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

Cell cultivation under different gravitational loads using a novel random positioning incubator ARTICLE Cell Cultivation Under Different Gravitational Loads Using a Novel Random Positioning Incubator 1,2 1 1,3 3 ¨ ¨ Tatiana Benavides Damm, Isabelle Walther, Simon L. Wuest, Jorg Sekler, Marcel Egli CC Aerospace Biomedical Science & Technology, Space Biology Group, Lucerne University of Applied Sciences and Arts (HSLU), Hergiswil, Nidwalden, Switzerland Institute for Biomechanics, Eidgeno¨ssische Technische Hochschule Zu¨ rich (ETHZ), Zu¨ rich, Switzerland Institute for Automation Engineering, University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Brugg-Windisch, Aargau, Switzerland KEYWORDS: partial gravity; random positioning machine; mechanical unloading; mechanotransduction; muscle cells; ABSTRACT: Important in biotechnology is the establishment lymphocyte activation 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 Introduction culture in simulated microgravity (0 g). In addition to Recent tissue engineering studies have revealed striking growing cells in a weightlessness-like environment, our RPI enables long-duration cell cultivation under various gravita- discrepancies between the behavior of cells cultured in 3D tional loads, ranging from close to 0 g to almost 1 g. This and 2D, where 3D cell cultures mimicked intact tissue in allows the study of the mechanotransductional process of terms of viability and gene expression (Baharvand et al., 2006; cells involved in the conversion of physical forces to an Sun et al., 2006). Indeed, 3D cell culture is one favorable appropriate biochemical response. Gravity is a type of approach for reducing the gap between artificial cultivation in physical force with profound developmental implications in cellular systems as it modulates the resulting signaling vitro and the in vivo physiological situation. The benefits of cascades as a consequence of mechanical loading. The 3D cell cultivation over 2D monolayers are featured due to experiments presented here were conducted on mouse the increased cell to cell contact, as well as cell to extracellular skeletal myoblasts and human lymphocytes, two types of matrix interaction, and the accumulation of nutrients and cells that have been shown in the past to be particularly growth factors (Saltzman et al., 1992). It has been shown that sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are cell growth in 3D can be achieved through the randomized conducted. The scientific data gathered may not only movement employed by rotating bioreactors which simulate improve the sustainment of human life in space, but also microgravity (Freed and Vunjak-Novakovic, 1995). Further- lead to the design of alternative countermeasures against more, this platform provides a constant circulation of diseases related to impaired mechanosensation and down- nutrient medium, removing the higher waste concentrations stream signaling processes on earth. that are usually found in the microenvironment adjacent to Biotechnol. Bioeng. 2014;111: 1180–1190. the cell surface (Rivera-Solorio and Kleis, 2006). 2013 The Authors. Biotechnology and Bioengineering The first instruments used to simulate microgravity were Published by Wiley Periodicals, Inc. clinostats, developed in 1879, a long time before the beginning of space exploration, by Julius von Sachs, a botanist who wanted to investigate gravitropism in plants This is an open access article under the terms of the Creative Commons Attribution- (van Loon, 2007). The uni-axial clinostat worked by slowly NonCommercial-NoDerivs License, which permits use and distribution in any medium, rotating the specimens around a longitudinal horizontal axis. provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. In this way, for example, the growing plant was experiencing Correspondence to: M. Egli a continuously reoriented gravity vector over a long period of Contract grant sponsor: European Space Agency (ESA) time. Later on, fast rotating clinostats were developed and Received 10 September 2013; Revision received 26 November 2013; Accepted 23 December 2013 introduced to negate the gravitational force on cells growing Accepted manuscript online 30 December 2013; in culture media. In order to study such cells, the diameter of Article first published online 22 January 2014 in Wiley Online Library the rotating part of the clinostat had to be small to avoid the (http://onlinelibrary.wiley.com/doi/10.1002/bit.25179/abstract). DOI 10.1002/bit.25179 generation of centrifugal forces, and the rotational speed had 1180 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014  2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. to be much faster (40–100 rpm). In the fast rotating clinostat, this machine, environments from close to 0 to almost 1 g can research on single mammalian cells and unicellular organ- be generated by several algorithms. A machine of this type isms could be performed since small samples no longer increases the variety of experiments which can be tested in experience the turning of gravity as they are in simulated general and additionally allows the exploration of effects on weightlessness (Block et al., 1986). Limitations of the fast biological samples in conditions which are difficult to create, rotating clinostat motivated the development of other such as the gravity field of the Moon or Mars. The data instruments that allowed larger samples to be cultivated reported in this study was gathered with mouse skeletal under simulated microgravity. The rotating wall vessel myoblasts (adherent cells) and human lymphocytes (non- (RWV) is one example of such a device that provides an adherent cells), in an attempt to better understand how environment with low-shear forces, allowing cells to grow deconditioning the cells through mechanical unloading by even as 3D aggregates (Schwarz et al., 1992). Since the late reducing gravity affects two major physiological mechanisms: nineties, a 3D clinostat called a random positioning machine the skeletal muscle and the immune system. These two (RPM) has been intensively used for ground-based research. mammalian cell types have repeatedly demonstrated high The RPM, originally developed by T. Hoson in Japan, applies sensitivity to simulated microgravity (Benavides Damm the principle of neutralizing gravity by vector averaging et al., 2013b; Chang et al., 2012; Sonnenfeld, 2012). As gravity (Hoson et al., 1997). Numerous experiments have already provides a stimulus for their maintenance, we hypothesized proven that this technique generates data that is indeed very that partial gravity exposure would produce changes in their similar to the data obtained in space (Herranz et al., 2010; behavior and sought to investigate whether this response was Infanger et al., 2006; Pietsch et al., 2012; Schwarzenberg proportionally linear, nonlinear, or set by a threshold with et al., 1999). decreasing gravity levels. The answer to this question is As access to real weightlessness in space is very limited essential, not only for aerospace physiology, but also and expensive, scientists have always been interested in necessary in order to comprehend the effects of mechanical having earth-bound tools available to perform pilot unloading associated with clinical scenarios on earth, such as studies in simulated microgravity. These instruments aging and illness arising from immobilization and prolonged are of great importance to test hardware and processes periods of bed rest. under microgravity approaching conditions and in studying biological systems under these specificsettings. Materials and Methods The RPM, though not totally equal to real space, is a good tool for preparing space experiments, for testing newly Random Positioning Incubator (RPI) developed instruments, and for studying biological systems of interest under simulated weightlessness, as The RPI equipment consists of two independently rotating well as for gathering enough valuable data that can be used gimbal-mounted frames, each axis being motorized by an for statistical purposes. Nevertheless, one of the major electrical drive to control random, or other positioning, constraints of the RPM so far has been that no simulated sequences. Through a dedicated algorithm the samples are partial gravity values could be attained. With the constantly reoriented with respect to the earth gravity vector. increasing interest of the scientific community to return This allows the distribution of the vector in space, leading mantothe Moon andtogofurther in theexploration of over time to an average gravity value smaller than 1 g. A Mars, studies performed with these two gravity fields unique feature of our present RPM, the RPI version, is the (0.16 g and 0.38 g, respectively) are becoming increasingly integration of a miniaturized CO incubator including a 14 L valuable. At the present time these types of studies have test chamber, mounted at the center of rotation of both been performed in head-up tilt studies, in body suspen- frames (Fig. 1). The specially functionalized incubator is sion devices, and for a short duration in parabolic flights supplied with electrical power and CO (and other (Cavagna et al., 2000; Dalmarco et al., 2006; Pavy-Le Traon experimental supplies) throughout the experiment’s dura- et al., 1997). Long-term experiments at a cellular level tion. Important data such as temperature, CO levels, and have not yet been possible except when performed in a mean gravity values are monitored automatically. Our centrifuge in real space such as during the IML-2 mission present RPI will be described in more details elsewhere in 1994 with the NIZEMI slow rotating microscope (manuscript under review). (Friedrich et al., 1996). As well as using partial gravity for space related studies, having various gravitational envi- Myoblasts ronments available for conducting research opens new approaches to investigate the mechanobiology of cells C2C12 mouse skeletal myoblasts were obtained from ATCC ranging from single mammalian cells to microorganisms. (American Type Culture Collection, LGC Standards S.a.r.l., In this paper, we present a newly designed and built RPM Molsheim Cedex, France) and cultured in a medium equipped with an integrated incubator, thus called random consisting of Dulbecco’s Modified Eagle Medium (Life positioning incubator (RPI) that in addition to generating Technologies, Lucerne, Switzerland) supplemented with simulated microgravity, offers new features such as partial 20% fetal bovine serum (PAA, Basel, Switzerland) and gravity simulation during an extended period of time. Using 2mM L-Glutamine (Life Technologies) in a humidified Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1181 Biotechnology and Bioengineering CFSE is a dye that passively diffuses into cells, reacting with intracellular amines and forming fluorescent conjugates. Following initial staining of the mother cells, subsequent rounds of cell division dilute the amount of dye inherited by daughter cells by one half with each division. For cell cycle analysis, cells were collected after 10 h of culture on the RPI or on the incubator and fixed. Samples were stored at 20 Cin 70% ice cold ethanol for at least 12–18 h prior to staining. Cell cycle was analyzed based on DNA content through propidium iodide (PI) with RNase staining by FACS. CFSE fluorescence intensity and cell cycle profiles were analyzed and quantified with the FlowJo flow cytometry analysis software (TreeStar, Inc., Ashland, OR). To quantify G0/G1, S, and G2/M populations, the Watson model was used to fit the histograms of single gated cells (Watson et al., 1987). FACS Calibur (BD Biosciences) was used for acquisition and appropriate settings for forward and side scatter gates were applied to examine 20,000 events per sample. The blue laser with excitation at 488 nm was used, with FL1 channel detector for CFSE and FL2 channel detector for PI staining. Data (means  standard deviations) were obtained from three or four