Micro-moulded non-adhesive hydrogels to form multicellular microtissues - the 3D Petri Dish®

This chapter provides an in-depth description of the technology, applications and protocols of the 3D Petri Dish®, a commercially available system for the production of multicellular, scaffold-free microtissues. The technology uses precision micromoulds to form agarose gels with arrays of small recesses. When cells are seeded onto these gels, they settle into the recesses, aggregate and self-assemble overnight, allowing the creation of hundreds of multicellular microtissues in a single gel. Assembled microtissues are then easily harvested for histology, immunostaining, Western blots and RT-PCR analyses. Microtissue size is controlled by micromould choice and the number of cells seeded. Cell mixtures can potentially self-sort into layers within a microtissue, replicating in vivo architectures. Microtissue shape is controlled by micromould design which, in addition to making spheroids, can direct cells to form toroids, rods and honeycombs. Useful applications include toxicity testing, phenotypic drug discovery, studies of drug uptake/diffusion, the scale-up of spheroid production, clonal growth of spheroids, quantification of cell adhesion and microtissue contraction, drug inhibition of self-assembly, encapsulation of microtissues and use of spheroids and honeycomb-shaped microtissues in tissue engineering.