Isolating and maintaining the appropriate stem cell for large scale cell culture is essential in tissue engineering or food production. For bovine satellite cells an optimized isolation and purification protocol is lacking and there is also no detailed understanding on the factors that maintain stemness of these cells. Here, we set up a fluorescence-activated cell sorting strategy to enrich bovine satellite cells. We found that p38-MAPK signalling is activated and PAX7 expression is gradually lost during satellite cell proliferation. The p38 inhibitor (SB203580) treatment maintained PAX7 expression but inhibited the fusion of satellite cells in a concentration-dependent way in short-term incubation. The mechanism of p38 inhibition was confirmed by inhibiting canonical p38 signalling, i.e. HSP27. Long-term culture with an appropriate concentration of p38i enhanced the proliferation and PAX7 expression, while the differentiation capacity recovered and was enhanced compared to vehicle control. These studies indicate that bovine satellite cells maintenance depends on cell purity and p38 MAPK signalling. Inhibition of p38 MAPK signaling is a promising strategy to facilitate large scale cell expansion of primary cells for tissue engineering and cultured meat purposes.

Cultured meat is meat for consumption produced in a more sustainable way. It involves cell harvesting and expansion, differentiation into myotubes, construction into muscle fibres and meat structuring. We isolated 5.3 × 10⁴ porcine muscle stem cells from 1 g of neonatal pig muscle tissue. According to calculations, we need to expand muscle stem cells 10⁶-10⁷ times to produce 100 g or 1 kg of cultured meat. However, the cells gradually lost the ability to express stemness and mature muscle cell markers (PAX7, MyHC). To tackle this critical issue and maintain cell function during cell expansion, we found that long-term culture with (100 μM) l-Ascorbic acid 2-phosphate (Asc-2P) accelerated cell proliferation while preserving the muscle cell differentiation. We further optimized a scalable PDMS mold. Porcine muscle stem cells formed structurally-organized myotubes similar to muscle fibres in the mold. Asc-2P enhanced porcine muscle cells grown as 3D tissue networks that can produce a relatively large 3D tissue networks as cultured meat building blocks, which showed improved texture and amino acid content. These results established a realistic workflow for the production of cultured meat that mimics the pork meat structurally and is potentially scalable for industry.