In biopharma research, accurately evaluating drug-induced cytotoxicity is crucial for developing effective therapies. This application note presents an innovative approach that integrates widefield microscopy, high-content screening (HCS), and artificial intelligence (AI) analysis for automated cell segmentation and viability quantification. By employing advanced imaging techniques on the ZEISS Celldiscoverer 7 and AI-driven data interpretation, researchers can gain comprehensive insights into how different drug concentrations impact cell viability and morphology. This methodology enhances the efficiency and precision of cytotoxicity studies and facilitates the identification of optimal treatment strategies, offering a powerful tool to transform drug development processes and contribute to the advancement of targeted therapies in the biopharmaceutical industry.

Drug development is a complex process that begins with identifying potential therapeutic targets and lead compounds. This involves extensive screening and optimization to find candidates with desired biological activity (lead discovery). Promising leads then undergo preclinical research to evaluate their safety and efficacy. A critical part of this is assessing the cytotoxicity of drug candidates, which provides essential insights into a drug's pharmacodynamics and helps researchers determine safe and effective dosing regimens. By characterizing the cytotoxic effects of drugs, scientists assess drug safety and efficacy, informing decisions about clinical trials and supporting regulatory submissions to ensure new therapies are safe and effective for patients. Cytotoxicity assays are vital in advancing drug discovery and development, particularly in oncology research.

This application note presents a protocol for utilizing High Content Screening and AI analysis in cytotoxicity assays. The case study involves the addition of varying concentrations of cisplatin to cells, followed by live imaging and evaluation of the effects of the compound on cell viability and morphology. The ZEISS Celldiscoverer 7 automated microscope system is used to streamline image acquisition and provide a controlled environment for long-term studies. The insights gained from this analysis can streamline the drug development process, facilitate informed decision-making regarding candidate selection, and support regulatory submissions. This application note aims to provide researchers with practical guidance on leveraging high-content imaging and AI analysis to enhance their studies in drug discovery and development.

HeLa cells were initially cultured in DMEM media supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin to promote optimal growth and prevent contamination. Once the cells reached 70-80% confluence in T-75 flasks, they were trypsinized and resuspended in fresh media to create a single-cell suspension. The cell density was adjusted 3,000 cells per well, and the cells were carefully seeded into a 96-well plate, allowed to adhere for 24 hours in a CO₂ incubator at 37°C, and then treated with varying concentrations of cisplatin, along with appropriate controls. All drug doses and controls were added to FluoBrite cell culture media, for optimal imaging.

Following cisplatin treatment, the cells were stained with DAPI to visualize the nuclei, incubating for an additional 5 minutes in the dark. The plate was then transferred to the Celldiscoverer 7. Utilizing the automated HCS workflow the type of plate is automatically recognized, and imaging regions are placed within the wells. Smart automation features such as autofocusing and automatic exposure setting streamline the process. The complete environmental control offered by Celldiscoverer 7, allows the sample to be continuously imaged for 48 hours. The cells were imaged in brightfield with Oblique illumination which angles the light onto the sample, significantly enhancing the contrast and creating a pseudo 3D appearance. Additionally, the DAPI label was imaged using fluorescence widefield microscopy, with excitation 385 nm LED. An area of 75% of each well was imaged allowing to ensure a statistically relevant sample size. Sample focus was maintained with automated SW features, ensuring the cells remain in focus of the entirety of the experiment.

Unlocking the power of automated cellular analysis, the Bio Apps Software, an integral component of the comprehensive ZEN suite, provides sophisticated cell counting capabilities. Leveraging advanced algorithms, the software meticulously identifies individual nuclei, whether through the precise detection of nuclear markers or the label-free approach of phase contrast imaging. Upon accurate nuclear identification, the analysis engine swiftly generates intuitive cell counting data presented in a visually engaging heatmap format.

These dynamic heatmaps offer an immediate and insightful overview, enabling researchers to rapidly pinpoint regions of elevated or diminished cell viability, effectively revealing critical correlations with varying cisplatin concentrations. In this example the end result of the experiment is visualized. Wells with the highest cisplatin concentrations (A-D;1-6) are depicted in darker blue color indicating lower cell numbers, whereas control wells are depicted in green and yellow tones, indicating higher cell numbers.

To quantify the cytotoxic effects, the IC50 value – representing the precise concentration of cisplatin required to inhibit 50% of the cell population – was determined by fitting the cell viability data to a dose-response curve. This crucial metric provides valuable insight into the drug's potency.Furthermore, the intuitive Bio Apps extend their analytical capabilities by enabling the visualization of data from each well across a time series. This longitudinal perspective empowers researchers to track cellular changes and responses dynamically. For streamlined data management and advanced analysis, the software facilitates the export of comprehensive statistical data from the entire plate into a single, readily accessible CSV file.

This application note demonstrates an easy and effective method for performing cytotoxicity assays, a standard research technique for evaluating the therapeutic efficacy of anti-cancer drugs, using the ZEISS Celldiscoverer 7. The integration of high-content screening, automated image acquisition, and AI-powered analysis on the Celldiscoverer 7 platform provides a powerful solution for streamlining drug discovery workflows.

By providing detailed information about drug effects at the cellular level, microscopy-based approaches contribute significantly to the development of safer and more effective therapies.