An Automation Workflow for High-Throughput Manufacturing and Analysis of Scaffold-Supported 3D Tissue Arrays.

Patient-derived organoids have emerged as a useful tool to model tumour heterogeneity. Scaling these complex culture models while enabling stratified analysis of different cellular sub-populations remains a challenge, however. One strategy to enable higher throughput organoid cultures is the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT allows the generation of flat, thin and dimensionally-defined microtissues in both 96- and 384-well plate footprints that are compatible with longitudinal image-based readouts. SPOT is currently manufactured manually however, limiting scalability. In this study, we optimize an automation approach to engineer tumour-mimetic 3D microtissues in SPOT using a liquid handler and show comparable within- and between-sample variation to standard manual manufacturing. Further, we develop a liquid handler-supported cell extraction protocol to support single-cell-based end-point analysis using high-throughput flow cytometry and multiplexed cytometry by time of flight. As a proof-of-value demonstration, we generate 3D complex tissues containing different proportions of tumour and stromal cells to probe the reciprocal impact of co-culture. We also demonstrate that we can incorporate primary patient-derived organoids into the pipeline to capture patient-level tumour heterogeneity. We envision that this automated 96/384-SPOT workflow will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets. This article is protected by copyright. All rights reserved.