Identifying effective anti-cancer drugs and combinations is essential for successful treatment. To support this goal, there's an urgent need for efficient methods to test drug efficacy and uncover new therapeutic targets.
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Three-dimensional cancer cell models, like tumoroids and spheroids, closely mimic the structure and behavior of tumors, making them powerful tools in cancer research and drug development.
However, manual 3D cell culture workflows are often time-consuming, inconsistent, and error-prone, hindering their adoption for high-throughput drug screening.
To expedite and standardize spheroid assays, automated 3D cell culture methods utilizing the CellXpress.ai™ Automated Cell Culture System have been developed. This system enables automation of the entire cell culture process, including automated processes for plating, media exchange, passaging and culture monitoring in response to compound treatment and endpoint assays.
The study discussed here outlines the automation of a colorectal cell culture workflow, specifically the culture and imaging of colorectal cancer 3D spheroids derived from HCT116 cell lines in U-shaped low-attachment plates.
HCT116 cells were expanded in two-dimensional culture, and spheroids were generated through automated dispensing of the cell suspension into U-shaped 96- or 384-well plates.
Following a 48-hour incubation, spheroids were treated with various anti-cancer compounds at multiple concentrations for 3–5 days, after which staining and imaging were conducted.
The processes of cell plating, compound addition, media exchange and staining were executed automatically by the CellXpress.ai system.
During the cell culture automation, spheroids were monitored using transmitted light to assess phenotypic changes, including growth inhibition and spheroid disintegration.
For the endpoint assay, spheroids were stained with a combination of Hoechst nuclear stain and viability dyes Calcein AM and EtHD. The spheroids were subsequently imaged and analyzed for size and live-dead cell scoring.
Additionally, ATP content was measured utilizing a CellTiter-Glo assay, with luminescent readouts obtained using the SpectraMax® iD3 Multi-Mode Microplate Reader.
A concentration-dependent decrease in ATP content was observed, as well as inhibition of spheroid growth and increased cell death in response to the anti-cancer compounds.
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Acknowledgments
Produced from material originally authored by Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Emilie Keidel, Felix Spira, Krishna Macha and Cathy Olsen from Molecular Devices.
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