Neutral Buoyancy as a Simple Approach to Simulated Microgravity

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. 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. 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. 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.