Balancing myogenic purity, expansion stability, and apoptotic resilience in Hanwoo muscle-derived cell sources for cultivated meat biomanufacturing.

The scalability of cultivated meat production depends on the establishment of bovine satellite cell (SC) populations that combine high myogenic purity with stable long-term expansion. This study compared three post-isolation fractionation strategies for expanded Hanwoo muscle-derived cells-fluorescence-activated cell sorting (FACS), pre-plating, and Percoll density gradient centrifugation-with respect to initial myogenic enrichment, proliferative dynamics, apoptotic susceptibility, and passage-dependent compositional stability. FACS provided the highest initial enrichment of PAX7-positive SCs, whereas pre-plating and Percoll F2 achieved moderate myogenic enrichment through differential adhesion and density-based separation, respectively. During serial passaging, the F2 fraction exhibited relatively greater cell expansion and lower mid-passage apoptosis, while the FACS group showed relatively higher PAX7 and MYOD1 expression at selected later passages. However, late-passage analyses suggested greater apoptotic susceptibility in myogenically enriched populations, whereas γH2AX-associated DNA damage signals were not a major determinant of this response. Flow cytometric phenotypic analysis further demonstrated a consistent decline in SC proportion from passage 3 to passage 9 across all fractionation methods, accompanied by expansion or persistence of smooth muscle/mesenchymal-like cells (SMMCs), while fibro-adipogenic progenitor-like populations showed no consistent directional trend. These results indicate that initial enrichment efficiency alone does not predict long-term myogenic utility, because prolonged culture progressively reshapes cellular composition and favors culture-adapted non-satellite populations. Collectively, the findings reveal a trade-off between initial myogenic purity and long-term culture stability, and suggest that fractionation strategy should be evaluated not only by early SC enrichment but also by maintenance of lineage composition, apoptotic resilience, and expansion behavior during industrially relevant cultivation.