independent series of experiments. Lymphocytes Whole peripheral blood was obtained from healthy human volunteers. Peripheral blood lymphocytes (PBL) were Figure 1. A recent version of the random positioning incubator (RPI) as designed isolated using Ficoll gradient (Histopaque; Sigma, Buchs, and developed by the project partner FHNW based on collaboration and requirement Switzerland), red blood cells were lysed and finally T-cells specifications of ETHZ. The RPI consists of two gimbal-mounted frames, which are were purified further using enrichment columns as per the driven by electrical motors, and a CO incubator mounted at the center of rotation of both frames. manufacturer’s instructions (R&D Systems, Abingdon, United Kingdom). T-cells were re-suspended into RPMI 1640 (Life Technologies) with 10% fetal calf serum (Life atmosphere at 37 Cin7%CO . Cells were passed every Technologies). The cells were loaded at a concentration of 1 2 days and were only used for experimentation before the to 2  10 cells/mL inside the “LYCIS” hardware, which has 15th passage. Myoblasts were seeded at 8,000 cells/cm in been developed specially for space experiments. This 25 cm flasks (Semadeni, Ostermundigen, Switzerland) 15 h hardware is based on the principle of moving pistons that before the start of each experiment. Immediately before being allows the loading of cells in the absence of air bubbles and placed on the RPI, the flasks were carefully filled with media, the injection of substances rapidly without opening the avoiding the formation of bubbles. Cell counting was hardware. The cells were preconditioned at each experi- performed manually with a hemocytometer (Biosystems, mental environment for 60 to 90 min prior to activation Nunningen, Switzerland) on trypan blue (Life Technologies) with a concanavaline A (conA)/anti CD28 mixture (BD treated cells to assess cell concentration and viability, Biosciences) for T-cells or only with conA (Sigma) for the according to the dye exclusion method (Yip and PBL. After 20 to 22 h of incubation at 37 Cunder the Auersperg, 1972). The counts were carried out in duplicate respective gravity conditions, the cells were harvested and per independent sample after 24 h of growth on the RPI or stained. The staining was carried out according to the inside an incubator under normal culture conditions (1 g supplier using PE-CD25 marker (Miltenyi Biotec, Bergisch control). Cell growth was also monitored with the CellTrace Gladbach, Germany) which is an early marker for CFSE Cell Proliferation Kit (Life Technologies), according to lymphocyte activation. Stained cells were analyzed on the the manufacturers’ protocol. Briefly, cells were stained with FACS Calibur using 10,000 events per sample. The FlowJo 5 mM carboxyfluorescein diacetate, succinimidyl ester (often software was used to gate the cells and fitthe histograms. called CFSE) in phosphate buffered saline þ 0.1% bovine The 1 g samples of each experiment were considered as serum albumin at 37 C for 10 min. Cells were plated and 100% activated, and were used to standardize the other collected after 24 h of growth under different gravitational samples among the different experiments. A non-activated conditions, and then re-suspended in 3.7% Cell Fix (BD sample wasusedasnegativecontrol.Data(means Biosciences, Allschwil, Switzerland) for overnight storage at standard deviations) were obtained from three independent 4 C before fluorescence activated cell sorting (FACS) analysis. series of experiments. 1182 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 Algorithms will result. There are numerous motion patterns possible to create simulated partial gravity by fulfilling the above Simulated microgravity on a RPM is achieved by gravity equations. We developed and used three different algorithms; vector averaging to zero as described earlier (Hoson all of them based on a random walk algorithm described et al., 1997). Thereby the earth gravity vector is distributed elsewhere (manuscript under review). In brief, this random over time by constantly reorienting the samples to random walk algorithm rotates both frames with constant rotational positions, where eventually the mean gravity will converge to velocity and inverts the direction of rotation (forward or a small value. As described below, we adjusted the backward) at random times. The velocity transition takes distribution of the gravity vector in order to obtain a desired place at a constant rotational acceleration. This unmodified mean gravity value. As a quality measure for the distribution random walk algorithm was employed for the 0 g experi- of orientation we use the mean gravity over time, defined by ments. The simulated partial gravity level that can be qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi achieved is limited to a certain maximum that depends on the 2 2 2 G ¼ ðG Þ þðG Þ þðG Þ ð1Þ mean X;mean Y;mean Z;mean algorithm used and the corresponding parameters. In addition, the accuracy and the time necessary to reach the Here, the mean gravity values in the three directions are desired partial gravity value is algorithm-dependent (Fig. 2). defined as follows: The stability of our applied algorithms was verified with P P n n numerical simulations (Fig. 3). In this study, we used the g g i¼1 X;i i¼1 Y;i G ¼ ; G ¼ ; G X;mean Y;mean Z;mean following three different algorithms: n n i¼1 Z;i ¼ ð2Þ Simulated Partial Gravity Based on Random Walk With Two Velocities (Two Velocities) If the pattern of random positions are chosen such that the earth vector spends more time in one particular area To achieve a particular mean gravity value, the local vector of (considered in the local frame of the sample), partial gravity one direction is enlarged by making the rotation velocity 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. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1183 Biotechnology and Bioengineering 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. position dependent. In principle, the RPM slows down, when high partial gravity values (>0.6 g) however, one has to keep this vector is pointing downward. Thus, the RPM spends in mind that the samples would be standing still for most of slightly more time in one direction than in the others. The the time. relation of the nominal to the slower velocity determines the resulting mean gravity. Since the random walk algorithm is Simulated Partial Gravity Based Random Walk and Fixed based on random numbers, two consecutive runs will not Static Intervals (Fixed Static Intervals) reproduce the same result. Therefore, the velocity relation is controlled by a predictive controller: While the nominal (or This algorithm works like the Flexible Static Intervals faster) velocity is fixed, the slower velocity is initially guessed algorithm, except that the time spent in random walk or based on numerical simulations. The RPM runs then for 1 h static position is fixed and constant throughout the such that the mean gravity value can be stabilized. Based on experiment. The relation of the rotating to the static time the deviation from the desired mean gravity, the value of the determines the resulting mean gravity value. For example, if lower velocity is refined. The RPM subsequently runs again the frames rotate for 40% of the time in random walk and are for 1 h, after which the slower velocity is again corrected. This static for 60% of the time, a mean gravity of 0.6 g will result. interval of letting the mean gravity stabilize for 1 h with a In our experiments we used a cycling period of 2.5 min (e.g., subsequent refinement of the slower velocity is repeated until for a 0.6 g experiment the RPM spends 1 min in random the end of the experiment. We were able to achieve up to 0.6 g walk, followed by 1.5 min standing still, where the cycle is reliably, which is close to the upper limit. This algorithm continuously repeated). This algorithm, again, allows reach- ensures that the frames are always in motion. However, it takes ing partial gravity values close to 1 g. However, the samples at least 2 h to achieve the desired partial gravity value (Fig. 2). would be standing still for most of the time at high partial gravity values (>0.6 g) as is the case when applying the Flexible Static Intervals algorithm. Simulated Partial Gravity Based Random Walk and Flexible For all three partial gravity algorithms and the unmodified Static Intervals (Flexible Static Intervals) random walk, the nominal rotational velocity was 60 /s for Since random walk reduces the mean gravity value towards the myoblast experiments and 40 /s for the lymphocyte zero, this algorithm interleaves the random walk with static experiments. phases, whereby the frames are stopped at one particular position. The timing of the static phases is controlled by the Results computer online, depending on the actual and the desired mean partial gravity value. The algorithm basically stops the Cell Proliferation is Decreased in Correlation With RPM at a predefined position if the mean gravity value is Reduced Partial Gravity Levels smaller than desired and it continuous in random walk if the mean gravity value is larger. In order to suppress too frequent One of the most prominent consequences of manned space switches between static and random walk, the RPM has to travel is muscle atrophy (Vandenburgh et al., 1999). It is spend a minimum amount of time in random walk until it therefore relevant to examine how muscle maintenance is can switch back again to static mode. This motion pattern compromised by prolonged exposure to reduced gravita- allows the simulation of partial gravity values close to 1 g. At tional forces. C2C12 mouse muscle cells were grown either 1184 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 under simulated microgravity (0 g), simulated partial gravity that the cells divide faster at higher partial gravity levels, (0.2, 0.4, and 0.6 g), or at terrestrial gravity (1 g), and their accordingly losing fluorescence intensity. However, with the total numbers were quantified 24 h afterwards. The three Fixed Static Intervals algorithm, there was no correlation with different algorithms for partial gravity simulation (see the partial gravity level of exposure, as the normalized MFI Materials and Methods) were compared to gain insights in varied between 176.53 25.19% at 0.2 g, 183.46 4.58% at their potential to influence the induction of biological effects 0.4 g, and 161.23  7.74% at 0.6 g. on the cells. By normalizing cell growth to the 1 g control, an increased proliferation rate is visible as the partial gravity level Simulated Partial Gravity Does Not Cause an Increase in gets bigger, where the values increased linearly from Cell Death Rate 43.08 5.71% at 0 g to 100% at 1 g when using the Two Velocities algorithm (Fig. 4A). Similarly, the Flexible Static The possibility of whether the decrease in cell propagation Interval algorithms also produced analogous results though observed in reduced partial gravity levels was due to an with smaller values. However, with the Fixed Static Interval increased cell death under these conditions was investigated. algorithm, the values varied between 52.13 6.44% at 0.2 g, After growing myoblasts under simulated partial gravity or at 48.71 2.72% at 0.4 g, and 54.70 5.16% at 0.6 g, thus no 1 g, no significant difference was observed between all the relation between the simulated partial gravity level and the cell samples, as they all exhibited nearly 100% viability, counts was established. These effects were confirmed by using irrespective of the type of algorithm used to simulate partial the alternative analysis method for cell proliferation, CFSE gravity (Fig. 5). In agreement with our previous results, this staining, where the median fluorescence intensity (MFI) was data indicates that another mechanism such as the cell cycle determined. The normalized MFI decreased linearly from progression plays a more substantial role in determining the 269.56  66.25% at 0 g to 100% at 1 g when using the Two cell growth pace under partial gravity, as the decrease in cell Velocities and Flexible Static Intervals algorithms (Fig. 4B). proliferation rate was not due to enhanced cell death in Thus, cells grown on the RPI at increasing partial gravity levels response to gravitational unloading (Benavides Damm exhibit a steadily decrease in fluorescence intensity, meaning et al., 2013b). 