An automation workflow for high-throughput manufacturing and analysis of scaffold-supported 3D tissue arrays

Abstract

The success rate of bringing novel cancer therapies to the clinic remains extremely low due to the lack of relevant pre-clinical culture models that capture the complexity of human tumours. Patient-derived organoids have emerged as a useful tool to model patient and tumour heterogeneity to begin addressing this need. 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 that also enables easy image-based analysis 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 and is compatible with tumour organoid culture and downstream image-based readouts. SPOT manufacturing is currently a manual process however, limiting the use of SPOT to perform larger-scale screening. In this study, we integrate and optimize an automation approach to generate tumour-mimetic 3D engineered microtissues in SPOT using a liquid handler, and show comparable within-sample and between-sample variation as the standard manual manufacturing process. Furthermore, we develop a liquid handler-supported whole-cell extraction protocol and as a proof-of-value demonstration, we generate 3D complex tissues containing different proportions of tumour and stromal cells and perform single-cell-based end-point analysis to demonstrate the impact of co-culture on the tumour cell population specifically. We also demonstrate we can incorporate primary patient-derived organoids into the pipeline to capture patient-level tumour heterogeneity. We envision that this automated workflow integrated with 96/384-SPOT and multiple cell types and patient-derived organoid models will provide opportunities for future applications in high-throughput screening for novel personalized therapeutic targets. This pipeline also allows the user to assess dynamic cell responses using high-content longitudinal imaging or downstream single-cell-based analyses.

mini-MEndR: A 96-well mixed species culture assay to co-evaluate human and mouse Pax7+cell-mediated skeletal muscle repair

“…The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524941 doi: bioRxiv preprint platform 18 , reported by our group, which we recently demonstrated is compatible with robotic liquid handler to enable massive increases in scale 19 .…”

“…This manual removal step, however would limit use of robots in fully automating our assay. If a larger scale screening capacity is desirable in the future, the MEndR assay could be further integrated with the SPOT platform 20 , reported by our group, which we recently demonstrated is compatible with robotic liquid handler to enable massive increases in scale 21 .…”

Digital light processing-based bioprinting of microtissue hydrogel arrays using dextran-induced aqueous emulsion ink

“…2 –4 Fortunately, advancements in tissue engineering technology have created more sophisticated in vitro 3D cellular models, such as microtissue arrays and 3D printing, which are becoming more commonly used. 5 –9 The widespread use of these 3D models provides a strong foundation for preclinical drug research. Among them, the microtissue array is a micro-scale three-dimensional bionic model carrying cells, 10,11 which can replicate the function of an organ at its most fundamental level.…”