Multiplexing spheroid volume, resazurin and acid phosphatase viability assays for high-throughput screening of tumour spheroids and stem cell neurospheres

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 10µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10µM 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.

与单层细胞培养相比，三维细胞培养具备诸多优势，而细胞球体（spheroid）被认为是目前体外模拟小型无血管肿瘤的最佳模型。然而，由于培养生长不均、重现性差以及缺乏适配三维模型的高通量分析方法，细胞球体在常规筛选项目中的应用一直受到限制。本研究旨在开发一种适用于肿瘤组织与人类胎儿脑组织的三维快速可靠抗癌药物筛选方法，以探究药物疗效与选择性细胞毒性作用。本研究采用商业化超低吸附96孔圆底板，以快速、重现性良好且可适配自动化操作的方式培养细胞球体。针对健康与药物处理后的细胞球体，本研究选取三种作用机制各异的细胞球体健康状态评估方法——细胞球体体积测定、代谢活性检测与酸性磷酸酶活性检测——与细胞计数结果进行了验证比对。本研究开发了自动化开源ImageJ宏脚本，以实现高通量的体积测量。尽管细胞球体体积测定法优于其他检测方法，但将其与刃天青还原（resazurin reduction）检测及磷酸酶活性检测联用，可更全面地反映细胞球体的状态。本研究依托依托泊苷（etoposide）处理UW228-3髓母细胞瘤细胞系与人类神经干细胞，验证了该方法区分恶性肿瘤与正常脑组织增殖组分的能力。当依托泊苷浓度低于10μM时，其对增殖态干细胞的毒性更强；而当浓度高于10μM时，肿瘤细胞球体受到的影响更为显著。该高通量检测流程采用预制底板与开源软件，且兼容标准酶标仪，因此可在大幅节省时间与实验成本的同时实现优异的预测效能。