Simulated microgravity: critical review on the use of random positioning machines for mammalian cell culture

Abstract

Random Positioning Machines (RPMs) have been used since many years as a ground-based model to simulate microgravity. In this review we discuss several aspects of the RPM. Recent technological development has expanded the operative range of the RPM substantially. New possibilities of live cell imaging and partial gravity simulations, for example, are of particular interest. For obtaining valuable and reliable results from RPM experiments, the appropriate use of the RPM is of utmost importance. The simulation of microgravity requires that the RPM's rotation is faster than the biological process under study, but not so fast that undesired side effects appear. It remains a legitimate question, however, whether the RPM can accurately and reliably simulate microgravity conditions comparable to real microgravity in space. We attempt to answer this question by mathematically analyzing the forces working on the samples while they are mounted on the operating RPM and by comparing data obtained under real microgravity in space and simulated microgravity on the RPM. In conclusion and after taking the mentioned constraints into consideration, we are convinced that simulated microgravity experiments on the RPM are a valid alternative for conducting examinations on the influence of the force of gravity in a fast and straightforward approach.

Figure 1

Random positioning incubator (RPI) featuring a fully integrated CO₂ incubator (developed by the Institute for Automation, University of Applied Science Northwestern Switzerland).

Figure 2

Mouse myoblasts (C2C12 cell line) were cultured until near confluence and subsequently exposed to a frequently passing air bubble. The culture chamber filled with medium was swinging upside down, such that the intentional air bubble frequently passed the same trajectory. The sample was fixed and stained for actin (green) and DNA (blue) thereafter. The cells in the trajectory of the air bubble got detached from the substrate (dark central area), while cells in the unaffected area kept proliferating (lateral green areas). Interestingly, detached cells could reattach to the opposite side of the culture chamber. Measuring bar 200 μm. (Due to the limited field of view, this image has been stitched together from five images.)

Figure 3

The worst-case peak centrifugal acceleration on an RPM depending on the distance to the center of rotation (a _pc ≈ 2.41 · ω ² · r). For example, a moderate rotational velocity of 60 deg/s (cyan line) and a distance of 10 cm from the center of rotation (vertical dashed line) results in a peak centrifugal acceleration of approximately 0.03 g (horizontal dashed line).

Figure 4

The worst-case tangential acceleration depending on the distance from the center of rotation (a _t = 2 · α · r). For a smooth velocity transition of, for example, 10 deg/s² (green line) and 10 cm distance from the center of rotation (vertical dashed line), a tangential acceleration of approximately 0.004 g is expected (horizontal dashed line).

Figure 5

The RPM rotation introduces fluid motion in the culture flask, leading to shear forces and enhanced convection. Therefore, a moderate rotational velocity needs to be chosen, and the velocity transitions have to be smooth in order to minimize the introduction of additional mechanical stimulation of the samples. In this numerical illustration, the fluid motion is shown if both frames rotate at 60 deg/s. This results in a periodic motion of 6 seconds. The four images indicate snapshots of the velocity at 0 s (a), 1.2 s (b), 2.2 s (c), and 4 s (d).

Figure 6

Thyrocytes cultured for seven days on the RPM organized to spheroid structures (arrow).