2.1 Fabrication of gut-liver axis chip and experimental set-up
The gut-liver axis chip was designed using Autocad (Autodesk, CA, USA). The embedded gut and liver on-a-chip master mold was made by two-step lithography process as previously reported [23, 24]. First, SU-8 100 photoresist (MicroChem Corp., MA, USA) was deposited and spin-coated with 2000 rpm for 30 s on a 4-inch silicon wafer and baked at 65 °C for 20 min, 95 °C for 1 h, respectively. Ultraviolet (UV) light was exposed for 40 s with UV aligner (MDA-400LJ, Midas System Co. Ltd, Daejeon, Korea) through a photomask and developed unexposed photoresist for 12 min to fabricated the microchannel. SU-8 100 photoresist was secondly deposited on the patterned wafer, spin-coated at 1000 rpm for 30 s, exposed to UV light for 60 s, and developed to fabricate the microwell array. The polydimethylsiloxane (PDMS)-based gut-liver axis chip mold was prepared using a 10:1 mixture of a silicone elastomer and curing agent (Sylgard 184, Dow Corning Corp., MI, USA). The elastomer mixture was placed in a vacuum desiccator (Lab Companion, Daejeon, Korea) for 30 min to remove air bubbles and polymerized at 80 °C for 1 h. The polymerized gut-liver axis chip molds were treated with in a plasma cleaner (Femto Science, Korea) to bond each other. For an osmotic pump, PDMS cubic chambers (1 × 1 × 1 cm) with one cellulose membrane face were fabricated to make the osmotic pump using conventional protocols as previously described [25]. The adhesion between the PDMS chamber and the cellulose membrane was adhered using the PDMS solution as an adhesive. In preliminary experiments, the osmotic experiments were conducted to evaluate the pumping ability of the osmotic pump. The deionized water was used as a buffer solution and polyethylene glycol (PEG) (Sigma-Aldrich, MO, USA; 2000 molecular weight) solution was used as a driving agent.
2.2 Computational fluid dynamic analysis
The flow distribution during the osmotic pumping was simulated via a computer-aided finite element analysis (FEA), which was constructed using the computational fluid dynamics module in COMSOL Multiphysics 6.0 (COMSOL, MA, USA). For our FEA, two-dimensional (2D) sketches were designed with AutoCAD (Autodesk, CA, USA) layer-by-layer and were subsequently imported to the COMSOL model builder to construct a 3D model. The geometric parameters of this 3D model were shown in Additional file 1: Table S1. The governing equation in the simulation was incompressible Navier–Stokes equations and continuity equation [26]:
$$\frac{\partial {\varvec{u}}}{\partial t}+\left({\varvec{u}}\cdot \nabla \right){\varvec{u}}= -\frac{1}{\rho }\nabla P+ \frac{\mu }{\rho }{\nabla }^{2}{\varvec{u}},$$
(1)
$$\nabla \cdot {\varvec{u}}=0,$$
(2)
where u is the velocity vector, P is the pressure, and ρ and μ are the density and the dynamic viscosity, respectively. The osmotic pressure was determined by the Van’t Hoff equation [27]:
where π is the osmotic pressure of the solution, C is the molar concentration of the solute in the solution, R is the molar gas constant [≈ 0.082 (L∙atm)/(K∙mol)], and T is the absolute temperature. Since we employed a PEG solution with a molar concentration of 0.36 M, the osmotic pressure of − 8.654 atm was applied to the outlet in a room temperature.
