Probing Dynamic Reassembly of Chemically-Etched 3D Embryonic Tissue

Abstract

Most embryonic development and tissue self-assembly requires coordinated cell movements within multiple cell layers composed of different cell types and integrated signaling networks in these 3D environments. The role of cell mechanics in tissue self-assembly has been demonstrated, but little is known about the mechanical responses of 3D multi-layer tissues to chemical cues. To investigate the collective movements within multilayered tissues, we developed a novel microfluidic technique capable of ablating strips of a specified width and depth from a composite tissue. We call this technique “3D tissue-etching” because it is analogous to techniques used in the microelectromechanical systems (MEMS) field where complex 3D structures are built by successively removing material from a monolithic solid through subtractive manufacturing. We used our microfluidic control system to deliver a variety of tissue ablation reagents (detergents, chelators, proteases, etc.) or “etchants” to specific regions of multilayered tissues microsurgically isolated from embryos of Xenopus laevis. Explanted embryonic Xenopus tissues provide an ideal model for 3D tissue etching. Long exposure to a narrow etchant stream cuts completely through cell-cell layers to expose the substrate while a shorter exposure time can remove a single layer. By varying the width of the etchant and the exposure time within a single experiment a broader strip of the surface layer may be removed and then a second narrow single narrow band within the band of exposed cells in the second layer. The ability to control 3D stimulation and the shape of multicellular tissues will extend the tools of tissue engineering to synthesize highly complex 3D integrated multicellular biosystems. Integration of tissue etching in our custom microfluidic system provides a “test-bed” where we can implement and test a range of hypotheses concerning the control and regulation of development and cell differentiation.