New Inks for 3D Bio-Printing based on Bio-orthogonal Click Chemistry

非技术摘要：三维（3D）打印允许用户从数字模型制作任何物理对象。这是通过一个接一个地铺设多层“墨水”来构建3D对象来实现的。开创性的工作已经证明，活细胞可以混合到一些墨水中打印组织，这被称为“生物打印”。生物打印有可能改变人们研究生物学和创造医学疗法的方式。然而，目前的生物打印油墨有两个关键缺陷。首先，它们使用可能破坏活细胞的化学反应。其次，目前的墨水无法定制以满足人体中发现的许多不同细胞类型的不同需求。因此，PI建议开发一种新的油墨系列，以克服这两个限制。在目标1中，PI将使用不损伤细胞的新型细胞相容性化学反应合成一系列墨水。Aim 2将定制墨水，以满足大脑中发现的两种不同细胞类型的独特要求：脑内皮细胞和成人神经干细胞。脑内皮细胞在大脑中形成血管，而成人神经干细胞可以在大脑受伤或疾病时产生新的神经细胞。在Aim 3中，PI将3D打印这两种细胞类型，每种细胞类型都有自己的定制墨水，并将其打印成单个组织。我们将使用这种打印的组织来研究这两种不同的细胞类型如何相互交流。展望未来，这些新开发的墨水可以定制用于体内任何类型的细胞，从而显着扩大3D生物打印的潜力。技术摘要：三维（3D）基于细胞的生物打印有望改变生物科学。然而，目前的生物油墨受到两个关键限制。首先，目前的生物墨水具有可能损害细胞功能的脱靶化学反应性。第二，目前的墨水对基质的最终机械、生物化学和降解性质没有独立的、可调的控制，所有这些都显著改变细胞表型。因此，为了实现功能性打印组织，迫切需要开发模块化生物墨水，其可以被定制以提供细胞类型特异性提示而不会不利地影响细胞功能。我们提出开发一种新的生物墨水家族，其（1）使用生物正交、点击化学进行两阶段的细胞相容性交联，以及（2）独立调整基质力学、细胞粘附和生物降解速率以进行细胞类型定制。作为概念证明，PI将使用脑内皮细胞（EC）和神经干细胞（NSC）评估对NSC小生境的体外生物打印模拟物的细胞反应。在目标1中，将建立一个工程生物墨水库，其机械和生物化学特性可独立调节。重要的是，这些生物墨水将通过点击反应交联，该反应不需要催化剂，具有快速动力学，并且与细胞上和培养基中的化学部分完全生物正交。在目标2中，PI将两种不同的生物降解机制设计到生物墨水中：通过水解的“全局”降解和通过细胞激活的蛋白水解裂解的“局部”降解，以定制用于维持EC和NSC的生物墨水。在目标3中，PI将评价促进EC-NSC细胞-细胞接触的定制生物油墨和印刷几何形状能够促进体外NSC静止，模拟体内天然NSC生态位的假设。展望未来，这些新开发的生物墨水可以定制用于与体内任何细胞类型的相互作用，从而显着扩大3D生物打印的应用范围。该奖项反映了NSF的法定使命，并通过使用该基金会的评估被认为值得支持智力优点和更广泛的影响审查标准。

Non-technical abstract: Three-dimensional (3D) printing allows the user to make any physical object from a digital model. This is achieved by laying down multiple layers of "ink", one after the other, to build up a 3D object. Pioneering work has demonstrated that living cells can be mixed into some inks to print tissue, which is called "bio-printing". Bio-printing has the potential to transform the way one studies biology and create medical therapies. However, current bio-printing inks have two critical flaws. First, they use chemical reactions that can damage living cells. Second, current inks cannot be customized to meet the differing needs of the many different cell types found in the human body. Therefore, the PI proposes the development of a new family of inks that overcome these two limitations. In Aim 1,the PI will synthesize a family of inks using novel, cell-compatible chemical reactions that do not damage cells. Aim 2 will customize the inks to meet the unique requirements of two different cell types found in the brain: brain endothelial cells and adult neural stem cells. Brain endothelial cells form blood vessels in the brain, while adult neural stem cells can produce new nerve cells if the brain has an injury or disease. In Aim 3, the PI will 3D print these two cell types, each one with their own customized ink, into a single tissue. We will use this printed tissue to study how the two different cell types communicate with each other. Looking forward, these newly developed inks can be customized for use with any cell type in the body, thereby significantly expanding the potential of 3D bio-printing.Technical abstract:Three-dimensional (3D) cell-based bio-printing is poised to transform biological science. However, current bio-inks suffer from two critical limitations. First, current bio-inks have off-target chemical reactivity that can damage cellular function. Second, current inks do not have independent, tunable control over the final mechanical, biochemical, and degradation properties of the matrix, all of which significantly alter cell phenotype. Thus, to achieve functional printed tissue, there is a critical need to develop modular bio-inks that can be customized to provide cell-type specific cues without adversely impacting cellular function. We propose the development of a new family of bio-inks that (1) undergo two-stage, cytocompatible crosslinking using bio-orthogonal, click chemistry and (2) independent tuning of matrix mechanics, cell-adhesion, and biodegradation rates for cell-type customization. As proof of concept, the PI will use brain endothelial cells (ECs) and neural stem cells (NSCs) to assess cellular responses to an in vitro, bio-printed mimic of the NSC niche. In Aim 1, a library of engineered bio-inks will be built with mechanical and biochemical properties that are independently tunable. Importantly, these bio-inks will be crosslinked via a click reaction that requires no catalyst, has rapid kinetics, and is fully bio-orthogonal to chemical moieties on cells and in culture medium. In Aim 2, the PI designs two different biodegradation mechanisms into the bio-inks: "global" degradation via hydrolysis and "local" degradation via cell-enabled proteolytic cleavage, to customize the bio-inks for maintenance of ECs and NSCs. In Aim 3, the PI will evaluate the hypothesis that customized bio-inks and print geometries that promote EC-NSC cell-cell contact will be capable of promoting NSC quiescence in vitro, mimicking the native in vivo NSC niche. Looking forward, these newly developed bio-inks can be customized for interactions with any cell type in the body, thereby significantly expanding the scope of applications for 3D bio-printing.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.