For 3D-HIM production, human intestinal myofibroblasts (H-InMyoFib) were purchased from Lonza and cultured in SMGMTM-2 BulletKitTM medium (Lonza). H-InMyoFibs at 3–5 passages were used. Human intestinal epithelial cells (Caco-2) were provided by American Type Culture Collection (ATCC) and were cultured in Dulbecco Modified Eagle Medium (DMEM, Microtech) with 10% of fetal bovine serum (FBS, Microtech), 100 μg mL⁻¹ L-glutamine, 100 U mL⁻¹ penicillin/streptomycin. For HepG2-μTPs production, hepatocellular carcinoma cells (HepG2) cells were purchased by ATCC and were cultured in Minimum Essential Medium Earle's Salt (MEM, Microtech), containing 10% fetal bovine serum, 100 μg mL⁻¹ L-glutamine, 100 U mL⁻¹ penicillin/streptomycin, 0.1 mM Non-Essential Amino Acid and 0.1 mM Sodium pyruvate. Cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.

Gelatine porous microscaffolds (GPMs) (75–150 μm diameter) were made-up by a slightly adapted double emulsion method (O/W/O) and stabilized with glyceraldehyde at 4% as previously described (Imparato et al., 2013). Briefly, 10 mL of water containing TWEEN 85 (6% w/v) were used to dissolve the gelatin powder (type B, Mw 176,654 Da) (Sigma Aldrich) at 60°C. Then, an oil in water (O/W) emulsion was produced by adding a solution of Toluene and SPAN 85 (3% w/v) to the aqueous gelatin solution (8% w/v) (Sigma Aldrich). To obtain gelatin micro-beads, 30 mL of additional toluene were added (O/W/O) and cooled to 5°C. At last, to obtain the toluene extraction and gelatin micro-beads stabilization, ethanol (20 mL) was poured. Dried GPMs were sterilized with ethanol, washed with PBS, and then inoculated (2 mg/mL of GPMs; 5 × 10³ beads/mg) with H-InMyoFibs (1.5 × 10⁵ cell/mL) and cultured in a dynamic conditions in spinner flask bioreactor (CELLSPIN, 250 ml, Integra Biosciences) to obtain Human Intestine-μTPs (HI-μTPs) (De Gregorio et al., 2018a). In order to promote H-InMyoFibs adhesion, spinner flask operated with and intermittent stirring for 6 h post inoculation (5 min shaking, 40 min stationary) and continuous stirring at 20 rpm was set for 10 days. Spinner flask bioreactor operated in the cell incubator at 37°C and 5% CO₂; the media replacement occurred every 2 days, and 2-O-alpha-D-Glucopyranosyl-L-ascorbic Acid 0.5 mM (TCI Europe) was added at each culture media change. After 10 days of spinner flask bioreactor culture, HI-μTPs were transferred into a maturation chamber bioreactor where HI-μTPs assembling took place forming disc-shaped (1 mm thick, 6 mm diameter) 3D Intestinal stroma (3D-ISs) (De Gregorio et al., 2018a). The maturation chamber was inserted into a spinner flask programmed at 60 rpm. After 2 weeks of culture, the maturation chamber was opened, the 3D-ISs were withdrawn and washed 2-folds with PBS solution, then transferred into a transwell insert (6.5 mm diameter; Corning), and left to dry for 5 min under laminar hood. Further, Caco-2 cells suspension (2 × 10⁵ cells in 50 μL) was seeded on each 3D-IS in order to produce the intestinal epithelium. The samples were incubated for about 2 h at 37°C, 5% CO₂ to permit Caco-2 cells adhesion onto the 3D-IS apical surface. Further, submerged culture was performed by adding 200 μL of DMEM on the apical side of the 3D-IS and 600 μL of DMEM in the baso-lateral side for 7 days. Then, to induce the epithelial cells polarization and differentiation, the samples were cultured for 2 weeks in an air-liquid interface culture. Three times per week the culture medium was replaced. At the end of the experiment, the full-thickness intestinal equivalents (3D-HIMs) composed by 3D-IS overlapped by intestinal epithelium were taken from the transwell insert for further investigation or were loaded into the Intestine chamber of the Intestine-on-Chip (In-OC) or Intestine-Liver-on-Chip (InLiver-OC).

