Supplementary Figure 9: Inter-cellular communication devices function more efficiently in a 3D cell culture than in a 2D monolayer setup.

a) HEK293T cells were engineered with genetic programs for cell D1, D2 and D3, and 1600 cells of D1 or 2300 cells of D2 were mixed with 5000 cells of D3 either in a 2D monolayer setup or in a 3D cell culture (untransfected wild-type cells were used to fill the cell mix to 10,000 total cells). Each cell mix was exposed to indicate inducer molecule combinations as shown in the truth table in a 100 μL cell culture volume and cultured for 48 h after secreted luciferase activity was quantified in the supernatant of cells. Partial data are reproduced from Supplementary Fig. 8c. b) HEK293T cells were engineered with genetic programs for cell H1, H2, H3 and H4. The sender cells H1 or H2 were titrated to respective receiver cells H3 and H4 (fixed amount of 6000 cells) and filled to a total of 10,000 cells using untransfected wild-type cells. Each cell mix was exposed to indicated inducer molecules in a 100 μL cell culture volume and cultured for 48 h in either a 2D monolayer setup or in a 3D cell culture after SEAP activity was quantified in the supernatant of cells. The data represent the means ± s.d. of n=3 independent experiments performed in triplicate.