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. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1185 Biotechnology and Bioengineering 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. Cells Accumulate at G2/M Phase in Relation to Partial samples. Subsequently, the effects we observed are caused Gravity Exposure merely by simulated partial gravity exposure. As simulated microgravity slows down the cell cycle Lymphocyte Activation Rate is Dependent on the Level of progression of myoblasts (Benavides Damm et al., 2013a), Partial Gravity Exposure we hypothesized that the decelerated proliferation rate observed in myoblasts grown under partial gravity is also It has been known for decades that the activation of due to retardation in the cell cycle. As the Two Velocities lymphocytes is severely impaired under microgravity con- algorithm showed the most consistent relation between the ditions in space (Bechler et al., 1992; Cogoli et al., 1984). We level of simulated gravity exposure and proliferation, only therefore investigated a potential relation between the this algorithm was tested for cell cycle analysis. In this case, different levels of simulated gravity ranging from 0.2 to the percentage of cells in G0/G1 and S phases increased 0.6 g (by using the three algorithms introduced) and the (Fig. 6A) while the percentage in G2/M phase decreased activation rate of lymphocytes. Cells activated in the presence (Fig. 6B) as the partial gravity values augmented. Specifically, of simulated 0.2 g using the Two Velocities algorithm were as the percentage of cells accumulated within the G2/M phase poorly activated as the 0 g samples, whereas the cells activated were 38.95  6.54% at 0 g, 31.68  4.97% at 0.2 g, at simulated 0.6 g responded similarly to the 1 g control, 26.95  4.87% at 0.4 g, 21.55  3.33% at 0.6 g, and standardized as 100% of activation (Fig. 7). Interestingly, the 16.40  1.98% in the 1 g control. By fitting the data into a activation level of the 0.4 g exposed cells was almost in the linear regression model (Fig. 6C), a correlation value equal to middle of the two other gravity levels (0.2 and 0.6 g). 0.98 was obtained, indicating the close relation between G2/ Although the level of gravity was correlated with the level of M phase accumulation and the simulated partial gravity level. lymphocyte activation in all three simulation modes, the It is remarkable that the same effect observed in cell effect of simulated partial gravity using the Two Velocities proliferation is also detected in the cell cycle analysis, where algorithm led to an analogous correlation pattern compared the magnitude of the effect depends on the extent of partial to the results gathered with the C2C12 cells. With this gravity exposure. As we have previously shown, the slowed algorithm, the percentage of activated cells went from proliferation observed in the mouse myoblasts might thus be 39.29  6.32% at 0.2 g, to 68.20  23.09% at 0.4 g, and explained by a delay in the G2/M phase progression after 10 h 80.60  21.29% at 0.6 g. of cultivation on the RPM (Benavides Damm et al., 2013b). To discard the possibility that the particular protocol Discussion followed when performing experiments on the RPI causing false positive results, two control experiments were con- Our upgraded RPI offers the possibility of studying cellular ducted at the same time with cells cultured at 1 g. Therefore, systems during simulated microgravity as well as partial additional samples were placed inside a standard laboratory gravity environments, an advantage over previous micro- CO incubator filled with culture media (Ctrl) and under gravity simulating devices like the classical RPM where a normal culture conditions (Inc). The analysis showed no variety of gravity levels was not possible. Mounting the difference between these two controls, indicating that our incubator on the rotating frame instead of placing a small experimental protocol does not lead to an artifact on the RPI RPM into an incubator brings several advantages not yet 1186 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 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 þ 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. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1187 Biotechnology and Bioengineering found with other products, such as: the volume available for relation between the proliferation rate and the gravity level in tests is larger, offering therefore more samples to be tested the myoblasts was observed only when using the Two under identical conditions at the same time; the sample Velocities and the Flexible Static Intervals algorithms. contamination originating from the machinery is completely Perhaps the Fixed Static Intervals algorithm triggers an effect avoided (e.g., through outgassing/offgassing from oil-grease in these cells that seems to be threshold dependent, where the lubrication, from the electronic circuitry and its cabling, and cells respond only after reaching this threshold, regardless of from wear debris); the test conditions can be controlled much the actual gravity value that is simulated by the RPI. The better as the essential parameters (like temperature, CO different reactions of the two types of cells (myoblasts and levels, etc.) are more homogeneously distributed because lymphocytes) might be due to the fact that they are adherent there is no heat dissipation and unplanned ventilation by a and free floating cells, respectively. Therefore, in the Fixed rotating motorized equipment in the incubator; the addition Static Intervals algorithm, the fixed time in static position of supplementary functions, such as daylight and UV might be more easily recognized by adherent cells than by free illumination, do not interfere with the machinery exigencies floating cells. In this case, it could be that the time the installed in the incubator. myoblasts spent at 1 g was already long enough to trigger the By illustrating the behavior of cells at different gravitational normal terrestrial response in a percentage of the cells, loads, our RPI helps to unravel how partial gravity affects the whereas the time spent at 0 g could trigger the opposite effect biological processes necessary to sustain life in different space on the remaining percentage of cells, averaging as a result of a scenarios, such as on the Moon or Mars. Furthermore, this time duration threshold. knowledge facilitates the comprehension of the mechano- Calcium signals control various cellular responses in skeletal biological processes that happen within each cell on earth, muscle as well as in lymphocytes (Bassel-Duby and from sensing the diverse gravitational forces to transducing Olson, 2006; Clapham, 2007; Lewis, 2001). Indeed, we the signals through various pathways to elicit an appropriate previously described how disturbed calcium entry through response. Defects in mechanotransduction may appear as mechanosensitive channels affects proliferation, cell cycle deficits in cellular structure and organization, impairing progression, and gene expression during simulated micro- mechanosensation, or as mutations or deregulations in the gravity (Benavides Damm et al., 2013b). The difference in proteins involved in the downstream signaling pathways, cellular responses between the three algorithms tested for directly affecting the normal mechanoresponse. In order to partial gravity could therefore be caused by diversions in improve our understanding of such faulty responses and calcium entry which impinge on downstream signaling consequently develop efficient therapeutic strategies against pathways. Thus on the one hand, the cells would sense the the related diseases it is important to identify the molecular partial gravity level at which they are exposed through calcium components encompassing normal and defective mechano- channels, and depending on the algorithm, this could be transduction. The omnipresent gravitational force can be enough to elicit a linear response according to the gravitational regarded as a physical force influencing cells, tissues, and force where cells respond gradually to the mechanical organs as other forces do, gravity thus exerts a mode of unloading. On the other hand, the stimulation could trigger mechanical input to cells that can have profound biological an effect on calcium entry only when a certain threshold is implications. Studying the behavior of cells cultured under reached, impounding the expected cellular response. different gravitational loads broadens our understanding of Though difficult to correlate with our results from single disorders related to mechanical stimulation relevant not only cell behavior, studies performed on whole organisms have for astronauts exposed to low gravity, but also for patients on shown a similar trend when measuring various parameters in earth suffering for example from muscle wasting disorders or different partial gravity environments. During parabolic a weak immune system. Our experimental data obtained here flights, human subjects showed that the external work from two different cell types (mouse skeletal myoblasts and required to walk a given distance is significantly less at lower human lymphocytes) demonstrated their sensitivity to the gravity (Cavagna et al., 2000). Consistently, a study done on gravitational environment by proving a strong correlation suspended subjects with a strapped harness reported that the between the different levels of simulated gravity exposure and vertical, forward, and total external work per stride also the observed biological effect in a reproducible manner. declined following a linear trend with diminishing gravity Three different algorithms for simulating partial gravity (Griffin et al., 1999). In a similar study performed on were tested and the cellular response was compared. On the suspended mice, the peak hind limb force was reduced as well one hand, the mouse myoblasts showed a reduction in the linearly with decreasing gravity values (Wagner et al., 2010). proliferation rate, while cells accumulated in the G2/M phase As for experiments performed on humans underwater with a of the cell cycle in parallel with decreasing values of simulated treadmill and an adjustable ballasting harness, the results are gravity (Figs. 4 and 6). On the other hand, the activation rate in agreement with the data mentioned above (Newman and of human lymphocytes dropped with a decline in gravity level Alexander, 1993). Research focusing on load-carrying in (Fig. 7). In addition, data obtained from the lymphocyte partial gravity also by underwater immersion confirmed that experiment demonstrated a strong correlation between the energy expenditure and stride rates dropped while the level of activation and the magnitude of the simulated gravity forward lean exhibited by subjects increased dramatically field regardless of the type of algorithm applied. In contrast, a with diminishing gravity levels (Wickman and Luna, 1996). 