Stratified tissue biofabrication by rotational internal flow layer engineering

The bioassembly of layered tissue that closely mimics human histology presents challenges for tissue engineering. Existing bioprinting technologies lack the resolution and cell densities necessary to form the microscale cell-width layers commonly observed in stratified tissue, particularly when using low-viscosity hydrogels, such as collagen. Here we present rotational internal flow layer engineering (RIFLE), a novel, low-cost biofabrication technology for assembling tuneable, multi-layered tissue-like structures. Using high-speed rotating tubular moulds, small volumes of cell-laden liquids added to the inner surface were transitioned into thin layers and gelled, progressively building macroscale tubes composed of discrete microscale strata with thicknesses a function of rotational speed. Cell encapsulation enabled the patterning of high-density layers (10⁸cells ml^-1) into heterogenous constructs. RIFLE versatility was demonstrated through tunica media assembly, encapsulating human smooth muscle cells in cell-width (12.5µm) collagen layers. Such deposition of discrete microscale layers, facilitates the biofabrication of composite structures mimicking the nature of native stratified tissue. This enabling technology has the potential to allow researchers to economically create a range of representative layered tissue.