An Efficient Method for Enrichment and In Vitro Propagation of Muscle Stem Cells Derived from Black Sea Bream (Acanthopagrus schlegelii) Skeletal Muscle

Abstract

Muscle stem cells (MSCs) play a crucial role in muscle growth, repair, and regeneration, offering potential applications in cell-mediated therapy, tissue engineering, and alternative food production. Despite significant advancements in isolating and enriching MSCs from mammalian tissues, research on fish MSCs remains limited. This study aimed to establish an optimized protocol for isolating, enriching, and propagating black sea bream (Acanthopagrus schlegelii) MSCs for potential biotechnological applications. Skeletal muscle tissues were enzymatically dissociated using various enzymes, with collagenase type II and pronase identified as the most effective combination for cell isolation and tissue debris removal. Differential plating (DP) on collagen type I effectively enriched MSCs, as evidenced by a significant increase in Pax7 expression in non-adhesive cells. Among several adhesion substrates tested, Matrigel-coated dishes best supported the maintenance and differentiation potential of enriched MSCs, enabling robust myotube formation. To mitigate the high cost of Matrigel, cells were transitioned to laminin- or gelatin-coated dishes after the early passages. Notably, Matrigel-conditioned cells maintained their survival and differentiation capacities on these more cost-effective substrates. After long-term culture on gelatin-coated dishes, the cell lines were stably maintained for more than 25 passages, and their myogenic differentiation potentials were well preserved, with variations observed between the cell lines. These findings provide a foundational framework for the efficient isolation, enrichment, and culture of fish MSCs, contributing to the development of scalable and cost-effective protocols for their application in muscle biology and biotechnology.