Goldfish regenerated scale culture at low temperatures improves osteoblast and osteoclast survival in scales without loss of the osteoblast and osteoclast response to changes in gravity

Biological samples that can be stored for long periods are desirable for experiments in space because of the potential for postponement of space vehicle launches. In this study, we determined whether culturing goldfish scales at lower temperatures increased survival of osteoblasts and osteoclasts in the scales without affecting their biological activities. After one-week storage of regenerated goldfish scales at 4 °C, both alkaline phosphatase (ALP)-positive osteoblasts and tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts (active type of osteoclasts) were detectable. Importantly, the multinucleated osteoclasts formed actin rings and expressed cathepsin K, indicating that the osteoclasts had resorptive activity. Additionally, a one-week storage of goldfish scales at 4 °C showed little effect on osteoblastic and osteoclastic activities in the scales. Therefore, after one-week storage at 4 °C, the regenerated scales were treated with vibrational acceleration due to gravity (3 G) for 10 min and then incubated at 15 °C for 6 h. ALP and TRAP activities in the regenerated scales sensitively responded to 3 G hypergravity: compared to unexposed scales, ALP activity significantly increased and TRAP activity significantly decreased in the regenerated scales. Next, the regenerated scales were stored at 4 °C and launched on the space shuttle Atlantis STS-132 (ULF4) to study the effects of vibration (maximum 6.8 G, 2 min) and subsequent hypergravity (maximum 3 G, 8 min 30 s) induced by the launch process. The results showed the vibration and subsequent hypergravity increased significantly ALP activity in the regenerated scales at 4 °C. In addition, our experiment with the Cell Biology Experiment Facility in outer space revealed that osteoblast activity in the regenerated scales decreased in response to microgravity after 6 days-storage of the scale at 4 °C. These findings show that goldfish scales can be stored at 4 °C for around one week, while maintaining the responsiveness of the osteoblast and osteoclast in the scales to changes in gravity.