Effects of Growth Factors on Acid-Induced Damage to Rat Gastric Epithelial Cells

Abstract Background: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.Results: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.Conclusions: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

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A novel method for isolation and culture of primary swine gastric epithelial cells

10.21203/rs.3.rs-42291/v3 ◽

2021 ◽

Author(s):

Henry Bautista-Amorocho ◽

Jorge Alexander Silva-Sayago ◽

Diego A. Goyeneche-Patino ◽

Tania Liseth Pérez-Cala ◽

Fabio Macías-Gómez ◽

...

Keyword(s):

Cell Adhesion ◽

Growth Factors ◽

Epithelial Cell ◽

Epithelial Cells ◽

Protease Inhibitors ◽

Cell Lines ◽

Cell Phenotype ◽

Swine Model ◽

Fundic Gland ◽

Gastric Epithelial Cells

Abstract Background: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach.Results: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37oC, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively.Conclusions: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

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

10.21203/rs.3.rs-42291/v1 ◽

2020 ◽

Author(s):

Henry Bautista-Amorocho ◽

Jorge Alexander Silva-Sayago ◽

Diego A. Goyeneche-Patino ◽

Tania Liseth Pérez-Cala ◽

Fabio Macías-Gómez ◽

...

Keyword(s):

Growth Factors ◽

Epithelial Cells ◽

Protease Inhibitors ◽

Swine Model ◽

Type I ◽

Fundic Gland ◽

Gastric Epithelial Cells ◽

Gland Area ◽

Fundic Gland Area ◽

Functional Phenotype

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.

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A novel method for isolation and culture of primary swine gastric epithelial cells

BMC Molecular and Cell Biology ◽

10.1186/s12860-020-00341-7 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Henry Bautista-Amorocho ◽

Jorge Alexander Silva-Sayago ◽

Diego A. Goyeneche-Patino ◽

Tania Liseth Pérez-Cala ◽

Fabio Macías-Gómez ◽

...

Keyword(s):

Cell Adhesion ◽

Growth Factors ◽

Epithelial Cell ◽

Epithelial Cells ◽

Protease Inhibitors ◽

Cell Lines ◽

Cell Phenotype ◽

Swine Model ◽

Fundic Gland ◽

Gastric Epithelial Cells

Abstract Background Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach. Results Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively. Conclusions A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

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