HepG2 cells were cultured on GPMs in spinner flasks (Integra), as previously reported with slight modifications (Corrado et al., 2019). Briefly, 35 mg of GPMs were loaded with 5.25 × 10⁶ cells (30 cell/GPM ratio). To promote cell seeding on GPMs an intermittent stirring regime (30 min at 0 rpm, 5 min at 30 rpm) was applied for 24 h. Afterwards, the stirring speed was kept at a continuous 20 rpm for up to 7 days. Culture medium was changed three times per week. All cultures were maintained at 37°C in a humidified 5% CO₂ incubator for 6–7 days, and then HepG2-μTPs were loaded into microfluidic Liver-On-Chip (Liver-OC) or into InLiver-OC.

The microfluidic InLiver-OC device was fabricated by a rapid prototyping procedure. The PMMA master mold was designed by AutoCAD and carved with micromilling machine (Minithech CNC Mini-Mill) making a relief positive geometry. A mixture of PDMS pre-polymer and curing agent 10:1 (w/w) was prepared, degassed under vacuum for 20 min to remove air bubbles, and then poured on PMMA master. The set-up was incubated at 80°C for 60 min, then peeled off from master molds. The device consists of two compartments, the intestine compartment (Intestine_c) and the liver compartment (Liver_c), which are connected by a central microchannel. The Intestine_c was designed with a central microchannel (1.2 mm wide × 40 mm long × 0.6 mm high), which transported the medium into an intestine chamber (9.5 mm diameter × 5 mm high). The Liver_c communicated with the Intestine_c by a central microchannel, which was separated from three parallels chambers (0.5 mm wide × 0.6 mm high × 1 mm long) by micro-pillars (0.100 mm diameter × 0.09 mm pillar interspace). A collection channel (0.5 mm wide × 0.3 high) was used to collect tissue supernatants. Culture medium inlet and outlet, as well as inlet for HepG2-μTPs loading in the Liver_c, were punched with a 2.5 mm biopsy punch (DifaCooper), while intestine chamber of the Intestine_c was punched using a 9.5-mm puncher (Am-Tech) (Figure S1). Then, a transwell insert was placed into the 9.5-mm holes of the biochip of the Intestine_c. To ensure the selective transport of fluid through the 3D-HIM, a silicon gasket and a PMMA cylinder were fabricated. In detail, PDMS gasket was fabricated by punching 1-mm-thick PDMS layer, first with a 4-mm puncher (for the inner hole) and then with a 6.5-mm puncher (for the outer hole). PMMA hollow cylinder (5 mm high, 1 mm thick) was made up with micromilling machine. The PDMS biochip was sterilized by autoclave, while PDMS gasket and PMMA cylinder were sterilized by UV light. Before the experiments, InLiver-biochip was washed twice with PBS and then was filled with cell culture medium without encapsulating air bubbles. Then, the 3D-HIM was inserted in the transwell insert of the Intestine_c, the PDMS gasket was placed on the surface of the 3D-HIM, and the PMMA cylinder was placed on the PDMS gasket. In addition, to avoid bacterial contaminations, the Intestine_c was closed by laying a 100-μm sterile PDMS sheet fabricated by spin coating of PDMS (750 rpm × 30 s) onto transwell insert of the Intestine_c. The entire set-up was connected to a syringe pump that worked at flow rate of 5 μL/min, a reservoir was connected to the basal Intestine_c, and another one was connected to the In-Liver outlet in order to collect the cell supernatants from each compartment. For each experiment, two parallel devices were used. To simulate oral ingestion and toxic damage to both Intestine_c and Liver_c, Et-OH dissolved into 300 μL of culture medium (400 mM) was added on the apical side of the Intestine_c. The InLiver-OCs were incubated for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. The control group InLiver-OC without Et-OH treatment was named “–Et-OH;” the 400-mM Et-OH treated sample was named “+Et-OH.” 3D-HIMs and HepG2-μTPs were collected and processed for immune-histotypical and molecular characterizations. In addition, the media collected were analyzed for metabolites produced on the apical side of the Intestine_c, on the lower side of the Intestine_c as well as on the outlet of the InLiver-OC.

