In the laboratory, we are studying the mechanism involved in skeletal muscle regeneration by using an in vitro model based on human primary myoblasts. These human primary myoblasts are obtained after purification of the muscle stem cells (MuSC) from different donors (age varies between 15 and 40 years) as previously described (Laumonier et al., 2017). Briefly, proliferating myoblasts are induced to differentiate into multinucleated myotubes with a particular medium (described in (Brunetti et al., 2021)). Interestingly, this differentiation process also allows to obtain mononucleated cells sharing the characteristics of MuSC, called reserve cells (RC). The aims of my master's project were, first, to improve the system allowing the maturation of myotubes into muscle fibers in vitro and, second, to investigate the interaction of the RCs with the maturated fibers obtained in vitro. Dr. Brunetti first developed the in vitro model and elaborated a 2D system to allow the culture and maturation of myotubes (Brunetti et al., 2021). This model involves unique dishes characterized by Nanopattern (Nano) surface: this support induces the alignment in parallel of plated cells, thus mimicking muscle cell in vivo organization. This particular pattern is coupled with two different layers of Matrigel® to limit cells detachment. These conditions allow maintaining the culture for up to ten days. During my master, I developed three different projects: 1) Characterization of the reserve cells population on the Nanopattern surface 2) Improving the level of maturation at ten days 3) Identification and tracking of the RCs Firstly, we demonstrated that the utilization of the Nanopatterned surfaces does not interfere with the establishment of the RCs (in vitro muscle stem cells) pool over time. In a second step, we tried to improve the maturation of the culture at ten days with the addition of various growth factors, hormones, or drugs. We have shown that adding 0.3% of horse serum (HS) improves the maturation of our culture. Finally, we developed robust tools to identify and track RCs in our model.