We developed a 96-well plate assay which allows fast, reproducible and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (PEG) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural read-outs.

Recent advances in immuno-oncology have significantly increased the therapeutic arsenal available for clinicians. However, being able to identify the responder to a given treatment remains difficult and time consuming. This is due to the lack of translational preclinical models that recapitulate the complete cellular and physical tumor-immune micro-environment (TME). We report for the first time a high throughput vascularized immunocompetent breast tumoroids model in standard multiwell culture plate (MW). The model uses micro physiological system (MPS) and microfabrication to recapitulate and precisely control the TME. The tumor model includes fibroblasts, tumor cells (MDA-MB-231), immune cells (CD81+, CD64+), endothelial cells (CD31+), and a collagen extra cellular matrix. These are essential for the TME and are often lacking in preclinical models, potentially biasing the observed therapeutic response. The physical microenvironment was recapitulated using Cherry Biotech’s MPS, CubiX, and 4DCell SmartSphero Plates (SSoP). A combined system, which to the best of our knowledge is the only one able to recreate the complete TME in a 24 MW with multiplexed and uniformized tumoroid sizes. The SSoP technology is based on microstructured hydrogels, where microwells with an anchoring point at the bottom, allow the formation of the tumoroids, and to keep them in place, making it easy to trace them. The non-adherent properties of the gels help maintain the tumoroids shape. The CubiX system allowed controlling the cell culture conditions: temperature (37°C) and medium perfusion (150 µL/min). The presence of an enriched gas mix with 5% CO2 and O2, was also provided to the tumoroids, and pH, lactate, glucose, O2 consumption were monitored along all culture periods. We focused on optimizing the recapitulation of the physiopathology of breast cancer in 3 aspects: growth rate; tumor cell migration; and oxygen gradient within the MW plate to mimic different depths of the tumor (normoxic to the hypoxic core). Those features are essential for accurate drug efficacy testing. We were able to grow and maintain up to 91 tumoroids per well in a 24MW. After tumoroids formation, we obtained a fully vascularized and immuno-competent model in 48h, the fastest to the best of our knowledge. The system enabled monitoring of the tumoroids growth rate, the differentiation of the endothelium cells (Kfl 2/4, Van Willebrand Factor, eNOS, -% actin fiber alignment), differentiation of CD81+ and CD64+ cells into Macrophage type 1 or 2. All cell types were kept viable for 7 days, and endothelial cells alignment was found physiological (80%). Furthermore, we were able to induce on demand the invasiveness phenotype of the tumoroids. We envision that this model will evolve into a vascularized immunocompetent patient derived tumor model that can be used routinely in precision oncology to predict the drug response of a given patient. Citation Format: Stijn Robben, Anais Peyron, Ana Rita Ribeiro, Divyasree Prabhakaran, Antoni Homs Corbera, Pierre Gaudriault, Patricia Davidson, Dario Fassini. A high throughput vascularized immunocompetent tumoroids model in a standard multiwell plate for precision oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 193.

We developed a 96-well plate assay which allows fast, reproducible and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (PEG) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural read-outs.

We developed a 96-well plate assay which allows fast, reproducible and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (PEG) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural read-outs.

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