Efficient expansion culture of bovine myogenic cells with differentiation capacity using muscle extract-supplemented medium

Abstract

The production of tissue-engineered skeletal muscles for cell-based therapy and cultivated meat production requires a large number of myogenic cells capable of differentiating into myotubes. A suspension culture with a stirred bioreactor is a system that is feasibly scalable, but scaling up the production of myogenic cells while maintaining differentiation capacity requires an efficient culture technique. This remains challenging in suspension culture. On the other hand, conventional meat processing generates substantial by-products, including meat trimmings containing effective substances for myogenic cell cultures. Efficiently utilizing these by-products can reduce environmental impact and disposal costs. Here, we describe an expansion culture method for bovine myogenic cells (BMCs) using Dulbecco's Modified Eagle's medium (DMEM) supplemented with muscle extracts. During the 6-day planar culture, the proliferation rate using the culture medium supplemented with muscle extracts (CMME) and 10% fetal bovine serum (FBS) was 18 ± 3.3, three times higher than that achieved using DMEM with 10% FBS. Furthermore, a combination of CMME supplemented with 10% FBS and an iMatrix-511-coated dish could maintain differentiation capacity through passaging culture. Finally, we demonstrated a suspension culture with microcarriers using CMME with 10% FBS. Through 3 passages, the cumulative proliferation rate was 191.7 ± 35.1, four times higher than that of produced DMEM with 10% FBS. Using CMME, the cells maintained differentiation capacity in comparison to that using DMEM. This approach will contribute to both the reduction of wasted by-products from conventional meat production and the development of a suspension culture system for maintaining high-quality myogenic cells.