developed actin network. The LSECs phenotype of the SK-HEP-1 barrier was confirmed by the positive staining for LSECs markers PECAM-1 and stabilin-2 (Figure 3B).
To evaluate the effects of coculture on the specific functions of HepG2/C3a, albumin secretion rates were quantified and found to be similar to those in monoculture (Figure 7B). After 48 h of culture, the albumin secretion levels were 127 ± 24 and 134 ± 28 ng/h in monoculture and coculture, respectively. The expression levels of several specific marker genes of HepG2/C3a cells (UGT2B7, UGT1A1, SULT1A2, CYP1A2 and CYP1A1) were also evaluated to determine whether the SK-HEP-1 barrier influenced the HepG2/C3a cultures. As shown in Figure 7C, there were no differences in expression levels in the selected genes between HepG2/C3a maintained as a monoculture and HepG2/C3a in coculture with SK-HEP-1.
To test the coculture model and demonstrate the crosstalk between the HepG2/C3a biochips and SK-HEP-1 barrier in the configuration of a drug study, we exposed the SK-HEP-1 barrier to APAP with and without coculture with the HepG2/C3a biochip. APAP was chosen because it is i) metabolised by HepG2/C3a cells, ii) widely studied with liver in vitro models, and iii) not adsorbed by the PDMS biochip (Bricks et al., 2015). APAP was introduced into the apical side of the SK-HEP-1 barrier at 1 mM, leading to a systemic theorical concentration of 100 µM after diffusion in the total circuit. For comparative purposes, HepG2/C3a monoculture in the IIDMP was also performed and APAP was deposited into the insert without SK-HEP-1.
The metabolism of APAP was then investigated in SK-HEP-1 and HepG2/C3a cocultures, or monocultures using HPLC coupled to MS. We used the culture medium collected in the basal compartment to confirm the passage of APAP through the SK-HEP-1 barrier. The ratios of APAP (compared to the initial systemic concentration of 100 µM) recovered at the end of the experiment are provided in Figure 9D. For the SK-HEP-1 monoculture, the APAP ratio at the end of the experiment was 1.02 ± 0.07, indicating that SK-HEP-1 did not metabolise APAP. The recovered ratio, corresponding to a concentration of 100 µM, confirmed the passage of APAP through the barrier, allowing the equilibrium of APAP concentration between the apical and basal sides. In the HepG2/C3a monoculture and SK-HEP-1/HepG2/C3a coculture, the APAP ratios were 0.83 ± 0.05 and 0.87 ± 0.08, respectively, illustrating metabolism (
Figure 9D). However, for both conditions, the paracetamol sulphate and paracetamol glucuronide concentrations were below detection limits.
3.5. Expression of inflammatory cytokines
The expression of inflammatory cytokines was evaluated in all culture conditions (with and without APAP) by analysing mRNA levels for TNFα, IL-1, IL-6 and IL-8 genes, and by quantifying IL-6 secretion in the culture medium. SK-HEP-1 cells expressed the four genes in all culture conditions (monoculture/coculture and APAP+/APAP-). Gene expression levels of IL8, IL6 and IL1 were similar, regardless of the culture conditions. There was a noticeable significant upregulation of TNFα in SK-HEP-1 cocultured with APAP (Figure 9E). Regarding HepG2C3a cells, there were no significant differences in expression levels of IL-8 in the conditions tested. On the other hand, there was a slight but significant overexpression of TNFα in HepG2/C3a cocultured without APAP when compared to monocultures (Figure 9F). IL-6 protein quantification in culture medium showed that it was only expressed by SK-HEP-1 cells and that HepG2/C3a monocultures with and without APAP did not produce detectable amounts of IL-6 (Figure 9G).
4. Discussion
Classic 2D in vitro coculture models consist of cells randomly mixed and heterogeneously distributed at the bottom of well-plates and dishes. However, in vivo, LSECs and hepatocytes are separated by the space of Disse which, in 3D models, is generally mimicked by a gel or collagen matrix which physically separates LSECs and hepatocytes (Bale et al., 2015). Furthermore, controlling the homotypic and heterotypic cell-to-cell interactions appears to be a key feature for maintaining and enhancing the hepatocyte phenotype (Bhatia et al., 1999; Bhatia 1997). In the present work, we have established a coculture model of liver sinusoidal endothelial cells with liver hepatocytes. Thanks to our platform which integrates a liver-on-chip solution and a barrier insert, we were able to propose technology that physically separated both cell types. In this model, cell-to-cell paracrine-like communication was made possible by exchanges through the insert membrane, as this model did not allow direct contact between LSECs and hepatocytes. Although this type of technology has already been presented for organ-to-organ models such as the intestine barrier-liver (Bricks et al., 2014), to our knowledge, only two other such dynamic LSECs barrier-hepatocyte coculture models have previously been described (van Grunsven 2017; Lauschke et al., 2019).
