Spheroids were dosed with the use of a 3D printed device as discussed previously.24 (link) Briefly, spheroids were placed into membrane inserts (0.4 μm pore diameter; Corning Incorporated, Corning, NY, USA) that fit into the 3D printed device. These inserts rest on top of a flow channel, which allows for the diffusion of small molecules. The device has 6 identical channels, which can accommodate 6 experiments at once and allows for 36 biological replicates in a single run. The spheroids were dosed using the clinical timescale for the FOLFIRI treatment regime over a 24 hour period, depicted in Figure 1.46 (link),47 (link) First, 11.3 μM folinic acid was added to the inserts that housed the spheroids within the 3D printed device for the first hour. At the start of the second hour, 20.6 μM irinotecan was manually added to the inserts. At the start of the third hour, 68.5 μM 5-fluorouracil was manually added to the inserts and flowed through the 3D printed device. The top of the inserts was covered to prevent evaporation.