Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia.
PLoS One. 2012;7(9):e44640. doi: 10.1371/journal.pone.0044640. Epub 2012 Sep 6.
In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures' response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.
与单层细胞相比，由于与实体瘤高度相似，MCTS 被认为是更适合研究药物渗透的候选物。然而，MCTS 的培养既繁琐又耗时，而且大多数技术都无法生成大小均匀的球体。因此，球体培养在高通量筛选中的应用受到了很大的限制。此外，缺乏适用于球体的经过良好验证的筛选方案方法也是造成这种限制的原因之一。在这里，我们报告了一种从 MCF-7 细胞培养同质 MCTS 培养物的简单方法，该培养物具有紧凑且刚性的结构。此外，我们还对标准 MTT 测定法进行了一些修改，以实现这些球体的高通量筛选。使用修改后的方案，他莫昔芬对 MCF-7 的 MCTS 培养物表现出细胞毒性作用，一致性很高。结果与 LDH 释放测定法评估的培养物反应相关性良好，但由于基础背景读数高，后者测定法不适合检测广泛的细胞毒性。MTT 测定法在检测细胞凋亡事件方面比 LDH 释放测定法更有优势。因此，我们在这里报告的球体生成方法和改良的 MTT 测定法可潜在地应用于 MCF-7 以及其他癌细胞系生成的球体培养物对细胞毒性刺激的反应的高通量筛选。
