Dissecting Acute Drug‐Induced Hepatotoxicity and Therapeutic Responses of Steatotic Liver Disease Using Primary Mouse Liver and Blood Cells in a Liver‐On‐A‐Chip Model

Abstract Metabolic dysfunction‐associated steatotic liver disease (MASLD) is hallmarked by hepatic steatosis, cell injury, inflammation, and fibrosis. This study elaborates on a multicellular biochip‐based liver sinusoid model to mimic MASLD pathomechanisms and investigate the therapeutic effects of drug candidates lanifibranor and resmetirom. Mouse liver primary hepatocytes, hepatic stellate cells, Kupffer cells, and endothelial cells are seeded in a dual‐chamber biocompatible liver‐on‐a‐chip (LoC). The LoC is then perfused with circulating immune cells (CICs). Acetaminophen (APAP) and free fatty acids (FFAs) treatment recapitulate acute drug‐induced liver injury and MASLD, respectively. As a benchmark for the LoC, multiplex immunofluorescence on livers from APAP‐injected and dietary MASLD‐induced mice reveals characteristic changes on parenchymal and immune cell populations. APAP exposure induces cell death in the LoC, and increased inflammatory cytokine levels in the circulating perfusate. Under FFA stimulation, lipid accumulation, cellular damage, inflammatory secretome, and fibrogenesis are increased in the LoC, reflecting MASLD. Both injury conditions potentiate CIC migration from the perfusate to the LoC cellular layers. Lanifibranor prevents the onset of inflammation, while resmetirom decreases lipid accumulation in hepatocytes and increases the generation of FFA metabolites in the LoC. This study demonstrates the LoC potential for functional and molecular evaluation of liver disease drug candidates.


Antibodies, dyes and microbeads
All antibodies, dyes and microbeads that were used for fluorescence detection and cell sorting are listed in Table S2.

Methods 1.2.1. All-from-one mouse primary liver cell isolation (see the isolation procedures in
Fig. 1A) 1.2.1.1.Mouse liver perfusion and digestion 1) Before starting, pre-warm EGTA / enzyme buffers in the 42°C water bath; assemble tubes and catheters in the pump.

NOTE:
The temperature can be adjusted to ensure solutions perfused in mouse liver at mouse body temperature.2) Sacrifice mice with the inhalation of isoflurane (60 µg/g weight) for 3 min.
3) Fix mouse feet on the operating board, with the abdominal side up.Spray the abdomen with 70% ethanol to sterilize the area and wet the fur.4) Open the abdominal cavity.Gently push intestines and colon to the right side, to expose the portal vein and inferior vena cava without punching into the organs.5) Cannulate the portal vein using an I.V catheter.Perfuse the liver with EGTA buffer (40 mL) at a flow rate of 2 mL/min.Section the vena cava at a distal position to allow for perfusate clearance.OPTIONAL: For blood collection, insert a needle into inferior vena cava prior to starting perfusion.Start perfusion and collect blood with a 1.5 mL Eppendorf tube.Pull out the needle after collection.6) CAUTION: It is critical to avoid bubbles during perfusion.Check for the presence of bubbles in the tubing prior to inserting the catheter.When switching perfusion reagents, make sure to stop the pump prior to transferring the tubing from one tube to the other.

NOTE:
The liver should appear homogeneously brighter within seconds after initiating the perfusion and cutting the inferior vena cava.If this is not the case, the perfusion is not optimal, and this will lead to difficulties during cell isolation.

NOTE:
During the perfusion, it is necessary to press inferior vena cava with a vessel lamp for 30-40 sec to check the perfusion quality.Liver lobes get inflating rapidly.7) Continue perfusion with the digestion buffer I (50 mL) at a flow rate of 2 mL/min.8) Stop the perfusion and remove the catheter.Carefully remove the whole liver (dissect and discard the gallbladder).9) Gently smash the liver tissue by vessel lamps for further isolation procedures.. OPTIONAL: Separate liver tissues if the multiple cell type isolation is required.For the HSC, KC and LSEC isolation, an extra digestion (in a 37℃ incubator with the mild shaking level for 25 -35 min) is necessary.1.2.1.2.Mouse primary hepatocyte isolation 1) Pass the liver cell suspension through a 70-µm strainer to a 50 mL Falcon tube.Fill up the tube with HBSS to 50 mL.Centrifuge the cell suspension at 4°C, 50 g for 5 min.2) Aspirate the supernatant and resuspend the hepatocyte pellet in 50 mL HBSS.Centrifuge again the cell suspension at 4°C, 50 g for 5 min.CAUTION: Keep the supernatant for MACS procedures if necessary.3) Aspirate the supernatant and resuspend the hepatocyte pellet in 50 mL Percoll-50% solution.Centrifuge cells at 4°C, 400 g for 10 min.4) Aspirate the supernatant and resuspend the hepatocyte pellet with 50 mL HBSS buffer.
Centrifuge the hepatocytes at room temperature, 50 g for 10 min.5) Resuspend the hepatocyte pellet in medium for counting and seeding.

