R40 - A Computational Approach to Tuning Orbital Shaker Conditions for Mechanobiological Experiments

Introduction: : Orbital shakers are widely used in endothelial cell (EC) mechanobiology to model both physiological and pathological shear environments. These platforms create complex, spatially varying flow profiles in multi-well plates that can replicate regions of laminar or disturbed flow, mimicking vascular conditions such as those found in arteries and branches. However, selecting orbital shaker parameters that reliably produce biologically relevant wall shear stress (WSS) while avoiding experimental artifacts, such as fluid spillage or exposing cells to air, is nontrivial. Variations in orbital radius (A), angular velocity (ω, in RPM), and initial fluid height (h0) all influence the shear profile and free surface behavior during shaking. While some groups rely on empirical rules-of-thumb to select conditions, a lack of quantitative guidance has hindered reproducibility and cross-study comparisons.

To address this gap, we used computational fluid dynamics (CFD) to systematically evaluate how orbital shaker parameters affect WSS distribution and fluid surface dynamics in standard 6-well plates. We focused on identifying operational bounds that (i) generate distinct peripheral and central flow regimes, resembling undisturbed (UF) and disturbed (DF) flow, respectively, and (ii) minimize the likelihood of adverse surface effects such as media uncovering or well overflow. Our results offer a tunable framework for matching desired flow regimes to safe and reproducible experimental settings, supporting the development of standardized protocols in EC mechanobiology. This study serves as a computational foundation for experimental optimization in shaker-based flow systems, enhancing the relevance, reproducibility, and interpretability of in vitro mechanotransduction research.

Materials and

Methods: : We employed CFD simulations using the Volume of Fluid (VOF) method to analyze orbital shaking in a 6-well plate. Simulations modeled incompressible, Newtonian flow in a rotating reference frame to capture both wall shear stress (WSS) distributions and free surface dynamics. We investigated two orbital radii: A = 5 mm and A = 10 mm, across a physiologically relevant range of angular velocities (RPM: 170–210) and initial fluid heights (h0: 2.6–4.1 mm). The working fluid properties (density and viscosity) were set to approximate standard culture media at 37°C.

Each simulation output included time-resolved surface shear stress maps and three-dimensional fluid surface deformation over several shaker cycles. WSS was evaluated at the well bottom to identify regions of undisturbed (UF, peripheral) versus disturbed (DF, central) flow. Minimum and maximum fluid height during orbital motion were quantified to assess the risk of cell monolayer exposure or well overflow.

We defined safe operational conditions as those that produced biologically relevant WSS (typically 10–15 dynes/cm2 in the periphery) while avoiding any location within the well dropping below 0.5 mm in fluid depth, considered a threshold for potential air exposure. CFD models were validated against analytical approximations for simple cases and cross-referenced with empirical data from literature where available. All simulations were performed using ANSYS Fluent with adaptive meshing to ensure accuracy near the fluid–air interface and wall surfaces. Simulation data were post-processed to generate shear stress profiles and extract shear ranges for different RPM and h0 combinations.

Results, Conclusions, and Discussions:: Simulation results revealed distinct shear environments and operational guidelines for both orbital radii. For A = 5 mm, a range of 190–210 RPM with h0 between 2.6 mm and 4.1 mm produced peripheral WSS of 11–14 dynes/cm2 and central WSS of 3–5 dynes/cm2. For A = 10 mm, we identified 170 RPM as optimal within the same h0 range, yielding peripheral WSS of ~10–11 dynes/cm2 and central WSS of 4–6 dynes/cm2. Across both radii, peripheral shear stress was relatively insensitive to h0, while central shear stress increased as h0 decreased, highlighting a controllable means to modulate disturbed flow without perturbing the undisturbed regions.

Free surface analyses demonstrated that maximum fluid height remained safely below the well edge in all tested conditions, reducing the risk of spillage. However, localized regions of minimal fluid height occasionally dropped near or below 0.5 mm, especially at higher RPM and lower h0. These regions were most prominent in the center of the well, coinciding with DF zones, underscoring the importance of balancing desired biological shear profiles with safety margins to prevent air exposure.

The ability to independently tune DF and UF zones by adjusting fluid height without compromising total shear exposure provides a powerful tool for experimental design. This feature enables studies comparing endothelial responses under different flow regimes within the same plate, improving throughput and consistency.

In conclusion, our CFD-based approach establishes a parameter space for safely and reproducibly generating heterogeneous flow environments on orbital shakers. These findings offer practical guidance for selecting operating conditions that align with specific mechanobiological goals and reduce experimental variability. Future work will focus on validating these simulations experimentally and correlating WSS distributions with cellular responses, laying the groundwork for standardized and biologically meaningful in vitro flow models.

Acknowledgements and/or References (Optional)::