This study aimed to establish an in vitro model for lipid synthesis in primary bovine mammary epithelial cells (pbMECs) extracted from milk and cultured on Transwell permeable supports (TW culture). The suitability of these cells as a functional model for lactation was assessed by measuring κ-casein (CSN3) and diacylglycerol acyl transferase 1 (DGAT1) gene expression, the presence of intracellular lipid droplets, and the concentration of triacylglycerol in the cell lysates. The functionality of the milk-derived pbMECs cultured under lactogenic conditions, with and without oleic acid supplementation, was evaluated by comparing the cells grown on Transwell supports to cells grown on an extracellular matrix (ECM) gel (3D culture) or a plastic surface (2D culture). Furthermore, the functionality of milk-derived cells was compared to pbMECs obtained from bovine mammary tissue. Here, we show that in both tissue and milk-derived pbMECs, 3D culture offered the most suitable in vitro environment and led to increased levels of CSN3 and DGAT1 gene expression along with increased intracellular triacylglycerol content. The TW culture conditions also resulted in increased DGAT1 gene expression compared to the 2D conditions and milk-derived pbMECs cultured on TW inserts showed the highest viability compared to cells grown under 2D or 3D treatments. However, this was not observed for tissue-derived pbMECs, suggesting that TW culture may offer a beneficial environment specifically for milk-derived cells. We suggest that with further optimization of the culture conditions, TW culture may present a suitable model for the study of milk lipid synthesis in pbMECs.

这项研究旨在建立体外模型，用于从牛奶中提取并在Transwell渗透性支持物上培养的原代牛乳腺上皮细胞（pbMECs）中进行脂质合成（TW培养）。通过测量κ-酪蛋白（CSN3）和二酰基甘油酰基转移酶1（DGAT1）基因表达，细胞内脂质小滴的存在以及细胞裂解液中三酰基甘油的浓度，评估了这些细胞是否适合作为泌乳功能模型。通过将在Transwell支撑物上生长的细胞与在细胞外基质（ECM）凝胶（3D培养物）或塑料表面上生长的细胞进行比较，评估了在生乳条件下添加和不添加油酸的牛奶衍生pbMEC的功能。 2D文化）。此外，将牛奶来源的细胞的功能与从牛乳腺组织获得的pbMEC进行了比较。在这里，我们显示在组织和源自乳汁的pbMECs中，3D培养均提供了最合适的体外环境，并导致CSN3和DGAT1基因表达水平增加以及细胞内三酰甘油含量增加。与2D条件相比，TW培养条件还导致DGAT1基因表达增加，与在2D或3D处理条件下生长的细胞相比，在TW插入物中培养的牛奶来源的pbMEC显示出最高的生存能力。但是，对于组织来源的pbMEC却没有观察到这一点，这表明TW培养可能会为牛奶来源的细胞提供专门的有益环境。我们建议，通过进一步优化培养条件，