The commercial CFD COMSOL Multiphysics vers. Five was used to verify the experimental setup, the three-dimensional velocity and the oxygen gradients in the InLiver-OC. In the CDF analysis, the entire InLiver-OC bioreactor was divided into two different parts, a fluid domain, indicated by “f” (culture medium), and a tissue domain, indicated by “t” (tissue equivalents). The oxygen transport and the three-dimensional velocity were evaluated by combining different physics. Fluid flow in tissue-free regions was modeled by using steady state Navier–Stokes Equation (1); fluid flow in tissue domains was modeled by using transport in porous media Equation (2); oxygen transport was modeled by using Mass Transport-Convection/Diffusion application mode Equation (3); the generation term for oxygen was modeled by using Michelis-Menten kinetic Equation (4). Reference pressure was considered at device outlet (p = 0 Pa); no slip condition was adopted at the walls; up to the intestine chamber a fixed concentration of O₂ was imposed to simulate an open chamber; equality for velocity and pressure was imposed at the Navier–Stokes (u, v, w, p)/Brinkman (u₂, v₂, w₂, p₂) interfaces. All values utilized in the simulation are reported in Table 1. Laminar flow with different flow rates was set at the inlet, and zero pressure was set at the outlet.

Where μ_f is the dynamic viscosity, ν_f is the fluid velocity, and P_f is the pressure. Brinkman Equation (2) was used to describe the flow through the porous medium:

Where K_t is the hydraulic permeability, μ_t is the viscosity of the tissue, and P_t is the pressure.

The oxygen concentration within the system was calculated using the following mass balance Equation (3):

Where C is the oxygen concentration, ν is the fluid velocity field that was set equal to νf in the domain “f” and ν_t in the domain “t,” respectively. D is the diffusion coefficient of the oxygen, set as D_f in the domain “f” and D_t in the domain “t,” respectively. R is the volumetric oxygen consumption rate expressed by the Michaelis–Menten law, according to the following Equation (4):

where V_max is the maximum oxygen consumption rate, and K_m is the concentration at which the oxygen consumption rate is half of V_max, ρ is the cell density in the perfusion chamber. For each tissue, corresponding values were considered. The HepG2 setting values were established from Weise et al. (2013). R was set to 0 only in the fluid domain, since cells are present only in the tissue domain.

Two culture media (DMEM and EMEM) and different supplements were used for 3D-HIMs and HepG2-μTPs, respectively. In order to determine the optimal medium composition to flow into InLiver-OC, different media were tested such as: In-medium (Enriched_DMEM), Liver-medium (Enriched_EMEM), and their combination at the following ratio 1:1, 1:2, and 2:1 ratio (v/v). MTT and LDH assays were used to establish the medium condition that guarantees the highest cell viability and lowest toxicity.

In order to evaluate the cell viability after Et-OH treatment in 2D as well as in HepG2-μTPs culture, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Lactate dehydrogenase (LDH) assays were used according to the manufacturer's instructions (Dojindo Molecular Technologies Inc., Rockville, MD). For MTT assay 2D cell cultures (HepG2 cells) were seeded at 5 x 10³ cells for each well in 96-well cell culture plate and cultured for 3 days prior to the start of the experiments. Based on the generation time of the HepG2, the total number of the cells at the beginning of the MTT test was ~ 2.52 × 10⁴. For 3D culture, HepG2-μTPs (~15 microtissues, 2.32 × 10⁴ total cells) were collected from the spinner flask at culture day 6 and loaded in 96-well cell culture plate. All samples were starved in culture medium with 0.2% FBS overnight and then, the medium with different Et-OH concentrations (0, 100, 200, 300, 400, 500, and 600 mM) was administered to 2D HepG2 and 3D HepG2-μTPs and were cultured for 24 h. For MTT assay, the optical density of each well sample was measured with a microplate spectrophotometer reader at 550 nm. Then, to evaluate the LDH release, the culture supernatants from 2D and 3D HepG2-Microtissues LDH activity was performed, using LDH Detection Kit (Sigma-Aldrich) according to the manufacturer's protocol. Briefly, 50 μL of sample or NADH standard were added to a 96-well. Then, reaction mix was added to each well, and after 2–5 min, the absorbance at 450 nm was measured using a microplate reader; in this way a T_initial was fixed. Subsequent measurements were performed once every 5 min. The final measurement [(A450) final] was the penultimate reading or the value before the most active sample was near or exceeds the end of the standard curve. The time of the penultimate reading was T_final. The LDH activity was evaluated by the following Equation (5):

Where B is the amount (nmole) of NADH generated between T_initial and T_final, reaction time is the difference between T_final and T_initial, and V is the sample volume. ADH was expressed as mUnit/mL.

An indirect estimation of the undigested Et-OH that crosses the 3D-HIM, and of the Et-OH metabolites produced by EtOH-treated, 3D-HIM were determined by collecting supernatants from apical (3D-HIM_{apical supernatants}) and basal side (3D-HIM_{basal supernatants}) of 3D-HIM treated or not with 400 mM Et-OH at T₀ or at 24 h. MTT was performed on HepG2-μTPs treated with the 3D-HIM supernatants. In addition, zone of Inhibition Test on L. rhamnosus were performed after the treatment of the bacteria with the 3D-HIM supernatants. For Zone of Inhibition assay, transparent zones of bacterial grown inhibition indicated the antibacterial activity of the Et-OH. Image J software was used for the measurement of the diameter of the inhibition zones.