2.3 Preparation and cell seeding on a gut-liver axis chip
The gut-liver axis chip was sterilized by autoclaving (120 °C for 30 min) and was dried in an oven. The gut chamber of gut-liver axis chip was coated with 1 mg/mL Poly-D-lysine (Sigma Aldrich, MO, USA) for overnight to improve cell adhesion and the liver chamber was coated with 3%(wt/vol) bovine serum albumin (BSA) blocking solution for overnight to improve the uniform-sized hepG2 spheroid generation and inhibit cell adhesion to the bottom PDMS surface. After coating, the gut-liver axis chip was rinsed with a deionized water more than three times, then placed in a dish at 80 °C in an oven at least 24 h. The culture medium was gently and slowly filled into each channel. The human intestinal epithelial cells (Caco-2) (ATCC clone HTB-37) and HepG2 cells were cultured in a modified Eagle’s medium with 10% fetal bovine serum, nonessential amino acids, L-glutamine, and penicillin–streptomycin in the absence of Calcium. Caco-2 cell and HepG2 cell suspension (30 μL, 7.5 × 105 cells/mL) were loaded into the microchannel using a micropipette. The cells in the suspension medium flowed into the microchannel by a gravity and were spontaneously trapped in the microchannel. As the cell suspension with homogenous density was applied in microchannel, the regular amounts of cells were allocated in each microchannel. We left the cells in the incubator for overnight without any treatment for stabilization of cells within the microchannel. After the cells were attached to the microchannel, the non-adherent cells were washed out. The outlet of the gut chamber is connected to the inlet of the filter channel, which is connected back to the inlet of the liver chamber. Each inlet and outlet were connected by a flexible polyurethane tube.
2.4 Immunofluorescence staining
The cells grown in the gut-liver axis chip were fixed with 4% (wt/vol) paraformaldehyde for 30 min, washed twice for 5 min with 0.1% BSA in phosphate buffer saline (PBS), and then permeabilized with 0.2% (vol/vol) Triton X-100 (Sigma Aldrich, MO, USA) for 30 min. After washing with 0.1% BSA in PBS, the cells were incubated with 3% (wt/vol) BSA blocking solution for 1 h. Subsequently, the cells were incubated with primary antibodies overnight at 4 °C, washed three times, incubated with secondary antibodies for 90 min, and washed three times with 0.1% BSA in PBS. The following antibodies were used for immunohistochemistry: rabbit anti-albumin polyclonal antibody (Invitrogen CA, USA, 1:500), Alexa Fluor 594-conjugated phalloidin (Invitrogen, USA, 1:250), and Donkey anti-rabbit Alexa Fluor 488 (Invitrogen, USA, 1:1000). Samples were then incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probe, OR, USA) to visualize cell nuclei before taking confocal microscopic images (Olympus, Japan).
2.5 Spheroid viability assay
HepG2 cells are seeded within a microwell array in a gut-liver axis chip and incubated in an incubator for 5 or 10 days to produce uniform-sized 3D spheroids. After the generation of spheroids, the spheroids were stained to measure viability using Live/Dead Cell Assay kit (Sigma-Aldrich, Bayswater, Australia) with staining for 20 min at 37 °C. Calcein-AM can permeate the plasma membrane and is hydrolyzed by the cytoplasmic esterase to form Calcein-AM that can emit the fluorescence. Propidium iodide (PI) is a nucleic acid-staining dye and it cannot permeate the plasma membrane. The stained hepG2 spheroids were imaged under a fluorescence microscope (IX73, Olympus, Japan).
2.6 Functional assessment
Albumin and urea secretion were analyzed by measuring the concentration of albumin and urea in the medium conditioned by culturing with microbiota-derived exosome and hepG2 spheroids. The hepG2 spheroids were cultured within microwell arrays in a fluidic-based gut-liver axis chip. After culturing for 5 and 10 days, the medium collected in the coiled tube to the osmotic pump was analyzed for albumin and urea concentration.
