鸡小肠类器官体外培养体系的优化

Optimization of the in vitro culture system for chicken small intestinal organoids

摘要:

为建立稳定的鸡小肠三维(three-dimensional, 3D)类器官体外培养平台，本研究从18胚龄的AA肉鸡小肠中收集隐窝细胞，在L-WRN条件培养基的基础上，通过调节烟酰胺、N-乙酰半胱氨酸、LY2157299、CHIR99021、Jagged-1、FGF等细胞因子的配比，对鸡小肠类器官培养条件进行优化，成功筛选出适合鸡小肠类器官长期稳定生长的培养基。优化结果显示，添加了1.5 µmol/L CHIR99021后，类器官的形成效率和类器官直径显著提高；添加0.5 µmol/L Jagged-1时，类器官出现少量芽样组织；添加50 ng/mL FGF-2后，类器官出芽率显著提高。鸡小肠器官培养基中添加1.5 µmol/L CHIR99021、0.5 µmol/L Jagged-1和50 ng/mL FGF-2能够起到协同作用，提高类器官的形成效率及增殖分化速度，细胞干性的维持效果显著提高。采用HE染色、透射电镜、实时荧光定量逆转录聚合酶链反应(reverse transcription quantitative real-time polymerase chain reaction，RT-qPCR)、间接免疫荧光和免疫组化方法对鸡小肠类器官进行形态学、含有的细胞类型和体外培养特性进行研究。结果表明，体外培养的鸡小肠3D类器官与鸡肠道组织在形态学上保持一致，并含有各种分化的上皮细胞。综上所述，本研究成功建立了鸡小肠类器官培养平台，为后续鸡肠道生理、病理、宿主-病原体相互作用机制和药物研究提供了新方法。

Abstract:

In order to establish a stable in vitro culture platform for chicken small intestine three-dimensional (3D) organoids, in this study, crypt cells were collected from the small intestine of 18-day-old embryos of AA broilers. On the basis of the L-WRN conditioned medium, we optimized the culture conditions of chicken small intestinal organoids by adjusting the proportions of nicotinamide, N-acetylcysteine, LY2157299, CHIR99021, Jagged-1, FGF, and other cytokines to select the medium suitable for the long-term stable growth of the organoids. The optimization results showed that the addition of 1.5 µmol/L CHIR99021 significantly improved the organoid formation efficiency and organoid diameter. When 0.5 µmol/L Jagged-1 was added, a small amount of bud-like tissue appeared in organoids. After the addition of 50 ng/mL FGF-2, the rate of organoid germination was significantly increased. The 1.5 µmol/L CHIR99021, 0.5 µmol/L Jagged-1, and 50 ng/mL FGF-2 added in the medium can cooperate with each other to improve the formation and speed up the proliferation and differentiation of organoids, while improving the stemness maintenance of cells. The morphology, cell types, and culture characteristics of chicken small intestinal organoids were studied by HE staining, transmission electron microscopy, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), indirect immunofluorescence, and immunohistochemistry. The results showed that the 3D organoids of the chicken small intestine cultured in vitro were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an in vitro culture system for chicken small intestinal organoids, providing a new method for the subsequent research on chicken intestinal physiology, pathology, and host-pathogen interaction mechanism and the development of relevant drugs.