A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.
Many tissues are comprised of multiple heterocellular layers, where interlayer cell communications between heterogeneous cell populations are essential to sustain normal tissue functions. Mimicking such natural organization presents a challenge to tissue engineering, and few methods are available to bioengineer heterocellular multilayer constructs in vitro. We have developed a bulk acoustic levitation (BAL) technique to assemble multilayer 3D neuronal constructs from homogenous neural progenitor l. We present here a novel approach to build heterocellular multilayer tissues by combining BAL and layer-by-layer assembly. Cells are levitated into several layers within fibrin prepolymer solution by bulk acoustic waves and immobilized in a fibrin hydrogel layer via gelation. Several hydrogel units can then be stacked subsequently, each unit with a distinct cell type, to form a single hydrogel construct. We demonstrate the bioengineering of a heterogenous multilayer construct composed of three different cell types (wild-type—mCherry and eGFP HeLa cells) spatially distributed in a single 3D hydrogel. This novel approach enables rapid formation 3D multilayer structures in minutes with tunable interlayer spacing, layer composition and thickness, and is potentially useful to bioengineer homogenous and heterogeneous multilayer cell organization for tissue engineering. 1 Bouyer et al. (2015) Adv. Mater. 28:161-7
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