A1153

Before cell culture, the fully assembled device was first sterilized by exposing it to ultraviolet light (Electro-lite ELC-500) for at least 30 min. To engineer vascularized organoids in OCTOPUS-EVO, we prepared 20 μl of cell suspension solution containing fibrinogen (5 mg ml⁻¹; F8630, Sigma), thrombin (1 U ml⁻¹; T7513, Sigma), aprotinin (0.15 U ml⁻¹; A1153, Sigma), human intestinal stem cells, primary human umbilical vein endothelial cells (HUVECs) (5 × 10⁶ cells per ml) and primary normal human lung fibroblasts (1 × 10⁶ cells per ml), and injected it into the open cell culture chamber through its inlet access port. The device was then left in a cell culture incubator at 37 °C and 5% CO₂ for 30 min. Upon gelation, IntestiCult media mixed with EGM-2 endothelial media were added to the medium reservoirs and the side microchannels.
Following the formation of a cell-laden hydrogel construct, the side microchannels were incubated with an FN solution (25 μg ml⁻¹ in PBS; 356008, Corning) for 2 h at 37 °C to create an ECM coating on the channel surface. Then, the channels were washed once with IntestiCult/EGM-2, and 10 μl of HUVEC suspension (1 × 10⁷ cells per ml) was introduced into both channels. The seeded cells were allowed to attach to the channel surface over a period of 1 h. Upon 1 h of incubation, pre-warmed medium was added to each medium reservoir.