Collection of organs and decellularization

Whole stomachs were collected from female piglets, weighing approximately 10 kg (obtained from Royal Veterinary College, Hawkshead Campus, UK). After sacrifice, the abdominal wall was disinfected with 70% ethanol (Sigma-Aldrich) in MilliQ water, and a midline incision was performed to expose the abdominal cavity. Then, stomachs were harvested, and their fundus and antrum were connected with Luer-lock connectors (Cole Parmer) using sutures with W792 Mersilk Suture (Size 00, Ethicon). Next, the stomachs were washed with abundant 1X phosphate-buffered saline (PBS; Sigma-Aldrich). After that, stomachs were decellularized by the continuous perfusion of the following reagents: 2% sodium deoxycholate (SDC; Sigma-Aldrich) for 5 h, distilled water (D.W.) overnight, DNase 30 µg/mL (EMD Millipore Corp) in 1X Hank’s Balanced Salt Solution (HBSS; Gibco) for 1 h, 1.5 mol/L sodium chloride in D.W. (NaCl, Sigma-Aldrich) for 30 min, and D.W. with 1% Penicillin/Streptomycin (P /S, Sigma-Aldrich) for 48 h. All the decellularization steps were carried out at room temperature (RT). The perfusion of decellularizing reagents was achieved with an iPump i150 peristaltic pump (iPumps) set to produce a flow rate of 14 mL/min. Decellularized stomachs were stored in 1X PBS with 1% P/S at 4℃. Finally, scaffolds were sterilized via gamma irradiation (3560 Gy).

Histological staining

Decellularized scaffolds were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) for 1 h at RT. After rinsing, they were embedded in optimal cutting temperature embedding medium (OCT; Thermo Scientific). Embedded samples were cryosectioned (Bright Instruments) to produce 7-µm-thick sections for histological staining. Tissue slides were stained with Haematoxylin and Eosin (H&E, Thermo Scientific), Masson’s Trichrome (MT) (RAL Diagnostic), Elastica Van Gieson (EVG, EMD Millipore corp.), and Alcian Blue (AB, Sigma-Aldrich) stains. Native porcine stomach tissue samples from fundus, body, and antrum regions were used as positive controls to ensure that histological stains were correctly performed.

Immunofluorescence staining

For immunofluorescence (IF) staining, tissue slides were dried and washed with 1X PBS to remove excess OCT. Tissue sections were quenched by incubating them in 50 mM NH4Cl (BDH Laboratory) for 1 h at RT. Subsequently, slides were blocked and permeabilized with 0.3% Triton X-100 (Sigma-Aldrich) in PBS (PBS-T) supplemented with 1% bovine serum albumin (BSA, Sigma-Aldrich) for 10 min at room temperature. Primary antibodies were diluted in 0.1% PBS-T supplemented with 1% BSA and applied overnight at 4 °C. Slides were incubated with Alexa Fluor secondary antibodies (Invitrogen) for 1 h at room temperature. Finally, slides were mounted with Anti-Fade Fluorescence Mounting Medium (Abcam). Immunofluorescence images were acquired on a Zeiss LSM710 confocal microscope (ZEISS). Primary antibodies used in this study were anti-Fibronectin (dilution = 1:500, Abcam, ab23751), and anti-Collagen-1 (dilution = 1:500, Novus Biologicals, NB600-450). Secondary antibodies used in this study were AlexaFluor® anti-rabbit 568 (dilution = 1:200, Thermo Fisher, A11011), AlexaFluor® anti-mouse 594 (dilution = 1:200, Thermo Fisher, A11012), and Hoechst 33,342 (Thermo Fisher, H1399) at 10 μg/mL.

DNA quantification

Tissue fragments, ranging from 15 to 25 mg, were cut from each tissue (decellularized and native porcine stomach). DNA was extracted with DNeasy® Blood & Tissue Kit (QIAGEN), according to the manufacturer’s instructions. DNA concentration was measured with NanoDrop One (Thermo Fisher).

Glycosaminoglycan quantification

Sulfated glycosaminoglycans content (sGAG) was extracted from native and decellularized tissues (weighing 15–25 mg) using the Blyscan GAG Assay Kit (Biocolor), according to the manufacturer’s instructions. The absorbance was measured at 636 nm using SpectraMax® i3x Multi-Mode Microplate Reader (Molecular devices).

