Animal sample collection
Animal experiments were approved by the Ethics Committee on Animal Experimentation of the University of Antioquia under approval number 121/2018, according to the Colombian Regulations for the Use of Laboratory Animals in Biomedical Research (Law 8430 of 1993 and Law 84 of 1989). Fresh stomach tissues were obtained from three young-adult male pigs (Sus scrofa domesticus) at ages of 30-34 weeks and an average weight of 80 kg (± 8 kg). These tissues were kindly donated by the owner of a private pig farm that has a local slaughterhouse named “Vijagual”. The approval for the use of pig tissue in this research was obtained through written informed consent by the farm owner. Animals were in good body conditions and disease-free, according to the veterinarian responsible for food safety and hygiene of the above slaughterhouse. About 25-100 g from the fundic gland region of the pig stomach were dissected and stored at 4oC in 50 mL conical centrifuge tubes for viral transport medium, which contained DMEM-HG (catalog number: 12100061, GIBCO, USA) supplemented with 200 U/mL penicillin, 200 μg/mL streptomycin (catalog number: 15140122- GIBCO, USA), 100 µg/mL gentamicin (catalog number: 15750078 - GIBCO, USA) and 5 μg/mL amphotericin B (catalog number: 15290018 - GIBCO, USA). Tissue samples were immediately shipped to the Biomedical Research Laboratory of the University of Santander - UDES.
Composition of the digestion medium
The digestion medium for tissue disaggregation and cell detachment contained a Hank's Balanced Salt Solution (HBSS) with calcium and magnesium (HBL03-Caisson, Smithfield, USA), supplemented with 200 U/mL collagenase type I (C0130-Sigma-Aldrich, St. Louis, USA), 1.2 U/mL dispase II (D4693-Sigma-Aldrich, St. Louis, USA), 0.01 mg/mL soybean trypsin inhibitor (STI) (29129-Chem Cruz, Dallas, USA), 1.25 mg/mL bovine serum albumin (BSA) (B005-Caisson, Smithfield, USA) and 0.1 mM Dithiothreitol (DTT) (A2948- PanReac, Barcelona, Spain). The solution was freshly prepared, filtered with 0.2 μM nylon membranes, stored at 4°C and used within the next 24 hours.
Composition of the proliferation medium
Different culture media preparations were evaluated to establish the best conditions for the isolation and growth of Gastric Epithelial Cells (GEC) in vitro. DMEM-HG, DMEM/F12 (DFP02--Caisson, Smithfield, USA), RPMI 1640 (catalog number: 11875119, GIBCO, USA) and William’s E (WML01-Caisson, Smithfield, USA) media were supplemented with 20% heat-inactivated fetal bovine serum (FBSi) (026-100- Cell Application, San Diego, CA, USA), 2.5 μg/mL amphotericin B, 100 U/mL penicillin, 100 μg/mL streptomycin, 25 μg/mL gentamicin, 1% L- glutamine (GLL01-Caisson, Smithfield, USA), 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (IVL01-Caisson, Smithfield, USA), 50 ng/mL epidermal growth factor (EGF) (RP1026AF- Cell Application, San Diego, CA, USA) and 4 μg/mL insulin (Apidra D-65926 -Sanofi-Aventis, Germany). The solutions were freshly prepared and filtered with 0.2 μM nylon membranes and stored at 4°C.
GEC isolation and culture
Fundic glandular tissues from the pig stomach (n=3) were transferred into 100‑mm cell culture plates containing 10 mL of fresh transport medium. Fat, connective and muscular tissues were mechanically removed by dissecting the mucous membrane layer (epithelium) using sterile tweezers and surgical scissors. The epithelial layer of the mucous membrane was peeled off through gentle scraping. Tissues were sectioned into pieces of approximately 1 mm3 in size, later transferred to 50 mL conical tubes and centrifuged at 80 g and 4°C for 10 min. Supernatants were discarded and tissue fragments were resuspended in the digestion medium, being constantly agitated in an orbital shaker at 150 rpm and 37ºC for 2 hours. After that, the resulting cell suspension was filtered through sterile gauze, rinsed three times to remove residual mucus and centrifuged at 80 g for 10 min. Supernatants were carefully discarded again so that GEC viability could be determined using the trypan blue dye exclusion test (T8154-Sigma-Aldrich, St. Louis, USA). Cells were seeded at a density of 3.5x105 cells/well in the proliferation medium into plastic 12-well plates that had been previously treated with a 400 μL bovine collagen type I coating solution (125-50 -Sigma-Aldrich, St. Louis, USA). Cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 for 24 hours. Non-adherent cells were removed by washing each well twice with pre-warmed HBSS the next day. Then, a fresh proliferation medium was immediately added and replaced every other day. By the eighth day of incubation, the proliferation medium was reduced to half of the initial concentration of EGF (25 ng/mL), insulin (2 μg/mL) and FBSi (10%). To avoid fibroblast contamination, the initial GEC monolayer was passaged with differential trypsinization strategies based on Jones’ protocol with some modifications . Cell cultures were washed once in PBS 1X and incubated with a 0.25% Trypsin/EDTA solution at 37°C for 3 min. Weakly adherent cells (fibroblasts) were immediately removed by discarding supernatants. GECs remained attached to the culture surface being later detached using a second Trypsin/EDTA solution for 5 min under the same conditions. Culture plates were thoroughly observed under a light microscope to ensure that only cells showing an epithelial phenotype remained attached after the initial trypsinization before subculturing.
