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Self-assembly and tissue fusion of toroid-shaped minimal building units.

A significant challenge of tissue engineering is to build tissues whose size is not limited by diffusion. We are investigating the use of scaffold-free lumen containing toroid-shaped microtissues as minimal building units. Monodispersed H35 cells, a rat hepatocyte cell line, were seeded onto micromolded agarose, forming self-assembled multicellular toroids within 48 h. Toroid and lumen diameter were easily controlled by micromold design, and toroid thickness was controlled by seeding density. When harvested, toroids were stable, but underwent predictable changes over time with their lumens narrowing. When brought into contact, these building units fused in the x-y plane, forming a double-lumen structure, as well as the z plane, forming a tubular structure, which completed within 72 h. Large, multi-luminal structures were assembled by multidimensional fusion of many toroids. Toroid settling was not entirely random, with most toroids lying flat with their lumens oriented along the z axis. The rapid production of toroid building units of controlled dimension and lumen size that undergo predictable changes and that can be fused to form larger structures is a step closer to tissue engineering large porous three-dimensional tissues with high cell density.