Animal sample collection
All animal procedures were approved by Ethics Committee for animal experimentation of Universidad de Antioquia (Approval Number 121/2018), conformed to the Colombian Regulations for Animal Use in Biomedical Research (Act 8430 of 1993 and 84 of 1989.). Fresh stomach tissues were obtained from three young adult male pigs (Sus scrofa domesticus) age of 30 - 34 weeks and a mean weight of 80 kg (± 8 kg). These animal tissues were kindly donated by a private porcine farm owner at a local slaughterhouse called “Vijagual”. Approval for pig research use was registered with the written inform consent from the farm owner. All the animals were in good body condition and considered disease free by the veterinary medical officer, responsible for the health and hygiene of the slaughterhouse “Vijagual”. From pig’s stomach, 25 - 100 grams of the fundic gland area were dissected and stored at 4oC in 50 mL conical centrifuge tubes with transport medium, which consisted of 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.
Digestion medium composition
Digestion medium for tissue disaggregation and cell detachment consisted of Hank's Balanced Salt Solution (HBSS) with calcium and magnesium (HBL03-Caisson, Smithfield, USA) supplemented with 200 U/mL of collagenase type I (C0130-Sigma-Aldrich, St. Louis, USA), 1.2 U/mL of dispase II (D4693-Sigma-Aldrich, St. Louis, USA), 0.01 mg/mL of soybean trypsin inhibitor (STI) (29129-Chem Cruz, Dallas, USA), 1.25 mg/mL of 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.
Proliferation medium composition
Several culture media preparations were evaluated to establish the best conditions to isolate and grow in vitro Gastric Epithelial Cells (GEC). DMEM HG (Catalog number: 12100061, GIBCO, USA), DMEM/F12 (DFP02--Caisson, Smithfield, USA), RPMI 1640 (Catalog number: 11875119, GIBCO, USA) and Williams’ (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 (Catalog number: 15290018- GIBCO, USA), 100 U/mL penicillin, 100 μg/mL streptomycin (Catalog number: 15140122- GIBCO, USA), 25 μg/mL gentamicin (Catalog number: 15750078- GIBCO, USA), 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 taken from swine stomachs (n=3) were transferred into 100‑mm cell culture plates containing 10 mL of fresh transport medium. Excessive fat, connective and muscular tissue was removed manually by dissecting the mucous membrane layer (epithelium) using sterile tweezers and surgical scissors. The epithelial layer of the mucous was peeled off by gentle scraping. Tissue was disrupted into pieces of approximately 1 mm3 in size, later transferred into 50 mL conical tubes and centrifuged at 80 g for 10 min at 4°C. Supernatants were discarded and tissue fragments were re-suspended in digestion medium and kept in agitation on a rotational shaker at 150 rpm for 2 hours at 37ºC. Thereafter, the resulting cell suspension was filtered by using sterile gauze and washed three times to eliminate mucous and centrifuged at 80 g for 10 min. Supernatant was removed carefully and GEC viability was evaluated with the trypan blue dye exclusion test (T8154-Sigma-Aldrich, St. Louis, USA). Cells were seeded at a density of 3.5x105 cells/well in proliferation medium onto 12-well plastic plates, previously treated with 400 μL bovine collagen type I coating solution (125-50 -Sigma-Aldrich, St. Louis, USA). Finally, cells were incubated for 24 hours at 37°C, under a 5% CO2 humidified atmosphere. On the next day, non-adherent cells were removed by washing each well twice with pre-warmed HBSS. After this, fresh proliferation medium was immediately added and replaced every other day. On the eighth day of incubation, proliferation medium was modified by using half of the initial concentration of EGF (25 ng/mL), insulin (2 μg/mL), and FBSi (10%).
GEC growth rate and proliferation kinetics
Once GEC conditions for in vitro culture were optimized, 1x105 cells were seeded in triplicate in Williams’ supplemented medium onto 12-well plastic plates previously treated with collagen type I and incubated for seven days at 37°C and 5% CO2. A growth curve was generated to identify the exponential and stationary phases by plotting out the number of viable cells. For this purpose, cells were collected every 24 hours, centrifuged and counted in a hemocytometer using trypan blue dye at 0.4% (T8154-Sigma-Aldrich, St. Louis, USA). Additionally, the proliferation kinetics was measured for up to 72 hours using WST-1 proliferation assay (5015944001-Sigma-Aldrich, St. Louis, USA). Briefly, 1x104 GEC were seeded onto 96-well plates, being previously treated and cultured as described above. Cells were harvested at 24, 48 and 72 hours, 10 µL of WST-1 reagent were added to each well and incubated at 37°C in 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)
Epithelial morphology of GEC was evaluated by H&E staining. 1 x 104 cells were harvested from a seven-day monolayer and seeded onto microscope slides and incubated for 24 hours at 37°C in 5% CO2. The slides were dipped into a Coplin jar containing Mayer’s hematoxylin dye for 30 seconds and rinsed twice with PBS during 1 min each; then, 1% eosin Y solution was added for 30 seconds. Images were taken using the Eclipse 2000 optical microscope (Nikon, Tokyo, Japan).
Mucin detection in GEC by Periodic Acid-Schiff (PAS) staining
GEC phenotype was confirmed by the Periodic Acid Schiff (PAS) staining in cell cultures collected on day 0, 7 and 15. Briefly, 1 x 104 cells were harvested at each time point, seeded onto microscope slides previously covered with collagen type I and incubated for 24 hours at 37°C in 5% CO2. Then, GEC were fixed with 4% paraformaldehyde dissolved in phosphate-buffered saline (PBS) for 15 min and rinsed with PBS. Slides were exposed for 5 min to 0.5% periodic acid solution and then stained with Schiff's reagent for 15 min. Schiff's reagent was removed and the slides were rinsed with running tap water for 10 min. Finally, cells were counterstained with hematoxylin solution for 5 min. All steps were performed at room temperature (RT). A homogeneous red-purple color inside the cytoplasm was considered positive for PAS staining. Images were taken using Eclipse 2000 optical microscope (Nikon, Tokyo, Japan).