GEC growth rate and proliferation kinetics
Once GEC conditions for in vitro culture were optimized, 1x105 cells were seeded in triplicate in 500 µL/well of supplemented William’s E medium on plastic 12-well plates that had been previously coated with collagen type I and incubated at 37°C, 5% CO2 for seven days. A growth curve was generated to identify the exponential and stationary phases by determining the number of viable cells. Cells were collected every 24 hours, centrifuged and counted using a hemocytometer with trypan blue dye at 0.4% (T8154-Sigma-Aldrich, St. Louis, USA). In addition, proliferation kinetics was measured for up to 72 hours with the Cell Proliferation reagent WST-1 (5015944001-Sigma-Aldrich, St. Louis, USA). Thus, 1x104 GECs were seeded on 96-well plates that had been previously coated and cultured as described above. Cells were harvested at 24, 48 and 72 hours, for which 10 µL reagent WST-1 was added to each well and incubated at 37°C, 5% CO2 for 2 hours. All samples were analyzed using the iMark Microplate Reader (Bio-Rad, Hercules, CA, USA) at 540 nm. These experiments were repeated twice under the same conditions.
Hematoxylin and eosin staining (H&E)
GEC morphology was assessed by H&E staining. 1 x 104 cells were harvested from a seven-day culture monolayer, then seeded onto microscope slides and incubated at 37°C, 5% CO2 for 24 hours. The slides were dipped in Mayer’s hematoxylin solution filled Coplin jar for 30 seconds and rinsed twice with PBS 1X for 1 min each. Then, a 1% eosin Y stock solution was added for 30 seconds. An Eclipse 2000 microscope was used for imaging (Nikon, Tokyo, Japan).
Mucin detection in GECs by Periodic Acid-Schiff (PAS) staining
GEC phenotype was confirmed by the Periodic Acid-Schiff (PAS) staining in cell cultures collected on days 0, 7 and 15. 1 x 104 cells were harvested at each time point, seeded onto microscope slides that had been previously coated with collagen type I and incubated at 37°C, 5% CO2 for 24 hours. Then, GECs were fixed with a 4% paraformaldehyde solution in phosphate-buffered saline (PBS) for 15 min and washed in PBS. The slides were treated in a solution of 0.5% periodic acid for 5 min and stained with Schiff's reagent for 15 min. After removing Schiff's reagent, the slides were rinsed with running tap water for 10 min. Finally, cells were counterstained with a hematoxylin solution for 5 min. All steps were performed at room temperature (RT). A uniform reddish-purple cytoplasm was considered positive for PAS staining. Imaging was made using a Nikon Eclipse 2000 microscope.
RT- PCR amplification of MUC1 and MUC20 genes
The expression of mucins 1 and 20 genes (MUC1, MUC20) in GECs was detected using RT-PCR. Total cellular RNA was isolated from 1 x 105 GECs harvested on days 0, 7 and 15. Cells were first washed twice with PBS and centrifuged at 80 g at RT for 10 min. 1 mL RiboZol RNA extraction reagent (VWR Life Science, Radnor, PE, USA) was added to 1.5 mL tubes and RNA extraction was performed following the manufacturer’s instructions. Purified RNA was measured using NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MS, USA). The isolated RNA was reverse transcribed and amplified sequentially using the OneTaq One-Step RT-PCR kit (E5315S - New England Biolabs MA, USA) with 0.4 ìL of each primer and 250 ng of total RNA in a 20 ìL reaction mixture volume, according to manufacturer’s instructions. Reverse transcription was carried out at 48oC for 20 min, followed by an initial denaturation at 94oC for 1 min. cDNA amplification of MUC1 and MUC20 genes included 40 cycles of denaturation at 94oC for 15 sec and annealing at 58oC for 30 sec, in addition to an extension at 68oC for 30 sec and a final step at 68oC for 5 min. RT-PCR amplicons were confirmed by adding 2% agarose gel stained with SYBR Safe DNA Gel Stain (S33102-Thermo Fisher Scientific, USA). MUC1 and MUC20 primers as target genes and β-actin (ACTB) as internal control were designed for covering exon-exon junctions based on annotated sequences of swine (Sus scrofa domesticus). Candidate gene primers were selected by bioinformatics analysis using Primer3 software (v. 0.4.0 at http://primer3.ut.ee) and checked for specific alignments using Primer-BLAST, the NCBI online tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) . Primers and RT-PCR amplification products are listed in Table 1.
