Guo J, Qiu Y, Hu C, Li D, DU Y
School of Biomedical Engineering, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
State Key Laboratory of Ultrasound Medical Engineering, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Dec 20;43(12):2095-2102. doi: 10.12122/j.issn.1673-4254.2023.12.14.
To establish a 3-dimensional tuberculosis spheroid model for studying the formation and characteristics of tuberculous granuloma .
Human myeloid leukemia mononuclear THP-1 cells and (BCG) were mixed in a 3D cell culture plate and co-cultured in the presence of PMA for 3 days. The growth of the spheroid was examined every 24 h, and the distribution of bacteria, cell survival rate, transformation of the monocytes into macrophages, and penetration of fluorescently labeled nanoparticles into the cell spheroids and tuberculosis spheroids were observed using confocal laser scanning microscopy. The BCG and cell architecture within the 3D tuberculosis spheroid was observed using transmission electron microscopy. Image-iT red hypoxia probe, HO test kit, and a waterproof pen PH meter were used to detect the differences in the microenvironment between BCG-infected and non-infected 3D tuberculous spheroids. The utility of this 3D tuberculous spheroids for assessing antibiotic effects of rifampicin and levofloxacin was evaluated by plate colony counting.
In the cell-bacterial suspensions, stable 3-D tuberculous spheroids (50-200 μm) occurred slowly, in which the cells adhered tightly with numerous bacteria in the center, and necrotic cells and monocytederived macrophages were seen within the spheroids. Drug penetration was difficult in the 3D tuberculous spheroids as compared with the non-infected cell spheroids. Transmission electron microscopy revealed the presence of cell necrosis and a large number of BCG in the macrophages in the tuberculous spheroids. The tuberculosis spheroid had a more hypoxic microenvironment than the non-infected cell spheroids with higher HO content and nearly a neutral PH. The tuberculous spheroid model was capable of evaluating the efficacy of anti-tuberculosis drugs, and among them rifampicin showed a stronger antibacterial effect.
The 3-D tuberculous spheroid model established in this study provides a useful platform for studies of tuberculous granuloma.
建立三维结核球模型，用于研究结核肉芽肿的形成及特征。
将人髓系白血病单核THP-1细胞与卡介苗（BCG）在三维细胞培养板中混合，并在佛波酯（PMA）存在的情况下共培养3天。每24小时检查球状体的生长情况，使用共聚焦激光扫描显微镜观察细菌分布、细胞存活率、单核细胞向巨噬细胞的转化以及荧光标记纳米颗粒进入细胞球状体和结核球状体的情况。使用透射电子显微镜观察三维结核球内的卡介苗和细胞结构。使用Image-iT红色缺氧探针、HO检测试剂盒和防水笔式pH计检测卡介苗感染和未感染的三维结核球状体之间微环境的差异。通过平板菌落计数评估该三维结核球状体在评估利福平和左氧氟沙星抗生素效果方面的实用性。
在细胞-细菌悬液中，稳定的三维结核球状体（50-200μm）形成缓慢，其中细胞紧密粘附，中心有大量细菌，球状体内可见坏死细胞和单核细胞衍生的巨噬细胞。与未感染的细胞球状体相比，三维结核球状体中的药物渗透困难。透射电子显微镜显示结核球状体中的巨噬细胞存在细胞坏死和大量卡介苗。结核球状体的微环境比未感染的细胞球状体更缺氧，HO含量更高，pH接近中性。结核球状体模型能够评估抗结核药物的疗效，其中利福平显示出更强的抗菌作用。
本研究建立的三维结核球状体模型为结核肉芽肿的研究提供了一个有用的平台。
