## 4 Discussion  
Cellular microenvironment parameters, such as mechanical FSS and hypoxia, play a central role in the physiological performance and the regenerative capacity of stem cells. Investigation of the cell niche is essential to gain insights into the physiological microenvironment, to optimize protocols for cell cultivation and differentiation in vitro, and to improve the therapeutic use of muscle stem cells for tissue regeneration. Muscle stem cells are a promising candidate for the treatment of musculoskeletal diseases, but their reduced regenerative potential after cultivation and low viability after transplantation are challenging aspects. To our knowledge, several mechanical stress protocols have been previously described, but there is no standard method for applying shear stress, making it difficult to compare the results of the different groups. Furthermore, little is known about the effect of long-term cultivation under either cyclic or intermittent FSS for muscle stem cells. In the present study, a dynamically oscillating FSS at a level between 1.68 and 2.3 mPa was applied either continuously or periodically for one hour per day for up to 7 days using a controlled mechanical rocking generator. A variety of different FSS delivery protocols have been extensively studied. These protocols vary in the form, strength, and duration of the FSS applied, as well as the total duration of the experiment. In the present study, a low FSS in the range of 1.68-2.3 mPa was applied to mimic the in vivo interstitial fluid flow in skeletal muscle as previously reported [24; 52; 55]. Oscillating FSS mimics in vivo conditions and is more appropriate due to the dynamic nature of interstitial fluid flow compared to a unidirectional flow [56]. It was found that a parallel plate flow chamber was used to create a laminar pulsating flow with an average shear stress between 0.4 and 1.4 Pa and a frequency of 1 Hz for 1 h [23]. Intermittent FSS could be applied using an FSS of 0.094 Pa for three cycles consisting of a 10 min stimulation followed by a 10 min pause with a 15 min pause between each cycle using a microfluidic system [57]. On the other hand, a continuous laminar FSS between 3 and 42 mPa for 5 d in conjunction with spatial confinement has also been documented using a microfluidic system [24]. In addition, a pulsed flow FSS between 0 and 1.2 Pa and a pulse frequency of 1 Hz for 1 h have been reported [58]. The present data showed an alteration in cell morphology under both CFSS and PFSS protocols, including a small, elongated cell body with numerous long and short cytoplasmic processes compared to a static culture. The data suggest that cells adapt their cytoskeletal components to minimize the effect of shear forces and establish a stable adhesion. In the same line, it has been documented that the actin cytoskeleton is a mechanosensitive component, and its integrity plays a critical role in the resistance to fluid-induced shear force [59]. Under chronic FSS, morphological changes including changes from a polygonal shape to a spindle shape, aligned with the direction of flow by establishing actin stress fibers, were observed in endothelial cells [58]. Consistent with our data, an investigation of the effect of FSS on renal epithelial cells showed a reorganization of the actin cytoskeleton from parallel F-actin bundles to peripheral bundles combined with an increase in the formation of cell junctions [60]. As a cellular response to FSS, one study observed the short-term contraction of cells followed by combined expansion and orientation to fluid flow, and the homogeneous distribution of F-actin. Consistent with our data, a study concluded that cells changed to a rounded morphology with multiple cytoplasmic processes to enhance their adhesion to overcome mechanical stress [61]. The level of shear force could affect the cell response, and one study showed no alignment of C2C12 myoblasts after 0.07-0.13 Pa of FSS for 6 h [62].