Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.
Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres.
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DOI：
10.1371/journal.pone.0103817
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Garnett MC
中科院分区：
文献类型：
--
作者：
Ivanov DP;Parker TL;Walker DA;Alexander C;Ashford MB;Gellert PR;Garnett MC
三维细胞培养比单层培养有许多优点，球形细胞被誉为目前最好的体外小型无血管肿瘤的代表。然而，由于培养生长不均衡、重复性差以及缺乏高通量的3D分析方法，它们在常规筛查项目中的采用受到了阻碍。本研究的目的是建立一种快速、可靠的肿瘤和人胎脑组织三维抗癌药物筛选方法，以考察药物的有效性和选择性的细胞毒作用。商业上可获得的超低附着性96孔圆底培养板被用来以符合自动化的快速、可重复的方式培养球体。一套三种不同的球体健康评估方法(球体体积、代谢活性和酸性磷酸酶活性)与健康球体和药物治疗后的球体的细胞数量进行了验证。开发了一个自动化的开源ImageJ宏，以实现高通量的体积测量。尽管球体体积测定优于其他检测方法，但将其与雷天青还原和磷酸酶活性相结合，可产生更丰富的球体状况。用依托泊苷在UW228-3髓母细胞瘤细胞系和人神经干细胞上测试了区分对正常脑的恶性和增殖成分的影响的能力。当依托泊苷浓度低于10微米时，对正在增殖的干细胞表现出较高的毒性，而当浓度高于10微米时，肿瘤球体受到的影响更大。高通量分析程序使用现成的板、开源软件，并与标准板读取器兼容，因此提供了高预测能力，节省了大量的时间和金钱。
Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.
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