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Background The innovative use of αβ T cells engineered with specific γδ TCR receptors shows promise for treating various cancers. For thorough preclinical evaluation, models that mimic the human tumor microenvironment (TME) are essential. Engineered γδ T cells’ ability to kill ovarian tumor models were assessed using Crown Bioscience’s 3D High Content Imaging (HCI) platform. Initial results from reconstituted co-cultures were followed by ex vivo testing using our 3D Ex Vivo Patient Tissue (EVPT) platform with fresh tumor tissue retaining its native TME. These methods provide valuable insights into the therapeutic potential of engineered γδ T cells in conditions closer to an in vivo environment.Methods In the reconstituted TME setup, OVCAR-3 and OVCAR-3 CD277 KO cells were seeded in 3D in a 384-well plate, and pre-stained engineered γδ T cells from two donors were added to the cultures, with or without a pathway-specific activator. In the EVPT assay, fresh ovarian cancer ascites samples were processed to isolate tumor clusters, embedded in hydrogel, and exposed to engineered γδ T cells with or without the activator. Both studies utilized automated phenotypic screening via our HCI analysis platform.Results In the reconstituted TME assay, OVCAR-3 and OVCAR-3 CD277 KO cells were exposed to allogeneic PBMCs, PBMCs with SEA as assay controls and engineered γδ T cells, and engineered γδ T cells with an activator. As expected, PBMCs alone and PBMCs with SEA treatment reduced tumoroid volume and count at 48 and 96 hours for both cell types. Engineered γδ T cells from two donors showed complete killing of OVCAR-3 tumors with the activator but had no effect on OVCAR-3 CD277 KO cells with the activator. In the EVPT assay recapitulating native TME, ovarian cancer ascites samples were treated with Staurosporine, SEA, and engineered γδ T cells, with and without the activator, for 6 days. All patient tissues responded strongly to Staurosporine and variably to SEA. γδ T cells led to tumor killing in 2 out of 3 samples, with increased tumor killing in all samples when combined with the activator. In certain conditions, increased γδ T cell counts, and tumor infiltration were observed.Conclusions This study demonstrated the critical importance of using advanced evaluation platforms for cell therapies in cancer treatment. Crown Bioscience’s native and reconstituted TME assays in the 3D HCI Platform were essential in providing biologically relevant, replicable, and scalable models to evaluate immuno-oncology therapies like engineered γδ T cells.