Individual cell lines (Caco-2, HT29, T-84 and SW620) were grown from frozen stocks, prepared at ca. 1 million/mL. Cells were seeded in a T-75 tissue culture flask, passaged once and matured to ca. 10 million/mL in a T-175 flask in complete media. Cells were rinsed twice with Ca 2+ /Mg 2+ -free PBS (25 mL, CAS D8537), trypsinised, the trypsin deactivated with P/S-free complete media, all four lineages mixed, pelleted (300 r.c.f., 5 min), and resuspended in alginate (sodium alginate, CAS 9005-38-3, 1% w/v in P/S-free complete media). The cell suspension was extruded dropwise into CaCl2 (0.3 M in phosphate buffered saline, PBS, CAS D8662, 15 °C) under stirring (100 r.p.m.). The cell spheres (Figures ) were removed from the CaCl2 solution, washed and stored in P/S-free complete media before further use.