We demonstrated the functionality of the coculture model using two human cell lines, SK-HEP-1 and HepG2/C3a. For this purpose, we optimised the culture medium, confirmed the innocuity of the fluid flow and coculture on the LSECs barriers, and characterised the cytokine crosstalk between cells. Establishing a coculture medium that is healthy for two or more types of cells is a critical step in in vitro physiological models (Vis et al., 2020), including liver cells (Lauschke et al., 2019).
Similarly, it was reported that LSECs are sensitive to serum components (Elvevold, et al., 2008). Our data demonstrated that the HepG2/C3a MEM-based medium which contained serum contributed to damaging the LSECs layer, whereas the conventional LSECs medium (also containing serum) did not. Interestingly, a mixture of the HepG2/C3a and SK-HEP-1 media led to both healthy LSECs and HepG2/C3a. Although we did not identify the specific factors leading to this result, we postulate that the presence of pro-angiogenic factors in EGM-2 medium played a part in stabilising the LSEC cultures. Interestingly, the present dynamic conditions did not affect the cell junctions or the expression levels of LSEC markers.
Endothelial cells are normally exposed to flow, and dynamic in vitro models have largely been reported as regulating their functions and physiology (Akbari et al., 2018, van Duinen et al, 2017). However, a decrease in endothelial barrier permeability was only reported in dynamic cultures coupled to high shear stress (0.7-1 Pa, van Duinen et al, 2017). In the present work, we did not observe significant variations in barrier permeability functions between static and dynamic SK-HEP-1 monocultures (Figure 4A and 6B). Conversely, the permeability was reduced in dynamic LSECs cocultures (Figure 6B), illustrating stronger cell junctions in the presence of HepG2/C3a, and suggesting that there is a synergistic effect of coculturing cells in our conditions. Furthermore, we found that the LSECs produced basal levels of the pro inflammatory cytokines IL6 and TNF without any significant morphology damage. The dynamic coculture also did not play a part in significantly increasing cytokine levels in LSECs. As high levels of production of pro-inflammatory cytokines in the liver by LSECs leads to fibrosis (DeLeve et al., 2015), our result illustrated the fact that the basal dynamic cocultures of LSECs were not pro-inflammatory.
Previous works reported an improved hepatocyte phenotype when cocultured with endothelial cells (Gugen Guillouzo et al., 2010; Xiao et al., 2015; Bale et al., 2015; Ortega‐Ribera et al., 2018). In the present model, we did not detect any striking benefit of the presence of LSECs on the HepG2/C3a phenotype (no albumin increase, no mRNA gene metabolism upregulation, no clear cytokine over secretion). In fact, the enhanced maturation of hepatocytes was mainly reported on primary hepatocytes that tend to rapidly dedifferentiate (Gugen Guillouzo et al., 2010). It is clear that the hepatocarcinoma HepG2/C3a cell line is probably not an ideal model for liver-on-chip approaches. Although it has been widely used in works related to cancer and liver disease (Donato et al., 2015), and shown that interactions between the liver endothelium (include SK-HEP-1) and this liver carcinoma were reported in studies investigating liver disorders (Thomann et al., 2020; Lee et al., 2022), it has a weak maturation profile. It is certainly a robust model for proof-of-principle studies, but the present on-chip approach would clearly benefit from being extended and refined using normal human primary hepatocytes.
