## 3 Results  
### Effect of Optimized Mechanical Fluid Shear Stress on the Cell Viability  
Skeletal muscle myoblasts exhibited a typical stem cell-like morphology for up to three days under static conditions. The cells had broad, spindle-shaped bodies with cytoplasmic processes (Figure 1a-c). In contrast, CFSS showed a small, elongated cell body with numerous long and short cytoplasmic processes (Figure 1d-f). Under PFSS, the cell body appeared wider without obvious cytoplasmic processes for up to 24 h, then marked increases in cell processes with a smaller body size were observed at day 3 compared to static culture (Figure 1g-i). To examine the effect of mechanical FSS on the physiological parameters of muscle cells, the MTT and SRB assays were carried out. The data analysis  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Gene** & **Forward** & **Reverse** & **Size (bp)** \\ \hline
18s & ATGCGCGCGCGTIATTCC & GCTCAATCTGCAATCCTGTCC & 204 bp \\ MyoD & GTGAAATGGCCTTGCAGAC & GAGCCTGCAGACCTTCGATG & 115 bp \\ Mtyogenin & GAAGAAAAGGACTGGGGAC & TCTTGAGCCTGCGCCTTCC & 136 bp \\ MHC1 & TCTTGACCCTGCGCCTTCTC & GCCACCAGACCTTGC & 141 bp \\ MHC7 & ACTATGCTGAGAGCTG & CTCTGTGAGAGCAGAC & 89 bp \\ mTOR & CGGCACACACATTGAAAAG & TCCATGCGTGATAGAGAC & 130 bp \\ VEGF & AGATCATGCGCGCATACAACC & TGACCCTTCTTCCTCTTCTC & 139 bp \\ HIF1a & GTGCTGATTTGAAACCCATTC & AAGTTCTTCCGGCCTCTAACC & 150 bp \\ NOS1 & TGGGGCGATCCATATGTG & GGATCTGAAAGTTGCGGTC & 131 bp \\ \hline \hline \end{tabular}
\end{table}
Table 1: Primer sequences used for RT-qPCR.  
revealed increased cell viability under both CFSS and PFSS (\(p<0.001\) and \(p<0.01\)) compared to static conditions after 3 h. A similar observation was found under CFSS and PFSS up to 24 h (\(p<0.01\) and \(p<0.001\)) compared to the matched static culture (Figure 1j). By analyzing the total protein contents for all experimental groups, an increased cell number was observed only under CFSS after 24 h compared to the matched static condition (Figure 1j). To assess the colony-forming potential of the myoblasts under both FSS and static cultures indicative of stemness capacity, the CFU assay was performed using three different cell concentrations: 1 \(\times\) 10\({}^{2}\), 5 \(\times\) 10\({}^{2}\), and 1 \(\times\) 10\({}^{3}\) cells/25 cm\({}^{2}\). The data analysis showed a decrease in the size of the cell colony under both CFSS and PFSS (\(p<0.05\) and \(p<0.001\)) compared to the static culture at a cell density of 1 \(\times\) 10\({}^{3}\), respectively (Figure 1l). An increase in the number of cell colonies at 1 \(\times\) 10\({}^{2}\) in the PFSS condition (\(p<0.001\) and \(p<0.01\)) compared to the CFSS and static culture conditions (Figure 1m) was observed. Similar increases in the number of cell colonies were observed at 5 \(\times\) 10\({}^{2}\) and 1 \(\times\) 10\({}^{3}\) cell densities under PFSS (\(p<0.001\) and \(p<0.01\)) compared to the CFSS condition (Figure 1n,o). Surprisingly, the data showed a marked reduction in the number of colonies under CFSS (\(p<0.01\) and \(p<0.05\)) compared with the control cells under static conditions at both 5 \(\times\) 10\({}^{2}\) and 1 \(\times\) 10\({}^{3}\) cell densities (Figure 1n,o).