MUC1 and MUC20 amplification by reverse transcription-polymerase chain reaction (RT-PCR)
Mucins 1 and 20 (MUC1, MUC20) expression in GEC was assessed by RT-PCR. Total cellular RNA was extracted from 1 x 105 GEC harvested on day 0, 7 and 15. Firstly, cells were washed twice with PBS and centrifuged at 80 g for 10 min at RT. Secondly, 1 mL of RiboZol RNA extraction reagent (VWR Life Science, Radnor, PE, USA) was added to 1.5 mL tubes and the extraction was performed according to the manufacturer’s instruction. Thirdly, purified RNA was measured using NanoDrop 2000C (Thermo Fisher Scientific, Waltham, MS, USA). The isolated RNA was reverse transcribed and amplified simultaneously using OneTaq One-Step RT-PCR kit (E5315S - New England Biolabs MA, USA) with 0,4 L of each primer and 250 ng of total RNA in a final volume of 20 L reaction mixture, according to manufacturer’s instructions. Reverse transcription was carried out at 48oC for 20 min followed by a denaturation step 94oC for 1 min. DNAc amplification of MUC1 and MUC20 genes included 40 cycles of an initial denaturation step at 94oC for 15 seconds, annealing at 58oC for 30 seconds and an extension at 68oC for 30 seconds, with a final step at 68oC for 5 min. RT-PCR amplicons were confirmed by adding 2% agarose gel, stained with SYBR Safe (S33102-Thermo Fisher Scientific, USA). Primers for MUC1 and MUC20 as target genes and Beta-actin (ACTB) as an internal control gene were designed for covering exon-exon junctions, based on annotated sequences of the porcine (Sus scrofa domesticus). Candidate primers were selected through bioinformatics analysis with Primer3 software (v. 0.4.0 at http://primer3.ut.ee) and checked for specific alignments with the NCBI online tool, Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) [28], primers and product amplification for RT-PCR are listed in Table 1.
MUC1 DNA sequencing
PCR product from muc1 was excised from 2% agarose gels after electrophoresis and purified with a commercially available QIAquick Gel Extraction Kit (Qiagen, USA), according to manufacturer’s instructions. Nucleotide sequences were directly determined from both strands by automated dideoxy sequencing with a genetic analyzer (Macrogen Inc, South Korea), using the primers described in Table 1. The alignment was carried out against MUC1 from Sus scrofa domesticus (Annotation XM_021089728.1 retrieved from GenBank) by Clustal W algorithm using MegAlign software (Lasergene 15.0, DNASTAR, USA).
Cytokeratin detection by Immunofluorescence
The expression of cytokeratin-18 (CK-18) was evaluated by immunofluorescence to confirm the epithelial phenotype of GEC. In short, 4 x 104 cell/well were seeded onto collagen type I pre-coated sterile microscope slides and incubated for 24 hours at 37°C and 5% CO2. Then, cells were fixed with 4% paraformaldehyde in PBS for 15 min at RT, rinsed three times with PBS and permeabilized for 20 min with 0.02% Triton X-100. GEC were blocked with 5% BSA and 2% goat serum in PBS during 1 hour at RT. Thereafter, GEC were incubated for 1 hours at RT with the CK-18 primary antibody (sc- 32329-Santa Cruz Biotechnology Dallas, TX, USA), diluted 1:100 in PBS 5% BSA, rinsed three times and then incubated another hour at RT with the Alexa Fluor 555 anti-mouse secondary antibody (A-21422, Thermo Fisher Scientific, USA), diluted 1:500 in PBS 5% BSA. After washing the slides three times with PBS, the samples were treated using UltraCruz Aqueous Mounting medium with DAPI (sc-24941, Santa Cruz Biotechnology Dallas, TX, USA). The slides were analyzed using the EVOS FL Cell Imaging System fluorescence microscope (Thermo Fisher Scientific, USA).
Epithelial markers detection by Immunohistochemical techniques
The expression of additional epithelial markers such as epithelial membrane antigen (EMA) and cytokeratin cocktail (AE1 / AE3) were assessed by immunohistochemical techniques. Cells were fixed with 4% paraformaldehyde in PBS, treated with 0.1% Triton x-100 in PBS for 15 min and blocked with BSA 2% for 2 hours at RT. Then, primary anti-EMA (MS-348-P, Thermo Scientific, USA) and anti- AE1/AE3 (MA5-13156, Thermo Scientific, USA) antibodies were incubated with cells at 4oC for 16 hours at 1:100 dilution. Finally, the slides were rinsed with PBS and incubated with a secondary anti-mouse IgG antibody HRP-conjugated for 45 min at RT. Immunocytochemical staining was performed using an avidin-biotin peroxidase complex kit. Images were taken using the Eclipse 2000 optical microscope (Nikon, Tokyo, Japan). Experiments were carried out in triplicates.
Mycoplasma spp contamination
To verify Mycoplasma spp contamination, 1x104 GEC were fixed with 4% paraformaldehyde in PBS for 15 min, washed three times with PBS and mounted with UltraCruz aqueous mounting medium with DAPI for 10 min at RT. Stained cells were analyzed using fluorescence microscopy for the presence of tiny nuclear bodies in the cytoplasm of cells associated with Mycoplasma infection.
Statistical analysis
SPSS IBM v.21 software was used for recording data and statistical analysis. All data are expressed as mean value and error bars denote ± standard deviation.