The development of in vitro liver models that mimic the key elements of the in vivo liver environment is very challenging due to the complexity of liver architecture and physiology. With the progress made in tissue engineering, bioprinting and microfluidics, several microfluidic-based in vitro liver models that reproduce a physiologically relevant microenvironment have been developed in recent years (Lee et al., 2021; Moradi et al., 2021). Although most of these models are based on hepatocyte monoculture, a growing number of groups are interested in developing microfluidic cocultures of different liver cells, especially hepatocytes and LSECs, to reproduce liver sinusoid and cell-cell interactions (Kang et al., 2015; Prodanov et al., 2016; Ortega‐Ribera et al., 2018; Du et al., 2017; Lee et al., 2021; Moradi et al., 2021). In these models, the different cell types are randomly mixed or organised in layers separated by a porous membrane, collagen layer or microstructures (Bale et al., 2015; Lee et al., 2021). The present model combines the advantages of the LSECs barrier and hepatocytes cultured in 3D spheroids thanks to the hyaluronic acid hydroscaffold functionalised with ECM components. A critical issue in microfluidic culture development is the balance between model relevance, complexity and practicality. For liver cell cocultures, all cell types are usually seeded in the same irreversibly sealed microfluidic device/membrane, which makes it extremely difficult to analyse the different cell types separately (Ma et al., 2018). Our model consists of two separate compartments easily assembled in IIDMP devices: hepatocytes in PDMS biochips and the LSECs barrier in commercially standard inserts. Each cell type can be cultured and characterised separately before being connected to an IIDMP device for coculture. At the end of the experiments, the inserts and biochips can also be easily removed for separate external analyses.
Conclusion
Author Contributions
Conceptualization, T.M., A.L.G., C.L., E.L. and R.J.; methodology, T.M., A.L.G., F.S., and R.J.; validation, A.L.G., F.S., C.L., E.L. and R.J.; formal analysis, T.M.; investigation, T.M.; resources, Z.S., F.M. and N.M.; data curation, A.L.G., F.S., E.L. and R.J.; writing original draft preparation, T.M.; writing review and editing, T.M., A.L.G., F.S., E.L., and R.J.; visualization, T.M.; supervision, A.L.G., E.L. and R.J.; project administration, C.L.; funding acquisition, C.L. All authors have read and agreed to the published version of the manuscript.
Acknowledgements
This work and T. Messelmani PhD were funded by the ANR (Agence Nationale de la Recherche, France) through the MIMLIVEROnChip project, grant number ANR-19-CE19-0020-01, and by a grant from the Contrat de Plan Etat-Région (CPER) Cancer 2015–2020. The authors would like to thank the JSPS Core-to-Core Program (JPJSCCA20190006), the Research Department at the Université de Technologie de Compiègne (Research Training Innovation Chair, DOT project - Disruptive Organoids Technologies) and CNRS (CNRS international research team, TEAMS project – Therapeutics and Engineering Against Metabolic Syndrome, between CNRS/UTC BMBI and CNRS/IIS LIMMS) for their support.
Conflict of Interest
HCS pharma is the BIOMIMESYS® Liver owner and is a partner of the ANR MimLiverOnChip (ANR-19-CE19-0020-01). Co-authors Z. Souguir and N. Maubon are employees of HCS Pharma.
T. Messelmani, A. Le Goff, F. Soncin, F. Merlier, C. Legallais, E. Leclerc and R. Jellali declare no conflict of interest.
Data Availability Statement
All data generated or analysed during this study are included in this published article and its additional files.
References
Akbari, E., Spychalski, G. B., Rangharajan, K. K., Prakash, S., & Song, J. W. (2018). Flow dynamics control endothelial permeability in a microfluidic vessel bifurcation model.
Lab on a Chip, 18(7), 1084–1093.
https://doi.org/10.1039/c8lc00130h.
Badmann, A., Langsch, S., Keogh, A., Brunner, T., Kaufmann, T., & Corazza, N. (2012). TRAIL enhances paracetamol-induced liver sinusoidal endothelial cell death in a Bim- and Bid-dependent manner.
Cell Death & Disease, 3(12), e447.
https://doi.org/10.1038/cddis.2012.185.
Bale, S. S., & Borenstein, J. T. (2018). Microfluidic Cell Culture Platforms to Capture Hepatic Physiology and Complex Cellular Interactions.
Drug Metabolism and Disposition, 46(11), 1638–1646.
https://doi.org/10.1124/dmd.118.083055.
Bale, S. S., Geerts, S., Jindal, R., & Yarmush, M. L. (2016). Isolation and co-culture of rat parenchymal and non-parenchymal liver cells to evaluate cellular interactions and response.
Scientific Reports, 6, 25329.
https://doi.org/10.1038/srep25329.
Bale, S. S., Golberg, I., Jindal, R., McCarty, W. J., Luitje, M., Hegde, M., Bhushan, A., Usta, O. B., & Yarmush, M. L. (2015). Long-term coculture strategies for primary hepatocytes and liver sinusoidal endothelial cells.
