Li Jing, Wang Liya, Ma Dingyun, Li Senyang, Li Juanfeng, Meng Qingda, Li Junqiang, Jian Fuchun
College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, Henan, China.
Sheng Wu Gong Cheng Xue Bao. 2024 Dec 25;40(12):4645-4659. doi: 10.13345/j.cjb.240331.
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 were morphologically consistent with the chicken intestinal tissue and contained differentiated epithelial cells. In summary, we successfully established an 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.
为建立稳定的鸡小肠三维（3D）类器官体外培养平台，本研究从18日龄AA肉鸡胚胎的小肠中收集隐窝细胞。在L-WRN条件培养基的基础上，通过调整烟酰胺、N-乙酰半胱氨酸、LY2157299、CHIR99021、Jagged-1、FGF等细胞因子的比例，优化鸡小肠类器官的培养条件，以筛选出适合类器官长期稳定生长的培养基。优化结果显示，添加1.5 µmol/L CHIR99021可显著提高类器官形成效率和类器官直径。添加0.5 µmol/L Jagged-1时，类器官中出现少量芽状组织。添加50 ng/mL FGF-2后，类器官发芽率显著提高。培养基中添加的1.5 µmol/L CHIR99021、0.5 µmol/L Jagged-1和50 ng/mL FGF-2可相互协作，促进类器官的形成，加速其增殖和分化，同时提高细胞的干性维持能力。通过苏木精-伊红（HE）染色、透射电子显微镜、逆转录定量实时聚合酶链反应（RT-qPCR）、间接免疫荧光和免疫组织化学等方法，对鸡小肠类器官的形态、细胞类型及培养特性进行了研究。结果表明，培养的鸡小肠3D类器官在形态上与鸡肠道组织一致，且含有分化的上皮细胞。综上所述，我们成功建立了鸡小肠类器官培养体系，为后续鸡肠道生理学、病理学、宿主-病原体相互作用机制研究及相关药物开发提供了新方法。
