摘要Background As the global population increases,the demand for protein sources is expected to increase,driv-ing the demand for cell-based cultivated meat.This study aimed to enhance the productivity of cultivated meat through optimization of the cell source and organization process.Results We engineered fibroblasts into myogenic cells via non-viral introduction of the MYOD1 gene,avoiding viral methods for safety.After confirming the stable derivation of myogenic cells,we combined knockout(KO)of MSTN,a negative regulator of myogenesis,with MYOD1-mediated myogenesis to improve cultivated meat production.Primary cells from MSTN KO cattle exhibited enhanced myogenic potential.Additionally,when tested in immortal-ized fibroblasts,myostatin treatment reduced MYOD1-induced myogenesis in two-dimensional cultures,while MSTN knockout increased it.To achieve muscle-like cell alignment,we employed digital light processing(DLP)-based three-dimensional(3D)bioprinting to organize cells into 3D groove-shaped hydrogels.These bioactive hydrogels supported stable cell proliferation and significantly improved muscle cell alignment.Upon differentiation into myo-tubes,the cells demonstrated an ordered alignment,particularly the MSTN KO cells,which showed highly efficient differentiation.Conclusions The integration of genetic modification and advanced DLP 3D bioprinting with groove-patterned hydrogels provides an effective strategy for producing high-quality,muscle-aligned cultivated meat.