Tissue engineering. Part C, Methods, 21(4), 413–422.
https://doi.org/10.1089/ten.TEC.2014.0152.
Beckwitt, C. H., Clark, A. M., Wheeler, S., Taylor, D. L., Stolz, D. B., Griffith, L., & Wells, A. (2018). Liver 'organ on a chip'.
Experimental Cell Research, 363(1), 15–25.
https://doi.org/10.1016/j.yexcr.2017.12.023.
Bhatia, S. N., Balis, U. J., Yarmush, M. L., & Toner, M. (1999). Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells.
FASEB journal, 13(14), 1883–1900.
https://doi.org/10.1096/fasebj.13.14.1883.
Bricks, T., Hamon, J., Fleury, M. J., Jellali, R., Merlier, F., Herpe, Y. E., Seyer, A., Regimbeau, J. M., Bois, F., & Leclerc, E. (2015). Investigation of omeprazole and phenacetin first-pass metabolism in humans using a microscale bioreactor and pharmacokinetic models.
Biopharmaceutics & drug disposition, 36(5), 275–293.
https://doi.org/10.1002/bdd.1940.
Bricks, T., Paullier, P., Legendre, A., Fleury, M. J., Zeller, P., Merlier, F., Anton, P. M., & Leclerc, E. (2014). Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines.
Toxicology in vitro, 28(5), 885–895.
https://doi.org/10.1016/j.tiv.2014.02.005.
Donato, M. T., Tolosa, L., & Gómez-Lechón, M. J. (2015). Culture and Functional Characterization of Human Hepatoma HepG2 Cells.
Methods in Molecular Biology, 1250, 77–93.
https://doi.org/10.1007/978-1-4939-2074-7_5.
Du, Y., Li, N., Yang, H., Luo, C., Gong, Y., Tong, C., Gao, Y., Lü, S., & Long, M. (2017). Mimicking liver sinusoidal structures and functions using a 3D-configured microfluidic chip.
Lab on a Chip, 17(5), 782–794.
https://doi.org/10.1039/c6lc01374k.
Elvevold, K., Smedsrød, B., & Martinez, I. (2008). The liver sinusoidal endothelial cell: a cell type of controversial and confusing identity.
American Journal of Physiology. Gastrointestinal and Liver Physiology, 294(2), G391–G400.
https://doi.org/10.1152/ajpgi.00167.2007.
González, L. T., Minsky, N. W., Espinosa, L. E., Aranda, R. S., Meseguer, J. P., & Pérez, P. C. (2017). In vitro assessment of hepatoprotective agents against damage induced by acetaminophen and CCl4.
BMC Complementary and Alternative Medicine, 17(1), 39.
https://doi.org/10.1186/s12906-016-1506-1.
Holt, M. P., Yin, H., & Ju, C. (2010). Exacerbation of acetaminophen-induced disturbances of liver sinusoidal endothelial cells in the absence of Kupffer cells in mice. Toxicology letters, 194(1-2), 34–41.
https://doi.org/10.1016/j.toxlet.2010.01.020.
Jellali, R., Bricks, T., Jacques, S., Fleury, M. J., Paullier, P., Merlier, F., & Leclerc, E. (2016). Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures.
Biopharmaceutics & Drug Disposition, 37(5), 264–275.
https://doi.org/10.1002/bdd.2010.
Jellali, R., Paullier, P, Fleury, M. J., & Leclerc, E., (2016) Liver and kidney cells cultures in a new perfluoropolyether biochip.
Sensors and Actuators B: Chemical, 229, 396-407.
https://doi.org/10.1016/j.snb.2016.01.141.
Kang, Y. B., Sodunke, T. R., Lamontagne, J., Cirillo, J., Rajiv, C., Bouchard, M. J., & Noh, M. (2015). Liver sinusoid on a chip: Long-term layered co-culture of primary rat hepatocytes and endothelial cells in microfluidic platforms.
Biotechnology and Bioengineering, 112(12), 2571–2582.
https://doi.org/10.1002/bit.25659.
Khetani, S. R., Berger, D. R., Ballinger, K. R., Davidson, M. D., Lin, C., & Ware, B. R. (2015). Microengineered liver tissues for drug testing.
