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共轭和 DNA 介导的细胞组装 快速创建具有可编程形状、尺寸的自组装多细胞支架的策略 和尺寸。几十年来，文化建设取得了长足进步 来自患者来源的角质形成细胞和成纤维细胞（即自体移植物）的上皮自体移植物（CEA），因为它们具有 免疫反应和宿主排斥的可能性最小。然而，新的皮肤组织必须生长并 在漫长的过程中形成层，可能会导致移植皮肤的机械性能较弱 与下面的伤口区域融合不良。此外，细胞天然粘附分子可促进 2D 结构在灵活性和合规性方面的优化很差，因此很难在 下面的基质或伤口。对于某些部位的伤口和烧伤，患者的移动通常是不可避免的 和/或对于烧伤率高的患者，这反过来又可能导致皮肤移植分层和失败。 结果，伤口部位可能会被感染并形成疤痕组织，在更极端的情况下，它们可能会 导致剧烈的痛苦，甚至死亡。 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、 具有预定最终细胞片厚度和可控细胞间间距的密堆积皮肤细胞阵列， 通过可逆、可编程的键连接在一起。这些细胞片将得到显着改善 机械性能超过当前最先进的水平，包括坚固性、顺应性、抗撕裂性和 甚至可以自我修复。通过将与细胞结合的 DNA 与在表面自由移动的互补 DNA 相结合， 互补DNA将充当“连接”链来桥接相邻细胞并驱动2- 和 3D 以形成密堆积单元阵列。让 DNA 连接体充当细胞之间的“粘合剂”应该会增加 形成的组织的机械稳定性，并且还允许自我修复。细胞上表达的DNA 膜还可用于设计细胞片，使其对底层基质具有可调节的粘附力，以 提高最终工程组织的整体机械强度。 为了将 DNA 结合到细胞膜上同时保持长期表达，PI 开发了一种新的 亲和介导共价光共轭 (AMCP) 细胞功能化方法，其中 PI 发现表皮生长因子受体（EGFR）的光交联蛋白标签允许附着 蛋白质绕过典型的蛋白水解途径并返回细胞膜。在拟议的研究中， PI 将利用皮肤细胞上丰富的 EGFR 来附着可光交联的亲和体-链霉亲和素 融合蛋白，进而与生物素-DNA 偶联，利用生物素-链霉亲和素的强相互作用 增加形成的细胞片的极限拉伸强度。该方法将允许调整两个数量 每个细胞的融合蛋白标签和 DNA 链密度，以保持健康的细胞内信号传导和增殖。

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.