3D-HIMs cultured in InLiver-OC were either treated or not treated with 400 mM Et-OH for 24 h, then supernatants were collected, and LDH was performed as reported in section Et-OH Curve and Cell Viability Assessment on 2D vs. 3D Liver Models. Moreover, 3D-HIMs (control groups and Et-OH-treated) were fixed in a solution of 10% neutral buffered formalin for 1 h, rinsed in PBS, dehydrated in an incremental series of alcohol (75, 85, 95, and 100% twice, each step 30 min at RT), then treated with xylene (30 min twice) and embedded in paraffin. Tissue sections of 5 μm thick were stained with Hematoxylin and Eosin (H&E) or Alcian blue, mounted with Histomount mounting solution (Bioptica) on coverslips, and the morphological features of 3D-HIMs were observed with a light microscope. To quantify the mucus secretion, samples were processed by using color deconvolution plugin (Image J®). Automatic thresholding was applied, and blue staining (indicating mucus secretion) was separated from other colors. Further, the mucus deposition was quantified as the mean percentage of blue-stained region. Ten sections were used, and at least five different fields were randomly examined in each section for each time point. For immunofluorescence assay, 3D-HIMs were withdrawn from the InLiver-OC and fixed with 4% paraformaldehyde for 20 min and then rinsed with PBS. The samples were then incubated with a permeabilization solution (0.2% Triton X-100 + 3% BSA + PBS) for 10 min. After blocked for 1 h at RT, primary antibody (Claudin-1, 1/40, Abcam Laminin V, 1/50, Abcam; MMP-9, 1/250, Abcam) was incubated for 1 h at RT. Then, secondary antibody incubation, donkey AlexaFluor 546-conjugated anti-rabbit IgG antibodies, for Claudin-1 and Laminin V and goat AlexaFluor 546-conjugated anti-mouse for MMP-9 were incubated for 1 h. Cells nuclei were detected by DRAQ5 staining (5 μm/mL, Sigma Aldrich).

HepG2-μTPs (control groups and Et-OH-treated) withdrawn from the Liver-OC or InLiver-OC were processed for histological and immunofluorescence analyses, as reported in section Cytotoxic and Immunohistotypical Analyses of 3D-HIM Cultured in InLiver-OC. For immunofluorescence assay, primary antibody (Claudin-1, 1/40, Abcam; P-gp, 1/50, Abcam) and secondary antibody (donkey AlexaFluor 546-conjugated anti-rabbit IgG antibodies for Claudin-1, goat Alexa Fluor 488-conjugated anti-mouse IgG antibodies for P-gp) were used. Cells nuclei were detected by DRAQ5 staining (5 μm/mL, Sigma Aldrich). Furthermore, in order to measure albumin and urea released by HepG2-μTPs, culture media were collected after Et-OH administration on HepG2-μTPs cultured in Liver-OC. Specifically, the supernatants were centrifuged at 2,000 g for 10 min in order to remove cell debris. They were then stored at −20°C before being analyzed. Albumin secretion in cell culture supernatants was quantified by a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Abcam), according to the manufacturer's instructions. All experiments were performed in triplicate. Urea levels in cell supernatants were determined by Quanti Chrom TM Urea Assay Kit (DIUR-500) according to the manufacturer's instruction.

Results were expressed as the mean ± standard deviation (s.d.) from three or more independent experiments (n ≥ 3). For section staining, three specimens were used for each experiment and five sections were stained per specimen, then about five ROIs were examined for each section. All results were then statistically analyzed by the Student's t-test. The differences between two or more groups were evaluated using one-way analysis of variance (ANOVA) followed by the Tukey's post-test. Statistical significance was set at a value of p < 0.05.

Immunofluorescence staining was performed to verify the expression and localization of a representative intestinal epithelial marker, such as villin. As depicted in Figure 1a, the positivity for villin signal suggested the Caco-2 differentiation toward enterocytes cells showing highly polarized epithelial cells. In addition, the presence of microvilli structure on the free surface of the epithelial cells was also evidenced by ultrastructural SEM analysis, as showed in Figure 1a inset, confirming the epithelial polarization. The morphological features of the HepG2-μTPs was investigated via immune-histochemical analysis. A detectable expression of the Cyt P450, a membrane-associated protein correlated with toxic compounds metabolism, was displayed (Figure 1b, ^*high magnification inset).