1188 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 Recently, the possibility of humans running in place on have been used so far to simulate partial gravity include water was investigated, where the subjects, suspended with a ballasted underwater immersion, parabolic flights, over-head harness, experienced different levels of partial gravity just suspension, off-loading pulley systems, and tilted bed rest above the water. The predicted available impulse and the studies (Louisy et al., 1994; Newman, 2000). However, all of number of successful subjects that could generate enough these have specific advantages and disadvantages. On the one muscle power to run in place over a wading pool decreased hand, the over-head suspension, off-loading pulley systems, linearly with increasing gravity levels (Minetti et al., 2012). and tilted bed rest studies are economical techniques, but Other studies performed on humans, also under suspension, they are not suitable for cell culture analyses and they restrict have focused on the performance of chest compressions the freedom of movement in humans and animals. While under partial and microgravity. In both reports, the mean underwater immersion has no constraints on movement, it frequency of external chest compression did not result in a entails an inconvenient hydrodynamic drag. On the other pattern relative to the level of gravity exposure in either male hand, parabolic flights offer freedom of movement, with no or female subjects (Dalmarco et al., 2006; Kordi et al., 2012). hydrodynamic drag, and are appropriate for cell culture On another area of research, investigators studied the human studies, but they are expensive and the partial gravity perception of verticality during a recent parabolic flight exposure only lasts for a short duration (ranging approxi- campaign of partial gravity. In this case, the authors found mately from 20 to 40 s). Moreover, on-board centrifuge out that the gravity level must exceed a certain threshold to be facilities inside a spacecraft have been suggested to enable recognized as a reference for verticality, which was of 0.3 g, investigators to study the effects of partial gravity (Wagner depicting an onset where the effect of gravity exposure was and Fulford-Jones, 2006). This comes, however, at an detected (de Winkel et al., 2012). expensive cost and is limited to a few experiments only, In summary, there are three different outcomes that can be restricting the amount of data that can be gathered. For these detected with changing partial gravity values: a linear effect, a reasons, our newly designed and built RPI is a breakthrough result that depends on a certain threshold, and no correlation tool that will enable scientists to investigate partial gravity on between the exposure level and the response. As whole cell cultures and small organisms for up to a long duration at organisms respond differently than single cells, an explana- the gravity field of interest, such as that of the Moon or Mars, tion for what we observe cannot be attained from the studies with countless reproducible experiments and at an economic mentioned above. For that reason, our new RPI uniquely cost. In addition to broadening the scope of available tools to provides the advantage of studying single cell behavior, study the behavior of biological systems affected by space something that was not possible until now. In this way, our travel, our RPI can be employed as a method to explore the RPI mediates a valuable tool to perform cell experiments effects of mechanical unloading on cells through partial under partial gravity environments where the exact mecha- gravity, being a relevant model system with which to better noresponse that gives rise to specific signals in each individual understand the cellular responses subsequent to mechano- cell can be investigated as a reaction from mechanical transduction. Studying the underlying mechanisms that unloading. regulate muscle development and the immune system activation under partial gravity exposure is crucial for the identification of the causes and countermeasures for trauma and earth-bound diseases as well as to improve the Conclusions sustainment of human life in space. We are introducing a novel instrument called RPI for 3D cell The authors wish to gratefully acknowledge the technical assistance of culturing that in addition to growing cells under simulated Dr Malgorzata Kisielow and Ms Anette Schütz from the Flow microgravity, allows the generation of a partial earth-like Cytometry Laboratory (ETHZ), and of Mr Stéphane Richard from the gravity field of various values. Therefore, studies of cell Space Biology Group (HSLU). We would also like to thank Prof growth behavior under a nullified gravity vector environment Dr. Ralph Müller from the Institute for Biomechanics (ETHZ) for insightful comments. This work was supported by the European as well as under different simulated gravity fields (from 0 to Space Agency (ESA). 1 g) are now possible. Such a technique opens new aspects of biotechnology and enables the study of fundamental cellular mechanisms modulating mechanically-regulated tissue de- References velopment and maintenance. Baharvand H, Hashemi SM, Kazemi Ashtiani S, Farrokhi A. 2006. In our frame of study there appears to be two different Differentiation of human embryonic stem cells into hepatocytes in responses to changes in partial gravity, one being set by a 2D and 3D culture systems in vitro. Int J Dev Biol 50(7):645–652. threshold from where the effect can be detected, and the other Bassel-Duby R, Olson EN. 2006. Signaling pathways in skeletal muscle one varying in proportion to the gravity exposure level. In remodeling. Annu Rev Biochem 75:19–37. Bechler B, Cogoli A, Cogoli-Greuter M, Muller O, Hunzinger E, Criswell SB. this report, we have unraveled the possibility of exploring the 1992. Activation of microcarrier-attached lymphocytes in microgravity. effects of partial gravity on biological samples at an earth- Biotechnol Bioeng 40(8):991–996. bound facility in a comparable manner. To our knowledge, Benavides Damm T, Franco-Obregon A, Egli M. 2013a. Gravitational force this is the first time simulated partial gravity has been modulates G 2/M phase exit in mechanically unloaded myoblasts. Cell achieved by using a RPM-like device. The techniques that Cycle 12(18):3001–3012. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1189 Biotechnology and Bioengineering Benavides Damm T, Richard S, Tanner S, Wyss F, Egli M, Franco-Obregon A. Minetti AE, Ivanenko YP, Cappellini G, Dominici N, Lacquaniti F. 2012. 2013b. Calcium-dependent deceleration of the cell cycle in muscle cells Humans running in place on water at simulated reduced gravity. PLoS by simulated microgravity. FASEB J 27(5):2045–2054. ONE 7(7):e37300. Block I, Briegleb W, Wohlfarth-Bottermann KE. 1986. Gravisensitivity of the Newman DJ. 2000. Life in extreme environments: How will humans perform acellular slime mold Physarum polycephalum demonstrated on the fast- on Mars? Gravit Space Biol Bull 13(2):35–47. rotating clinostat. Eur J Cell Biol 41:44–50. Newman DJ, Alexander HL. 1993. Human locomotion and workload for Cavagna GA, Willems PA, Heglund NC. 2000. The role of gravity in human simulated lunar and Martian environments. Acta Astronaut 29(8):613–620. walking: Pendular energy exchange, external work and optimal speed. J Pavy-Le Traon A, Allevard AM, Fortrat JO, Vasseur P, Gauquelin G, Guell A, Physiol 528(Pt 3):657–668. Bes A, Gharib C. 1997. Cardiovascular and hormonal changes induced Chang TT, Walther I, Li CF, Boonyaratanakornkit J, Galleri G, Meloni MA, by a simulation of a lunar mission. Aviat Space Environ Med 68(9):829– Pippia P, Cogoli A, Hughes-Fulford M. 2012. The Rel/NF-kappaB 837. pathway and transcription of immediate early genes in T cell activation Pietsch J, Sickmann A, Weber G, Bauer J, Egli M, Wildgruber R, Infanger M, are inhibited by microgravity. J Leukoc Biol 92(6):1133–1145. Grimm D. 2012. Metabolic enzyme diversity in different human thyroid Clapham DE. 2007. Calcium signaling. Cell 131(6):1047–1058. cell lines and their sensitivity to gravitational forces. Proteomics 12(15– Cogoli A, Tschopp A, Fuchs-Bislin P. 1984. Cell sensitivity to gravity. Science 16):2539–2546. 225(4658):228–230. Rivera-Solorio I, Kleis SJ. 2006. Model of the mass transport to the surface of Dalmarco G, Calder A, Falcao F, de Azevedo DF, Sarkar S, Evetts S, Moniz S, animal cells cultured in a rotating bioreactor operated in micro gravity. Russomano T. 2006. Evaluation of external cardiac massage performance Biotechnol Bioeng 94(3):495–504. during hypogravity simulation. Conf Proc IEEE Eng Med Biol Soc Saltzman WM, Parkhurst MR, Parsons-Wingerter P, Zhu WH. 1992. Three- 1:2904–2907. dimensional cell cultures mimic tissues. Ann NY Acad Sci 665:259–273. de Winkel KN, Clement G, Groen EL, Werkhoven PJ. 2012. The perception of Schwarz RP, Goodwin TJ, Wolf DA. 1992. Cell culture for three-dimensional verticality in lunar and Martian gravity conditions. Neurosci Lett modeling in rotating-wall vessels: An application of simulated 529(1):7–11. microgravity. J Tissue Cult Methods 14(2):51–57. Freed LE, Vunjak-Novakovic G. 1995. Cultivation of cell-polymer tissue Schwarzenberg M, Pippia P, Meloni MA, Cossu G, Cogoli-Greuter M, Cogoli constructs in simulated microgravity. Biotechnol Bioeng 46(4):306–313. A. 1999. Signal transduction in T lymphocytes—A comparison of the Friedrich UL, Joop O, Putz C, Willich G. 1996. The slow rotating centrifuge data from space, the free fall machine and the random positioning microscope NIZEMI–a versatile instrument for terrestrial hypergravity machine. Adv Space Res 24(6):793–800. and space microgravity research in biology and materials science. J Sonnenfeld G. 2012. Editorial: Space flight modifies T cellactivation-role of Biotechnol 47(2–3):225–238. microgravity. J Leukoc Biol 92(6):1125–1126. Griffin TM, Tolani NA, Kram R. 1999. Walking in simulated reduced gravity: Sun T, Jackson S, Haycock JW, MacNeil S. 2006. Culture of skin cells in 3D Mechanical energy fluctuations and exchange. J Appl Physiol 86(1):383– rather than 2D improves their ability to survive exposure to cytotoxic 390. agents. J Biotechnol 122(3):372–381. Herranz R, Benguria A, Laván DA, López-Vidriero I, Gasset G, Javier Medina van Loon JJWA. 2007. Some history and use of the random positioning F, Van L, Jwa J, Marco R. 2010. Spaceflight-related suboptimal conditions machine, RPM, in gravity related research. Adv Space Res 39(7):1161– can accentuate the altered gravity response of Drosophila transcriptome. 1165. Mol Ecol 19(19):4255–4264. Vandenburgh H, Chromiak J, Shansky J, Del Tatto M, Lemaire J. 1999. Space Hoson T, Kamisaka S, Masuda Y, Yamashita M, Buchen B. 1997. Evaluation of travel directly induces skeletal muscle atrophy. FASEB J 13(9):1031– the three-dimensional clinostat as a simulator of weightlessness. Planta 1038. 203(Suppl):S187–S197. Wagner EB, Fulford-Jones TR. 2006. Sensorimotor investigations for the Infanger M, Kossmehl P, Shakibaei M, Bauer J, Kossmehl-Zorn S, Cogoli A, Mars Gravity Biosatellite: A rotating spacecraft for partial gravity Curcio F, Oksche A, Wehland M, Kreutz R, Paul M, Grimm D. 2006. research. Brain Res 1091(1):75–78. Simulated weightlessness changes the cytoskeleton and extracellular Wagner EB, Granzella NP, Saito H, Newman DJ, Young LR, Bouxsein ML. matrix proteins in papillary thyroid carcinoma cells. Cell Tissue Res 2010. Partial weight suspension: A novel murine model for investigating 324(2):267–277. adaptation to reduced musculoskeletal loading. J Appl Physiol Kordi M, Kluge N, Kloeckner M, Russomano T. 2012. Gender influence on 109(2):350–357. the performance of chest compressions in simulated hypogravity and Watson JV, Chambers SH, Smith PJ. 1987. A pragmatic approach to the analysis microgravity. Aviat Space Environ Med 83(7):643–648. of DNA histograms with a definable G1 peak. Cytometry 8(1):1–8. Lewis RS. 2001. Calcium signaling mechanisms in T lymphocytes. Annu Rev Wickman LA, Luna B. 1996. Locomotion while load-carrying in reduced Immunol 19:497–521. gravities. Aviat Space Environ Med 67(10):940–946. Louisy F, Guezennec CY, Guell A. 1994. Leg vein hemodynamics during Yip DK, Auersperg N. 1972. The dye-exclusion test for cell viability: Persistence bedrests simulating lunar trip. J Gravit Physiol 1(1):P100–P101. of differential staining following fixation. In Vitro 7(5):323–329. 1190 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biotechnology and Bioengineering Pubmed Central

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

Biotechnology and Bioengineering , Volume 111 (6) – Jan 22, 2014

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© 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.