2.7 Bacterial cell culture and live staining
Lactobacillus paracasei HY7014 (HY7014) and Lactobacillus casei HY7207 (HY7207) probiotics were supplied by hy Co., Ltd. (Yongin-si, Korea). Lactobacillus paracasei type strain ATCC25302 (ATCC25302) and Lactobacillus casei type strain ATCC393 (ATCC393) from the American Type Culture Collection (ATCC; Manassas, VA, USA) was used a reference strain. L. paracasei and L. casei strains were grown in Man, Rogosa and Sharp (MRS) broth (BD, Franklin Lakes, NJ, USA) at 37 °C for 24 h. Subsequently, the bacterial cells were harvested by centrifugation (4000 rpm, 10 min, 4 °C), washed three times with PBS, and resuspended in cell culture medium at 109 CFU/mL before each assay. When bacterial cells were harvested by centrifugation (5000 g, 15 min) the cells were resuspended with 2 mL of raw medium. After suspension, the dye mixture of equal volumes (3 μL to each milliliter) of SYTO® 9 and PI (LIVE/DEAD® BacLight™ Bacterial Viability Kit, L7012, Thermo Fisher Scientific) was added. The cells with dye mixtures were incubated at room temperature in the dark for 15 min. After the two-time centrifugation and washing, they loaded to the Caco-2 cell chamber.
2.8 Bacterial-derived EVs purification: Tangential flow filtration (TFF)
L. paracasei HY7014 and L. casei HY7207 were grown in 1L MRS broth of 37 °C for 24 h and were pelleted by sequential centrifugation at 15,970 g\(\times\) at 10 °C for 15 min. The culture supernatants of HY7014 and HY7207 were filtered through a 0.45 μm filter membrane. Bacterial-derived EVs isolation was performed using the KrosFlo® KR2i TFF System from Repligen (Spectrum Labs, Los Angeles, CA, United States) and 500 kDa cutoff TFF filter module (C02-E500-10-N, Spectrum Labs., MicroKros). Briefly, the feed flow rate and transmembrane pressure (TMP) were kept constant at 400 mL/min and 0.5 bar, respectively. The retentate was concentrated into a final volume of 20 mL for 50-fold concentrations.
2.9 Tunable resistive pulse sensing assessment
Quantification and size characterization of EVs were measured using a tunable resistive pulse sensing (TRPS) instrument (Exoid; IZON Science Ltd, Christchurch, New Zealand). Two different nanopores (NP250, NP400, IZON Science Ltd.) were used to assessment in the size range. Carboxylate polystyrene calibration particles (CPC200 and CPC 500, IZON Science Ltd.) were used with NP200 and NP400 nanopores to ensure optimization conditions (e.g., size, concentration). All calibrations and sample measurements were run under the same conditions recommended by the manufacturer and a minimum of 500 particles was recorded at three different pressures. The acquired data was analyzed using Izon Control Suite software (Izon Control Suite version 3.2.2.268, Izon Science Ltd.).
2.10 Preparation and treatment of EVs and Cytotoxicity test
Both microbiota-derived EVs were made up as a 10 × 1010 particles/mL stock solution in a culture medium. Then both EVs were diluted in culture medium to give an appropriate final concentration. Briefly, hepG2 spheroids cultured within microwell arrays were treated 0.1, 1, and 10 × 109 particles/mL concentrations until 10 days. After incubation, the cell culture medium transferred into corresponding wells which were optically clear with a 96-well flat bottom plate. LDH activity was measured using a Cytotoxicity Detection Kit (ThermoFisher, MA, USA) according to the manufacturer’s procedure. Finally, the OD at 490 nm with a reference wavelength of 690 nm for each sample was measured. LDH is a soluble cytosolic enzyme that is released into the culture medium following the loss of membrane integrity. LDH activity can be used as an indicator of cell viability. The percentage of LDH release was expressed as the proportion of LDH released into the medium as compared to the total amount of LDH presented in cells that could treat with 2% Triton X-100. It was estimated in the following way:
$$Cytotoxicity \left(\%\right)= \frac{Treated \,group - Low \,control}{High \,control - Low \,control} \times 100$$
(4)
2.11 Statistical analysis
Data are presented as means ± standard error (SEM). P-values were analyzed using Student's t test or one-way ANOVA followed by Tukey's post hoc test (GraphPad Prism version 8.0, GraphPad Software Inc. San Diego, CA, USA). Differences between control groups and experimental groups were considered statistically significant (*p < 0.05, **p < 0.01). Difference between wild type and experimental probiotics were considered statistically significant (†p < 0.05, ††p < 0.01). Additionally, difference between microbiota-derived EVs were considered statistically significant (§p < 0.05).