Journal of Laboratory Automation, 20(3), 216–250.
https://doi.org/10.1177/2211068214566939.
Lauschke, V. M., Shafagh, R. Z., Hendriks, D. F. G., & Ingelman-Sundberg, M. (2019). 3D primary hepatocyte culture systems for analyses of liver diseases, drug metabolism, and toxicity: emerging culture paradigms and applications.
Biotechnology Journal, 14(7), e1800347.
https://doi.org/10.1002/biot.201800347.
LeCluyse, E. L., Witek, R. P., Andersen, M. E., & Powers, M. J. (2012). Organotypic liver culture models: meeting current challenges in toxicity testing.
Critical Reviews in Toxicology, 42(6), 501–548.
https://doi.org/10.3109/10408444.2012.682115.
Lee, S. Y., Kim, D., Lee, S. H., & Sung, J. H. (2021). Microtechnology-based in vitro models: Mimicking liver function and pathophysiology.
APL Bioengineering, 5(4), 041505.
https://doi.org/10.1063/5.0061896.
Lee, D., Park, J. S., Kim, D., & Hong, H. S. (2022). Substance P hinders bile acid-induced hepatocellular injury by modulating oxidative stress and inflammation.
Antioxidants, 11(5), 920.
https://doi.org/10.3390/antiox11050920.
Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method.
Methods, 25(4), 402–408.
https://doi.org/10.1006/meth.2001.1262.
Ma, L. D., Wang, Y. T., Wang, J. R., Wu, J. L., Meng, X. S., Hu, P., Mu, X., Liang, Q. L., & Luo, G. A. (2018). Design and fabrication of a liver-on-a-chip platform for convenient, highly efficient, and safe in situ perfusion culture of 3D hepatic spheroids.
Lab on a Chip, 18(17), 2547–2562.
https://doi.org/10.1039/c8lc00333e.
Merlier, F., Jellali, R., & Leclerc, E. (2017). Online monitoring of hepatic rat metabolism by coupling a liver biochip and a mass spectrometer.
The Analyst, 142(19), 3747–3757.
https://doi.org/10.1039/c7an00973a.
Messelmani, T., Le Goff, A., Souguir, Z., Maes, V., Roudaut, M., Vandenhaute, E., Maubon, N., Legallais, C., Leclerc, E., & Jellali, R. (2022). Development of liver-on-chip integrating a hydroscaffold mimicking the liver's extracellular matrix.
Bioengineering, 9(9), 443.
https://doi.org/10.3390/bioengineering9090443.
Messelmani, T., Morisseau, L., Sakai, Y., Legallais, C., Le Goff, A., Leclerc, E., & Jellali, R. (2022). Liver organ-on-chip models for toxicity studies and risk assessment.
Lab on a Chip, 22(13), 2423–2450.
https://doi.org/10.1039/d2lc00307d.
Milner, E., Ainsworth, M., McDonough, M., Stevens, B., Buehrer, J., Delzell, R., Wilson, C., & Barnhill, J. (2020) Emerging three-dimensional hepatic models in relation to traditional two-dimensional in vitro assays for evaluating drug metabolism and hepatoxicity.
Medicine in Drug Discovery, 8, 100060.
https://doi.org/10.1016/j.medidd.2020.100060.
Odeyemi, S., & Dewar, J. (2019). Repression of acetaminophen-induced hepatotoxicity in HepG2 cells by polyphenolic compounds from Lauridia tetragona (L.f.) R.H. Archer.
Molecules, 24(11), 2118.
https://doi.org/10.3390/molecules24112118.
Ortega-Ribera, M., Fernández-Iglesias, A., Illa, X., Moya, A., Molina, V., Maeso-Díaz, R., Fondevila, C., Peralta, C., Bosch, J., Villa, R., & Gracia-Sancho, J. (2018). Resemblance of the human liver sinusoid in a fluidic device with biomedical and pharmaceutical applications.
Biotechnology and bioengineering, 115(10), 2585–2594.
https://doi.org/10.1002/bit.26776.
Polidoro, M. A., Ferrari, E., Marzorati, S., Lleo, A., & Rasponi, M. (2021). Experimental liver models: From cell culture techniques to microfluidic organs-on-chip.
Liver International, 41(8), 1744–1761.
https://doi.org/10.1111/liv.14942.