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0006-3592
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10.1002/bit.25179
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Abstract

ARTICLE Cell Cultivation Under Different Gravitational Loads Using a Novel Random Positioning Incubator 1,2 1 1,3 3 ¨ ¨ Tatiana Benavides Damm, Isabelle Walther, Simon L. Wuest, Jorg Sekler, Marcel Egli CC Aerospace Biomedical Science & Technology, Space Biology Group, Lucerne University of Applied Sciences and Arts (HSLU), Hergiswil, Nidwalden, Switzerland Institute for Biomechanics, Eidgeno¨ssische Technische Hochschule Zu¨ rich (ETHZ), Zu¨ rich, Switzerland Institute for Automation Engineering, University of Applied Sciences and Arts Northwestern Switzerland (FHNW), Brugg-Windisch, Aargau, Switzerland KEYWORDS: partial gravity; random positioning machine; mechanical unloading; mechanotransduction; muscle cells; ABSTRACT: Important in biotechnology is the establishment lymphocyte activation 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 Introduction culture in simulated microgravity (0 g). In addition to Recent tissue engineering studies have revealed striking growing cells in a weightlessness-like environment, our RPI enables long-duration cell cultivation under various gravita- discrepancies between the behavior of cells cultured in 3D tional loads, ranging from close to 0 g to almost 1 g. This and 2D, where 3D cell cultures mimicked intact tissue in allows the study of the mechanotransductional process of terms of viability and gene expression (Baharvand et al., 2006; cells involved in the conversion of physical forces to an Sun et al., 2006). Indeed, 3D cell culture is one favorable appropriate biochemical response. Gravity is a type of approach for reducing the gap between artificial cultivation in physical force with profound developmental implications in cellular systems as it modulates the resulting signaling vitro and the in vivo physiological situation. The benefits of cascades as a consequence of mechanical loading. The 3D cell cultivation over 2D monolayers are featured due to experiments presented here were conducted on mouse the increased cell to cell contact, as well as cell to extracellular skeletal myoblasts and human lymphocytes, two types of matrix interaction, and the accumulation of nutrients and cells that have been shown in the past to be particularly growth factors (Saltzman et al., 1992). It has been shown that sensitive to changes in gravity. Our novel RPI will expand the horizon at which mechanobiological experiments are cell growth in 3D can be achieved through the randomized conducted. The scientific data gathered may not only movement employed by rotating bioreactors which simulate improve the sustainment of human life in space, but also microgravity (Freed and Vunjak-Novakovic, 1995). Further- lead to the design of alternative countermeasures against more, this platform provides a constant circulation of diseases related to impaired mechanosensation and down- nutrient medium, removing the higher waste concentrations stream signaling processes on earth. that are usually found in the microenvironment adjacent to Biotechnol. Bioeng. 2014;111: 1180–1190. the cell surface (Rivera-Solorio and Kleis, 2006). 2013 The Authors. Biotechnology and Bioengineering The first instruments used to simulate microgravity were Published by Wiley Periodicals, Inc. clinostats, developed in 1879, a long time before the beginning of space exploration, by Julius von Sachs, a botanist who wanted to investigate gravitropism in plants This is an open access article under the terms of the Creative Commons Attribution- (van Loon, 2007). The uni-axial clinostat worked by slowly NonCommercial-NoDerivs License, which permits use and distribution in any medium, rotating the specimens around a longitudinal horizontal axis. provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. In this way, for example, the growing plant was experiencing Correspondence to: M. Egli a continuously reoriented gravity vector over a long period of Contract grant sponsor: European Space Agency (ESA) time. Later on, fast rotating clinostats were developed and Received 10 September 2013; Revision received 26 November 2013; Accepted 23 December 2013 introduced to negate the gravitational force on cells growing Accepted manuscript online 30 December 2013; in culture media. In order to study such cells, the diameter of Article first published online 22 January 2014 in Wiley Online Library the rotating part of the clinostat had to be small to avoid the (http://onlinelibrary.wiley.com/doi/10.1002/bit.25179/abstract). DOI 10.1002/bit.25179 generation of centrifugal forces, and the rotational speed had 1180 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014  2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. to be much faster (40–100 rpm). In the fast rotating clinostat, this machine, environments from close to 0 to almost 1 g can research on single mammalian cells and unicellular organ- be generated by several algorithms. A machine of this type isms could be performed since small samples no longer increases the variety of experiments which can be tested in experience the turning of gravity as they are in simulated general and additionally allows the exploration of effects on weightlessness (Block et al., 1986). Limitations of the fast biological samples in conditions which are difficult to create, rotating clinostat motivated the development of other such as the gravity field of the Moon or Mars. The data instruments that allowed larger samples to be cultivated reported in this study was gathered with mouse skeletal under simulated microgravity. The rotating wall vessel myoblasts (adherent cells) and human lymphocytes (non- (RWV) is one example of such a device that provides an adherent cells), in an attempt to better understand how environment with low-shear forces, allowing cells to grow deconditioning the cells through mechanical unloading by even as 3D aggregates (Schwarz et al., 1992). Since the late reducing gravity affects two major physiological mechanisms: nineties, a 3D clinostat called a random positioning machine the skeletal muscle and the immune system. These two (RPM) has been intensively used for ground-based research. mammalian cell types have repeatedly demonstrated high The RPM, originally developed by T. Hoson in Japan, applies sensitivity to simulated microgravity (Benavides Damm the principle of neutralizing gravity by vector averaging et al., 2013b; Chang et al., 2012; Sonnenfeld, 2012). As gravity (Hoson et al., 1997). Numerous experiments have already provides a stimulus for their maintenance, we hypothesized proven that this technique generates data that is indeed very that partial gravity exposure would produce changes in their similar to the data obtained in space (Herranz et al., 2010; behavior and sought to investigate whether this response was Infanger et al., 2006; Pietsch et al., 2012; Schwarzenberg proportionally linear, nonlinear, or set by a threshold with et al., 1999). decreasing gravity levels. The answer to this question is As access to real weightlessness in space is very limited essential, not only for aerospace physiology, but also and expensive, scientists have always been interested in necessary in order to comprehend the effects of mechanical having earth-bound tools available to perform pilot unloading associated with clinical scenarios on earth, such as studies in simulated microgravity. These instruments aging and illness arising from immobilization and prolonged are of great importance to test hardware and processes periods of bed rest. under microgravity approaching conditions and in studying biological systems under these specificsettings. Materials and Methods The RPM, though not totally equal to real space, is a good tool for preparing space experiments, for testing newly Random Positioning Incubator (RPI) developed instruments, and for studying biological systems of interest under simulated weightlessness, as The RPI equipment consists of two independently rotating well as for gathering enough valuable data that can be used gimbal-mounted frames, each axis being motorized by an for statistical purposes. Nevertheless, one of the major electrical drive to control random, or other positioning, constraints of the RPM so far has been that no simulated sequences. Through a dedicated algorithm the samples are partial gravity values could be attained. With the constantly reoriented with respect to the earth gravity vector. increasing interest of the scientific community to return This allows the distribution of the vector in space, leading mantothe Moon andtogofurther in theexploration of over time to an average gravity value smaller than 1 g. A Mars, studies performed with these two gravity fields unique feature of our present RPM, the RPI version, is the (0.16 g and 0.38 g, respectively) are becoming increasingly integration of a miniaturized CO incubator including a 14 L valuable. At the present time these types of studies have test chamber, mounted at the center of rotation of both been performed in head-up tilt studies, in body suspen- frames (Fig. 1). The specially functionalized incubator is sion devices, and for a short duration in parabolic flights supplied with electrical power and CO (and other (Cavagna et al., 2000; Dalmarco et al., 2006; Pavy-Le Traon experimental supplies) throughout the experiment’s dura- et al., 1997). Long-term experiments at a cellular level tion. Important data such as temperature, CO levels, and have not yet been possible except when performed in a mean gravity values are monitored automatically. Our centrifuge in real space such as during the IML-2 mission present RPI will be described in more details elsewhere in 1994 with the NIZEMI slow rotating microscope (manuscript under review). (Friedrich et al., 1996). As well as using partial gravity for space related studies, having various gravitational envi- Myoblasts ronments available for conducting research opens new approaches to investigate the mechanobiology of cells C2C12 mouse skeletal myoblasts were obtained from ATCC ranging from single mammalian cells to microorganisms. (American Type Culture Collection, LGC Standards S.a.r.l., In this paper, we present a newly designed and built RPM Molsheim Cedex, France) and cultured in a medium equipped with an integrated incubator, thus called random consisting of Dulbecco’s Modified Eagle Medium (Life positioning incubator (RPI) that in addition to generating Technologies, Lucerne, Switzerland) supplemented with simulated microgravity, offers new features such as partial 20% fetal bovine serum (PAA, Basel, Switzerland) and gravity simulation during an extended period of time. Using 2mM L-Glutamine (Life Technologies) in a humidified Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1181 Biotechnology and Bioengineering CFSE is a dye that passively diffuses into cells, reacting with intracellular amines and forming fluorescent conjugates. Following initial staining of the mother cells, subsequent rounds of cell division dilute the amount of dye inherited by daughter cells by one half with each division. For cell cycle analysis, cells were collected after 10 h of culture on the RPI or on the incubator and fixed. Samples were stored at 20 Cin 70% ice cold ethanol for at least 12–18 h prior to staining. Cell cycle was analyzed based on DNA content through propidium iodide (PI) with RNase staining by FACS. CFSE