The designed InLiver-OC device allowed the co-culture of the two 3D organ models (3D-HIM in the Intestine_c and HepG2-μTPs in the Liver_c) into separate compartments connected by means of microfluidic channel allowing an efficient unidirectional transport from the 3D-HIM to the HepG2-μTPs, mimicking the first-pass metabolism (Figures 2A,B). The Intestine_c presents a central hole to accommodate a commercially available transwell insert in order to obtain two separate Intestine side (apical and basal). The Liver_c was located downstream to the Intestine_c and was connected through a microchannel to the basal side of the Intestine_c. 3D-HIM and HepG2-μTPs were produced as previously reported (De Gregorio et al., 2018a; Corrado et al., 2019), and when the best performance in terms of viability and functionality (5 weeks for 3D-HIM and 5–7 days for HepG2-μTPs) were detected, 3D-HIM was aseptically transferred into the apical side of the Intestine_c, and HepG2-μTPs were loaded in each chamber of the Liver_c by pipetting. In order to assure the optimal conditions for cell survival, CFD (Figure 2) was used for simulating fluid dynamic conditions and oxygen concentration profile inside the InLiver-OC device. The flow rate at inlet (5 μL/min) assured the oxygen supply to both 3D models. As showed in Figure 2C, the O₂ concentration in the 3D-HIM was 0.16 mol/m³, while the minimum O₂ concentration in the HepG2-μTPs was 0.12 mol/m³. In Figure 2D, the velocity profile was shown, the maximum value of the fluid flow along the central channel was 5.4 × 10⁻⁴ m/s which is safe for cell viability according to literature (Sibilio et al., 2019).

Preliminary experiments were carried out in order to select the optimal media combination that did not cause the metabolic waste accumulation into the Liver_c. MTT and LDH assays (Figures 3A,B, respectively) were performed to establish the best medium combinations. We found that when 3D-HIM was cultured with the Liver-medium (Enriched_EMEM) or with InLiver-media (Enriched_DMEM:Enriched_EMEM at ratio 1:2), the viability of 3D-HIM was marginally reduced (86 ± 7.12% and 88 ± 6.22%, respectively), and low value of LDH was detected (0.2 ± 0.04 and 0.16 ± 0.02 munits/mL, respectively). At the same media combination, the HepG2-μTPs viability was slightly affected (87 ± 4.44% and 90.5 ± 4.2%, respectively), and a small but significant amount of LDH (0.35 ± 0.18 and 0.21 ± 0.07 munits/mL, respectively) was detected, indicating that the 3D-HIM probably produced a small amount of metabolic waste that slightly induced the epithelial cell injury of the HepG2-μTPs. In addition, when HepG2-μTPs were cultured with In-medium (Enriched_DMEM) or with InLiver-media (Enriched_DMEM:Enriched_EMEM at ratio 2:1), a slight amount of LDH was revealed (0.12 ± 0.02 and 0.11 ± 0.04 munits/mL, respectively), although these values were not indicative of cellular damage. Therefore, the optimal media combination resulted in the InLiver-media (Enriched_DMEM:Enriched_EMEM at ratio 1:1) that did not affect viability (96 ± 8.3% for 3D-HIM, 98 ± 6.5% for HepG2-μTPs) and did not lead to the production of LDH (0.04 ± 0.01 for 3D-HIM and 0.02 ± 0.005 munits/mL for HepG2-μTPs, respectively) as reported in Figures 3A,B, respectively.

