小鼠气管–支气管上皮细胞的气–液界面培养

Air-liquid interface culture of mouse tracheal-bronchial epithelial cells

目的建立一种小鼠气管–支气管上皮细胞气–液界面(ALI)培养及纤毛摆动频率测量的方法,最大程度还原气道上皮的生理功能。方法利用1 mg/mLⅪⅤ型链蛋白酶冷消化的方法获取BALB/c小鼠气管–支气管上皮细胞,筛选出最佳消化时长以保证所得细胞的数量及活力。差速贴壁法去除成纤维细胞后,接种于Ⅰ型鼠尾胶原预包被的Transwell小室,用不同培养基进行增殖期和ALI分化期培养。结果采用链蛋白酶冷消化12 h、14 h和16 h所得细胞数目分别为(1.78±0.33)×10^5、(1.93±0.26)×10^5和(2.01±0.28)×10^5,经台盼蓝染色活细胞率分别为(96.86±0.25)%、(94.73±1.63)%和(86.87±5.95)%。1周左右细胞铺满小室,继续ALI培养2~3周后光学显微镜下可见纤毛节律性摆动,电镜及免疫荧光均证实纤毛结构。培养所得细胞纤毛摆动频率与小鼠气管在体纤毛摆动频率与一致。结论本实验建立的气管–支气管上皮细胞分离、ALI培养及纤毛摆动频率测量体系简便、稳定、高效、可靠,为探索气道疾病的致病和治疗机制创造了基础,亦可为其他物种气道上皮和(或)其他器官上皮的培养提供参考依据。

Objective To establish a method of air-liquid interface culture and ciliary beat frequency measurement of mouse tracheal-bronchial epithelial cells to simulate the physiological function of airway epithelium.Methods BALB/c mouse tracheal-bronchial epithelial cells were obtained by digestion with 1 mg/mL protease in cold temperature overnight, and the digestion time was optimized to ensure the quantity and viability of the obtained cells.After removing fibroblasts by differential velocity adhesion method, the cells were cultured into collagen coated Transwell inserts. Proliferating phase and air-liquid interface culture were promoted with different culture media. Results Cell numbers obtained by cold protease overnight digestion for 12 h, 14 h and 16 h were(1.78±0.33)×10~5,(1.93±0.26)×10~5 and(2.01±0.28)×10~5, respectively. Cell viability by trypan blue staining were(96.86±0.25)%,(94.73±1.63)% and(86.87±5.95)%,respectively. Cells were 100% confluent in Transwell chamber after 1-week proliferation, and the ciliary beat frequency was observed under microscope after 2-3 weeks of air-liquid interface culture. The cilia structure was confirmed by hematoxylin-eosin staining, electron microscopy and immunofluorescence. Ciliary beat frequency of the cells obtained by this method was consistent with that of mouse trachea in vivo, which further demonstrated its capacity in simulating the physiological function of airway epithelium. Conclusions The separation and air-liquid interface culture system as well as the ciliary beat frequency measurement method established in this experiment is simple, stable, efficient and reliable,which establishes a substantial foundation for exploring the pathogenesis and treatment mechanism of airway diseases. It can also provide reference for the culture of epithelium in the airway of other species and/or other organs.