Optimizing Drug Efficacy Testing in Cancer Research Using 3D Cell Culture Automation

The development of efficient anti-cancer drugs and drug combinations as well as optimized and representative testing methods for the discovery of viable therapeutic targets is crucial for successful cancer therapies.

3D cancer cell models, such as tumoroids, have emerged as highly valuable tools in this aspect for cancer research and drug development. By mimicking the three-dimensional structure and microenvironment of actual tumors, tumoroids provide a realistic and predictive platform for evaluating drug responses.

Yet, despite their potential, manual 3D cell culture workflows are time-consuming, error prone, and inconsistent, posing significant challenges for their adoption in high-throughput drug screening. The CellXpress.ai™ Automated Cell Culture System helps expedite and standardize the spheroid assay by automating key processes including plating, passaging, media exchange and organoid monitoring in response to compound treatments.

By helping automate these processes, the CellXpress.ai Automated Cell Culture System expedites the 3D cell culture processes while enhancing research reproducibility and accuracy which provides for reliable anti-cancer drug discovery.

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Automation of a colorectal cell culture workflow using 3D spheroids from HCT116 cell lines in U-shape low attachment plates
Automation of entire 3D cell culture process, from assay set-up screening and imaging to data analysis
Increasing confidence in drug screening readouts
Generating actionable insights for high-throughput drug discovery and precision medicine applications

Cell culture automation of the 3D cancer spheroid assay using the CellXpress.ai™ Automated Cell Culture System (1)

APPLICATION NOTE

Cell culture automation of the 3D cancer spheroid assay with the CellXpress.ai cell culture system

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, 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

Benefits

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
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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.
CellXpress.ai Automated Cell Culture System
- Automated liquid handler
- Scheduling and analysis software
- Automated incubator
- Automated imager
  Enables development of complex protocols including but not limited:
- Cell seeding/feeding
- Cell passaging
- Imaging and monitoring
- Planning-scheduling protocols

Figure 1. The CellXpress.ai cell culture system components and functionality

Automation of 3D spheroid/tumoroid assay

Tumor cells

Cell plating into U-shape low attachment plates

Spheroid formation

Feeding, differentiation,

compound addition Tumoroid plate

Widefield imaging and AI analysis

Confocal imaging, image analysis

and cell staining

2D culture 3D culture Endpoint assay prep

Media exchange

Monitoring

Advanced endpoint analysis

ImageXpress Confocal HT.ai

CellXpress.ai Automated Cell Culture System

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

Main steps of spheroid workflow:

Cells plated into U-ULA plates
Formation of spheroids
Media exchange
Monitoring, imaging
Staining and compound treatment
- Workflow is suitable for cancer spheroid assays
- Cardiac organoids, neuro-spheroids
  1
- Cell suspension in media pipetted from deep well reservoir
  3
- Seeded 100µL 10,000 cells into 96well U-bottom plates (Greiner)
  2
- Move to incubator for 48h
- Monitor spheroid formation with imaging
- Full workflow protocol set in "phases" defined by user:
- Plating, feeding/monitoring, staining, imaging
- Each step defines main attributes (source and destination plates, pipetting protocols, volumes, media, imaging settings, analysis protocols
- Main steps can be modified by fine-tuning parameters, that allow further optimization
  5
- Decisions for the process can be made automatically based on imaging analysis
- Spheroids imaged using CellXpress with the DAPI, FITC and Texas Red channels, 10X or 4X objective. Image analysis was performed with CME (IN Carta).
- Removal of media
- Add fresh media
  4
- Protocol set for repeated media exchange every "X" hours
- Periodic imaging of plate is defined by user
- On-the-fly image analysis done by using pre-trained deep-learning based model, or customized protocol
- Data analytics tools allow to view growth of organoids over time
- Staining organoids and washes were done by using "media exchange" protocol step
- FL imaging of spheroids was done after treatment with compounds and staining with Calcein AM, MitoTracker and Hoechst nuclear stain.

Hoechst - Blue Calcein AM - Green EtHD - Red

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.

Screen-shot of compound treatment
Effects of anti-cancer compounds
- Staining: Hoechst, Calcein AM (live, green), EtHD (dead, read)
- Image analysis: Custom module editor in IN Carta software: define spheroids via Find blobs using Hoechst stain mask, then measure average intensities in Calcein AM and EtHD, use ratio to evaluate Live/Dead cell content.

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.

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