Initial experiments were focused on understanding how the HepG-2 cells respond to different concentration of Et-OH. In this experimental phase, the cytotoxic effect of Et-OH was assessed on standard culture dishes (2D HepG-2 monolayer) or in 3D configuration (3D HepG2-μTPs) (Figure 3C) by MTT viability assay. In detail, the 2D HepG-2 monolayer were seeded in 96 multiwell plate for 60 h in order to obtain 2.5 × 10⁴ cells for each well. Instead, the 3D HepG2-μTPs were cultured in a spinner flask in dynamic conditions in order to obtain 1,550 cell/μTPs; then on the sixth day, 5 HepG2-μTPs were added for each liver chamber for a total of 15 HepG2-μTPs (2.32 × 10⁴ total number of cells), as shown in the Figure S3. For 2D HepG-2 monolayer, a dose-dependent reduction of cell viability was reported from 0 to 600 mM Et-OH. For 3D HepG2-μTPs, a slight reduction of viability was revealed by adding 200 mM Et-OH, but an increase in cell cytotoxicity was shown at concentration over 300 mM. Specifically, when 2D HepG-2 monolayer was treated with Et-OH 400 mM, a significant reduction in cell vitality (44 ± 4.08%) compared to 3D HepG2-μTPs treated with the same concentration (61.9 ± 5.02%) was revealed. In the groups treated with 500 mM Et-OH, the viability percentage of the cells decreased at 32 ± 4.3% in 2D HepG-2 monolayer and at 49.07 ± 5.32% in 3D HepG2-μTPs. Finally, the cells treated with 600 mM showed a mortality that exceeds 80% in 2D HepG-2 monolayer and was around 60% in 3D HepG2-μTPs. In order to perform the experiment in conditions that guarantee the cells functionality by preserving more than 50% of cell viability, the Et-OH concentration of 400 mM was selected.

In order to determine the liver functionality, the response of the HepG2-μTPs to Et-OH treatment at 24 h was analyzed by measuring albumin and urea production. As shown in Figures 3E,F, the untreated samples (–Et-OH) exhibited high levels of albumin (51.84 ± 3.9 ng) and urea (20.52 ± 2.3 μg). In contrast, there was a significant decrease in the metabolic activity in 400 mM Et-OH-treated samples (+Et-OH). In particular, albumin production decreased at value of 13.56 ± 3.81 ng and urea at 8.39 ± 1.48 μg, demonstrating that the HepG2-μTPs responded to the toxic stimulus by reducing their metabolic activity.

An indirect estimation of the undigested Et-OH crossing the 3D-HIM was evaluated by treating HepG2-μTPs and L. rhanmosus with tissue supernatants in order to perform cell viability assay (MTT) and Zone of Inhibition Test, respectively (Figures 4A,B). Specifically, HepG2-μTPs were treated with the 3D-HIM supernatants collected from apical (3D-HIM_{apical supernatants}) or basal side (3D-HIM_{basal supernatants}) of the Intestine_c at T₀ or after 24 h of 400 mM Et-OH treatment. The results indicated that at T₀ the 3D-HIM_{apical supernatants} slightly reduced HepG2-μTPs viability, resulting in cell vitality of 62.5 ± 5.2%. This value corresponded in the Et-OH dose response curve to an Et-OH concentration of 400 mM (Figure S4). No significant reduction in cell viability was induced by treating HepG2-μTPs with 3D-HIM_{basal supernatants} harvested at T₀. HepG2-μTPs treated with 3D-HIM_{apical supernatants} collected at 24 h, revealed a very slight reduction of cell viability (87 ± 5.4% cell viability), while HepG2-μTPs treated with 3D-HIM_{basal supernatants} harvested after 24 h of Et-OH treatment, presented a more pronounced reduction in cell viability (74 ± 5.3% cell viability). The latter value corresponded in the dose response curve (Figure 3D) to an Et-OH concentration of 350 mM, suggesting that a small amount of Et-OH was metabolized by the 3D-HIM (Figure 4A) at 24 h. Further, the antibacterial action of Et-OH was evaluated by analyzing the supernatants collected from 3D-HIM_apical treated or not treated with 400 mM Et-OH at T₀ and 24 h. The maximum zone of L. rhamnosus strain inhibition growth (2.1 ± 0.51 cm) was found by using supernatants collected at T₀ from Et-OH treated 3D-HIM_apical while a relatively lower (0.8 ± 0.66 cm) growth inhibition zone was found by using supernatants collected from Et-OH treated 3D-HIM_apical at 24 h. Moreover, there was no antibacterial effect of the 3D-HIM_{basal supernatants} at T₀, but an inhibition of the growth zone (1.5 ± 0.5 cm) was observed when 3D-HIM_{basal supernatants} at 24 h was used. Taken together, these results demonstrate that Et-OH or Et-OH products such as acetaldehyde reduce the growth of L. rhamnosus strain (Figure 4B).