1.2.1.3.Mouse primary hepatic stellate cell isolation
1) Filter the liver cell suspension through a 70-um strainer.Fill up the tube with GBSS/B buffer to 50ml.Centrifuge the cell suspension at 4°C, 580 g for 10 min.2) Aspirate the supernatant until 10 mL remains in the tube.Add 100 μL of DNase I solution and fill it up to 50 mL with GBSS/B buffer.Centrifuge again at 580g for 10 min at 4 °C.3) Aspirate the supernatant until 10 mL remains in the tube.Add 100 μL of DNase I solution and fill it up to 32 mL with GBSS/B buffer.Add 16 mL Nycodenz solution and mix thoroughly.Split 48 mL cell-Nycodenz suspension equally into four 15-ml Falcon tubes.4) Gently overlay with cell-Nycodenz suspension with 1.5 mL of GBSS/B buffer using a 3ml syringe with a 26-gauge needle attached.Centrifuge the suspension at 4 °C, 1,380g with no brake for 17 min.CAUTION: Make sure to overlay the GBSS/B buffer gently above the cell-Nycodenz suspension to create a discontinuous gradient.A clear separation should be observed.5) Use a 5-ml pipette to collect the cells and transfer them into a new 50-ml Falcon tube.
Resuspend the cell pellets by MACS buffer for the magnetic cell sorting.OPTIONAL: Repeat the procedure (3 -5) with cell pellets to obtain more cells.6) Fill up the Falcon tube to 50 mL with GBSS/B buffer.Gently resuspend the collected HSCs.Centrifuge at 4 °C, 580g for 10 min.7) Resuspend the cell pellet in medium for counting and seeding.
3) Respectively add 10 µL /10 7 cells magnetic beads (Anti-F4/80 for KCs or Anti-CD146 for LSECs) into the cell suspension.Mix well and incubate on ice for 15 min.4) Add 900 µL MACS buffer per 10 7 cells and centrifuge at 4°C, 300 g for 10 min.5) Discard the supernatant and resuspend cells in 500 µL of MACS buffer.Place the MS column in the magnetic separator and rinse it with 500 µL of MACS buffer.Add the cell suspension into the column.Collect the flow-through containing unlabeled cells for a next magnetic cell sorting.OPTIONAL: LS column can be used if higher amount of cell sorting is required.6) Wash column with 3x 500 µL of MACS buffer.Move the column out from the magnetic separator.Add 1 mL MACS buffer into the column.Firmly flush out the buffer from the column by the plugger.Collect the labelled retained cells by a 15-mL Falcon tube.
Centrifuge the cell suspension at 4°C, 400 g for 10 min.7) Resuspend the cell pellet in medium for counting and seeding.

1.2.1.5.Mouse Peripheral Blood Mononuclear Cell (circulating immune cells, or CIC)
isolation NOTE: This section refers to blood collected from the vena cava during the liver perfusion.If needed, more blood may be collected from the heart using a 1 mL syringe containing 0.5 mL EGTA buffer before or during the liver perfusion.1) Centrifuge blood samples at 4°C, 400 g for 10 min.
2) Aspirate supernatant and resuspend cell pellets with 1 mL 1X red blood cell lysing buffer.
3) Incubate cell suspension on ice for 15 min.4) Centrifuge at 4°C, 400 g for 10 min.5) Aspirate supernatant and resuspend the cell pellet in cell culture medium for counting and seeding.

