Gastric Fundic Mucous Epithelial Cells Isolated from Omeprazole-Treated Guinea Pigs Have Increased Basal and Hormone-Stimulated Growth Rates

Rutten, Michael J.; Harmon, Patricia

doi:10.1159/000200734

Cited by 1 publication

(5 citation statements)

References 25 publications

(33 reference statements)

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“…Several enzymatic protocols for isolating GEC have been described, which include enzymes such as collagenase type 1 [26,27,39] or in combination with pronase [24,25] and dispase [23]. However, previous methodologies do not include the use of protease inhibitors to neutralize the effects of proteolytic enzymes and antioxidants to preserve the cellular redox environment.…”

Section: Discussionmentioning

confidence: 99%

“…This medium includes higher glucose concentrations, essential amino acids, vitamins, among others, that allowed inhibiting apoptosis and supporting long-term cell culture. In addition, GEC had been previously isolated using well-described media such as Ham's F12 [39], DMEN [27], DMEM/F12 [23] and RPMI 1640 [25].…”

Section: Discussionmentioning

confidence: 99%

“…Additionally, insulin helps cells to metabolize glucose and amino acids stimulating growth and proliferation of rabbit gastric epithelial cells [44]. In addition, there is evidence that the combination of insulin and EGF improves the proliferation of gastric epithelial cells [27]. Therefore, the isolation of GEC Williams' medium supplemented with mitogens (EGF and insulin) is proposed, improving cell viability and proliferation as well as preserving the phenotype, which makes this model reliable for experiments that need long-time observations.…”

Section: Discussionmentioning

confidence: 99%

“…Mucins are glycoproteins covering gastric epithelium highly expressed in the stomach [27]. In order to con rm the phenotype of GEC isolated from the fundic gland area of the porcine stomach, the detection of MUC1 and MUC20 genes by RT-PCR on days 0, 7 and 15 of culture was performed.…”

Section: Mucins Expression In Gecmentioning

confidence: 99%

“…These primary cells were used to evaluate gene expression patterns, drug susceptibility and cell physiology [22]. However, as for swine stomach, few protocols to isolate and culture gastric cells have been described, but there have not been improvements for phenotype conservation [23][24][25][26][27]. Therefore, the objective of this research was to develop a new methodology for establishing a primary cell culture derived from the fundic gland area of the porcine stomach.…”

Section: Introductionmentioning

confidence: 99%

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A Novel Methodology For Isolation and Primary Culture of Swine Gastric Epithelial Cells.

Bautista-Amorocho

Silva-Sayago

Goyeneche-Patino

et al. 2020

Preprint

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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.

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