Abstract: Organoids are widely accepted as surrogates of native tissues thanks to their unique properties. These 3D structures are derived from stem cells that can self-organize and differentiate like their in-vivo counterparts. Since the development of the first mouse intestinal organoids in 2009, a large variety of organoid models have been established. However, these cultures heavily rely on the use of solid extracellular matrix (ECM), which introduces a high level of heterogeneity, thus hampering robust assay development. To standardize their use as well as increase their adoption in the industry, we developed Gri3D®, a ready-to-use platform for high-throughput and reproducible organoid culture. Based on a standard SBS 96 microtiter plate format, each well contains an array of ultra- dense U-bottom-shaped microwells in a cell-repellent hydrogel. The platform enables the generation of a single organoid in each microcavity in suspension-like conditions, without the need for a solid ECM. Our approach solves key challenges related to disease modelling and compound assessment at a larger scale using organoids. Immune therapies are positioned amongst the most promising approaches to fight cancer as they harness the immune system as a targeting tool against tumor cells. With the recent developments in 3D cell culture and organoid technology, various T-cell killing assays have been described to test T-cell functionality and efficacy or off-target effects of immunotherapies in vitro. However, current assay systems are limited in throughput and are difficult to handle. Establishing a controlled co-culture system is cumbersome due to the difficulty of precisely controlling effector to target ratio, resulting in low reproducibility and high variability of results. We demonstrate how Gri3D® is used to establish a robust and scalable workflow to study immune cell functionality on 3D organoids. Gri3D® enables the generation of tumoroids of very defined sizes that are then added with the immune cells at various effector to target (E:T) ratios. Using this concept, we could efficiently assess the specific toxicity of different CAR-T cell clones against patient-derived stomach organoids. We demonstrate that both the cell ratios and the different CAR-T cell clones have varying toxic effects on human stomach organoids, validating the assay to address human antigen-specific as well as dose-specific off-target toxicity. Our approach demonstrates superiority to current state-of-the-art methods as it is robust by tightly controlling effector to target ratios, it maximizes contact between T-cells and tumoroids, and is scalable through the ability to evaluate hundreds of tumoroids in a single well at single organoid resolution.