Soluble collagen quantification

Native and decellularized tissues (weighing 10–20 mg) were used for quantifying the soluble collagen. Sircol™ Soluble Collagen Assay Kit (Biocolor) was used according to the manufacturer’s instructions. The absorbance was measured at 556 nm using SpectraMax® i3x Multi-Mode Microplate Reader (Molecular devices).

Elastin quantification

Elastin content was extracted from native and decellularized tissues (5–10 mg) with Fastin™ Elastin Assay Kit (Biocolor), according to the manufacturer’s instructions. The absorbance was measured at 513 nm with SpectraMax® i3x Multi-Mode Microplate Reader (Molecular devices).

Mechanical tensile test

Mechanical tensile tests were performed to detect Young’s elastic modulus (MPa), ultimate tensile strength (MPa), and strain at break (%) of decellularized tissues and further highlight the effect of the decellularization process on tissue’s mechanical properties. Instron 5565 tensile testing system (Instron) was used to detect the mechanical parameters. Native and decellularized gastric tissues were cut into 30 mm x 6 mm tissue stripes. Stripes were pinched from both sides so the final working tissue size was 15 mm × 6 mm. The mechanical testing speed was set to 10 mm/min. Mechanical tests were carried out for the stomach’s body regions only.

HepG2 cell culture

HepG2 cells were cultured on tissue culture plates coated with 0.1% gelatin embryo culture water (Millipore corp.), supplied by Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco supplemented with 10% foetal bovine serum (FBS, Gibco), 1% L-glutamine (Gibco), and 1% Penicillin–Streptomycin (Sigma-Aldrich). HepG2 cells were split 1:3 using TrypPLE reagents (Gibco) and passaged on coated culture plates. HepG2 cells were applied to check the cytocompatibility of the decellularized samples.

Indirect cytocompatibility

Decellularized scaffolds were processed with a biopsy punch (diameter = 4 mm, Stiefel) to obtain decellularized gastric mucosal discs. They were incubated with HepG2 culture medium at 37 degree for 72 h with agitation. The scaffold-conditioned medium was used to assess the indirect effect of the scaffolds in terms of HepG2 cell viability and proliferation. In particular, the culture medium supernatant (conditioned medium) was further used for the culture of HepG2 cells in gelatinized tissue culture plates as previously described (“Test” condition). A medium containing 20% dimethyl sulphoxide (Sigma-Aldrich)  and an unconditioned medium were used as positive and negative controls, respectively. At 24 h and 96 h of culture, cellular viability was assessed using the live/dead viability/cytotoxicity assay (Molecular Probes, Invitrogen Corp.). This assay utilizes the fluorescent dyes ethidium homodimer and calcein. Ethidium homodimer (red) increases in fluorescence intensity upon binding to DNA. Calcein (green) is hydrolysed by intracellular hydrolases found in living cells and subsequently undergoes an increase in fluorescence intensity. Therefore, in this assay, viable cells fluoresce green while non-viable cells fluoresce red. Live/dead assay was performed as per the manufacturer’s instructions. Imaging was carried out with Axio Observer A1 (Zeiss) and images were further processed with ImageJ (Version Windows 32-bit) software to count the percentage of live/dead cells. The 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) assay (Sigma-Aldrich) was also used to assess cellular proliferation via quantification of the enzyme mitochondrial dehydrogenase. MTT assay was performed according to the manufacturer’s instructions. The absorbance at 570 nm was measured by the SpectraMax® i3x Multi-Mode Microplate Reader (Molecular devices).

 Direct cytocompatibility

The decellularized gastric mucosal discs were top-seeded with 20 µL of HepG2 cells (1.5 × 105). Repopulated gastric discs were cultured in tissue culture plates supplied with HepG2 culture medium for 72 h at 37, 5% CO2. They were then stained by H&E staining to check the cellular attachment. Cellular proliferation was also confirmed by performing immunofluorescence staining for the Ki67 marker (dilution 1: 200, Abcam, ab15580).

Statistical methods

For three parameters, a one-way ANOVA was performed using GraphPad Prism 9 (National Institutes of Health, V1.52). In the case of two parameters, a T-test was performed with GraphPad Prism 9. In all the presented figures, statistical significance is expressed as ***p < 0.0001, **p < 0.01, *p < 0.05. Quantitative results are expressed as mean ± standard deviation (SD).