Freestanding stacked mesh-like hydrogel sheets enable the creation of complex macroscale cellular scaffolds.

Hydrogel-based bottom-up tissue engineering depends on assembly of cell-laden modules for complex three-dimensional tissue reconstruction. Though sheet-like hydrogel modules enable rapid and controllable assembly, they have limitations in generating spatial microenvironments and mass transport. Here, we describe a simple method for forming large-scale cell-hydrogel assemblies via stacking cell-embedded mesh-like hydrogel sheets to create complex macroscale cellular scaffolds. Freestanding stacked hydrogel sheets were fabricated for long-term cell culturing applications using a facile stacking process where the micropatterned hydrogel sheets (8.0 mm × 8.7 mm) were aligned using a polydimethylsiloxane drainage well. The stacked hydrogel sheets were precisely aligned so that the openings could facilitate mass transport through the stacked sheets. Despite the relatively large height of the stacked structure (400-700 μm), which is larger than the diffusion limit thickness of 150-200 μm, the freestanding cell-ydrogel assemblies maintained cell viability and exhibited enhanced cellular function compared with single hydrogel sheets. Furthermore, a three-dimensional co-culture system was constructed simply by stacking different cell-containing hydrogel sheets. These results show that stacked hydrogel sheets have significant potential as a macroscale cell-culture and assay platform with complex microenvironments for biologically relevant in vitro tissue-level drug assays and physiological studies.