Heart valve disease is a serious and growing public health problem for which 
prosthetic replacement is most commonly indicated. Current prosthetic devices 
are inadequate for younger adults and growing children. Tissue engineered living 
aortic valve conduits have potential for remodeling, regeneration, and growth, 
but fabricating natural anatomical complexity with cellular heterogeneity remain 
challenging. In the current study, we implement 3D bioprinting to fabricate 
living alginate/gelatin hydrogel valve conduits with anatomical architecture and 
direct incorporation of dual cell types in a regionally constrained manner. 
Encapsulated aortic root sinus smooth muscle cells (SMC) and aortic valve 
leaflet interstitial cells (VIC) were viable within alginate/gelatin hydrogel 
discs over 7 days in culture. Acellular 3D printed hydrogels exhibited reduced 
modulus, ultimate strength, and peak strain reducing slightly over 7-day 
culture, while the tensile biomechanics of cell-laden hydrogels were maintained. 
Aortic valve conduits were successfully bioprinted with direct encapsulation of 
SMC in the valve root and VIC in the leaflets. Both cell types were viable (81.4 
± 3.4% for SMC and 83.2 ± 4.0% for VIC) within 3D printed tissues. Encapsulated 
SMC expressed elevated alpha-smooth muscle actin, while VIC expressed elevated 
vimentin. These results demonstrate that anatomically complex, heterogeneously 
encapsulated aortic valve hydrogel conduits can be fabricated with 3D 
bioprinting.