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

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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.