Engineering 3D in vitro niches to reveal fundamentals of cellular biomechanics

描述(由申请人提供) 摘要：罗斯·格兰维尔·哈里森于1907年开发的组织培养技术被誉为医学十大发现之一，并使生物学认识取得了不朽的进步。尽管体外培养在现代生物医学中的重要性经久不衰，但自1940年S以来，哺乳动物细胞培养技术在很大程度上保持不变：细胞培养在坚硬、平坦的底物上，周围环绕着均匀的培养液，几乎不会重建体内发现的精致微环境。众所周知，细胞对其体内生态位中发现的多种信号做出反应，例如，可溶和系留生物化学物质的浓度梯度、基质刚性、基质配体的模式以及与其他类型细胞的相互作用；然而，在体外细胞培养研究中，很少有方法概括这些信号。为了解决这些限制，我建议创建多功能的、三维的体外壁龛，具有细胞线索的精确空间和时间分辨率。这些三维微环境将使用创新和跨学科的方法来构建，这些方法将蛋白质工程、生物材料和微流体技术的进步与传统的细胞生物学方案相结合。作为一个模型系统，这些体外生态位将被用来定量研究调节神经前体细胞(NPC)迁移的细胞生物力学和信号机制。假设鼻咽癌在可溶性因子的梯度内的趋化作用是与背景相关的，并依赖于来自3D基质的额外生物力学线索。如果神经前体细胞被诱导迁移到需要的地方，那么在大脑特定的壁龛中的神经前体的存在开启了一种诱人的可能性，即如果神经前体细胞被诱导到需要的地方，成年中枢神经系统可能能够在受伤或疾病后再生。体内生态位的定量体外模拟的发展将对生物医学研究产生深远影响，因为它使科学家能够测试关于不同细胞及其三维微环境之间相互作用的全新假说。 与公共健康相关：尽管组织培养技术在现代生物医学中的重要性经久不衰，但哺乳动物细胞培养技术自1940年代的S以来基本保持不变：细胞培养在坚硬、平坦的底物上，周围环绕着几乎不能重建体内精致微环境(称为微环境)的培养液。为了解决这些局限性，我建议创建具有细胞线索精确空间和时间分辨率的三维壁龛的多功能模拟。作为一个模型系统，这些工程化的生态位将被用来定量研究调节神经前体细胞(NPC)迁移的细胞生物力学和信号机制，为未来的中枢神经系统再生治疗打开大门。

DESCRIPTION (Provided by the applicant) Abstract: The development of tissue culture techniques by Ross Granville Harrison in 1907 has been cited as one of the ten greatest discoveries in medicine and enabled monumental advances in biological understanding. Despite the enduring importance of in vitro culture in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940's: cells are cultured on hard, flat substrates and surrounded by homogeneous solutions of medium that do little to recreate the exquisite microenvironments found in vivo. Cells are well known to respond to multiple cues found within their in vivo niches, e.g., concentration gradients of soluble and tethered biochemicals, matrix rigidity, patterns of matrix ligands, and interactions with other cell types; however, few methods exist to recapitulate these cues in in vitro cell culture studies. To address these limitations, I propose creating versatile, three-dimensional in vitro niches with precise spatial and temporal resolution of cellular cues. These three-dimensional microenvironments will be fabricated using innovative and transdisciplinary approaches that combine advances in protein engineering, biomaterials, and microfluidics with traditional cell biology protocols. As a model system, these in vitro niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration. NPC chemotaxis within gradients of soluble factors is hypothesized to be contextual and reliant on additional biomechanical cues from the 3D matrix. The presence of NPCs within specific niches of the brain opens up the tantalizing possibility that the adult central nervous system may be able to regenerate following injury or disease if NPCs were induced to migrate to sites of need. The development of quantitative, in vitro mimics of in vivo niches will have a profound impact on biomedical research by enabling scientists to test entirely new hypotheses about the interactions between different cells and their three-dimensional microenvironments. Public Health Relevance: Despite the enduring importance of tissue culture techniques in modern biomedicine, the technology of mammalian cell culture has remained largely unchanged since the 1940's: cells are cultured on hard, flat substrates and surrounded by solutions of medium that do little to recreate the exquisite microenvironments (called niches) found inside the body. To address these limitations, I propose creating versatile mimics of three-dimensional niches with precise spatial and temporal resolution of cellular cues. As a model system, these engineered niches will be used to quantitatively study the cellular biomechanics and signaling mechanisms regulating neural progenitor cell (NPC) migration, opening the door to future therapies for regeneration of the central nervous system.