fluorescence intensity and cell cycle profiles were analyzed and quantified with the FlowJo flow cytometry analysis software (TreeStar, Inc., Ashland, OR). To quantify G0/G1, S, and G2/M populations, the Watson model was used to fit the histograms of single gated cells (Watson et al., 1987). FACS Calibur (BD Biosciences) was used for acquisition and appropriate settings for forward and side scatter gates were applied to examine 20,000 events per sample. The blue laser with excitation at 488 nm was used, with FL1 channel detector for CFSE and FL2 channel detector for PI staining. Data (means  standard deviations) were obtained from three or four independent series of experiments. Lymphocytes Whole peripheral blood was obtained from healthy human volunteers. Peripheral blood lymphocytes (PBL) were Figure 1. A recent version of the random positioning incubator (RPI) as designed isolated using Ficoll gradient (Histopaque; Sigma, Buchs, and developed by the project partner FHNW based on collaboration and requirement Switzerland), red blood cells were lysed and finally T-cells specifications of ETHZ. The RPI consists of two gimbal-mounted frames, which are were purified further using enrichment columns as per the driven by electrical motors, and a CO incubator mounted at the center of rotation of both frames. manufacturer’s instructions (R&D Systems, Abingdon, United Kingdom). T-cells were re-suspended into RPMI 1640 (Life Technologies) with 10% fetal calf serum (Life atmosphere at 37 Cin7%CO . Cells were passed every Technologies). The cells were loaded at a concentration of 1 2 days and were only used for experimentation before the to 2  10 cells/mL inside the “LYCIS” hardware, which has 15th passage. Myoblasts were seeded at 8,000 cells/cm in been developed specially for space experiments. This 25 cm flasks (Semadeni, Ostermundigen, Switzerland) 15 h hardware is based on the principle of moving pistons that before the start of each experiment. Immediately before being allows the loading of cells in the absence of air bubbles and placed on the RPI, the flasks were carefully filled with media, the injection of substances rapidly without opening the avoiding the formation of bubbles. Cell counting was hardware. The cells were preconditioned at each experi- performed manually with a hemocytometer (Biosystems, mental environment for 60 to 90 min prior to activation Nunningen, Switzerland) on trypan blue (Life Technologies) with a concanavaline A (conA)/anti CD28 mixture (BD treated cells to assess cell concentration and viability, Biosciences) for T-cells or only with conA (Sigma) for the according to the dye exclusion method (Yip and PBL. After 20 to 22 h of incubation at 37 Cunder the Auersperg, 1972). The counts were carried out in duplicate respective gravity conditions, the cells were harvested and per independent sample after 24 h of growth on the RPI or stained. The staining was carried out according to the inside an incubator under normal culture conditions (1 g supplier using PE-CD25 marker (Miltenyi Biotec, Bergisch control). Cell growth was also monitored with the CellTrace Gladbach, Germany) which is an early marker for CFSE Cell Proliferation Kit (Life Technologies), according to lymphocyte activation. Stained cells were analyzed on the the manufacturers’ protocol. Briefly, cells were stained with FACS Calibur using 10,000 events per sample. The FlowJo 5 mM carboxyfluorescein diacetate, succinimidyl ester (often software was used to gate the cells and fitthe histograms. called CFSE) in phosphate buffered saline þ 0.1% bovine The 1 g samples of each experiment were considered as serum albumin at 37 C for 10 min. Cells were plated and 100% activated, and were used to standardize the other collected after 24 h of growth under different gravitational samples among the different experiments. A non-activated conditions, and then re-suspended in 3.7% Cell Fix (BD sample wasusedasnegativecontrol.Data(means Biosciences, Allschwil, Switzerland) for overnight storage at standard deviations) were obtained from three independent 4 C before fluorescence activated cell sorting (FACS) analysis. series of experiments. 1182 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 Algorithms will result. There are numerous motion patterns possible to create simulated partial gravity by fulfilling the above Simulated microgravity on a RPM is achieved by gravity equations. We developed and used three different algorithms; vector averaging to zero as described earlier (Hoson all of them based on a random walk algorithm described et al., 1997). Thereby the earth gravity vector is distributed elsewhere (manuscript under review). In brief, this random over time by constantly reorienting the samples to random walk algorithm rotates both frames with constant rotational positions, where eventually the mean gravity will converge to velocity and inverts the direction of rotation (forward or a small value. As described below, we adjusted the backward) at random times. The velocity transition takes distribution of the gravity vector in order to obtain a desired place at a constant rotational acceleration. This unmodified mean gravity value. As a quality measure for the distribution random walk algorithm was employed for the 0 g experi- of orientation we use the mean gravity over time, defined by ments. The simulated partial gravity level that can be qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi achieved is limited to a certain maximum that depends on the 2 2 2 G ¼ ðG Þ þðG Þ þðG Þ ð1Þ mean X;mean Y;mean Z;mean algorithm used and the corresponding parameters. In addition, the accuracy and the time necessary to reach the Here, the mean gravity values in the three directions are desired partial gravity value is algorithm-dependent (Fig. 2). defined as follows: The stability of our applied algorithms was verified with P P n n numerical simulations (Fig. 3). In this study, we used the g g i¼1 X;i i¼1 Y;i G ¼ ; G ¼ ; G X;mean Y;mean Z;mean following three different algorithms: n n i¼1 Z;i ¼ ð2Þ Simulated Partial Gravity Based on Random Walk With Two Velocities (Two Velocities) If the pattern of random positions are chosen such that the earth vector spends more time in one particular area To achieve a particular mean gravity value, the local vector of (considered in the local frame of the sample), partial gravity one direction is enlarged by making the rotation velocity 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. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1183 Biotechnology and Bioengineering 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. position dependent. In principle, the RPM slows down, when high partial gravity values (>0.6 g) however, one has to keep this vector is pointing downward. Thus, the RPM spends in mind that the samples would be standing still for most of slightly more time in one direction than in the others. The the time. relation of the nominal to the slower velocity determines the resulting mean gravity. Since the random walk algorithm is Simulated Partial Gravity Based Random Walk and Fixed based on random numbers, two consecutive runs will not Static Intervals (Fixed Static Intervals) reproduce the same result. Therefore, the velocity relation is controlled by a predictive controller: While the nominal (or This algorithm works like the Flexible Static Intervals faster) velocity is fixed, the slower velocity is initially guessed algorithm, except that the time spent in random walk or based on numerical simulations. The RPM runs then for 1 h static position is fixed and constant throughout the such that the mean gravity value can be stabilized. Based on experiment. The relation of the rotating to the static time the deviation from the desired mean gravity, the value of the determines the resulting mean gravity value. For example, if lower velocity is refined. The RPM subsequently runs again the frames rotate for 40% of the time in random walk and are for 1 h, after which the slower velocity is again corrected. This static for 60% of the time, a mean gravity of 0.6 g will result. interval of letting the mean gravity stabilize for 1 h with a In our experiments we used a cycling period of 2.5 min (e.g., subsequent refinement of the slower velocity is repeated until for a 0.6 g experiment the RPM spends 1 min in random the end of the experiment. We were able to achieve up to 0.6 g walk, followed by 1.5 min standing still, where the cycle is reliably, which is close to the upper limit. This algorithm continuously repeated). This algorithm, again, allows reach- ensures that the frames are always in motion. However, it takes ing partial gravity values close to 1 g. However, the samples at least 2 h to achieve the desired partial gravity value (Fig. 2). would be standing still for most of the time at high partial gravity values (>0.6 g) as is the case when applying the Flexible Static Intervals algorithm. Simulated Partial Gravity Based Random Walk and Flexible For all three partial gravity algorithms and the unmodified Static Intervals (Flexible Static Intervals) random walk, the nominal rotational velocity was 60 /s for Since random walk reduces the mean gravity value towards the myoblast experiments and 40 /s for the lymphocyte zero, this algorithm interleaves the random walk with static experiments. phases, whereby the frames are stopped at one particular position. The timing of the static phases is controlled by the Results computer online, depending on the actual and the desired mean partial gravity value. The algorithm basically stops the Cell Proliferation is Decreased in Correlation With RPM at a predefined position if the mean gravity value is Reduced Partial Gravity Levels smaller than desired and it continuous in random walk if the mean gravity value is larger. In order to suppress too frequent One of the most prominent consequences of manned space switches between static and random walk, the RPM has to travel is muscle atrophy (Vandenburgh et al., 1999). It is spend a minimum amount of time in random walk until it therefore relevant to examine how muscle maintenance is can switch back again to static mode. This motion pattern compromised by prolonged exposure to reduced gravita- allows the simulation of partial gravity values close to 1 g. At tional forces. C2C12 mouse muscle cells were grown either 1184 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 under simulated microgravity (0 g), simulated partial gravity that the cells divide faster at higher partial gravity levels, (0.2, 0.4, and 0.6 g), or at terrestrial gravity (1 g), and their accordingly losing fluorescence intensity. However, with the total numbers were quantified 24 h afterwards. The three Fixed Static Intervals algorithm, there was no correlation with different algorithms for partial gravity simulation (see the partial gravity level of exposure, as the normalized MFI Materials and Methods) were compared to gain insights in varied between 176.53 25.19% at 0.2 g, 183.46 4.58% at their potential to influence the induction of biological effects 0.4 g, and 161.23  7.74% at 0.6 g. on the cells. By normalizing cell growth to the 1 g control, an increased proliferation rate is visible as the partial gravity level Simulated Partial Gravity Does Not Cause an Increase in gets bigger, where the values increased linearly from Cell Death Rate 43.08 5.71% at 0 g to 100% at 1 g when using the Two Velocities algorithm (Fig. 4A). Similarly, the Flexible Static The possibility of whether the decrease in cell propagation Interval algorithms also produced analogous results though observed in reduced partial gravity levels was due to an with smaller values. However, with the Fixed Static Interval increased cell death under these conditions was investigated. algorithm, the values varied between 52.13 6.44% at 0.2 g, After growing myoblasts under simulated partial gravity or at 48.71 2.72% at 0.4 g, and 54.70 5.16% at 0.6 g, thus no 1 g, no significant difference was observed between all the relation between the simulated partial gravity level and the cell samples, as they all exhibited nearly 100% viability, counts was established. These effects were confirmed by using irrespective of the type of algorithm used to simulate partial the alternative analysis method for cell proliferation, CFSE gravity (Fig. 5). In agreement with our previous results, this staining, where the median fluorescence intensity (MFI) was data indicates that another mechanism such as the cell cycle determined. The normalized MFI decreased linearly from progression plays a more substantial role in determining the 269.56  66.25% at 0 g to 100% at 1 g when using the Two cell growth pace under partial gravity, as the decrease in cell Velocities and Flexible Static Intervals algorithms (Fig. 4B). proliferation rate was not due to enhanced cell death in Thus, cells grown on the RPI at increasing partial gravity levels response to gravitational unloading (Benavides Damm exhibit a steadily decrease in fluorescence intensity, meaning et al., 2013b). 