Schaaf Kaitlyn, Smith Samuel R, Hayley Virginia, Kutsch Olaf, Sun Jim
Department of Medicine, University of Alabama at Birmingham.
Department of Medicine, University of Alabama at Birmingham;
J Vis Exp. 2017 Mar 24(121):55453. doi: 10.3791/55453.
The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.
抗结核药物的早期研发过程受到阻碍，原因在于具有体外活性的化合物在临床环境中转化为有效性的效率低下。这可能是由于未考虑受感染宿主中存在的生理相关细胞渗透屏障。我们最近建立了一种替代感染模型，该模型可生成大型巨噬细胞聚集体结构，其核心包含紧密堆积的结核分枝杆菌（Mtb），适用于药物敏感性测试。这种感染模型成本低廉、速度快，最重要的是符合生物安全二级（BSL-2）标准。在此，我们描述了生成Mtb/巨噬细胞聚集体结构的实验程序，该结构将产生经巨噬细胞传代的Mtb用于药物敏感性测试。特别是，我们展示了这种感染系统如何能够直接适用于96孔板形式，显示出针对Mtb筛选化合物库的通量能力。总体而言，由于其简单性、成本效益和可扩展性，该检测方法是当前可用的Mtb药物发现工具箱中的一项有价值的补充。
