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。这是通过一层接一层地铺设多层“墨水”来构建3D对象来实现的。开创性的工作证明，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”。生物印刷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，它们使用的化学反应会损害活细胞。其次，目前的墨水不能被定制以满足人体内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家族来克服这两个限制。在目标1中，PI将使用不损害细胞的新颖的、细胞兼容的化学反应来合成一系列墨水。Aim 2将定制墨水，以满足大脑中两种不同细胞类型的独特需求：脑内皮细胞和成人神经干细胞。大脑内皮细胞在大脑中形成血管，而成年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将这两种细胞类型进行3D打印，每种细胞都有自己定制的墨水，打印到单个组织中。我们将使用打印的组织来研究这两种不同类型的细胞是如何相互沟通的。展望未来，这些新开发的墨水可以定制用于人体内的任何细胞类型，从而显著扩展3D生物打印的潜力。技术摘要：基于三维(3D)细胞的生物打印有望改变生物科学。然而，目前的生物墨水受到两个关键限制。首先，目前的生物墨水具有偏离目标的化学反应能力，可能会损害细胞功能。其次，目前的油墨不能独立、可调节地控制基质的最终机械、生化和降解特性，所有这些都会显著改变细胞的表型。因此，为了实现打印组织的功能，迫切需要开发模块化生物墨水，该墨水可以被定制以提供细胞类型的特定提示而不会对细胞功能产生不利影响。我们建议开发一种新的生物墨水家族，这种墨水(1)使用生物正交、点击化学进行两步细胞相容的交联，(2)独立调整基质力学、细胞粘附性和生物降解率，以实现细胞类型的定制。作为概念的证明，PI将使用脑内皮细胞(ECs)和神经干细胞(NSCs)来评估细胞对体外生物打印模拟NSC利基的反应。在目标1中，将建立一个具有可独立调节的机械和生化特性的工程生物墨水库。重要的是，这些生物墨水将通过点击反应进行交联，这种反应不需要催化剂，具有快速的动力学，并且与细胞和培养液中的化学成分完全生物正交。在目标2中，PI在生物墨水中设计了两种不同的生物降解机制：通过水解性“全局”降解和通过细胞激活的蛋白质降解裂解“局部”降解，以定制用于维持ECs和NSCs的生物墨水。在目标3中，PI将评估这样一种假设，即促进EC-NSC细胞接触的定制生物墨水和打印几何形状将能够促进NSC在体外的静止，模仿体内天然的NSC生态位。展望未来，这些新开发的生物墨水可以为人体内任何类型的细胞相互作用而定制，从而显著扩大3D生物打印的应用范围。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.