Directed Self Assembly of Complex Branching Micro-Tissues

我们开发了一种基于微模塑非粘附性琼脂糖凝胶的新技术来引导3D微组织的自组装。单分散的细胞被输送到微型模具中，它们固定在成型的特征中，无法结合到非粘附性琼脂糖凝胶上，因此自发地自组装3D微型组织。通过在微型模具中制造复杂的特征，我们首次展示了细胞将自组装复杂的结构，如杆、环状和蜂窝。人们对支配这种定向细胞自组装过程的规则知之甚少，特别是对于复杂的分支结构，因此本提案的首要目标是定义和量化这些规则。我们的假设是，不同类型细胞的自组装规则不同，除了表面粘附力外，细胞骨架介导的张力/收缩有助于细胞特异性差异，并显著影响复杂微组织的定向自组装。为了验证这一假设并确定定向自组装的规则，我们建议使用五种细胞系及其混合物，即正常人成纤维细胞(NHF)、肝细胞系(H35)、平滑肌细胞(SMC)和两种内皮细胞系；人脐静脉内皮细胞(HUVEC)和小牛肺动脉内皮细胞(CPAE)来测试以下特定目标：目的1.确定定向自组装的细胞类型差异及其产生复杂形状的能力。目的2.量化细胞骨架介导的收缩和细胞表面黏附蛋白在定向自组装中的各自作用。 已修改

We've developed a new technology based on micro-molded nonadhesive agarose to guide the self assembly of 3D micro-tissues. Mono-disperse cells are pipetted into the micro-mold, they settle into the molded features, are unable to bind to the nonadhesive agarose and so spontaneously self assembly 3D micro-tissues. By making complex features in the micro-molds, we've shown for the first time that cells will self assembly complex structures such as rods, toroids and honeycombs. Little is understood about the rules that govern this directed cellular self-assembly process, especially for complex branching structures and so the overarching goal of this proposal is to define and quantify these rules. Our hypothesis is that the rules of selfassembly differ between specific cell types and that in addition to surface adhesive forces, cytoskeletal mediated tension/contraction contribute to cell specific differences and significantly influence the directed self assembly of complex micro-tissues. To test this hypothesis and define the rules of directed self assembly, we propose to use five cell lines and their mixtures, normal human fibroblasts (NHF), a hepatocyte cell line (H35), smooth muscle cells (SMC) and two endothelial cell lines; human umbilical cord vein endothelial cells (HUVEC) and calf pulmonary artery endothelial cells (CPAE) to test the following specific aims: Aim 1. To determine cell type differences in directed self assembly and their ability to generate complex shapes. Aim 2. To quantify the respective roles of cytoskeletal mediated contraction and cell surface adhesion proteins on directed self-assembly. Modified