A reliable supply of muscle cells without the need to slaughter additional cattle is needed for the commercialization of cultured beef. For this purpose, active MSCs (which have self-renewal characteristics) can be used, but their yield from skeletal muscle tissue needs to be improved. Here, we report that the yield was maximized by incubating rump tissue for 30 minutes in 0.2% (w/v) collagenase type II in HG-DMEM followed by 1% (w/v) pronase in HG-DMEM for 5 minutes. Finally, the dissociated tissue was subjected to DP in HG-DMEM supplemented with 10% (v/v) FBS and 5 ng/mL bFGF.
Skeletal muscle tissue is composed of organized, bundled muscle fibers enclosed within an extracellular matrix formed by the interactions of collagen types I, III, and IV [17]. Most prior studies involving pigs have used a single dissociation enzyme, such as collagenase type I, III, or IV [18], or have conducted treatments with other enzymes, such as trypsin or dispase [16, 18]. By contrast, we used collagenase type II followed by pronase to dissociate bovine skeletal muscle tissue. Collagenase type II targets a peptide sequence in collagen bounded by serine and arginine residues [19, 20]. Despite its absence in the extracellular matrix of skeletal muscle tissue, collagenase type II breaks down collagen types I, III, and IV, leading to disruption of tissue. Subsequently, pronase promotes the dissociation of tissue into single cells. Pronase cleaves proteins at random sites, enabling the complete dissociation of small fragments into single cells. In this study, the sequential application of collagenase type II and pronase under the optimal conditions maximized the yield of active MSCs; the yield was higher than would be the case using conventional methods [20].
Muscle tissue includes a variety of cell types, including myocytes, adipocytes, and hematocytes. Active MSCs have been isolated from primary cells by fluorescence-activated cell sorting (FACS). FACS has the advantage of yielding active MSCs with 100% purity; however, it is prohibitively expensive. An alternative method for cell separation is DP, which relies on cell attachment. Cells attach to a surface at different times, enabling the isolation of cell populations of interest. Although it is more cost-effective and straightforward than FACS, DP yields cell populations of low purity. Research aiming to overcome this drawback is ongoing.
Active MSCs have been isolated at high yield from mouse skeletal muscle tissue by DP in Ham’s F-10 nutrient mixture supplemented with 15% (v/v) FBS and 2.5 ng/mL bFGF [20]. However, the optimal DP solution, when applied to beef, results in a low yield of MSCs (data not shown). In this study, HG-DMEM supplemented with 10% (v/v) FBS and 5 ng/mL bFGF was the optimal DP solution. The use of HG-DMEM resulted in the largest proportion of active MSCs adherent to the bottom of culture dishes (Supplementary Fig. 1). This was likely because the amino acid and glucose concentrations in HG-DMEM are three- and fourfold, respectively, those in Ham’s F-10 nutrient mixture and in LG-DMEM. Indeed, an increased amino acid or glucose concentration stimulates the production of extracellular matrix components [21–23].
Skeletal muscle tissues that experience a high level of physical activity are subjected to more damage than those that experience a low level of physical activity, resulting in an increased number of active MSCs [24]. The yield of active MSCs differed depending on the source of the muscle tissue (Fig. 4), and rump tissue was the best source. It is likely that muscles in the rump experience more physical activity than those in chuck and brisket, leading to an increased number of active MSCs. Further research on the isolation of active MSCs from muscle tissues that experience higher levels of physical activity than rump is needed.
In conclusion, we developed a high-yield protocol for isolating active MSCs from bovine skeletal muscle tissue by determining the optimal enzymatic treatment conditions, optimal medium for DP, and the optimal muscle type as a source of active MSCs. This simple and convenient technique will overcome the major technical obstacles to the mass production of bovine muscle cells, potentially enabling the commercialization of cultured beef.