Engineering a genomically stable porcine myogenic cell line for serum-free cultured meat production

Genetically engineered cell lines hold promise for cultured meat production, yet are hindered by safety concerns, regulatory issues, and unproven serum-free adaptability. Here, we establish the porcine myogenic cell line PM166 via co-overexpression of telomerase reverse transcriptase (TERT) and cyclin-dependent kinase 4 (CDK4) to assess its proliferative and myogenic capacity, genomic stability and serum-free culture performance. The PM166 cell line achieved a doubling time of less than 24 h, sustained expansion beyond 200 population doublings, and robust myogenic differentiation after extensive passaging. Critically, PM166 maintained a normal diploid karyotype and genomic stability during prolonged culture, with whole-genome sequencing revealing a conserved mutational landscape and intact key cancer-associated loci. The absence of anchorage-independent growth further underscored its non-tumorigenic phenotype. Notably, PM166 was successfully adapted to serum-free conditions, maintaining a stable proliferation rate and myogenic identity. Moreover, following differentiation in 3D hydrogels, serum-free expanded PM166 cells formed myotubes, demonstrating the capacity to generate muscle tissues for cultured meat. This study presents PM166 as a genomically stable, serum-free compatible cell resource potentially for cultured meat production and safety assessment.