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. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1185 Biotechnology and Bioengineering 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. Cells Accumulate at G2/M Phase in Relation to Partial samples. Subsequently, the effects we observed are caused Gravity Exposure merely by simulated partial gravity exposure. As simulated microgravity slows down the cell cycle Lymphocyte Activation Rate is Dependent on the Level of progression of myoblasts (Benavides Damm et al., 2013a), Partial Gravity Exposure we hypothesized that the decelerated proliferation rate observed in myoblasts grown under partial gravity is also It has been known for decades that the activation of due to retardation in the cell cycle. As the Two Velocities lymphocytes is severely impaired under microgravity con- algorithm showed the most consistent relation between the ditions in space (Bechler et al., 1992; Cogoli et al., 1984). We level of simulated gravity exposure and proliferation, only therefore investigated a potential relation between the this algorithm was tested for cell cycle analysis. In this case, different levels of simulated gravity ranging from 0.2 to the percentage of cells in G0/G1 and S phases increased 0.6 g (by using the three algorithms introduced) and the (Fig. 6A) while the percentage in G2/M phase decreased activation rate of lymphocytes. Cells activated in the presence (Fig. 6B) as the partial gravity values augmented. Specifically, of simulated 0.2 g using the Two Velocities algorithm were as the percentage of cells accumulated within the G2/M phase poorly activated as the 0 g samples, whereas the cells activated were 38.95  6.54% at 0 g, 31.68  4.97% at 0.2 g, at simulated 0.6 g responded similarly to the 1 g control, 26.95  4.87% at 0.4 g, 21.55  3.33% at 0.6 g, and standardized as 100% of activation (Fig. 7). Interestingly, the 16.40  1.98% in the 1 g control. By fitting the data into a activation level of the 0.4 g exposed cells was almost in the linear regression model (Fig. 6C), a correlation value equal to middle of the two other gravity levels (0.2 and 0.6 g). 0.98 was obtained, indicating the close relation between G2/ Although the level of gravity was correlated with the level of M phase accumulation and the simulated partial gravity level. lymphocyte activation in all three simulation modes, the It is remarkable that the same effect observed in cell effect of simulated partial gravity using the Two Velocities proliferation is also detected in the cell cycle analysis, where algorithm led to an analogous correlation pattern compared the magnitude of the effect depends on the extent of partial to the results gathered with the C2C12 cells. With this gravity exposure. As we have previously shown, the slowed algorithm, the percentage of activated cells went from proliferation observed in the mouse myoblasts might thus be 39.29  6.32% at 0.2 g, to 68.20  23.09% at 0.4 g, and explained by a delay in the G2/M phase progression after 10 h 80.60  21.29% at 0.6 g. of cultivation on the RPM (Benavides Damm et al., 2013b). To discard the possibility that the particular protocol Discussion followed when performing experiments on the RPI causing false positive results, two control experiments were con- Our upgraded RPI offers the possibility of studying cellular ducted at the same time with cells cultured at 1 g. Therefore, systems during simulated microgravity as well as partial additional samples were placed inside a standard laboratory gravity environments, an advantage over previous micro- CO incubator filled with culture media (Ctrl) and under gravity simulating devices like the classical RPM where a normal culture conditions (Inc). The analysis showed no variety of gravity levels was not possible. Mounting the difference between these two controls, indicating that our incubator on the rotating frame instead of placing a small experimental protocol does not lead to an artifact on the RPI RPM into an incubator brings several advantages not yet 1186 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 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 þ 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. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1187 Biotechnology and Bioengineering found with other products, such as: the volume available for relation between the proliferation rate and the gravity level in tests is larger, offering therefore more samples to be tested the myoblasts was observed only when using the Two under identical conditions at the same time; the sample Velocities and the Flexible Static Intervals algorithms. contamination originating from the machinery is completely Perhaps the Fixed Static Intervals algorithm triggers an effect avoided (e.g., through outgassing/offgassing from oil-grease in these cells that seems to be threshold dependent, where the lubrication, from the electronic circuitry and its cabling, and cells respond only after reaching this threshold, regardless of from wear debris); the test conditions can be controlled much the actual gravity value that is simulated by the RPI. The better as the essential parameters (like temperature, CO different reactions of the two types of cells (myoblasts and levels, etc.) are more homogeneously distributed because lymphocytes) might be due to the fact that they are adherent there is no heat dissipation and unplanned ventilation by a and free floating cells, respectively. Therefore, in the Fixed rotating motorized equipment in the incubator; the addition Static Intervals algorithm, the fixed time in static position of supplementary functions, such as daylight and UV might be more easily recognized by adherent cells than by free illumination, do not interfere with the machinery exigencies floating cells. In this case, it could be that the time the installed in the incubator. myoblasts spent at 1 g was already long enough to trigger the By illustrating the behavior of cells at different gravitational normal terrestrial response in a percentage of the cells, loads, our RPI helps to unravel how partial gravity affects the whereas the time spent at 0 g could trigger the opposite effect biological processes necessary to sustain life in different space on the remaining percentage of cells, averaging as a result of a scenarios, such as on the Moon or Mars. Furthermore, this time duration threshold. knowledge facilitates the comprehension of the mechano- Calcium signals control various cellular responses in skeletal biological processes that happen within each cell on earth, muscle as well as in lymphocytes (Bassel-Duby and from sensing the diverse gravitational forces to transducing Olson, 2006; Clapham, 2007; Lewis, 2001). Indeed, we the signals through various pathways to elicit an appropriate previously described how disturbed calcium entry through response. Defects in mechanotransduction may appear as mechanosensitive channels affects proliferation, cell cycle deficits in cellular structure and organization, impairing progression, and gene expression during simulated micro- mechanosensation, or as mutations or deregulations in the gravity (Benavides Damm et al., 2013b). The difference in proteins involved in the downstream signaling pathways, cellular responses between the three algorithms tested for directly affecting the normal mechanoresponse. In order to partial gravity could therefore be caused by diversions in improve our understanding of such faulty responses and calcium entry which impinge on downstream signaling consequently develop efficient therapeutic strategies against pathways. Thus on the one hand, the cells would sense the the related diseases it is important to identify the molecular partial gravity level at which they are exposed through calcium components encompassing normal and defective mechano- channels, and depending on the algorithm, this could be transduction. The omnipresent gravitational force can be enough to elicit a linear response according to the gravitational regarded as a physical force influencing cells, tissues, and force where cells respond gradually to the mechanical organs as other forces do, gravity thus exerts a mode of unloading. On the other hand, the stimulation could trigger mechanical input to cells that can have profound biological an effect on calcium entry only when a certain threshold is implications. Studying the behavior of cells cultured under reached, impounding the expected cellular response. different gravitational loads broadens our understanding of Though difficult to correlate with our results from single disorders related to mechanical stimulation relevant not only cell behavior, studies performed on whole organisms have for astronauts exposed to low gravity, but also for patients on shown a similar trend when measuring various parameters in earth suffering for example from muscle wasting disorders or different partial gravity environments. During parabolic a weak immune system. Our experimental data obtained here flights, human subjects showed that the external work from two different cell types (mouse skeletal myoblasts and required to walk a given distance is significantly less at lower human lymphocytes) demonstrated their sensitivity to the gravity (Cavagna et al., 2000). Consistently, a study done on gravitational environment by proving a strong correlation suspended subjects with a strapped harness reported that the between the different levels of simulated gravity exposure and vertical, forward, and total external work per stride also the observed biological effect in a reproducible manner. declined following a linear trend with diminishing gravity Three different algorithms for simulating partial gravity (Griffin et al., 1999). In a similar study performed on were tested and the cellular response was compared. On the suspended mice, the peak hind limb force was reduced as well one hand, the mouse myoblasts showed a reduction in the linearly with decreasing gravity values (Wagner et al., 2010). proliferation rate, while cells accumulated in the G2/M phase As for experiments performed on humans underwater with a of the cell cycle in parallel with decreasing values of simulated treadmill and an adjustable ballasting harness, the results are gravity (Figs. 4 and 6). On the other hand, the activation rate in agreement with the data mentioned above (Newman and of human lymphocytes dropped with a decline in gravity level Alexander, 1993). Research focusing on load-carrying in (Fig. 7). In addition, data obtained from the lymphocyte partial gravity also by underwater immersion confirmed that experiment demonstrated a strong correlation between the energy expenditure and stride rates dropped while the level of activation and the magnitude of the simulated gravity forward lean exhibited by subjects increased dramatically field regardless of the type of algorithm applied. In contrast, a with diminishing gravity levels (Wickman and Luna, 1996). 