In order to evaluate the harmful effect of 400 mM Et-OH on 3D-HIM, the cell damage was assessed by LDH assay. The Et-OH was given on the apical side of the 3D-HIM, and at this dose, an LDH release of 0.92 ± 0.07 mU/mL at 24 h was found (Figure 4C), suggesting slight Et-OH-induced cell damage. This Et-OH concentration also impacted 3D-HIM's morphology and functional performance. In order to evaluate the effect in terms of the intestinal barrier integrity, intestinal tight junctions were investigated. High intensity of Claudin-1 signal, a tight junction marker, was observed on untreated samples (–Et-OH) compared to the Et-OH-treated ones (+Et-OH), in which the higher amount of acetaldehyde compromises the tight junction between the Caco-2 cells, determining an increase in barrier permeability (Figures 5a,b, respectively). Quantitative immunofluorescence analysis showed that the number of the tight junctions found on the untreated samples (189 ± 14.8, number of tight junctions per field) was higher compared to Et-OH-treated 3D-HIM (82 ± 8.01, number of tight junctions per field) (Figure 5c). Further, immunofluorescence staining for the Laminin V along the interface between stroma and epithelium indicated the presence of an intact basement membrane with a polarized epithelium on the untreated sample (-Et-OH, Figure 5d). In contrast, Et-OH treatment compromised the basement membrane integrity, as shown by pixelated signal of Laminin V and revealed the separation of the supra-basal epithelium from the basal lamina, which ultimately could cause the epithelium rupture (Figure 5e). In addition, an altered epithelial organization was also detected in Et-OH-treated 3D-HIM. TEER measurements were performed in order to confirm the damage at epithelium permeability. In agreement with immunofluorescence results, the untreated 3D-HIM (–Et-OH) expressed higher TEER values (4.72 kΩ) compared to Et-OH-treated 3D-HIM (43.4Ω) (Figure 5f), confirming that Et-OH treatment affected paracellular junctions. On the contrary, the capacitance measurements on untreated and treated samples resulted comparable (1.1 and 1.34 μF, respectively), suggesting that the membranes of the epithelial cells remain intact. Furthermore, in order to evaluate the mucus secretion on untreated (–Et-OH) and Et-OH-treated samples (+Et-OH), Alcian blue staining was performed. 3D-HIM respond to Et-OH treatment by overproducing mucus on Et-OH-treated 3D-HIM compared with the untreated one (Figures 5g,h, respectively). Quantification analysis of the Alcian blue images further confirmed the qualitatively observation of the histological images (Figure 5i). Moreover, analysis of the composition and architecture of the ECM was performed. Figure 6 reported the Second Harmonic Generation (SHG) images rising from newly formed collagen signal on the untreated (–Et-OH, Figure 6a) and Et-OH-treated 3D-HIM (+Et-OH, Figure 6b). The SHG images were exploited to estimate the CF, the CAD, and the correlation length of collagen network in the ECM (Figures 6c–e). Statistically significative differences were found between the values of untreated and Et-OH-treated samples. Both CF and CAD were affected by Et-OH treatment, 43.64 ± 6.6% vs. 32.48 ± 5.0%, the former, and 31.00 ± 7.6 vs. 24.43 ± 3.0 a.u, the latter. In addition, the correlation length highlights differences in collagen texture, reporting a value for untreated samples of 52.34 ± 2.50 a.u. and a value for Et-OH-treated samples of 37.40 ± 2.10 a.u (Figure 6e). Lastly, Et-OH treatment induced MMP-9 overexpression, as reported in Figures 6f,g.

In order to determine the intestine contribution to Et-OH metabolism, 400 mM Et-OH was administrated in three configurations of the device: Control group samples (untreated), Et-OH-treated Liver-OC (HepG2-μTPs without 3D-HIM), and Et-OH-treated InLiver-OC (device with both HepG2-μTPs and 3D-HIM). Histological characterization of untreated samples using H&E staining displayed a homogenous cell distribution around the microbeads surface of the HepG2-μTPs with the typical liver-like histotypic features, such as cuboidal hepatocyte cell shape with tight cell–cell contacts. In the Liver-OC configuration, HepG2-μTPs, although preserving the 3D epithelial organization, showed a high amount of lipid accumulation, as indicated by the black arrowheads. In contrast, in InLiver-OC configuration, HepG2-μTPs showed a small amount of lipid accumulation with a well-polarized epithelium (Figures 7a–c). Immunofluorescence staining showed that the Claudin-1 expression was higher in control groups than in Liver-OC, in which a completely damaged tight junction network was detected (Figures 7d,e, respectively). In contrast, a continuous network of tight junction was found in some area of HepG2-μTPs in InLiver-OC (Figure 7f). Moreover, in order to better characterize the liver functionality, HepG2-μTPs were stained with P-Glycoprotein (P-gp) antibody—a membrane's protein that guides the transport of the substance across the epithelium—and a qualitative analysis of the P-gp expression for untreated samples, Liver-OC, and InLiver-OC was performed, as highlighted in Figures 7g–i. Specifically, as depicted in Figure 7g, the formation of canalicular-like structures was evidenced in untreated samples. However, a slight signal of P-gp was detected in Liver-OC, which indicated no assembly of the membrane protein (Figure 7h) in this condition, while the InLiver-OC showed higher expression of this protein, well-organized both in the core and on the external surface of the microtissue (Figure 7i). Furthermore, in order to determine the lipid accumulation on HepG2-μTPs, oil red staining was assessed directly into the biochip (Figure S5) or cross-sections after withdrawing the sample from the device (Figures 7j–l). In particular, untreated samples showed a small amount of lipid accumulation. In contrast, high amount of lipid drops (stained in red, black arrowheads) were displayed in Liver-OC, and a small quantity of lipid drops was found in InLiver-OC cross-sections, as reported in Figures 7j–l. Finally, ROS were found to be over-expressed in Liver-OC compared to untreated and InLiver-OC. Figure S6 shows that the ROS expression on the Liver-OC was higher than on the InLiver-OC.

