高通量兼容检测，以评估对巨噬细胞传代药物疗效<em>结核分枝杆菌</em
Summary
New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.
Abstract
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.
Introduction
结核病（TB），尽管依然抗结核化疗方案超过40年1可用性一个严重的全球健康威胁。这是部分原因是由于使用多种药物组合为6个月以上长期治疗期的要求，从而导致病人不遵守2。耐药结核病近年来的出现进一步加剧的一个领域的问题，其中的临床批准的药物研制成功是几乎不存在3。事实上，尽管详尽的抗结核药物研发，只有一个单一的药物已被FDA批准用于在过去40年4临床使用。因此，迫切需要抗结核药物的新世代来解决这个问题。
结核病药物研发的一个关键问题是缺乏从化合物在体外活性在临床上成功转让给疗效=“外部参照”> 5，6，7。最初，基于目标方法被用于筛选抗Mtb的药物5，其未能转化为整个细菌细胞。即使使用的Mtb的细胞时，常常使用肉汤生长的培养物，不准确预测在体内 8,9药效进行。这些问题已被确认，并根据包含的Mtb或潜的Mtb的巨噬细胞的药物筛选测定法已成功地建立了8个 ，10个 ，11个 ，12个 。然而，即使是这些更先进的分析不该药物在非血管肺部病变遇到的渗透屏障给予充分的考虑，并在感染部位的坏死灶。确实，即使对于一线TB药利福平，亚最佳剂量已被质疑，由于体内组织和脑脊髓液（CSF） 中不足渗透13，14，15以及功效抗胞内结核杆菌 8,9下降。因此，新的模型和试验，将在早期的领先开发过程中考虑到这些参数，无疑会提高结核病药物研发努力。
为了满足这一需求，我们最近成立了一个廉价，快速，BSL-2兼容的替代感染结核分枝杆菌的药物疗效测试16的模型。产生密集的大巨噬细胞聚集结构内挤满结核杆菌这种感染模型，概括生理学相关蜂窝普及率障碍和产生的巨噬细胞传代<eM>的Mtb具有改变的生理状况相似潜结核杆菌 。从这种感染模型导出结核杆菌与刃天青酶标法（REMA）相结合，以评估药物疗效，其产生的结果与其他细胞感染模型一致并与之共同结核病药物，以达到相对的血药浓度高CSF浓度的报道能力很好的相关性16。
在这里，我们详细描述的Mtb /巨噬细胞聚集体结构的产生，以产生巨噬细胞传代的Mtb适于使用REMA药敏试验。特别是，我们表明这种感染系统如何适合于96孔格式用于与候选抗结核药物通量筛选相容性。
Protocol
Representative Results
Discussion
这里，我们已经详细适于药物功效测试的替代的Mtb感染模型说明。该模型考虑到应在早期结核病药物开发过程被给予更多的考虑的两个关键因素：生理相关障碍药物渗透和感染期间的Mtb的代谢变化的存在。虽然我们先前已经显示了感染模型的好处，并提出扩大感染吞吐量兼容性16的可能性，这里，我们证明，这是通过直接调整系统到96孔板形式确实可以实现的。产生…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We thank Dr. Frank Wolschendorf for access to the Cytation 3 automated imaging plate reader. This work was funded in part by NIH grant R01-AI104499 to OK. Parts of the work were performed in the UAB CFAR facilities and by the UAB CFAR Flow Cytometry Core/Joint UAB Flow Cytometry Core, which are funded by NIH/NIAID P30 AI027767 and by NIH 5P30 AR048311.
Materials
| 7H9 | BD Difco | 271310 | Follow manufacturer's recommendations |
| Middlebrook OADC | BD Biosciences | 212351 | |
| Tyloxapol | Sigma | T8761 | Prepare 20% stock solution in H2O; filter sterilize |
| D-Pantothenic acid hemicalcium salt | Sigma | P5710 | Prepare 24 mg/ml stock solution in H2O; filter sterilize |
| L-leucine | MP Biomedicals | 194694 | Prepare 50 mg/ml stock solution in H2O; filter sterilize |
| Hygromycin B | EMD Millipore | 400051 | Prepare 200 mg/ml stock solution in H2O |
| Nalgene Square PETG media bottle | Thermo Fisher | 2019-0030 | |
| RPMI 1640 media | Hyclone | SH30027.01 | |
| Fetal Bovine Serum | Atlanta Biologicals | S12450H | |
| L-glutamine | Corning | MT25005CI | |
| HEPES | Hyclone | SH30237.01 | |
| Cytation 3 plate reader | Biotek | Interchangable with any fluorescent plate reader and microscope | |
| Gen5 Software | Biotek | Recording and analysis of rezasurin coversion | |
| Rifampicin | Fisher Scientific | BP2679250 | Prepare 10 mg/ml stock solution in H2O |
| Moxifloxacin Hydrochloride | Acros Organics | 457960010 | Prepare 10 mg/ml stock solution in H2O |
| Resazurin Sodium Salt | Sigma | R7017 | Prepare 800 μg/mL stock solution in H2O; filter sterilize |
| Tween-80 | Fisher Scientific | T164500 | Prepare 20% stock solution in H2O; filter sterilize |
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