Reproducible Manufacturing of SPOT as a High-throughput Scaffold-based Culture Platform

Ruonan Cao; Nancy T. Li; Chantelle B. Shing; Zoe Kutulakos; Cassidy M. Tan; Alison P. McGuigan

doi:10.3791/68405

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Method Article

Reproducible Manufacturing of SPOT as a High-throughput Scaffold-based Culture Platform

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DOI:

10.3791/68405

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July 29th, 2025

Ruonan Cao^*¹ , Nancy T. Li^*² , Chantelle B. Shing¹ , Zoe Kutulakos³ , Cassidy M. Tan² , Alison P. McGuigan¹^,²

¹Institute of Biomedical Engineering, University of Toronto, ²Department of Chemical Engineering and Applied Chemistry, University of Toronto, ³Division of Engineering Science, University of Toronto

^* These authors contributed equally

Summary

This protocol documents the fabrication and assembly of SPOT, a high-throughput scaffold-based culture platform, in 96- and 384-well formats. It also details the seeding process for SPOT, both manual and using a commercially available liquid handler.

Abstract

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.

Introduction

Despite advancements in biomedical research, developing physiologically relevant in vitro assays remains challenging. An ideal assay should accommodate a diverse range of cell types, treatment formulations, timescales, commercial instrumentation, and analytical readouts to meet the needs of researchers across various disciplines. However, due to the lack of suitable tools, researchers often rely on simplistic models, such as 2D monolayers of immortalized cell lines, which fail to recapitulate the complexity and clinical relevance, particularly in cancer research¹^,². To address this, large-scale tumor ....