O26 - Advancing Precision Oncology: Bio-printed Cancer Organoids for High-Throughput Drug Screening Platforms

Introduction: : High attrition rates in drug development remain a significant challenge in the pharmaceutical industry, often driven by preclinical models that fail to mimic human physiology accurately [1]. While tissue-engineered models, such as organoids, offer physiologically relevant alternatives, their widespread adoption is hindered by issues of scalability, practicality, and reproducibility [2]. Addressing these gaps is essential to improve the translational success of drug discovery pipelines. Three-dimensional (3D) bioprinting presents a promising solution to these limitations by enabling tissue models' rapid, precise, and reproducible fabrication [3]. In this study, we leverage a nanodrop inkjet bioprinter to generate a high-throughput model of colorectal cancer (CRC) organoids in a xenol-free bioactive matrix with controlled stiffness and composition, aiming to reduce preparation time, enhance reproducibility, and facilitate scalability of organoid models for drug screening. We hypothesize that this platform will improve the efficiency and consistency of organoid handling while ensuring physiological relevance, facilitating more reliable preclinical testing.

Materials and

Methods: : CRC organoids (ISO68, Molecular Devices) were thawed and bioprinted using the RASTRUM nanodrop 3D printer at a seeding density of 19 organoids per well in a 96-well plate. A xeno-free matrix with 1.1 kPa stiffness containing Collagen I (Inventia Life Science) was used. To mitigate shear force effects during printing, organoids were maintained in media supplemented with 10 μM ROCK inhibitor (Y-27632) and allowed a 48-hour recovery before initiating treatments. As a control, a subset of organoids was manually seeded in Matrigel (Corning) domes. Printed organoids were treated with a panel of anticancer compounds, including cisplatin, romidepsin, trametinib, 5-FU, Keytruda, irinotecan, and DK419. Viability was assessed using luminescent ATP detection (CellTiter-Glo 3D, Promega). Organoids were also stained with LIVE/DEAD™ viability dyes (Invitrogen) and imaged using the ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices). Brightfield images were captured over time to monitor morphological changes such as size and shape. High-content image analysis was conducted using MetaXpress software, and statistical analysis was performed using JMP® software (SAS).

Results, Conclusions, and Discussions:: Bioprinted 30 μm-diameter colorectal cancer (CRC) organoids showed consistent morphology and uniform distribution across wells, reducing seeding time from 1 minute per well manually to 20 seconds with bioprinting, and improving reproducibility. The optimized recovery protocol yielded a high post-printing. After 10 days, they grew to 200 μm and developed necrotic centers, which is expected due to limited nutrient flow. This shows that bioprinting offers a reliable and faster way to seed organoids, though longer cultures still need splitting or passaging like standard methods. Drug treatments produced measurable responses, with metabolic and morphological changes aligning with known drug mechanisms. High-content imaging and analysis helped reduce variability and improve data quality. Early results suggest that combining bioprinted organoids with automated imaging offers a scalable and reliable platform for high-throughput drug screening. This has the potential to streamline preclinical testing and improve translational outcomes. Future work will expand this approach to additional cancer models, as well as hepatocyte and liver organoids, to further demonstrate its utility in precision oncology and drug metabolism studies.

Acknowledgements and/or References (Optional):: Acknowledgements: We thank Inventia Life Science team for supporting our lab in all 3D printing endeavors and Molecular Device team for their support when generating high-content screening protocols for image processing.

References: [1] D. Sun, et al. 2022, Acta Pharm Sin B. [2] E. Heinzelmann et al. 2024, Curr Res Toxicol. [3] E.H.Y. Lam et al. 2023. Int J Mol Sci.