LoC model assembly and disease modeling
(see the biochip structure in Fig. 2B) 1) Sterilize the surface and inner cavities of biochip by pipetting in 70% ethanol and incubating for 40 min.2) Wash cavities 3 times with DPBS buffer.
3) Pipet 500 mL collagen coating buffer in each cavity and incubate for 15 min.4) Wash cavities 3 times by DPBS buffer.Fill 500 μL medium in each cavity.Block connections between cavities by plugs.5) Cell seeding and intervention strategies: a) On Day 1, seed 300,000 LSECs and 100,000 liver macrophages in the lower cavity.

1.2.4.3.Biochip perfusion
hLoC was perfused on both sides for 24 hours with a flow rate of 50 µL / min using a peristaltic pump (Cole-Parmer™, Ismatec™ Reglo ICC Digital). 1 mL medium containing 100,000 CICs (total PBMCs) was circulated at the lower cavity containing HUVEC / macrophages.Medium containing FFAs, FFAs + 20 μM lanifibranor, and 10% BSA was added to the medium perfusing both sides of the membrane.After perfusing chips with CICs for 30 minutes, flow cytometry analysis of the flow through medium containing nonadherent CICs was performed.Lipid accumulation was measured at 24h after starting FFA treatment.

1.2.4.4.Staining of CICs and flow cytometry
Cells were collected from perfusate by centrifugation and stained for viability using Zombie UV™ Fixable Viability Kit (BioLegend, 423107) for 10 minutes at 4℃.Cells were subsequently stained with antibodies provided in Table S1, fixed with 4% PFA in PBS for 10 minutes at 4℃ and cells analyzed using a BD LSR Fortessa flow cytometer (Becton Dickinson).

1.2.4.7.Ethics
The study with hLoC and the isolation and use of PBMCs and HUVECs was approved by the ethics committee of the Jena University Hospital (2020-1684, 3939-12/13).All donors were informed about the aim of the study and gave written consent.

Analysis of APAP metabolization by HPLC coupled to high-resolution mass spectrometry (HRMS)
LoC perfusates and 2D cell culture supernatants were centrifuged at 10,000 g for 10 min and then subjected to HPLC-HRMS analysis.Chromatographic separation was achieved on a 1290 Infinity II HPLC (Agilent Technologies) equipped with a Zorbax Eclipse Plus C18 column (2.1 x 50 mm, 1.8 μm; Agilent Technologies).A mobile phase system consisting of water and acetonitrile, both acidified with 0.1% formic acid, was used for gradient elution at a flow rate of 0.35 mL/min.The HPLC eluate was directed to an electrospray ionization interface operated in positive mode (ESI+).Ions were analyzed using a 6550 iFunnel quadrupole time-of-flight (QTOF) mass spectrometer (Agilent Technologies).QTOF analysis was performed in scan mode in the range of m/z 50 to 1,700 with a scan rate of 3 spectra/s.The reference masses m/z 121.0509 and m/z 922.0098 were continuously supplied by an isocratic pump.The identity of the APAP metabolites was confirmed by the mass error (Δm/z) of less than 3 ppm and the isotopic pattern, both determined using MassHunter Qualitative Analysis software (version 10.0, Agilent Technologies).
buffer: Prepare the solution by dissolving the components of the recipe given below in 1 L of ddH 2 O. Adjust the pH to 7.4 and filter the solution through a 0.2-μm bottle-top filter.The ready-to-use solution can be stored at 4°C for up to 6 months.

