Henry Bautista-Amorocho
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Jorge Alexander Silva-Sayago
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Diego A. Goyeneche-Patino
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Tania Liseth Pérez-Cala
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Fabio Macías-Gómez
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Abstract
Background: Culture of primary epithelial cells has a great advantage over tumor-derived or immortal cells lines since functional phenotype and genetic makeup are preserved. Swine model has proved to be helpful and reliable as a surrogate model in human diseases. Several porcine cell lines have been established from a variety of tissues and shown to extensively contribute to the current understanding of several pathologies, including cancer. However, few protocols for the isolation and culture swine gastric epithelial cells with phenotype preservation have been described. Therefore, the objective of this research was to develop a new methodology for establishing a primary cell culture from the fundic gland area of the porcine stomach.Results: Enzymatic disaggregation of gastric tissue by using a combination of collagenase type I and dispase II, protease inhibitors (soybean trypsin inhibitor and bovine serum albumin), and antioxidants (Dithiothreitol) allowed the isolation of gastric epithelial cells from the fundic gland area with viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM HG, and DMEM/F12 media did not lead to cell adhesion, cluster formation and cell proliferation. By contrast, Williams’ medium supplemented with growth factors supports the confluence and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC in a 5% CO2 incubator. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining as well as the expression of MUC1 and MUC20 genes by RT-PCR and DNAc sequencing. Swine Gastric epithelial cells also showed origin-specific markers such as epithelial membrane antigen (EMA), cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) detected by immunohistochemistry and immunofluorescence, respectively. Conclusions: A new methodology was successfully established for the isolation of primary gastric epithelial cells from the fundic gland area in a swine model, based on a combination of tissue specific proteases, protease inhibitors, and antioxidants. The formulation of Williams’ medium with growth factors for epithelial cells supports the adherence and maintains the functional phenotype of primary cells, which was confirmed by mucin production and expression of typical epithelial markers in the long term.
Gastric Ulcers in the Fundic Gland Area