In order to determine the 3D-HIM as well as the HepG2-μTPs response to the Et-OH treatment in the device, three configurations were explored: In-OC (device loaded with 3D-HIM without HepG2-μTPs), Liver-OC (device loaded with HepG2-μTPs without 3D-HIM), and InLiver-OC (device with both HepG2-μTPs and 3D-HIM). In the In-OC configuration, the tissue supernatants were collected from the apical compartment of the Intestine_c and from the sampling channel between the Intestine_c and Liver_c. In the Liver-OC configuration, the tissue supernatants were collected downstream to the Liver_c. And, in the InLiver-OC configuration, the tissue supernatants were collected only downstream to the Liver_c (Figure 8A). The detection of ADH activity was used as indication of acute cell damage (Figure 8B). As deduced from the results obtained after treatment with 400 mM Et-OH, a high amount of ADH was detected in the Intestine_apical (In_apical) (2.42 ± 0.65 mU/mL), indicating the enzyme activity of 3D-HIM. In addition, a slight but significant release of ADH was revealed in the sampling channel (In_basal) (1.33 ± 0.32 mU/mL). Further, a higher amount of ADH (4.27 ± 0.42 mU/mL) was detected downstream to the Liver_c in the Liver-OC configuration, mimicking what occurs during acute alcoholic liver injury. At last, InLiver-OC showed the highest ADH value (7.2 ± 0.56 mU/mL) compared to the other configuration, suggesting a synergic activity of both intestinal and hepatic tissue co-cultured into the device. Meanwhile, experiments were performed to evaluate whether the Et-OH injury induced MDRs gene expression modulation. 3D-HIM as well as the HepG2-μTPs samples cultured in Liver-OC or InLiver-OC were processed to quantify the MDR gene expression by means of molecular analysis. The results indicated that MDR4 expression on 3D-HIM decreased after Et-OH injury (5.3 ± 0.6 a.u.) compared to untreated samples (7.9 ± 2.4 a.u.) (Figure 8C). Likewise, Et-OH-treated HepG2-μTPs cultured in Liver-OC displayed low values of the MDR4 gene expression (3.8 ± 0.9 a.u.) compared to untreated samples (7.2 ± 1.5). On the other hand, when HepG2-μTPs were cultured in InLiver-OC, they displayed higher values in the MDR4 gene expression (8.1 ± 1.6 a.u.) compared to Et-OH-treated Liver-OC, suggesting a protective action of intestine on hepatic function. Furthermore, an increase of P-gp gene expression (9.8 ± 2.6) was revealed on 3D-HIM after Et-OH injury, in agreement with the in vivo situation. P-gp gene expression slightly increased on Et-OH-treated Liver-OC (7.9 ± 1.7) but reached higher values on Et-OH treated InLiver-OC (9.2 ± 2.8) compared to untreated samples (7.5 ± 1.7) (Figures 8D,E). Lastly, the biomass production after 400 mM Et-OH treatment was measured, showing the amount of cell debrides collected in the tissue supernatants. In particular, Et-OH-treated Liver-OC showed highest biomass compared to the other conditions, indicating the major cell debrides accumulation due to the cell damage in this configuration. The biomass quantification of InLiver-OC Et-OH-treated samples correspond to the sum of the two organs contributions: 20% of Liver-OC debrides (extrapolated from the HepG2-μTPs viability assay) and In-OC debrides (Figure S7).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.