识别影响培养哺乳动物细胞在低到中度通量下细胞生长和生存的化合物的策略
Summary
通常有必要评估培养细胞中一组化合物的潜在细胞毒性。在这里，我们描述了一种以 96 井格式可靠地筛选有毒化合物的策略。
Abstract
细胞毒性是一个关键参数，在研究可能具有治疗效果的药物时需要量化。因此，许多药物筛选分析利用细胞毒性作为单个化合物的关键特征之一。培养中的细胞是评估细胞毒性的有用模型，然后再在更昂贵和劳动密集型的动物模型中对有前途的铅化合物进行跟踪。我们描述了一种策略，用于识别表达人类神经干细胞（NSC）线的tdTomato中影响细胞生长的化合物。该策略使用两个互补检测来评估细胞数量。一种检测通过减少3-（4，5-二甲基硫硫-2-yl）-2，5-二苯基溴化二甲苯（MTT）作为细胞数的代理，另一个直接计数表达NSC的tdTomato。两种检测可以在单个实验中同时进行，并且不耗费大量人力、快速且价格低廉。本演示中描述的策略以 96 孔板格式在探索性初级屏幕中测试了 57 种化合物的毒性。三个命中在六点剂量响应中进一步特征，使用与主屏幕相同的测定设置。除了提供极好的毒性佐证外，两种测定结果的比较可能有效识别影响细胞生长其他方面的化合物。
Introduction
对于具有治疗潜力的化合物，需要确定的最重要特征之一是其对动物细胞的毒性。这一特点将决定药物是否是更广泛的研究的好候选者。在大多数情况下，寻求毒性最小的化合物，但在某些情况下，具有杀死特定细胞类型的化合物是值得关注的，例如抗肿瘤药物。虽然整个动物是确定系统毒性的最佳模型系统，但当需要测试多个化合物时，所涉及的成本和人工是令人望而却步的。因此哺乳动物细胞培养一般用作最有效的替代1，2。小中度药物筛是评估细胞培养中毒性的重要方式。这些屏幕可用于查询针对单个信号通路的带过号库。这种屏幕的一般格式是首先在探索性原发毒性筛查中以单剂量（一般为10μM）测试库中的所有化合物，然后执行深度二次剂量反应屏幕以完全表征毒性主屏幕的点击配置文件。此处将介绍实施此战略的方法，并提供一种快速、高效和廉价的方式来识别和描述有毒化合物。
已经开发出多种方法来评估哺乳动物细胞中小化合物和纳米物质的细胞毒性3，4。需要注意的是，某些材料可以与提供误导性结果的检测结果相互作用，在描述毒性屏幕4的命中时，应测试这种相互作用。细胞毒性测定包括锥形蓝排除5、乳酸脱氢酶（LDH）释放测定6、阿拉马尔蓝测定7、钙化乙氧甲基酯（AM）8、ATP测定9。所有这些测定测量细胞代谢的各个方面，可以作为细胞数的代理。虽然所有提供的好处， 四氧化二氮盐基的测定，如 3-（4，5-二甲基硫硫-2-yl）-2，5-二苯四甲二甲二苯溴化 （MTT）， 2，3-bis （2-甲基-4-硝基-5-磺胺）-2H-四氧化二氮-5-卡博基尼酰胺内盐 （XTT）-1Iodophenyl®-2-[4-硝基]-2H-5-四聚二代）-1，3-苯二苯二醇（WST-1）10，11以低成本提供良好的精度和易用性。MTT，将被用于这个演示，通过线粒体还原酶减少到不溶性对马赞，这种转换率与细胞数密切相关。这种测定在小规模和筛选库与多达2000个化合物12常规使用。通过标记标记直接计数细胞提供了另一种评估细胞数的方法，与 MTT 测定不同，它可以提供有关细胞生长动态的其他信息。几个公开可用的算法可用于执行自动细胞计数分析，还有专有算法，是成像读取器13，14的软件包的一部分。在此方法描述中，经过基因编辑的人类神经干细胞 （NSC）行将用作测试线，用于比较 MTT 测定和自动细胞计数之间的细胞存活结果在屏幕中评估57种试验化合物的毒性。虽然这项战略的主要目标是识别和鉴定有毒化合物，但它具有潜在识别生长抑制和生长增强化合物的额外益处，从而为识别药物提供了一种有效的方法可以调节细胞生长。
Protocol
Representative Results
Discussion
本文的主要目标是描述一种策略，该策略可以高效、廉价地识别在低通量到中度筛选中影响细胞生长的化合物。使用两种正交技术来评估细胞数量，以提高对结论的信心，并提供如果只使用一次测定就无法提供的其他见解。其中一种检测使用荧光细胞成像器直接计数tdTomato阳性细胞，第二种检测依赖于线粒体将MTT切向非马扎人，从而作为细胞10的代理。在这次演示中，共评估了57?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了NINDS内学研究计划的支持。
Materials
| B-27 (50X) | ThermoFisher Scientific | 17504001 | Neural stem cell medium component. |
| BenchTop pipettor | Sorenson Bioscience | 73990 | Provides ability to pipette compound library into a 96-well plate in one shot. |
| BioLite 96 well multidish | Thermo Scientific | 130188 | Any 96 well cell culture plate will work. We use these in our work. |
| Cell culture microscope | Nikon | Eclipse TS100 | Visual inspection of cells to ensure proper density. |
| Cytation 5/ Imaging reader | BioTek | CYT3MFV | Used for cell imaging and absorbance readings. |
| DMSO | Fisher Scientific | 610420010 | Solvent for compounds used in screen. Dissolves MTT precipitates to facilitate absorbance measurements. |
| FGF-basic | Peprotech | 100-18B | Neural stem cell medium component. |
| GelTrex | ThermoFisher Scientific | A1413202 | Neural stem cell basement membrane matrix. Allows cells to attach to cell culture plates. |
| Gen5 3.04 | BioTek | Analysis software to determine cell counts for tdTomato expressing cells. | |
| Glutamine | ThermoFisher Scientific | 25030081 | Neural stem cell medium component. |
| Microtest U-Bottom | Becton Dickinson | 3077 | Storage of compound libraries. |
| MTT | ThermoFisher Scientific | M6494 | Active assay reagent to determine cellular viability. |
| Multichannel pippette | Rainin | E8-1200 | Column-by-column addition of cell culture medium, MTT, or DMSO. |
| Neurobasal medium | ThermoFisher Scientific | 21103049 | Neural stem cell base medium. |
| RFP filter cube | BioTek | 1225103 | Filter in Cytation 5 used to image tdTomato expressing cells. |
| TrypLE | ThermoFisher Scientific | 12605036 | Cell dissociation reagent. |
References
- National Research Council. . Toxicity Testing in the 21st century: A Vision and a Strategy. , (2007).
