Three-dimensional (3D) bioprinting has emerged as a promising approach for the development of functional tissues and organs, including the heart valves. In this study, we investigated the interaction of 3D printed collagen scaffolds with H9c2(2–1) and NIH/3T3 cells to improve heart-valve repair strategies. Type I collagen was extracted from rat tails, characterized using SDS-PAGE and Raman spectroscopy, and used as a biomaterial ink for 3D printing. The rheological properties were evaluated. The FRESH technique was used to support the printed construct. In vitro assessments were performed to determine the cell viability and distribution within the scaffold. These results demonstrated the successful extraction and characterization of Type I collagen, which exhibited suitable rheological properties for 3D bioprinting. The printed collagen scaffolds supported the growth and distribution of H9c2(2–1) and NIH/3T3 cells, indicating their potential application in heart valve repair. This study highlights the importance of collagen as a biomaterial in 3D bioprinting and provides insights into the development of advanced strategies for heart valve repair.