Development of High Cell Density Bioinks for FRESH 3D Bioprinting of Muscle Constructs

The need for heart muscle suitable for transplant has never been greater as the disparity between the supply and need of tissue for transplant broadens every year. Bioprinted muscle tissues are typically fabricated from bioinks composed of cells suspended in a hydrogel which entraps the cells in the printed filaments after deposition. While specific requirements for the bioink materials properties vary based on bioprinting method being employed, cellular bioinks for extrusion based bioprinting generally exhibit shear thinning rheological properties, are viscous enough to prevent cell sedimentation, and do not adversely affect cell viability prior to deposition. However, utilizing embedded extrusion bioprinting processes such as Freeform Reversible Embedding of Suspended Hydrogels (FRESH) rather than direct ink write bioprinting redefines the bioink formulation requirements. FRESH 3D bioprinting provides physical support to extruded filaments and enables the use of hydrogel precursor bioinks with low yield stress, various modes of gelation, and low viscosity to build high resolution (<200 µm feature) to build soft material scaffolds. In this work, I developed a cellular bioink with cell densities more than 300x10⁶ cells/mL to fabricate skeletal and cardiac muscle tissues in vitro utilizing FRESH bioprinting, which better recapitulates the cell density and properties of muscle tissue in vivo. To achieve this goal, I (1) developed a method for FRESH bioprinting acellular fibrin scaffolds, (2) developed a high cell density fibrinogen bioink for FRESH 3D bioprinting, and (3) investigated the how printing cardiac aggregate vs single cell bioinks affects tissue remodeling and function. To develop a method to print fibrinogen, I first characterized the gel strength of acellular bioinks known to be compatible with FRESH bioprinting then evaluated the effect of fibrinogen concentration on gel strength to select a bioink concentration with sufficient gel strength for FRESH bioprinting. I then investigated how print pathing, bath thrombin concentration, and bath fluid phase density affect fibrin microstructure, filament resolution, and shape fidelity of printed scaffolds. I then applied these findings to develop a cell laden fibrinogen bioink. I demonstrated that increasing bioink cell concentration had minimal impact on the bioinks terminal gel storage modulus. When I observed that cells interacted with the fibrinogen to form undesirable separation of the bioink, I developed a treatment which maintains bioink homogeneity and printability for up to 3 hours after preparation. The developed treatment has been successfully applied to at least 8 cell lines including: C2C12 murine myoblasts, 3T3 murine fibroblasts, primary embryonic chick muscle, normal human cardiac fibroblasts, human umbilical vein endothelial cells, mesenchymal stem cells, various human induced pluripotent stem cell (hiPSC) lines, and MIN6. I used FRESH 3D bioprinting to fabricate muscle strip models from C2C12 and cardiac bioinks to create engineered muscle strips with aligned infill. Skeletal muscle strips with low infill density and perpendicular infill layers showed that myotubes formed in the direction of the infill. Printed cardiac muscle tissues began beating spontaneously after several days in culture, but muscle alignment created during strip fabrication deteriorated over time as cell mediated remodeling occurred. Finally, I explored how the bioink formulation could be applied to printing of tissues from cell aggregates. I began by establishing a methodology for forming pluripotent stem cell aggregates (embryoid bodies), prepared them into a bioink and differentiated the tissues after FRESH 3D bioprinting. The in-situ differentiation of printed constructs was inconsistent batch to batch and within individual tissues. Considering this finding, we transitioned to comparing how fabricating cardiac tissues from aggregate and single cell bioinks alters cell-mediated remodeling of the printed tissue at the macro and macroscale in addition to its effect on tissue electrophysiology. Printing tissues from self-assembled cardiac aggregate microtissues composed purified cardiomyocytes and cardiac fibroblasts connected and synchronously beat after 14 days in culture similarly to tissues printed from single cell bioinks. This preliminary result opens the door to using aggregates produced in automated bioreactors, that is a commercially viable pathway to scale up tissues to the size of human organs. 

In total, the work established a pathway to creating tissues from a single material bioink that breaks down over time to create tissues composed of the cell type of interest and the extracellular matrix produced by those cells. This is an important property for tissues manufactured in vitro to translate to clinical adoption as the immune response can be reduced or eliminated by producing the scaffold entirely from the patient’s own cells.