Cell cultivation under different gravitational loads using a novel random positioning incubator

Abstract

Important in biotechnology is the establishment of cell culture methods that reflect the in vivo situation accurately. One approach for reaching this goal is through 3D cell cultivation that mimics tissue or organ structures and functions. We present here a newly designed and constructed random positioning incubator (RPI) that enables 3D cell culture in simulated microgravity (0 g). In addition to growing cells in a weightlessness-like environment, our RPI enables long-duration cell cultivation under various gravitational loads, ranging from close to 0 g to almost 1 g. This allows the study of the mechanotransductional process of cells involved in the conversion of physical forces to an appropriate biochemical response. Gravity is a type of physical force with profound developmental implications in cellular systems as it modulates the resulting signaling cascades as a consequence of mechanical loading. The experiments presented here were conducted on mouse skeletal myoblasts and human lymphocytes, two types of cells that have been shown in the past to be particularly sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are conducted. The scientific data gathered may not only improve the sustainment of human life in space, but also lead to the design of alternative countermeasures against diseases related to impaired mechanosensation and downstream signaling processes on earth.

Keywords: lymphocyte activation; mechanical unloading; mechanotransduction; muscle cells; partial gravity; random positioning machine.

Figure 1

A recent version of the random positioning incubator (RPI) as designed and developed by the project partner FHNW based on collaboration and requirement specifications of ETHZ. The RPI consists of two gimbal-mounted frames, which are driven by electrical motors, and a CO₂ incubator mounted at the center of rotation of both frames.

Figure 2

Timeline of the mean gravity values for the algorithms. The Two Velocities algorithm (top track) achieves partial gravity through position dependent rotation velocity, whereby the frames are permanently rotating. The mean gravity value is controlled with a predictive controller. In contrast, the Flexible Static Intervals algorithm (middle track) controls the mean gravity value by particular intervals between random walk and static phases. The interleaving timing of the random walk and static phases is flexible and controlled online. The Fixed Static Intervals algorithm (bottom track) works similarly but has fixed periods of random walk and static phases. The nominal rotational velocity was 60°/s for the three algorithms. Note: for presentation reasons, the time scale is not identical.

Figure 3

Verification of the stability of the algorithms through numerical simulations; all of the algorithms are based on a random walk pattern which depends on a sequence of random numbers. The stability was verified by 500 numerical runs. The resulting mean gravity value was taken following a simulated experiment of 20 h in the case of Two Velocities algorithm (left), and 5 h in the case of Flexible Static Intervals (middle) and Fixed Static Intervals (right) algorithm. The nominal rotational velocity was 60°/s for the three algorithms.

Figure 4

Cell proliferation decreases with reduced partial gravity levels. Cell proliferation was assessed by counting cells manually (A) or by CFSE staining (B). The counts and median fluorescence intensity (MFI) of cells grown on the RPI after 24 h were normalized to the values of cells grown inside an incubator under normal culture conditions (1 g). Three different algorithms for partial gravity simulation (see Materials and Methods) were compared in the following order: Two Velocities, Flexible Static Intervals, and Fixed Static Intervals. Means ± standard deviations were obtained from three independent series of experiments.

Figure 5

Cell viability is not affected by partial gravity. Cell viability was assessed by counting trypan blue treated cells manually, following 24 h of growth on the RPI or inside an incubator under normal culture conditions (1 g). The three different algorithms for partial gravity simulation (see Materials and Methods) were compared in the following order: Two Velocities, Flexible Static Intervals, and Fixed Static Intervals. Means ± standard deviations were obtained from three independent series of experiments.

Figure 6

Partial gravity causes cells to accumulate at G2/M phase. Cells were collected 10 h after cultivation under partial gravity (RPI) or at 1 g (Ctrl and Inc). The Two Velocities algorithm was used to obtain partial gravity (see Materials and Methods). The cell cycle was analyzed and the percentage of cells in G0/G1 + S phase (A) and in the G2/M phase (B) is displayed, as well as the linear regression model with data from G2/M phase cell percentages (C). Means ± standard deviations were obtained from four independent series of experiments.

Figure 7

Lymphocyte activation depends on partial gravity exposure. Cells were treated after an adaptation period of 60 to 90 min with an activator solution and then cultured for 20 to 22 h on the RPI. Three different algorithms for partial gravity simulation (see Materials and Methods) were compared in the following order: Two Velocities, Flexible Static Intervals, and Fixed Static Intervals. The 1 g samples of each experiment were considered as 100% activated, and were used to standardize the other samples among the different experiments. A non-activated sample was used as a negative control. Means ± standard deviations were obtained from three independent series of experiments.