Abstract 6029: Long-term three-dimensional (3D) tumor culture models using a universal well-plate platform
Authors: Jeong Yoon , Tin Ashley , Irudayaraj Joseph 
Published: June 2022

Abstract
Generating 3D tumor spheroid in vitro has been a straightforward method not only to replicate in vivo like cytoarchitecture and also to study complex interactions to the 3D milieu. Although various approaches have been developed to date, most of the in vitro techniques were hampered by the limitations like small-volume, non-uniformity, irregular sphericity, short-term culture, labor-intensive and studies’ complexities. Here, we have developed very intuitive methods to generate 3D tumor spheroid models and scaffold-based 3D culture models in a single well of universal plate for long-term cultivation.
Experimental Procedures: The prototype of 3D printed chamber was designed by Sketchup software (Trimble) and printed at Illinois MakerLab (http://makerlab.illinois.edu) for this study. The human colon cancer cell (HCT116), obtained from American Type Culture Collection (ATCC), were cultured in DMEM medium containing 10% FBS and stored in a humidified incubator of 5% CO 2 at 37 °C. Five universal 96 well plates (Corning, Thermo Scientific, CytoOne, Advangene, and Genesee Scientific), Elisa 96 well strip (Corning), Black 96-well plate (Thermo scientific) were used without any modification (Not a type of ultra-low attachment (ULA) plate). The prototype designs are flexible to be modified and will be utilized to develop a product beyond conceptual frameworks in the market.
Results: Recently, microfabricated approaches have been intensively studied for micro 3D tumor models (&lt; 300 µm in diameter) implemented with high-throughput analysis. However, the micro tumor model incubated in a short-term might not effectively develop tumor heterogeneity such as a non-perfusion core, diffusion gradient and increased interstitial pressure. We intentionally generated a tumor spheroid model incubated over 1 month with appropriate media replacement to supply nutrients. The spheroid in the system grew over 1.5mm of diameter with an excellent sphericity (&gt; 0.95). Besides, our methodology can perform various in-situ spheroid assays in basic cancer research and also generate scaffold-free/based 3D tumor culture models in the system. To replicate in vivo like cytoarchitecture more physiologically relevant, decellularized tissue scaffold-based 3D culture was conceptually demonstrated.
Conclusions: We developed a simple and effective process that generates 3D tumor culture models in millimeter size and allows long-term incubation to develop local heterogeneity. Importantly, this approach does not require additional hidden costs and any specialized instruments. We believe the present strategy satisfies all essential criteria (i.e., easiness, accessibility, reproducibility, effectiveness, and applicability) to appeal to general researchers compared to other available methods by far.
Citation Format: Yoon Jeong, Ashley Tin, Joseph Irudayaraj. Long-term three-dimensional (3D) tumor culture models using a universal well-plate platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6029.