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

拟议的研究计划将开发创新的生物结合和dna介导的细胞组装。 快速创建具有可编程形状、大小、 和维度。在过去的几十年里，在发展人工养殖方面取得了巨大的进步 来自患者来源的角质形成细胞和成纤维细胞的自体上皮移植(CEA)(即自体移植)，因为它们有 免疫反应和宿主排斥反应的可能性最小。然而，新的皮肤组织必须生长并 在漫长的过程中形成多层，并可能导致移植皮肤的机械性能较弱 与底层创面结合不良。此外，细胞自然产生的黏附分子会促进 2D结构在灵活性和合规性方面优化不佳，因此很难在 下层基质或伤口。对于某些部位的伤口和烧伤，病人的活动往往是不可避免的。 和/或烧伤百分率高的患者，这反过来可能导致皮肤移植剥离和失败。 结果，伤口部位会受到感染，形成疤痕组织，在更极端的情况下，它们可能 会导致剧烈的痛苦甚至死亡。 这项拟议的研究将开发一种DNA介导的自下而上的方法来快速生成大面积、 具有预定的最终细胞片厚度和可控制的细胞-细胞间距的紧密堆积的皮肤细胞阵列， 通过可逆的、可编程的键连接在一起。这些电池板将会有显著的改进 机械性能超过当前最先进的水平，包括坚固性、顺应性、抗撕裂和 甚至可以自我修复。通过将结合到细胞上的DNA与在表面自由移动的互补DNA结合， 互补的DNA将作为连接相邻细胞和驱动相互作用的‘连接物’链，在2- 和3D以形成紧密堆积的细胞阵列。让DNA连接物充当细胞之间的粘合剂应该会增加 形成的组织具有机械稳定性，并且还允许自我愈合。细胞上表达的DNA 膜也可以用来设计具有可调节的粘附力的细胞片，以 提高最终工程化组织的整体机械强度。 为了将DNA连接到细胞膜上，同时保持长期表达，PI已经开发出一种新的 亲和力介导的共价光共轭(AMCP)细胞功能化方法 发现将蛋白质标签光交联到表皮生长因子受体(EGFR)上可以使附着的 蛋白质绕过典型的蛋白质分解途径，返回细胞膜。在拟议的研究中， PIS将利用皮肤细胞上丰富的EGFR连接光交联体-链霉亲和素 融合蛋白，它将与生物素-DNA偶联，利用生物素-链霉亲和素的强烈相互作用来 提高成形单元板的极限抗拉强度。此方法将允许调整 每个细胞的融合蛋白标签和DNA链密度，以保护健康的细胞内信号和增殖。

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.