## 4 Discussion  
The weightlessness experienced as a result of the lower levels of gravity on board the ISS has negative impacts on astronauts' organs and tissues. Skeletal muscle is one of the most affected tissues during long stays in space, undergoing atrophy as a result of loss of mass due to a decrease of muscle fiber size [20] and alterations in the myofiber composition [21]. The adaptive pathophysiological changes that astronauts experience during space flights are similar to an accelerated aging process and to certain diseases [22]. While the average loss of muscle tissue with age is about 1% per year starting from peak development at about 24-30 years of age, with significant cumulative loss with advancing years, astronauts in space lose about 1% of muscle mass a month. The muscle atrophy, which occurs over a period of weeks to months of exposure to weightlessness with an overall 7-20% size decrease [23], can be only partially contained with adequate in-flight physical exercise and, in due course, is mostly reversible after landing. Indeed, despite exercise countermeasures [24], the crewmembers experienced muscle atrophy, particularly in leg muscles, in long-duration (6 months) missions [25; 26]. Investigating the molecular mechanisms of muscle atrophy induced by microgravity is essential for establishing clinical approaches and interventions to counteract this process and promote the growth and maintenance of muscle mass, not only during long-term space flights, but also in the atrophying processes linked to aging and several diseases. An advantage of the studies performed in microgravity conditions is that microgravity induces muscle atrophy much faster than aging. SCs, and the MPCs they provide, play a fundamental role in the maintenance of muscle mass in adulthood and in the muscle regeneration process following damage [15]. Muscle atrophy occurring during space flights is thought to be related, at least in part, to a deficit of these cells. Various studies have shown that cells grown in simulated microgravity (with the use of a random positioning machine) undergo morphological and molecular alterations [4; 27; 28]. However, very few studies were carried out on cells under real microgravity on board the ISS. These include the MyofGravity project whose aim was to study the functional alterations induced by real microgravity in huMPCs. This kind of study requires the use of dedicated devices in which several culture parameters have to be adapted, especially in the case of adherent cell types. We used specifically designed EUs (Kayser Italia, Livorno, Italy) and defined the optimal culture conditions in terms of coverslip coating, HEPES concentration in the culture medium, and cell density at plating. Moreover, we evaluated the resilience of huMPCs to T variations occurring during the rocket launch and flight to the ISS, and identified the best kit to isolate the RNAs from huMPCs inside the EUs. Importantly, we performed several analyses showing that the use of the set parameters allows culturing huMPCs in the EUs without significantly affecting their proliferation rate and differentiation extent in comparison with standard cultivation on plates. Indeed, the developed protocol reduces at the minimum the differences between huMPC cultures on plates and in EUs, in terms of population doubling level, fusion index, expression of muscle specific factors (_PAX7_, _MYOD1_ and _MYOG_), and myomiRNAs (miR-1, miR-133a, miR-133b and miR-206), thus suggesting that the EUs are useful devices to study the biological responses of huMPCs to real microgravity on board the ISS.