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

通过细胞膜上 DNA 的长期表达实现机械鲁棒性皮肤组织的可控 2 和 3D 组装

• 批准号: 10328551
• 负责人: Jennifer N Cha
• 金额: $ 18.93万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-13 至 2022-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10328551
• 关键词: 3-Dimensional; Adhesions; Affinity; Area; Autologous Transplantation; Binding; Biochemical; Biotin; Burn injury; Bypass; Cadherins; Cell Adhesion Molecules; Cell membrane; Cell surface; Cells; Cessation of life; Chimeric Proteins; Cicatrix; Collagen; Complementary DNA; Complex; Coupled; DNA; DNA Binding; DNA Sequence; DNA annealing; Dermis; Development; Dimensions; Engineering; Epidermal Growth Factor Receptor; Epithelial; Failure; Fibroblasts; Glass; Glues; Goals; Health; Immune response; Lead; Length; Linker DNA; Lipids; Location; Mechanics; Mediating; Methods; Movement; Nanostructures; Pathway interactions; Patients; Pattern; Polymers; Process; Property; Proteins; Publishing; Receptor Cell; Research; Resistance; Scheme; Shapes; Signal Transduction; Site; Skin; Skin Tissue; Skin Transplantation; Streptavidin; Structure; Surface; Tensile Strength; Thick; Time; Tissue Engineering; Tissues; Transplantation; Work; cell assembly; density; extracellular; flexibility; healing; improved; innovation; keratinocyte; mechanical properties; migration; preservation; programs; scaffold; trafficking; wound; wound bed

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.

拟议的研究计划将开发创新的生物结合和DNA介导的细胞组件 快速创建具有可编程形状，大小， 和尺寸。在过去的几十年中，在发展文化方面取得了巨大的进步 来自患者衍生的角质形成细胞和成纤维细胞（即自体移植）的上皮自体移植（CEA），因为它们具有 免疫反应和宿主排斥的最小机会。但是，新的皮肤组织必须生长，并且 在漫长的过程中形成层，可能导致移植皮肤的机械性能弱 与潜在伤口区域的整合不佳。另外，促进的细胞天然粘合分子 2D结构对灵活性和合规性的优化程度不佳，因此很难操纵 基础基板或伤口。患者运动通常是不可避免的，在某些地方受伤和燃烧 和/或对于高燃烧百分比的患者，这又可能导致皮肤移植分层和衰竭。 结果，伤口部位可以被感染并形成疤痕组织，在更极端的情况下，它们可能 导致严重的痛苦甚至死亡。 拟议的研究将开发一种DNA介导的自下而上的方法，以迅速产生大区域， 封闭的皮肤细胞阵列，预定的最终细胞板厚度和受控的细胞间距， 通过可逆的，可编程的债券加在一起。这些细胞表将大大改善 当前最新的机械性能，包括鲁棒性，合规性，抵抗撕裂和 甚至是自我修复。通过将与细胞结合的DNA与表面自由移动的完整DNA结合在一起， 完整的DNA将充当“接头”链，以桥接相邻细胞并驱动两个2-的相互作用 和3D形成封闭的电池阵列。使DNA接头充当细胞之间的“胶水”应增加 形成的组织的机械稳定性，还可以进行自我修复。在细胞上表达的DNA 膜也可用于用可调的粘合力来设计细胞表，以使底层底物 提高最终工程组织的整体机械强度。 为了在保留长期表达的同时将DNA与细胞膜结合，PIS已经发展出一种新的 亲和介导的共价光结合（AMCP）细胞功能化方法，其中PI 发现光链接蛋白标签与表皮生长因子受体（EGFR）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EGFR在皮肤细胞上的抽象来附加可光叠链接抗体 - 链霉替蛋白 融合蛋白又将与生物素-DNA结合，使用强生物素 - 链霉亲和素相互作用 增加形成的细胞表的最终拉伸强度。此方法将允许调整两个 融合蛋白标签每个细胞和DNA链密度，以保留健康的细胞内信号传导和增殖。