Cao Ruonan, Li Nancy T, Shing Chantelle B, Kutulakos Zoe, Tan Cassidy M, McGuigan Alison P
Institute of Biomedical Engineering, University of Toronto.
Department of Chemical Engineering and Applied Chemistry, University of Toronto.
J Vis Exp. 2025 Jul 29(221). doi: 10.3791/68405.
Patient-derived organoids (PDOs) are becoming increasingly used in the field of cancer research to model tumors. They combine the benefits of in vivo and traditional in vitro models, recapitulating tissue complexity and heterogeneity while still consisting of human cells. The rise of physiologically representative PDO models has prompted a need for devices that enable straightforward analysis of organoid behavior. This protocol explains how to assemble and use the Scaffold-supported Platform for Organoid-based Tissues (SPOT), a high-throughput, three-dimensional (3D) organoid culture device. This platform eliminates the meniscus typically formed by seeded hydrogels to allow accurate, quick imaging, similar to the imaging process for two-dimensional (2D) cultures. As a result, organoids and co-cultures seeded in SPOT can easily be imaged using high-throughput microscopy. Designed for off-the-shelf use, SPOT is accessible to researchers with basic tissue culture experience and is compatible with both manual and automated seeding workflows. This protocol details the fabrication of SPOT in 96- and 384-well formats, including step-by-step instructions for assembly, seeding, quality control, and troubleshooting. While fabrication requires 3-4 h, excluding idle time, multiple plates can be produced simultaneously, improving scalability. By streamlining PDO culture and analysis, SPOT provides a robust tool for high-throughput drug screening and translational cancer research.
患者来源的类器官（PDO）在癌症研究领域越来越多地用于肿瘤建模。它们结合了体内模型和传统体外模型的优点，既能再现组织的复杂性和异质性，又仍由人类细胞组成。具有生理代表性的PDO模型的兴起促使人们需要能够直接分析类器官行为的设备。本方案介绍了如何组装和使用基于类器官组织的支架支撑平台（SPOT），这是一种高通量的三维（3D）类器官培养设备。该平台消除了接种水凝胶时通常形成的弯月面，从而能够进行准确、快速的成像，类似于二维（2D）培养的成像过程。因此，接种在SPOT中的类器官和共培养物可以很容易地使用高通量显微镜进行成像。SPOT设计为现货供应，有基本组织培养经验的研究人员即可使用，并且与手动和自动接种工作流程均兼容。本方案详细介绍了96孔和384孔格式SPOT的制作方法，包括组装、接种、质量控制和故障排除的分步说明。虽然制作过程需要3-4小时（不包括闲置时间），但可以同时制作多个板，提高了可扩展性。通过简化PDO培养和分析，SPOT为高通量药物筛选和转化癌症研究提供了一个强大的工具。
