Fig. 1: Optimisation of the co-culture setup for CCA organoids and immune cells.

Bright field images on days 4–5 show that CCA organoids lose 3D morphology in culture without BME and PBMCs are not able to interact well with CCA organoids in BME domes (a). Co-culture in a 10% BME in medium suspension (b) can sustain 3D organoid morphology and allows PBMC–organoid interaction. CCA organoids and PBMCs were exposed to different medium compositions to find a medium in which both survive and function. Flow cytometry for Ki-67, HLA-DR, and CD137 protein expression in CD4⁺ and CD8⁺ T cells after 7 days of culture in T cell medium without and with anti-CD3/CD28 coated beads (TM, TM+), organoid medium without and with anti-CD3/CD28 coated beads (OM, OM+), and organoid medium without specified components in the presence of anti-CD3/CD28 coated beads (c) demonstrates an inhibitory effect of forskolin and nicotinamide on T cells (n = 3 different PBMC donors). Statistical significance is depicted for OM+, -forsk and -nic compared to TM+. ATP quantification shows that CCA organoid survivability is not affected by removal of forskolin or nicotinamide from the organoid medium, but removal of both decreases viability (d) (n = 3 technical replicates). CCA viability was not affected by the addition of human serum to organoid medium as determined by ATP quantification assay (e) (n = 3 technical replicates). Overlay of bright field (grey scale) and RFP (red) demonstrates that the killing potency of anti-CD3/CD28 bead pre-activated PBMCs in both T cell medium (TM) and organoid medium without nicotinamide (OM-nic) is comparable (f). Scale bar: 500 µm (a and left panel of b), 100 µm (right panel of b), 200 µm (f). All values with error bars represent mean with SEM. *p < 0.05, **p < 0.01, ***p < 0.001.