reproducible-manufacturing-spot-as-high-throughput-scaffold-based

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

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 ...

University of Toronto

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, however, remains a challenge. One strategy to enable higher throughput organoid cultures is the scaffold-supported platform for organoid-based tissues (SPOT). 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, an automation approach to engineer tumour-mimetic 3D microtissues in SPOT using a liquid handler is optimized and comparable within- and between-sample variation to standard manual manufacturing is shown. Further, 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 is developed. As a proof-of-value demonstration, 3D complex tissues containing different proportions of tumour and stromal cells are generated to probe the reciprocal impact of co-culture. It is also demonstrated that primary patient-derived organoids can be incorporated into the pipeline to capture patient-level tumour heterogeneity. It is envisioned that this automated 96/384-SPOT workflow will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets.

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

Pancreatic ductal adenocarcinoma (PDAC) is a high-mortality cancer characterized by its aggressive, treatment-resistant phenotype and a complex tumour microenvironment (TME) featuring significant hypoxia. Bulk transcriptomic analysis has identified the "classical" and "basal-like" transcriptional subtypes which have prognostic value; however, it is not well-established how microenvironmental heterogeneity contributes to these transcriptional signatures. Here, we exploited the TRACER platform to perform single cell transcriptome analysis of organoids at specific spatial locations to explore the effect of oxygen and other cell-generated microenvironmental gradients on organoid heterogeneity. We found that the microenvironmental gradients present in TRACER significantly impact the distribution of organoid transcriptional phenotypes and the enrichment of gene sets linked to cancer progression and treatment resistance. More significantly, we found that microenvironmental gradients, predominantly oxygen, drive changes in the expression of classical and basal-like transcriptional subtype gene signatures. This work suggests that hypoxia contributes to determining transcriptional subtypes in PDAC tumour cells independent of additional cells in the TME and broadly highlights the importance of considering microenvironmental gradients such as oxygen in organoid-based studies.

Article	Year
An Automation Workflow for High-Throughput Manufacturing and Analysis of Scaffold-Supported 3D Tissue Arrays. Advanced healthcare materials\| PubMed ID: 37086259	2023
Analysis of an engineered organoid model of pancreatic cancer identifies hypoxia as a contributing factor in determining transcriptional subtypes. Scientific reports\| PubMed ID: 40604231	2025

Cassidy M. Tan

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