Application Note
Cell culture automation of the 3D cancer spheroid assay with the CellXpress.ai cell culture system
- Automate the entire 3D cell culture process, from assay set-up to screening, imaging, and data analysis
- Increase confidence in drug screening readouts
- Generate actionable insights for high-throughput drug discovery and precision medicine applications
Oksana Sirenko, Angeline Lim, Astrid Michlmayr, Emilie Keidel,
Felix Spira, Krishna Macha, Cathy Olsen | Molecular Devices
Introduction
Finding efficient anti-cancer drugs and drug combinations is critical for therapy success. Accordingly, there is a critical need to develop methods for efficient drug efficacy testing to discover new viable therapeutic targets. Being highly representative of the structure and behavior of tumors, 3D cancer cell models, such as tumoroids, are highly valuable tools for cancer research and drug development. However, manual 3D cell culture workflows are time-consuming, error-prone, and inconsistent, making their adoption for high-throughput drug screening cumbersome.
To expedite and standardize the spheroid assay, we developed 3D cell culture automation methods using the CellXpress.ai™ Automated Cell Culture System to provide automated plating, passaging, media exchange, and organoid monitoring in response to compound treatment, and endpoint assays. In this study, we describe the automation of a colorectal cell culture workflow, where we automated the culture and imaging of colorectal cancer 3D spheroids formed from HCT116 cell lines in U-shape low attachment plates.
HCT116 cells were expanded in 2D and spheroids were formed after automated dispensing of the cell suspension into U-shape 96 or 384-well plates. After 48h, spheroids were treated with several anti-cancer compounds at multiple concentrations for 3–5 days, followed by staining and imaging. Cell plating, compound addition, media exchange, and staining were performed automatically by the CellXpress.ai system. During cell culture automation, the spheroids were monitored using transmitted light to analyze phenotypic changes, including inhibition of growth and spheroid disintegration. For the endpoint assay, spheroids were stained with a combination of the Hoechst nuclear stain and viability dyes Calcein AM and EtHD. The spheroids were then imaged and analyzed for spheroid size and live-dead cell scoring. In addition, we measured ATP content using a CellTiter-Glo assay. Luminescent read- outs were obtained using the SpectraMax® iD3 Multi-Mode Microplate Reader. We observed a concentration-dependent decrease in ATP content, inhibition of spheroid growth, and cell death in response to anti-cancer compounds.
Methods
Automation of organoid workflow
The new the CellXpress.ai cell culture system automates the entire cell culture process with an integrated incubator, liquid handler, and image-based decision-making. This hands-off solution manages demanding feeding and passaging schedules by monitoring the development of cell cultures with periodic imaging and analysis, and leverages machine learning to initiate the next processing step or troubleshoot issues.
Cell culture protocols
3D spheroids were formed from HCT116 colorectal cells (ATCC) using U-shaped ultra-low attachment 96-well plates (Greiner). Cell culture steps including seeding, media exchange, compound addition and stainings. a well as imaging and analysis, were done using the CellXpress.ai system. Spheroid imaging was done during culture in transmitted light (TL) using 4X magnification. End point assay imaging was done using 10x magnification using florescent imaging (FL) and analysis for organoid size and intensities of viability markers based on staining with Calcein AM, EtHD and Hoechst nuclear stain. Analysis in TL was done using IN Carta® Image Analysis Software‘s SINAP model and analysis in FL was done using CME analysis protocol.
Figure 1. The CellXpress.ai cell culture system components and functionality
Automation of 3D spheroid/tumoroid assay
Figure 2. Schematic diagram of automated spheroid culture
Brief description of spheroid protocol
Protocol was started from cells suspension with media placed into a deep well reservoir (we used 12 well reservoir). Cell suspension was dispensed into the 96 well U-bottom low attachment plates (Greiner or Corning). Plates were placed into the incubator, incubation was continued with imaging every 12h or 24h using 4x or 10x magnification, in TL. Image analysis was run on the fly detecting spheroids and evaluating size and density.
Compound addition: After 48hours after plating 50µl of media was removed and add 2x concentrations of compounds in the volume of 50µl were added using “different media” protocol. Compounds were pre-diluted in the 96 well deep well block, then each compound was added from a single column of the block into 4 columns of the 96-well plate. Each compound was added as a separate step that required defining a plate map for each compound then triggered a compound addition step.
Staining spheroids: After 3 days organoids were stained using premixed solution of 3 viability dyes. Staining was done as a media exchange step. Important: to avoid cross- contamination, tips need to be discarded and cannot be re-used. Staining incubation was done in the incubator for 1h.
Washing step (one step seemed sufficient) was done using media exchange (feeding) phase with PBS.
Spheroids imaging was done using pre-defined protocol with DAPI, FITC, TexasRed. 10–15 steps, 10–15 µm apart, with offset 50–100, Z-stacks around focus, Best focus projections were used for analysis. CME analysis protocol in IN Carta® Image Analysis Software was used to define spheroids sizes and intensities with different channels. Changes in spheroid area and average intensities, as well as ratios of live/dead average intensities were used to evaluate of compound effects. For more detailed phenotypic characterization of cells, the ImageXpress® Micro Confocal system is recommended.
Automated spheroid culture and analysis
Figure 3. Steps for the spheroid culture and analysis workflow.
Results
Phenotypic evaluation of compound effects
We demonstrated here the workflow enabling formation spheroids/tumoroids using example of HCT116 colorectal carcinoma cells. Spheroids were formed and maintained using automated culture by CellXpress.ai instrument.
After compound treatment for 3 days with anti-cancer compounds (staurosporine, doxorubicin, paclitaxel) spheroids were stained with cell viability dyes Calcein AM (live cells) and EtHD (dead cells), and nuclear stain Hoechst. Spheroids were imaged using appropriate fluorescent channels DAPI, FITC and Texas Red, using best focus projection images from Z-track of images taken at 10X magnification. Fluorescence intensities and the ratios of live/dead staining were evaluated. The figure 5 shows decrease of the ratios of live/dead marker intensities with increasing concentrations of compounds. In addition, the decrease in cell viability was evaluated by CellTiter Glow reagent (measure of ATP content). Decrease in ATP content was observed with increase of compound concentrations.
Figure 4. A, B Representative images (10X) of organoid culture taken after treatment with indicated compounds and staining. C. Graphical presentation of spheroid analysis: Ratios of the Ave Intensities of spheroids for Calcein AM and EtHD, maximum concentrations, and across concentration range. Averages and STDEV calculated from quadruplicates.
Figure 5. After compound treatment for 3 days, spheroid samples were tested for ATP content using CellTiterGlo reagent for 3D samples. EC50 for Staurosporin (red) was 0.05µM, for Paclitaxel (blue) 0.5µM. Data for Doxorubicin (green) were ambiguous due to the possible contribution of compound into Lumi signal.
Summary
There is a great unmet need to develop more effective and personalized approaches to cancer treatment. The transition from 2D to 3D cell biology is considered a game-changer in cancer modeling and drug screening, as 3D models better resemble tissue structure and functionality with more predictive responses to drug effects. However, the complexity of 3D cell culture protocols intercepts the large-scale culturing of cell lines and their wide biomedical applications.
CellXpress.ai Automated Cell Culture System streamlines the entire process, from cell culture to assay set-up, screening, cell culture imaging, and data analysis. Thus, automation makes 3D cell culture workflows reproducible at scale, greatly increasing confidence in the drug screening readouts and generating insights for high-throughput drug discovery and precision medicine applications.