- Llorens, J., Li, A. A., Ceccatelli, S., Sunol, C. Strategies and tools for preventing neurotoxicity: to Test, to predict, and how to do it. Neurotoxicology. 33 (4), 796-804 (2012).
- Adan, A., Kiraz, Y., Baran, Y. Cell proliferation and cytotoxicity assays. Current Pharmaceutical Biotechnology. 17 (14), 1213-1221 (2016).
- Ciofani, G., Danti, S., D’Alessandro, D., Moscato, S., Menciassi, A. Assessing cytotoxicity of boron nitride nanotubes: interference with the MTT assay. Biochemical and Biophysical Research Communications. 394 (2), 405-411 (2010).
- Tennant, J. R. Evaluation of the trypan blue technique for determination of cell viability. Transplantation. 2 (6), 685-694 (1964).
- Korzeniewski, C., Callewaert, D. M. An enzyme-release assay for natural cytotoxicity. Journal of Immunological Methods. 64 (3), 313-320 (1983).
- Ahmed, S. A., Gogal, R. M., Walsh, J. E. A new rapid and simple nonradioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay. Journal of Immunological Methods. 170 (2), 211-224 (1994).
- Neri, S., Mariani, E., Meneghetti, A., Cattini, L., Facchini, A. Calcein-acetyoxymethyl cytotoxicity assay: Standardization of a method allowing additional analyses on recovered effector cells and supernatants. Clinical and Diagnostic Laboratory Immunology. 8 (6), 1131-1135 (2001).
- Crouch, S. P., Kozlowski, R., Slater, K. J., Fletcher, J. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. Journal of Immunological Methods. 160 (1), 81-88 (1993).
- Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assay. Journal of Immunological Methods. 65 (1-2), 55-63 (1983).
- Berridge, M. V., Herst, P. M., Tan, A. S. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnology Annual Review. 11, 127-152 (2005).
- Malik, N., et al. Compounds with species and cell type specific toxicity identified in a 2000 compound drug screen of neural stem cells and rat mixed cortical neurons. Neurotoxicology. 45, 192-200 (2014).
- Schneider, C. A., Rasband, W. S., Eliceiri, K. W. NIH Image to ImageJ: 25 years of image analysis. Nature Methods. 9 (7), 671-675 (2012).
- Carpenter, A. E., et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology. 7 (10), 100 (2006).
- Cerbini, T., et al. Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluriopotent stem cell (iPSC) and neural stem cell (NSC) lines. PLoS One. 10 (1), 0116032 (2015).
- Lundholt, B. K., Scudder, K. M., Pagliaro, L. A simple technique for reducing edge effect in cell-based assays. Journal of Biomolecular Screening. 8 (5), 566-570 (2003).
- Qie, S., et al. Glutamine depletion and glucose depletion trigger growth inhibition via distinctive gene expression reprogramming. Cell Cycle. 11 (19), 3679-3690 (2012).
- Muelas, M. W., Ortega, F., Breitling, R., Bendtsen, C., Westerhoff, H. V. Rational cell culture optimization enhances experimental reproducibility in cancer cells. Scientific Reports. 8 (1), 3029 (2018).
- Carmichael, J., DeGraff, W. G., Gazdar, A. F., Minna, J. D., Mitchell, J. B. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Recherche en cancérologie. 47 (4), 936-942 (1987).
- Romijn, J. C., Verkoelen, C. F., Schroeder, F. H. Application of the MTT assay to human prostate cancer cell lines in vitro: establishment of test conditions and assessment of hormone-stimulated growth and drug-induced cytostatic and cytotoxic effects. Prostate. 12 (1), 99-110 (1988).
- Jo, H. Y., et al. The unreliability of MTT assay in the cytotoxic test of primary cultured glioblastoma cells. Experimental Neurobiology. 24 (3), 235-245 (2015).
- Kalinina, M. A., Skvortsov, D. A., Rubtsova, M. P., Komarova, E. S., Dontsova, O. A. Cytotoxicity test based on human cells labeled with fluorescent proteins: photography, and scanning for high-throughput assay. Molecular Imaging and Biology. 20 (3), 368-377 (2018).
