This study explores the use of 3D bioprinting to fabricate living aortic valve conduits with heterogeneous cell types, specifically smooth muscle cells (SMC) and interstitial cells (VIC). The authors developed an alginate/gelatin hydrogel system to encapsulate these cells and print them into complex structures that mimic the natural anatomy of the aortic valve. Key findings include: 1. **Cell Viability and Differentiation**: Encapsulated VIC and SMC remained viable over 7 days in culture, with high viability rates (81.4±3.4% for SMC and 83.2±4.0% for VIC). VIC expressed both αSMA and vimentin, indicating a fibroblastic phenotype, while SMC expressed more αSMA, suggesting a myofibroblastic phenotype. 2. **Hydrogel Biomechanics**: Cell-free alginate/gelatin hydrogels exhibited reduced modulus, ultimate strength, and peak strain over 7 days, while cell-laden hydrogels maintained their mechanical properties, likely due to the secretion of extracellular matrix proteins by the encapsulated cells. 3. **3D Bioprinting Accuracy**: The bioprinting process was accurate, with a printing accuracy of 84.3±10.9% for a grid pattern design. The printed structures maintained their geometry and mechanical integrity after crosslinking. 4. **3D Valve Conduit Fabrication**: Aortic valve conduits were successfully bioprinted with SMC in the valve root and VIC in the leaflets, recapitulating key anatomical features such as sinuses and coronary ostia. 5. **Conclusion**: The study demonstrates the feasibility of using 3D bioprinting to fabricate heterogeneous aortic valve conduits with high cell viability and good mechanical properties, making it a promising strategy for tissue-engineered living valve replacements. This research addresses the challenges of fabricating anatomically complex and functionally heterogeneous heart valve replacements, offering a potential solution for younger adults and growing children who currently lack suitable prosthetic options.This study explores the use of 3D bioprinting to fabricate living aortic valve conduits with heterogeneous cell types, specifically smooth muscle cells (SMC) and interstitial cells (VIC). The authors developed an alginate/gelatin hydrogel system to encapsulate these cells and print them into complex structures that mimic the natural anatomy of the aortic valve. Key findings include: 1. **Cell Viability and Differentiation**: Encapsulated VIC and SMC remained viable over 7 days in culture, with high viability rates (81.4±3.4% for SMC and 83.2±4.0% for VIC). VIC expressed both αSMA and vimentin, indicating a fibroblastic phenotype, while SMC expressed more αSMA, suggesting a myofibroblastic phenotype. 2. **Hydrogel Biomechanics**: Cell-free alginate/gelatin hydrogels exhibited reduced modulus, ultimate strength, and peak strain over 7 days, while cell-laden hydrogels maintained their mechanical properties, likely due to the secretion of extracellular matrix proteins by the encapsulated cells. 3. **3D Bioprinting Accuracy**: The bioprinting process was accurate, with a printing accuracy of 84.3±10.9% for a grid pattern design. The printed structures maintained their geometry and mechanical integrity after crosslinking. 4. **3D Valve Conduit Fabrication**: Aortic valve conduits were successfully bioprinted with SMC in the valve root and VIC in the leaflets, recapitulating key anatomical features such as sinuses and coronary ostia. 5. **Conclusion**: The study demonstrates the feasibility of using 3D bioprinting to fabricate heterogeneous aortic valve conduits with high cell viability and good mechanical properties, making it a promising strategy for tissue-engineered living valve replacements. This research addresses the challenges of fabricating anatomically complex and functionally heterogeneous heart valve replacements, offering a potential solution for younger adults and growing children who currently lack suitable prosthetic options.