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. 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. 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.

中文翻译：

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一种分离培养原代猪胃上皮细胞的新方法

培养原代上皮细胞比肿瘤衍生或永生化细胞系具有主要优势，只要它们的功能表型和基因组成主要得以维持。当用作人类疾病的替代模型时，猪模型已被证明是有用和可靠的。已经基于多种组织建立了多种猪细胞系，这些细胞系已显示出对当前对多种病理学，尤其是癌症的理解做出了广泛贡献。然而，保留细胞表型的猪胃上皮细胞的分离和培养方案相当有限。我们旨在开发一种新方法，用于从猪胃的胃底腺区域建立原代上皮细胞培养物。通过结合 I 型胶原酶和 II 型胶原酶、蛋白酶抑制剂和抗氧化剂，胃组织的机械和酶解成为可能，这使得从猪胃底腺分离上皮细胞成为可能，在潜伏期显示细胞活力 > 90%。在 RPMI 1640、DMEM-HG 和 DMEM/F12 培养基中培养的胃上皮细胞对细胞粘附、簇形成和细胞增殖的贡献不足。相比之下，在 37 °C、5% CO2 条件下培养 10 天后，补充有生长因子的 William's E 培养基支持纯上皮细胞单层的融合和增殖。初级分离株的粘蛋白产生细胞表型通过 PAS 染色确认，MUC1 通过免疫组织化学确认，以及 MUC1 和 MUC20 基因的表达通过 RT-PCR 和 cDNA 测序确认。猪胃上皮细胞还分别使用免疫组织化学和免疫荧光方法显示出起源特异性标志物，例如细胞角蛋白混合物 (AE1/AE3) 和细胞角蛋白 18 (CK-18)。基于组织特异性蛋白酶、蛋白酶抑制剂和机械细胞解离后的抗氧化剂的组合，成功建立了一种新方法，用于通过猪模型从胃底腺区分离原代胃上皮细胞。含有上皮细胞生长因子的 William's E 培养基配方有助于细胞粘附并保留功能性原代细胞表型，这一点通过粘蛋白的产生和典型上皮标志物随时间的表达得到证实。