(1173-B) Automation of the full workflow for 3D cancer spheroid assays with CellXpress.ai

Abstract: Finding efficient drug combinations to treat cancer patients is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient testing drug efficacy to discover new therapeutic targets. 3D cancer models are highly valuable tools for cancer research and drug development, however, the complexity of performing 3D assays remains a hurdle for the wide adoption of these methods for compound screening.
We developed cell culture automation methods using CellXpress.ai instrument. The instrument enables full automation of 2D or 3D assays for prolonged complex workflows. The CellXpress.ai Automated Cell Culture System provides automated plating, passaging, media exchanges, monitoring organoids, alone with compound treatment and the end point assays. In the present study we describe automation of cell culture process and end-point assays which enables scaling up complex 3D cell-based assays and compound screening.
As an example of 3D protocol, we automated culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell line in U-shape low attachment plates. HCT116 cells were expanded in 2D, then spheroids were formed after automated dispensing cell suspension into U-shape 96 or 384 plates. After 48h spheroids were treated with several anti-cancer compounds, at multiple concentrations for 3-5 days, then stained and imaged. Cell plating, compound additions, media exchange, and staining were performed automatically by CellXpress.ai instrument. During culture spheroids were monitored using transmitted light, with analysis of phenotypic changes, including inhibition of growth or spheroid disintegration. For endpoint assay spheroids were stained with combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD, then spheroids were imaged and analyzed for spheroid size and live-dead cell scoring. In addition, we measured ATP content by using CellTiter-Glo assay. Luminescent read-outs were done by using SpectraMax iD3 Multi-Mode Microplate Reader. We observed concentration-dependent decrease in ATP content, inhibition of spheroid growth and cell death in response to anti-cancer compounds and evaluated effective concentrations for compound effects.
Cell culture process automation powered by imaging has a potential to bring 3D biology into another level, allowing to increase throughput and reproducibility, and enabling variety of high throughput drug discovery and precision medicine applications.