Layer-by-layer microfluidics for biomimetic three-dimensional structures

Due to the complex structures of living systems, with size scales spanning from the micron to millimeter range, the use of microtechnology to recreate in vivo-like architecture has exciting potential applications. However, most microscale systems are two-dimensional, and few three-dimensional (3-D) systems are being explored. We have developed a versatile technique, combining surface engineering with layer-by-layer microfluidics technology, to create a 3-D microscale hierarchical tissue-like structure. The process involves immobilization of a cell–matrix assembly, cell–matrix contraction, and pressure-driven microfluidic delivery. An aminopropyltriethoxysilane-glutaraldehyde activated chip is used to effectively immobilize the cell–matrix assemblies while maintaining cell viability. Pressure-driven microfluidics is applied to transport cells-matrices with controlled flow rates, determined from dynamic flow imaging. By taking advantage of the contraction of the biopolymer matrices by cells, layer-by-layer microfluidics can be used to build multilayers of cell–matrix inside a microchannel and the thickness of each layer can be controlled down to microscale dimensions. Confocal and electron microscopy images of the final structure show a hierarchical layered cellular configuration composed of heterogeneous biomimetic materials. For a model system, a biomimetic arterial structure is formed using three types of vascular cells to mimic the 3-tunic structure found in vivo. This approach provides solutions to fabricate hierarchical “neotissues” with controlled microarchitectures and 3-D configurations of multiple cell types.