Controllable 2- and 3D Assembly of Mechanically Robust Skin Tissue Via Long Term Expression of DNA on Cell Membranes

The proposed research plan will develop innovative bioconjugation and DNA-mediated cell assembly strategies for rapid creation of self-assembled multicellular scaffolds with programmable shapes, sizes, and dimensions. Over the past several decades, enormous strides have been made in developing cultured

epithelial autografts (CEA) from patient-derived keratinocytes and fibroblasts (i.e. autografts) because they have the smallest chance of immune response and host rejection. However, the new skin tissue must be grown and formed into layers in a lengthy process and the weak mechanical properties of the transplanted skin may result

in poor integration with the underlying wound area. In addition, the cells natural adhesion molecules that promote 2D structure are poorly optimized for flexibility and compliance, making it difficult to manipulate onto an underlying substrate or a wound. Patient movement is often inevitable for wounds and burns at certain locations

and/or for patients with high burn percentages, which in turn can lead to skin transplant delamination and failure. As a result, the wound sites can become infected and form scar tissue, and in more extreme cases they may lead to intense suffering and even death. The proposed research will develop a DNA mediated bottom-up approach to rapidly generate large-area,

close-packed skin cell arrays with predetermined final cell sheet thickness and controllable cell-cell spacing, joined together by reversible, programmable bonds. These cell sheets will boast significantly improved mechanical properties over current state-of-the-art, including robustness, compliance, resistance to tearing, and

even self-healing. By combining DNA bound to the cells with complementary DNA freely mobile on the surface, the complementary DNA will act as ‘linker’ strands to bridge neighboring cells and drive interactions in both 2- and 3D to form close packed cell arrays. Having DNA linkers act as a ‘glue’ between cells should increase the

mechanical stability of the formed tissues and also allow for self-healing. The DNA expressed on the cell membranes can also be used to engineer cell sheets with tunable adhesion forces to an underlying substrate to improve the overall mechanical strength of the final engineered tissue. To conjugate DNA to cell membranes while retaining long-term expression, the PIs have developed a new

Affinity-Mediated Covalent Photoconjugation (AMCP) cell functionalization method where the PIs discovered that photocrosslinking protein tags to epidermal growth factor receptor (EGFR) allowed the attached proteins to bypass typical proteolytic pathways and return to the cell membrane. In the proposed research, the

PIs will take advantage of the abundance of EGFR on skin cells to attach photocrosslinkable affibody-streptavidin fusion proteins, which in turn will be coupled with biotin-DNA, using the strong biotin-streptavidin interactions to increase ultimate tensile strength of the formed cell sheets. This method will allow tuning of both the number of

fusion protein tags per cell and DNA strand density to preserve healthy intracellular signaling and proliferation.