Supplementary Figure 10: 3D cell culture formation, z-stack imaging settings and micrograph preparation.

a) Schematic illustration of one well of a 96-well GravityTRAP plate comprising 3D cell culture. The diameter of the bottom of each well is 1 mm. b) Representative brightfield micrographs illustrating the tissue formation process over time. HEK293T cells were seeded onto GravityTRAP plates at time point t=0 and monitored every 6 h until t=48 h. c) Schematic illustration showing the microscopy settings used to image 3D 3D cell culture using a z-stack. The tissue was resolved using 22 slices in z with a distance of 15 μm. d) Representative fluorescent micrographs illustrating 22 slices of an exemplary 3D cell culture and its corresponding maximum intensity projection in z. e) Example of image processing for all fluorescent micrographs shown in the present study using three conditions of the I1-I2 multicellular consortium. Brightfield images are shown, illustrating the 1 mm diameter of the GravityTRAP plate in which the 3D cell culture was cultured and monitored. A square region around the cell culture was cropped, and the cell culture is indicated with a white circle. The original fluorescent micrograph contains a lookup table (LUT) from 1-65,000. The linear LUT was set to 3000-20,000 for all images and fluorescent channels. Scale bars: 100 μm.