Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

Adequate mechanical stimulation is essential for cellular health and tissue maintenance, including articular cartilage, which lines the articulating bones in joints. Chondrocytes, which are the sole cells found in articular cartilage, are responsible for matrix synthesis, maintenance and degradation. It is generally believed that chondrocytes require mechanical stimuli through daily physical activity for adequate cartilage homeostasis. However, to date, the molecular mechanisms of cellular force sensing (mechanotransduction) are not fully understood. Among other mechanisms, the cytoskeleton is thought to play a key role. Despite that gravity is a very small force at the cellular level, cytoskeletal adaptations have been observed under altered gravity conditions of a parabolic flight in multiple cell types. In this study, we developed a novel hardware which allowed to chemically fix primary bovine chondrocytes at 7 time points over the course of a 31-parabola flight. The samples were subsequently stained for the microtubules and vimentin network and microscopic images were acquired. The images showed a large heterogeneity among the cells in morphology as well as in the structure of both networks. In all, no changes or adaptions in cytoskeleton structure could be detected over the course of the parabolic flight.

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