Prodanov, L., Jindal, R., Bale, S. S., Hegde, M., McCarty, W. J., Golberg, I., Bhushan, A., Yarmush, M. L., & Usta, O. B. (2016). Long-term maintenance of a microfluidic 3D human liver sinusoid.
Biotechnology and Bioengineering, 113(1), 241–246.
https://doi.org/10.1002/bit.25700.
Prot, J. M., Bunescu, A., Elena-Herrmann, B., Aninat, C., Snouber, L. C., Griscom, L., Razan, F., Bois, F. Y., Legallais, C., Brochot, C., Corlu, A., Dumas, M. E., & Leclerc, E. (2012). Predictive toxicology using systemic biology and liver microfluidic "on chip" approaches: application to acetaminophen injury.
Toxicology and applied pharmacology, 259(3), 270–280.
https://doi.org/10.1016/j.taap.2011.12.017.
Ruoß, M., Vosough, M., Königsrainer, A., Nadalin, S., Wagner, S., Sajadian, S., Huber, D., Heydari, Z., Ehnert, S., Hengstler, J. G., & Nussler, A. K. (2020). Towards improved hepatocyte cultures: Progress and limitations.
Food and Chemical Toxicology, 138, 111188.
https://doi.org/10.1016/j.fct.2020.111188.
Schepers, A., Li, C., Chhabra, A., Seney, B. T., & Bhatia, S. (2016). Engineering a perfusable 3D human liver platform from iPS cells.
Lab on a Chip, 16(14), 2644–2653.
https://doi.org/10.1039/c6lc00598e.
Soldatow, V. Y., Lecluyse, E. L., Griffith, L. G., & Rusyn, I. (2013). In vitro models for liver toxicity testing.
Toxicology Research, 2(1), 23–39.
https://doi.org/10.1039/C2TX20051A.
Son, Y. W., Choi, H. N., Che, J. H., Kang, B. C., & Yun, J. W. (2020). Advances in selecting appropriate non-rodent species for regulatory toxicology research: Policy, ethical, and experimental considerations.
Regulatory Toxicology and Pharmacology, 116, 104757.
https://doi.org/10.1016/j.yrtph.2020.104757.
Souguir, Z., Vidal, G., Demange, E., & Louis, F., (2016) WO Pat., 2016166479A1.
Thomann, S., Weiler, S. M. E., Marquard, S., Rose, F., Ball, C. R., Tóth, M., Wei, T., Sticht, C., Fritzsche, S., Roessler, S., De La Torre, C., Ryschich, E., Ermakova, O., Mogler, C., Kazdal, D., Gretz, N., Glimm, H., Rempel, E., Schirmacher, P., & Breuhahn, K. (2020). YAP orchestrates heterotypic endothelial cell communication via HGF/c-MET signaling in liver tumorigenesis.
Cancer Research, 80(24), 5502–5514.
https://doi.org/10.1158/0008-5472.CAN-20-0242.
van Duinen, V., van den Heuvel, A., Trietsch, S. J., Lanz, H. L., van Gils, J. M., van Zonneveld, A. J., Vulto, P., & Hankemeier, T. (2017). 96 perfusable blood vessels to study vascular permeability in vitro.
Scientific Reports, 7(1), 18071.
https://doi.org/10.1038/s41598-017-14716-y.
Vis, M. A. M., Ito, K., & Hofmann, S. (2020). Impact of Culture Medium on Cellular Interactions in in vitro Co-culture Systems.
Frontiers in Bioengineering and Biotechnology, 8, 911.
https://doi.org/10.3389/fbioe.2020.00911.
Xiao, W., Perry, G., Komori, K., & Sakai, Y. (2015). New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells.
Integrative Biology, 7(11), 1412–1422.
https://doi.org/10.1039/c5ib00170f.
Yu, F., Deng, R., Hao Tong, W., Huan, L., Chan Way, N., IslamBadhan, A., Iliescu, C., & Yu, H. (2017). A perfusion incubator liver chip for 3D cell culture with application on chronic hepatotoxicity testing.
Scientific Reports, 7(1), 14528.
https://doi.org/10.1038/s41598-017-13848-5.
Zeller, P., Legendre, A., Jacques, S., Fleury, M. J., Gilard, F., Tcherkez, G., & Leclerc, E. (2017). Hepatocytes cocultured with Sertoli cells in bioreactor favors Sertoli barrier tightness in rat.
Journal of Applied Toxicology, 37(3), 287–295.
https://doi.org/10.1002/jat.3360.