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Astronauts traveling or researching in space are subject to a harsh environment that includes high levels of ionizing radiation and microgravity. Ionizing radiation is dangerous to overall health because it causes double-stranded DNA breaks, increases the production of free radicals or reactive oxygen species, and leads to an enhanced risk of cardiovascular disease and cancer over a person’s lifetime. Similarly, microgravity is hazardous to astronaut health because humans require gravity to maintain a healthy heart and muscular and vascular systems. Both ionizing radiation and microgravity have been studied individually in previous research, but there is a lack of information on how these stressors work in combination to damage cellular health. To simulate the spaceflight radiation environment, Utah State University’s Space Survivability Test Chamber was used to irradiate mouse skeletal muscle cells with doses similar to what astronauts will be exposed to on a Mars mission. Microgravity was simulated within the radiation test chamber by designing a custom miniature rotary cell culture system. This device rotates muscle cells growing on polystyrene microcarrier beads so that the cell clusters remain suspended at their terminal settling velocity. The mouse muscle cells irradiated with a long-term spaceflight dosage exhibited significant amounts of double-stranded DNA damage as well as significantly increased production of reactive oxygen species compared to the short-term mission models. The combined model of ionizing radiation and microgravity designed here is more representative of the spaceflight environment than either condition alone. Our model is a valuable platform that can be used to study the effects of space travel on a variety of cell types and test potential treatments.

Muscular atrophy, defined as the loss of muscle tissue, is a serious issue for immobilized patients on Earth and in human spaceflight, where microgravity prevents normal muscle loading. Developing countermeasures for atrophy in spaceflight will require extensive screening of pharmaceuticals for efficacy, safety, contraindications, and dosage schedule. Due to the cost of spaceflight and limited crew time aboard the International Space Station, high throughput screening of pharmaceuticals under real microgravity conditions is not feasible. While traditional ground-based atrophy studies using the rodent hind-limb unloading model are effective at inducing physiological changes similar to spaceflight, they are not suited for first round screening of novel therapeutics due to resource and regulatory challenges. Additionally, these ground-based studies do not account for the high levels of ionizing radiation experienced in spaceflight that may increase atrophy. Here, we present a protocol to combine both microgravity and radiation using an in vitro ground-based model of microgravity with a rotary cell culture system combined with Utah State University’s Space Survivability Test Chamber (SSTC). To model the levels of ionizing radiation received by astronauts in space, murine C2C12 cells were cultured in standard tissue culture well plates and exposed to Strontium 90 in both their undifferentiated and differentiated states using the SSTC. We hypothesize that cells will show a significant loss of viability when exposed to a dosage of approximately 4.0 Gy, the LD50 for most human tissue. Changes after exposure to radiation were indicated by cell viability counts and morphology characterization. To model the microgravity conditions on the International Space Station, a custom rotary cell culture system was designed to be compatible with the SSTC. We hypothesize that combining both microgravity and radiation will lead to a greater biosimilarity for ground-based atrophy models. Contrary to expectations, undifferentiated cells exposed to radiation did not experience a significant loss in viability withstanding doses up to 38 Gy. Irradiated differentiated cells followed a lethal dosage curve as expected to reach 50% viability at approximately 4.0 Gy. Ground-based simulation of microgravity and radiation will provide a valuable platform for drug discovery and an understanding of the multiple mechanisms underlying muscular atrophy.