Spontaneously immortalized porcine cells support scalable cultured pork production in serum-free suspension

Abstract Cell lines suitable for serum-free suspension culture remain limited for cultured meat production. Here, we established a spontaneously immortalized porcine muscle-derived cell line by adapting a satellite-cell-enriched primary culture to serum-free suspension without genetic modification. Cells completed more than 255 population doublings by day 300. Longitudinal single-cell transcriptomics revealed shifts from myogenic to mesenchymal stromal-like and then adipogenic-like states during adaptation. The cells showed little basal lipid accumulation but inducible adipogenesis, with no detectable myogenic differentiation. They retained DNA damage recovery capacity and exhibited no detectable tumorigenicity. Compared to fed-batch or cell-retention perfusion culture, semi-continuous culture improved medium-use efficiency, requiring less dry medium per unit of wet biomass. Scale-up in a 2,000-litre bioreactor yielded 97.18 kg wet biomass with a medium conversion ratio of 0.63:1 (dry medium/wet biomass). These results provide a porcine cell source and a semi-continuous process for scalable production of cultured pork biomass.