3D bioprinting enables the fabrication of engineered tissues, but cell damage during printing and limitations in long-term preservation hinder practical applications. Traditional cryoprotectants, such as DMSO, introduce cytotoxicity and require complex removal, restricting immediate tissue usability. Here, we present an integrated extrusion-based bioprinting and DMSO-free antifreeze hydrogel strategy to produce cell-laden constructs with high post-thaw viability and proliferative capacity. Systematic optimization of bioink composition (6% L-proline with varying GelMA), extrusion parameters, and crosslinking conditions enabled high-fidelity scaffold fabrication while preserving cell viability and proliferation. Numerical simulations guided the maximum printable heights for fibers of different diameters, supporting construct scalability. Storing cell-laden 3D-printed scaffolds in cryovials at −80 °C effectively maintained high cell viability. Cells in 3D scaffolds exhibited superior post-thaw proliferation compared with 2D culture, and the platform was validated with C2C12 myoblasts, achieving high survival and robust recovery of proliferative capacity. This study establishes a practical and versatile framework for integrating bioprinting and cryopreservation to support the generation of cell-laden constructs with preserved viability and structural integrity for regenerative medicine applications.