Establishment and validation of cell pools using primary muscle cells derived from satellite cells of pig skeletal muscle

Abstract

Primary cell cultures derived from satellite cells of skeletal muscle provide an appropriate in vitro model for proliferating myoblasts and differentiating myotubes for muscle biological research. These cell cultures may consist of harvested cells per animal or of a cell pool made of cells from several animals. However, cell pooling reduces the biological variability of the different cell donors. On the other hand, the use of cell pools offers an opportunity to use less donor tissue and to perform longterm projects with a broad spectrum of analysis and replications. In the literature, information about the donors of cell pools, the procedure used for pooling, and the characterization/validation of cell pools is often lacking. In this study, we established three cell pools consisting of M. rhomboideus or M. longissimus from ten or six piglets, each with one gender and medium birth weight. Real-time impedimetric monitoring was used to evaluate the proliferative growth behavior of myoblasts for the cell pools in comparison to their corresponding unpooled cells over a period of 72 h, with a measurement being taken every 30 min. For each of the tested cell pools, cell index, slope, and doubling time did not differ between the cell pool and the unpooled cells of the donor animals. Differentiation capacity and mRNA expression of PAX7, MYOD and MYOG remained unchanged between the cell pool and the unpooled cells. Current results support that the use of cell pools is an appropriate method to reflect the average proliferative growth behavior of unpooled cells.

Characterisation of intracellular molecular mechanisms modulated by carnosine in porcine myoblasts under basal and oxidative stress conditions

“…Satellite cells were isolated from the skeletal muscles of 3 to 4-day old crossbred male piglets (n = 34) using a Percoll gradient method as previously described in Metzger et al [ 18 ]. This method was chosen because it provides highly pure satellite cells in rats (> 90%) [ 19 ] and pigs (>95%) [ 18 ].…”

“…Satellite cells were isolated from the skeletal muscles of 3 to 4-day old crossbred male piglets (n = 34) using a Percoll gradient method as previously described in Metzger et al [ 18 ]. This method was chosen because it provides highly pure satellite cells in rats (> 90%) [ 19 ] and pigs (>95%) [ 18 ]. Moreover, satellite cells isolated with this method conserve their myogenic phenotype, with expression of paired Box 7 ( PAX7 ) in satellite cells and expression of myogenic differentiation 1 ( MYOD ) and myogenin ( MYOG ) genes after 72 hours of proliferation [ 18 ].…”

“…This method was chosen because it provides highly pure satellite cells in rats (> 90%) [ 19 ] and pigs (>95%) [ 18 ]. Moreover, satellite cells isolated with this method conserve their myogenic phenotype, with expression of paired Box 7 ( PAX7 ) in satellite cells and expression of myogenic differentiation 1 ( MYOD ) and myogenin ( MYOG ) genes after 72 hours of proliferation [ 18 ]. In addition, these cells are also able to differentiate with relatively high fusion index.…”

“…It also offers the possibility to conduct long-term projects with multiple experiments and replications, which was the case for the current study. In a recent paper by Metzger et al [ 18 ] it was clearly demonstrated that cell pools can be used as a relevant method to reflect the cellular behavior of unpooled cells. The percentage of myogenic cells was determined as described in Mau et al [ 20 ] using a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA).…”

Targeted over-expression of PPARγ in pig skeletal muscle improves oxidative fiber formation and intramuscular fat deposition

“…Primary myoblasts were isolated from the hind limb skeletal muscles of five-day piglets. Primary myoblasts were isolated and cultured according to the previously published method (Metzger et al, 2020). Histology staining.…”