Validation for our low gravity simulator was particularly tricky because it was difficult to find a way where we could get unbiased quantitative data from the interior of the vessel during operation. Using literature, we adapted some qualitative tests that can indicate that low gravity may be present within the vessel.
Qualitative Validation
Qualitative Physical Test --- Neutrally Buoyant Sphere
As a qualitative check, we will place a neutrally buoyant, water-filled sphere at the center of the reactor chamber during operation. The logic is that if the simulated microgravity is effective, the sphere should remain suspended relatively motionless and not exhibit a consistent settling direction over time. Any sudden force would displace the sphere In effect, the gravitational vector should be “averaged out” by the continuous reorientation, such that no net buoyant or gravitational settling dominates. An ordinary air-filled ball would not be valid, because buoyant forces would dominate its motion relative to the fluid medium, and it would float or rise, confounding the test.
Failure of this “floating sphere” test would be a clear red flag: it would indicate that the rotation scheme is insufficient to cancel or randomize gravity effectively, or that residual accelerations or asymmetries are biasing the system. In the literature on microgravity simulators, neutral buoyancy has been used (e.g., in astronaut training environments, such as the Neutral Buoyancy Laboratory) to approximate weightlessness underwater by carefully balancing buoyant and gravitational forces. [1] While that is a very different scale and context, the underlying principle of neutral buoyancy as a check remains relevant.
It is worth noting that commonly used simulators like the Random Positioning Machine (RPM) rely on gravity-vector averaging, or continuously reorienting the sample so that over time the gravitational vector is randomized and does not produce a persistent bias. [2] In those devices, the presence of residual accelerations (sometimes called “g-jitter”) or asymmetric mass distribution can introduce unwanted bias or perturbations. [2] Thus, this neutrally buoyant sphere test helps ensure that the net effect is sufficiently isotropic.
To complement the physical test, we will run computational fluid dynamics (CFD) simulations of the reactor’s fluid environment under rotation. The goal is to estimate residual accelerations, flow fields, shear stresses, and convective effects within the vessel under different rotation speeds, rotation sequences, and design variants. These simulations will help us interpret what we see in the qualitative test, and also guide design refinements without having to rely purely on trial and error.
CFD is widely used in bioreactor design and scale-up to understand mixing, turbulence, shear, mass transfer, and concentration gradients --- parameters that are difficult to measure directly.[3] In our context, the CFD model will specifically simulate the rotational dynamics and fluid behavior under simulated microgravity, allowing us to predict how well gravitational forces are being canceled and how secondary fluid motions (e.g. due to centrifugal or Coriolis effects) might impact the culture.
By combining the neutrally buoyant sphere test and CFD modeling, we create a robust validation framework:
The sphere test gives a qualitative, visual confirmation that gravity is being sufficiently randomized (or canceled) in the real, physical system.
The CFD model provides quantitative insights into residual accelerations, velocity fields, shear, and other fluid dynamic effects that may not be directly visible.
Cross-comparison between what is observed in the real setup and what the simulation predicts allows us to detect discrepancies, assess whether residual effects are within tolerances, and further tune rotation parameters (speeds, sequences, balancing) if needed. In sum, this dual approach ensures that the bioreactor is not only qualitatively behaving like a reduced-gravity analog, but also quantitatively consistent with expectations. That gives us higher confidence that downstream biological experiments conducted in the system will meaningfully reflect low-gravity culture conditions.
2. Wuest SL, Richard S, Kopp S, Grimm D, Egli M. Simulated Microgravity: Critical Review on the Use of Random Positioning Machines for Mammalian Cell Culture. Biomed Res Int [Internet]. 2015 [cited 2025 Oct 1];2015:971474. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4310317/