1 )
/A buffer: Prepare the solution by dissolving the components of the recipe given below in 1 L of ddH 2 O. Adjust the pH to 7.4 and filter the solution through a 0.2-μm bottle-top filter.The constituted solution can be stored at 4°C for up to 6 months./B buffer: Add 8 g/L NaCl into 1 L of GBSS/A buffer described above to make GBSS/B buffer.Adjust the pH to 7.35 -7.4 and filter the solution through a 0.2-μm bottle-top filter.Constituted solution can be stored at 4°C for up to 6 months.20) Digestion buffers: Prepare the solution by dissolving the components of the recipe given below.Buffers need to be freshly made for each experiment.Filter the buffer with a 0.22μm filter before use.a) Digestion buffer I: Dissolve 4.4U collagenase type 4 in 50 mL enzyme buffer.b) Digestion buffer II: Dissolve 4.4U collagenase type 4, 40 µg DNase I and 4.5 mg pronase in 50 mL enzyme buffer.21) DNase I solution: Dissolve 100 mg in 100 mL PBS to make 1 mg/mL solution.22) Percoll-50% solution: Dissolve 10.8 mL Percoll-100% and 1.2 mL 10X-PBS in 14.5 mL 1X-PBS to make Percoll-50% solution.Mix it thoroughly.23) Nycodenz solution: Dissolve 4.94 g nycodenz in 15ml GBSS/A buffer and filter it through a 0.22-μm filter.Adjust the solution to 17ml.Optional: Add phenol red to indicate gradient layers.24) Magnetic-activated cell sorting (MACS) buffer: Dissolve 250 μg BSA and 37.2 μg EDTA in 50 mL DPBS.Filter it through a 0.22-μm filter.The constituted solution can be stored at 4°C for up to 2 weeks.25) Collagen-coating buffer: Dissolve 660 µL collagen-I in 50 mL DPBS containing 0.12% acetic acid.Filter it through a 0.22-μm filter.26) Blocking buffer: 5% normal goat serum and 0.3% Triton-X in DPBS.27) Antibody buffer: 1% BSA and 0.3% Triton-X in DPBS.28) Free fatty acid (FFA) solution: Dissolve PA or OA in DPBS containing 1% BSA, Ratios of PA and OA in FFA mixture can be adjusted for different perspectives.Final working FFA concentration is 30 mM. 29) Blocking buffer: Respectively dilute NGS and TritonX-100 into the DPBS to make 5% NGS and 0.3% TritonX-100 as final concentrations.30) Antibody buffer: Respectively dilute BSA and TritonX-100 into the DPBS to make 1% BSA and 0.3% TritonX-100 as final concentrations.31) VectaMount AQ Aqueous Mounting Medium (Vectorlabs, cat.no.H-5501) Microfluidic biochips were made from polybutylene terephthalate (PBT) and obtained

CAUTION:
Bubbles should be avoided at all time during cell seeding into the biochip, medium changing and CIC perfusion.7) Sampling strategies: a) Apply live cell dye on cells and observe under microscope.b) Harvest the perfusion medium and centrifuge at 4°C, 400g for 10 min.Collect CICs in the cell pellets.Keep the supernatant for further analyses (e.g.transaminases, cytokine levels, see corresponding sections).c) Cut off membranes to harvest resident cells for cell visualization or RNA extraction.

Figure S1 .
Figure S1.Multiplex immunostaining used to characterize the changing immune landscape in the mouse acute acetaminophen-induced liver injury model.Mice received a single APAP injection and tissues were collected 24 hours later.(A) Representative fields of view depicting liver tissue sections subjected to multiplex immunostaining.(B) Heat-maps representing cellular density distribution across the samples shown in panel (A).Cell counts and individual cell measurements were collected and are depicted as (C) a matrix or (D-F) in individual graphs.Sample sizes: n(Veh) = 4, n(APAP) = 4. Abbreviations: APAP:

Figure S2 .
Figure S2.Multiplex immunostaining used to characterize the changing immune landscape in the mouse CDAHFD steatosis model.Mice were fed a CDAHFD diet for 8

Figure S3 .
Figure S3.In vitro investigations on primary hepatocyte culture with cultivation and gene expression alterations in the presence of CICs.(A) Relative cell amount of in-vitro

Figure S4 .
Figure S4.All-from-one primary liver cell isolation (mouse) procedure and multicellular layout of LoC model.(A) Primary hepatocytes, HSCs, KCs and LSECs are sequentially isolated from mouse liver using centrifugation or MACS methods.CICs are isolated from mouse blood using red blood lysis and centrifugation methods.Abbreviations: HSC: hepatic stellate cell; KC: Kupffer cell; LSEC: liver sinusoidal cell; CIC: circulating immune cell; MACS: Magnetic-activated cell sorting.