MUC1 cDNA sequencing
Amplified MUC1 RT-PCR products were separated by electrophoresis in 2% agarose gel and purified using a commercially available QIAquick Gel Extraction Kit (Qiagen, USA), following the manufacturer’s instructions. Nucleotide sequences were directly determined from two strands by automated Sanger dideoxy sequencing using a genetic analyzer (Macrogen Inc, South Korea) and primers described in Table 1. A sequence alignment of MUC1 was carried against Sus scrofa domesticus (GenBank Accession number XM_021089730.1) by the ClustalW alignment algorithm of the MegAlign software (Lasergene 15.0, DNASTAR, USA).
Cytokeratin detection by Immunofluorescence
The expression of cytokeratin-18 (CK-18) was detected by immunofluorescence to confirm the epithelial GEC phenotype. 4 x 104 cells/wells were cultured on pre-coated collagen type I on sterile glass microscopic slides and incubated at 37°C, 5% CO2 for 24 hours. Then, cells were fixed with 4% paraformaldehyde in PBS for 15 min at RT, washed three times in PBS 1X and permeabilized in 0.02% Triton X-100 for 20 min. GECs were blocked with 5% BSA and 2% goat serum in PBS for 1 hour at RT. Thereafter, GECs were incubated at RT for 1 hour using the CK-18 primary antibody (sc- 32329-Santa Cruz Biotechnology Dallas, TX, USA), diluted 1:100 in PBS with 5% BSA, washed three times and then incubated at RT for another hour with the Alexa Fluor 555 goat anti-mouse secondary antibody (A-21422, Thermo Fisher Scientific, USA), diluted 1:500 in PBS with 5% BSA. After washing the slides three times in PBS 1X, the samples were treated using the UltraCruz Aqueous Mounting medium with DAPI (sc-24941, Santa Cruz Biotechnology Dallas, TX, USA). Human epithelial cell lines, known as HeLa cells (ATCC® CCL-2), and human embryonic kidney 293T cell lines (ATCC® CRL-3216) were used as positive and negative controls, respectively. The slides were analyzed using the EVOS FL Cell Imaging Station (Thermo Fisher Scientific, USA).
Epithelial markers detection by immunohistochemistry
The expression of additional epithelial markers such as transmembrane mucin 1 (MUC1), also known as epithelial membrane antigen (EMA), and cytokeratin cocktail (AE1/AE3) were detected by immunohistochemistry methods. Cells were fixed with 4% paraformaldehyde in PBS 1X, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with 2% BSA in PBS at RT for 2 hours. Then, primary anti-EMA antibodies (MS-348-P, Thermo Scientific, USA) and anti-AE1/AE3 (MA5-13156, Thermo Scientific, USA) were incubated with cells at 4oC for 16 hours at 1:100 dilution. Finally, the slides were washed in PBS and incubated with an HRP conjugated goat anti-mouse IgG secondary antibody at RT for 45 min. Immunocytochemical staining was performed using an avidin-biotin complex peroxidase standard staining kit. HeLa and AGS cell lines (ATCC CRL-1739) were used as positive controls for AE1/AE3 and MUC1 markers, respectively, whereas hematopoietic U-937 cell lines (ATCC® CRL-1593.2™) were used as a negative control. Imaging was made using a Nikon Eclipse 2000 microscope (Nikon, Tokyo, Japan). All experiments were performed in triplicate.
Contamination with Mycoplasma spp
To confirm the absence of mycoplasma spp contamination, 1x104 GECs were fixed with a 4% paraformaldehyde solution in PBS for 15 min, washed three times in PBS and treated using a consistent size at RT for 10 min. Stained cells were analyzed using a fluorescence microscope to detect the presence of small nuclear bodies in the cell cytoplasm associated with Mycoplasma infections.
IBM SPSS Statistics v21 was used for data collection and statistical analysis. All data were expressed as mean values. Error bars represent ± standard deviation (SD).