Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Abstract

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.

Methods: Using Three-Dimensional Culture (Spheroids) as an In Vitro Model of Tumour Hypoxia

“…A more complex version of this for images with poor spheroid contrast has been published elsewhere [40].…”

“…Cell viability of intact spheroids can be an efficient method of analysis of a large number of conditions and can be analysed by a number of assays including Resazurin assay [40] MTT assay [42] and acid phosphatase assay [40,43]. Resazurin reduction assay, acid phosphatase assay and cell counts can be carried out on the same spheroids sequentially as described in [40].…”

“…Resazurin reduction assay, acid phosphatase assay and cell counts can be carried out on the same spheroids sequentially as described in [40]. Similar assays have also been performed including the cell titre glo assay to measure ATP levels [31].…”

Phosphonium Polymethacrylates for Short Interfering RNA Delivery: Effect of Polymer and RNA Structural Parameters on Polyplex Assembly and Gene Knockdown

“…The effect of polymer (4d) on the cell viability was assessed using a Resazurin metabolic cell assay 49 ( Figure 6C). HeLa cells were exposed to …”

Fully Automated One-Step Production of Functional 3D Tumor Spheroids for High-Content Screening

“…[2][3][4] Recent progress in specialized imaging cytometers and the analysis of phase contrast images has opened up multiplexing of plate assays with high-throughput lowresolution microscopy of spheroids, providing complementary simple endpoints concerning spheroid numbers and size. [5][6][7] As yet, however, spheroids have been rarely used in multiparametric high-content screening (HCS) campaigns, the only strategy that can provide functional insights into the spatial pattern of response within individual spheroids and an understanding of the mechanisms that underlie response (Suppl . Table S1).…”