Figure S6 .
Figure S6.Primary liver cell response to acetaminophen, FFAs and lanifibranor in conventional 2-dimensional culture.Primary liver cells were seeded in conventional 2dimensional culture and exposed to acetaminophen, FFAs and lanifibranor.(A) Cell death was evidenced by Apopxin.(B) Apopxin staining in hepatocytes was quantified.(C) lipid vesicle accumulation was evidenced by BODIPY.Gene expression was analyzed in (D) HSCs and (E) LSECs.Sample size: n = 4 per group.Abbreviations: APAP: acetaminophen; FFAs: free fatty acids; HSC: hepatic stellate cell; KC: Kupffer cell; Lanif: lanifibranor; LSEC: liver sinusoidal endothelial cell; Veh: vehicle.Unpaired student's t-tests or one-way ANOVA and Tukey's multiple comparison were performed.*p<0.05 as indicated.

Figure S8 .
Figure S8.The presence of CICs influences the in vitro cytokine milieu upon FFA treatment of primary hepatocytes.Primary mouse hepatocytes were treated with APAP, FFA and lanifibranor in the presence or absence of CICs.Culture medium was collected and cytokine levels were measured.Cytokines levels were measured after (A) APAP, or (B) FFA and lanifibranor treatments.Data is depicted as arbitrary units due to the low range of the measurements.Sample size: n = 4 per group.Abbreviations: A: acetaminophen; C: circulating immune cells; F or FFAs: free fatty acids; HPLC-ESI-MS/MS: liquid chromatography-electrospray ionization tandem mass spectrometry; L or Lani: lanifibranor; MFI: mean fluorescence intensity; V or Veh: vehicle.One-way ANOVA and Tukey's multiple comparison were performed.*p<0.05 as indicated.

Figure S9 .
Figure S9.CIC migration to the chip membrane and survival kinetic of primary mouse CICs.CICs were collected from 20-week-old wild-type mice (1 mL blood/mouse) and perfused through a cell-free chip system (CICs of 200 µL blood/chip).CIC accumulating in blank chips were recorded (A) and quantified (B) at 0, 0.5 and 24 h.(C) Survival of CIC cultivated in petri dishes was assessed at 0, 24, 48 and 72 h using the CCK-8 assay.Survival of CIC cultivated in ultra-low-attachment flasks (D) or perfused in blank chips (E) were assessed at 0, 0.5 and 24 h using the CCK-8 assay.Sample size: (A, B) n = 3 per group, (C-E) n = 4 per group.Abbreviations: CIC: circulating immune cell; LoC: liver-on-a-chip.One-way ANOVA and Tukey's multiple comparison were performed.*p<0.05 as indicated.

Figure S10 .
Figure S10.Multispectral flow cytometry to decipher the immune cell migration to the LoC.CICs were isolated from healthy mouse blood and analyzed by flow cytometry either before or after perfusion into the LoC.(A) Gating strategy used to identify the distinct immune cell populations.(B) Comparison of the relative immune cell composition between fresh (unperfused) or perfused (vehicle, APAP, FFAs and lanifibranor) CICs.(C) Migration scores after perfusing fresh CICs into a control LoC.(D) Cell death of CICs was assessed on both fresh/unperfused and perfused samples.Sample size: (B, C) n = 3 per group, (D) n = 4

Figure S11 .
Figure S11.Recapitulation of liver steatosis and therapeutic efficacy in the human LoC.Human cell lines and primary blood cells were isolated and seeded in the human LoC.Liver steatosis and therapeutic efficacy were demonstrated upon vehicle, FFAs and FFAs + lanifibranor treatments.(A) Scheme of human LoC.(B) Lipid vesicle accumulation in LoC

Figure S12 .
Figure S12.Gene expression analysis to assess the immune cell migration to the LoC.(A-D) Gene expression analysis of on-membrane cells of LoC upon APAP, FFAs and lanifibranor administration.Sample size: n = 4 per group.Abbreviations: APAP: acetaminophen; CIC: circulating immune cell; FFAs: free fatty acids; Lani: lanifibranor; Veh: vehicle.Unpaired student's t-tests were performed.
If necessary, mount cells by the VectaMount AQ aqueous mounting medium and cover cells with glass slides.Immersed into DPBS, glass slides can be gently detached in 30 min.Therefore, cells or membranes can be recycled for IF staining or other perspectives.
OPTIONAL: Cells labelled by live cell dyes can be processed to ICC approaches after fixation.Permeabilize cells and block unspecific membrane antigens by the blocking buffer at the room temperature for 30 -60 min.b) Freshly dilute primary antibodies into the antibody buffer following manufacturers' manuals.Apply antibody solutions on cells and incubate at 4℃ overnight or at room