Simulating microgravity using a random positioning machine for inducing cellular responses to mechanotransduction in human osteoblasts

Abstract

The mechanotransduction pathways that mediate cellular responses to contact forces are better understood than those that mediate response to distance forces, especially the force of gravity. Removing or reducing gravity for significant periods of time involves either sending samples to space, inducing diamagnetic levitation with high magnetic fields, or continually reorienting samples for a period, all in a manner that supports cell culturing. Undesired secondary effects due to high magnetic fields or shear forces associated with fluid flow while reorienting must be considered in the design of ground-based devices. We have developed a lab-friendly and compact random positioning machine (RPM) that fits in a standard tissue culture incubator. Using a two-axis gimbal, it continually reorients samples in a manner that produces an equal likelihood that all possible orientations are visited. We contribute a new control algorithm by which the distribution of probabilities over all possible orientations is completely uniform. Rather than randomly varying gimbal axis speed and/or direction as in previous algorithms (which produces non-uniform probability distributions of orientation), we use inverse kinematics to follow a trajectory with a probability distribution of orientations that is uniform by construction. Over a time period of 6 h of operation using our RPM, the average gravity is within 0.001 23% of the gravity of Earth. Shear forces are minimized by limiting the angular speed of both gimbal motors to under 42 °/s. We demonstrate the utility of our RPM by investigating the effects of simulated microgravity on adherent human osteoblasts immediately after retrieving samples from our RPM. Cytoskeletal disruption and cell shape changes were observed relative to samples cultured in a 1 g environment. We also found that subjecting human osteoblasts in suspension to simulated microgravity resulted in less filamentous actin and lower cell stiffness.

FIG. 1.

Hardware design of the RPM. (a) CAD model of the RPM depicting the secured payload to the central frame. (b) Photo of the RPM showing connected wiring and the center panel. (c) CAD drawing of the cross section of the motor driving rotation of the inner frame. (d) Cross section of the motor driving rotation of the outer frame.

FIG. 2.

RPM model and simulation. (a) A MATLAB-generated figure showing the configuration of the RPM and definitions for the angles q₁ and q₂ when displaced. (b) Points in a uniform random distribution on a unit sphere. Among candidate points (orange) lying within a wedge of angle η and radius Δs, one point is selected (green) and its distance from the previous point is normalized before being added to the trajectory (red). (c) Trigonometric construction for determining the maximum geodesic curvature from the wedge parameters η and Δs. (d) A sample of the trajectory path created by the algorithm. (e) Decreasing effective gravity experienced by the system as a function of the number of points generated by the program. 10 000 points corresponds roughly to 1 h of run time. The inset shows the first 10 000 points.

FIG. 3.

Fluorescence images of fixed, adhered hFOB cytoskeleton and quantifications of cell morphology changes of cells under 1 g and cells under simulated microgravity. (a) Representative images of F-actin stained with acti-stain 488 phalloidin, immunofluorescently labeled microtubule, and the nucleus of hFOB cells under 1 g condition or experiencing simulated microgravity for 3 or 6 h. Scale bar: 10 μm. (b) Measurement of the cell spreading area between the three conditions indicated in (a). N = 51. (c) Quantification of circularity for conditions in a scored within 0–1 range, where 1 represents a perfect circle and 0 represents an increasingly elongated polygon (N = 51).

FIG. 4.

Mechanical characterization of 1 g and simulated microgravity-subjected hFOB cells in suspension using the μFPA device. (a) Schematic of the microfluidic device connected to a pressure generator at the inlet of the device. (b) Cell viability test, for cells in suspension, for the indicated conditions as measured by PI staining using flow cytometry. Three independent replicates were performed for all experiments. Plots indicate mean and standard error of the mean. **p < 0.01. (c) Fluorescence images of aspirated hFOB cells under 1 g condition and following 3 and 6 h simulated microgravity treatment. Edges of the cell are contoured using dashed lines. Scale bar: 10 μm. (c) Stiffness measurements for each condition. N = 10. (d) Young’s modulus changes of hFOB cells under 1 g and simulated microgravity conditions determined using the μFPA device. *p < 0.05.