The TrueGel3D® HTS Hydrogel Plate is a ready-to-use solution to easily establish 3D cell cultures using fully synthetic hydrogels in a simple and automation-compatible manner. The 96-well polystyrene glass bottom plates contain precasted synthetic functionalized PEG based hydrogels. These innovative hydrogels contain gradually increasing crosslinking densities throughout the well. Users can proceed directly with seeding cells without any hydrogel preparation or encapsulation steps. After cell seeding, cells will gradually infiltrate the hydrogel and establish a 3D cell culture environment within days. Furthermore, this newly engineered hydrogel surface gives the user the possibility to sequentially establish co-culture systems seeding different cell populations at different time-points in the same hydrogel.