Neutral Buoyancy as a Simple Approach to Simulated Microgravity.

BACKGROUND: It is well recognized that interesting biological phenomena occur in various organisms in microgravity. However, real microgravity research is limited by cost and accessibility. Furthermore, current ground-based microgravity simulators often cause shear stress and vibration, which restrict the accurate reproduction of a real microgravity environment. This study aimed to develop a simple, low-cost, and reproducible simulated microgravity system based on neutral buoyancy to reproduce an environment similar to that of real space. METHODS: A neutral buoyancy medium (NBM) was created by adjusting the density of conventional cell culture medium through mixing with density gradient medium (Ficoll-Paque™, Percoll™, and Optiprep™). The buoyancy stability of human bone marrow-derived mesenchymal stem cell (hBMSC) spheroids was examined experimentally and by computational fluid dynamics (CFD). The effects of neutral buoyancy-based simulated microgravity (3D-sim-μg) on hBMSC stemness and trilineage differentiation (osteogenic, adipogenic, and chondrogenic) were compared with normal gravity. RESULTS: Optiprep-based NBM (Optiprep™/cell culture medium, 20/80 v/v) maintained a stable suspension of hBMSC spheroids for 14 days. CFD analysis confirmed near-zero static pressure under neutral buoyancy, reproducing a microgravity-like environment. hBMSC spheroids in 3D-sim-μg showed enhanced expression of pluripotency markers and suppressed osteogenic differentiation, with increased adipogenic and chondrogenic expression compared to normal gravity. CONCLUSION: The neutral buoyancy-based system effectively simulates key microgravity-associated cellular behaviors, including maintenance of stemness and lineage-specific differentiation. This approach provides a simple and accessible platform for various microgravity research endeavors.