Tissue engineering has great potential to provide a functional de novo living 
valve replacement, capable of integration with host tissue and growth. Among 
various valve conduit fabrication techniques, three-dimensional (3-D) 
bioprinting enables deposition of cells and hydrogels into 3-D constructs with 
anatomical geometry and heterogeneous mechanical properties. Successful 
translation of this approach, however, is constrained by the dearth of printable 
and biocompatible hydrogel materials. Furthermore, it is not known how human 
valve cells respond to these printed environments. In this study, 3-D printable 
formulations of hybrid hydrogels are developed, based on methacrylated 
hyaluronic acid (Me-HA) and methacrylated gelatin (Me-Gel), and used to bioprint 
heart valve conduits containing encapsulated human aortic valvular interstitial 
cells (HAVIC). Increasing Me-Gel concentration resulted in lower stiffness and 
higher viscosity, facilitated cell spreading, and better maintained HAVIC 
fibroblastic phenotype. Bioprinting accuracy was dependent upon the relative 
concentrations of Me-Gel and Me-HA, but when optimized enabled the fabrication 
of a trileaflet valve shape accurate to the original design. HAVIC encapsulated 
within bioprinted heart valves maintained high viability, and remodeled the 
initial matrix by depositing collagen and glyosaminoglycans. These findings 
represent the first rational design of bioprinted trileaflet valve hydrogels 
that regulate encapsulated human VIC behavior. The use of anatomically accurate 
living valve scaffolds through bioprinting may accelerate understanding of 
physiological valve cell interactions and progress towards de novo living valve 
replacements.