Employing PEG crosslinkers to optimize cell viability in gel phase bioinks and tailor post printing mechanical properties

The field of 3D bioprinting has rapidly grown, yet the fundamental ability to manipulate material properties has been challenging with current bioink methods. Here, we change bioink properties using our PEG cross-linking (PEGX) bioink method with the objective of optimizing cell viability while retaining control of mechanical properties of the final bioprinted construct. First, we investigate cytocompatible, covalent cross-linking chemistries for bioink synthesis (e.g. Thiol Michael type addition and bioorthogonal inverse electron demand Diels-Alder reaction). We demonstrate these reactions are compatible with the bioink method, which results in high cell viability. The PEGX method is then exploited to optimize extruded cell viability by manipulating bioink gel robustness, characterized by mass flow rate. Below a critical point, cell viability linearly decreases with decreasing flow rates, but above this point, high viability is achieved. This work underscores the importance of building a foundational understanding of the relationships between extrudable bioink properties and cell health post-printing to more efficiently tune material properties for a variety of tissue and organ engineering applications. Finally, we also develop a post-printing, cell-friendly cross-linking strategy utilizing the same reactions used for synthesis. This secondary cross-linking leads to a range of mechanical properties relevant to soft tissue engineering as well as highly viable cell-laden gels stable for over one month in culture.