Designing a Scaffold-Free Bio-Orthogonal Click Chemistry Method of Cell Assembly for Application in Tissue Engineering

Tissue engineering is a growing field of science that relies on the use of material chemistry, engineering, genetics, and cell biology to produce functional tissues for use in transplantation, drug testing and disease modelling. Presently, there is an urgent need for a technology which would enable assembly of cells into 3-dimensional multilayered tissues. Current cell-assembly technologies rely on biodegradable polymer scaffolds to assemble cells into 3D structures and to support the cell mass of the growing tissue. The presence of these materials in tissues, however, lowers the cell density and the process of scaffold biodegradation results in accumulation of monomer byproducts within the tissue. To overcome these issues we developed a scaffold free method of cell-assembly based on bio-orthogonal ligation reactions between oxyamine and ketone groups to form a stable oxime bond. The reaction is quick, specific and occurs under physiological conditions without a catalyst. To deliver the bio-orthogonal functionalities onto cell surfaces, ketone- and oxyamine- functionalized lipids were incorporated into liposomes which were subsequently fused with cell membranes. The surface engineered cells were assembled into three-dimensional tissues. Using this approach, we were able to produce functional cardiac and liver tissues with variable thicknesses and cell orientations for drug testing as well as the complex 3D co-cultures of stem cells to study stem cell differentiation. The rapid bio-orthogonal cell ligation process also enables assembly of cells into co-culture spheroids in flow, inside a microchannel. The introduction of a bi-functional oxyamine crosslinker molecule allowed for the rapid crosslinking of ketone-functionalized cells into 3D tissues. This bio-orthogonal click chemistry technology can be used with different cell types to produce customized tissues for applications in drug development and regenerative medicine.