1188 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014 Recently, the possibility of humans running in place on have been used so far to simulate partial gravity include water was investigated, where the subjects, suspended with a ballasted underwater immersion, parabolic flights, over-head harness, experienced different levels of partial gravity just suspension, off-loading pulley systems, and tilted bed rest above the water. The predicted available impulse and the studies (Louisy et al., 1994; Newman, 2000). However, all of number of successful subjects that could generate enough these have specific advantages and disadvantages. On the one muscle power to run in place over a wading pool decreased hand, the over-head suspension, off-loading pulley systems, linearly with increasing gravity levels (Minetti et al., 2012). and tilted bed rest studies are economical techniques, but Other studies performed on humans, also under suspension, they are not suitable for cell culture analyses and they restrict have focused on the performance of chest compressions the freedom of movement in humans and animals. While under partial and microgravity. In both reports, the mean underwater immersion has no constraints on movement, it frequency of external chest compression did not result in a entails an inconvenient hydrodynamic drag. On the other pattern relative to the level of gravity exposure in either male hand, parabolic flights offer freedom of movement, with no or female subjects (Dalmarco et al., 2006; Kordi et al., 2012). hydrodynamic drag, and are appropriate for cell culture On another area of research, investigators studied the human studies, but they are expensive and the partial gravity perception of verticality during a recent parabolic flight exposure only lasts for a short duration (ranging approxi- campaign of partial gravity. In this case, the authors found mately from 20 to 40 s). Moreover, on-board centrifuge out that the gravity level must exceed a certain threshold to be facilities inside a spacecraft have been suggested to enable recognized as a reference for verticality, which was of 0.3 g, investigators to study the effects of partial gravity (Wagner depicting an onset where the effect of gravity exposure was and Fulford-Jones, 2006). This comes, however, at an detected (de Winkel et al., 2012). expensive cost and is limited to a few experiments only, In summary, there are three different outcomes that can be restricting the amount of data that can be gathered. For these detected with changing partial gravity values: a linear effect, a reasons, our newly designed and built RPI is a breakthrough result that depends on a certain threshold, and no correlation tool that will enable scientists to investigate partial gravity on between the exposure level and the response. As whole cell cultures and small organisms for up to a long duration at organisms respond differently than single cells, an explana- the gravity field of interest, such as that of the Moon or Mars, tion for what we observe cannot be attained from the studies with countless reproducible experiments and at an economic mentioned above. For that reason, our new RPI uniquely cost. In addition to broadening the scope of available tools to provides the advantage of studying single cell behavior, study the behavior of biological systems affected by space something that was not possible until now. In this way, our travel, our RPI can be employed as a method to explore the RPI mediates a valuable tool to perform cell experiments effects of mechanical unloading on cells through partial under partial gravity environments where the exact mecha- gravity, being a relevant model system with which to better noresponse that gives rise to specific signals in each individual understand the cellular responses subsequent to mechano- cell can be investigated as a reaction from mechanical transduction. Studying the underlying mechanisms that unloading. regulate muscle development and the immune system activation under partial gravity exposure is crucial for the identification of the causes and countermeasures for trauma and earth-bound diseases as well as to improve the Conclusions sustainment of human life in space. We are introducing a novel instrument called RPI for 3D cell The authors wish to gratefully acknowledge the technical assistance of culturing that in addition to growing cells under simulated Dr Malgorzata Kisielow and Ms Anette Schütz from the Flow microgravity, allows the generation of a partial earth-like Cytometry Laboratory (ETHZ), and of Mr Stéphane Richard from the gravity field of various values. Therefore, studies of cell Space Biology Group (HSLU). We would also like to thank Prof growth behavior under a nullified gravity vector environment Dr. Ralph Müller from the Institute for Biomechanics (ETHZ) for insightful comments. This work was supported by the European as well as under different simulated gravity fields (from 0 to Space Agency (ESA). 1 g) are now possible. Such a technique opens new aspects of biotechnology and enables the study of fundamental cellular mechanisms modulating mechanically-regulated tissue de- References velopment and maintenance. Baharvand H, Hashemi SM, Kazemi Ashtiani S, Farrokhi A. 2006. In our frame of study there appears to be two different Differentiation of human embryonic stem cells into hepatocytes in responses to changes in partial gravity, one being set by a 2D and 3D culture systems in vitro. Int J Dev Biol 50(7):645–652. threshold from where the effect can be detected, and the other Bassel-Duby R, Olson EN. 2006. Signaling pathways in skeletal muscle one varying in proportion to the gravity exposure level. In remodeling. Annu Rev Biochem 75:19–37. Bechler B, Cogoli A, Cogoli-Greuter M, Muller O, Hunzinger E, Criswell SB. this report, we have unraveled the possibility of exploring the 1992. Activation of microcarrier-attached lymphocytes in microgravity. effects of partial gravity on biological samples at an earth- Biotechnol Bioeng 40(8):991–996. bound facility in a comparable manner. To our knowledge, Benavides Damm T, Franco-Obregon A, Egli M. 2013a. Gravitational force this is the first time simulated partial gravity has been modulates G 2/M phase exit in mechanically unloaded myoblasts. Cell achieved by using a RPM-like device. The techniques that Cycle 12(18):3001–3012. Benavides Damm et al.: Cell Culture at Simulated Partial Gravity 1189 Biotechnology and Bioengineering Benavides Damm T, Richard S, Tanner S, Wyss F, Egli M, Franco-Obregon A. Minetti AE, Ivanenko YP, Cappellini G, Dominici N, Lacquaniti F. 2012. 2013b. Calcium-dependent deceleration of the cell cycle in muscle cells Humans running in place on water at simulated reduced gravity. PLoS by simulated microgravity. FASEB J 27(5):2045–2054. ONE 7(7):e37300. Block I, Briegleb W, Wohlfarth-Bottermann KE. 1986. Gravisensitivity of the Newman DJ. 2000. Life in extreme environments: How will humans perform acellular slime mold Physarum polycephalum demonstrated on the fast- on Mars? Gravit Space Biol Bull 13(2):35–47. rotating clinostat. Eur J Cell Biol 41:44–50. Newman DJ, Alexander HL. 1993. Human locomotion and workload for Cavagna GA, Willems PA, Heglund NC. 2000. The role of gravity in human simulated lunar and Martian environments. Acta Astronaut 29(8):613–620. walking: Pendular energy exchange, external work and optimal speed. J Pavy-Le Traon A, Allevard AM, Fortrat JO, Vasseur P, Gauquelin G, Guell A, Physiol 528(Pt 3):657–668. Bes A, Gharib C. 1997. Cardiovascular and hormonal changes induced Chang TT, Walther I, Li CF, Boonyaratanakornkit J, Galleri G, Meloni MA, by a simulation of a lunar mission. Aviat Space Environ Med 68(9):829– Pippia P, Cogoli A, Hughes-Fulford M. 2012. The Rel/NF-kappaB 837. pathway and transcription of immediate early genes in T cell activation Pietsch J, Sickmann A, Weber G, Bauer J, Egli M, Wildgruber R, Infanger M, are inhibited by microgravity. J Leukoc Biol 92(6):1133–1145. Grimm D. 2012. Metabolic enzyme diversity in different human thyroid Clapham DE. 2007. Calcium signaling. Cell 131(6):1047–1058. cell lines and their sensitivity to gravitational forces. Proteomics 12(15– Cogoli A, Tschopp A, Fuchs-Bislin P. 1984. Cell sensitivity to gravity. Science 16):2539–2546. 225(4658):228–230. Rivera-Solorio I, Kleis SJ. 2006. Model of the mass transport to the surface of Dalmarco G, Calder A, Falcao F, de Azevedo DF, Sarkar S, Evetts S, Moniz S, animal cells cultured in a rotating bioreactor operated in micro gravity. Russomano T. 2006. Evaluation of external cardiac massage performance Biotechnol Bioeng 94(3):495–504. during hypogravity simulation. Conf Proc IEEE Eng Med Biol Soc Saltzman WM, Parkhurst MR, Parsons-Wingerter P, Zhu WH. 1992. Three- 1:2904–2907. dimensional cell cultures mimic tissues. Ann NY Acad Sci 665:259–273. de Winkel KN, Clement G, Groen EL, Werkhoven PJ. 2012. The perception of Schwarz RP, Goodwin TJ, Wolf DA. 1992. Cell culture for three-dimensional verticality in lunar and Martian gravity conditions. Neurosci Lett modeling in rotating-wall vessels: An application of simulated 529(1):7–11. microgravity. J Tissue Cult Methods 14(2):51–57. Freed LE, Vunjak-Novakovic G. 1995. Cultivation of cell-polymer tissue Schwarzenberg M, Pippia P, Meloni MA, Cossu G, Cogoli-Greuter M, Cogoli constructs in simulated microgravity. Biotechnol Bioeng 46(4):306–313. A. 1999. Signal transduction in T lymphocytes—A comparison of the Friedrich UL, Joop O, Putz C, Willich G. 1996. The slow rotating centrifuge data from space, the free fall machine and the random positioning microscope NIZEMI–a versatile instrument for terrestrial hypergravity machine. Adv Space Res 24(6):793–800. and space microgravity research in biology and materials science. J Sonnenfeld G. 2012. Editorial: Space flight modifies T cellactivation-role of Biotechnol 47(2–3):225–238. microgravity. J Leukoc Biol 92(6):1125–1126. Griffin TM, Tolani NA, Kram R. 1999. Walking in simulated reduced gravity: Sun T, Jackson S, Haycock JW, MacNeil S. 2006. Culture of skin cells in 3D Mechanical energy fluctuations and exchange. J Appl Physiol 86(1):383– rather than 2D improves their ability to survive exposure to cytotoxic 390. agents. J Biotechnol 122(3):372–381. Herranz R, Benguria A, Laván DA, López-Vidriero I, Gasset G, Javier Medina van Loon JJWA. 2007. Some history and use of the random positioning F, Van L, Jwa J, Marco R. 2010. Spaceflight-related suboptimal conditions machine, RPM, in gravity related research. Adv Space Res 39(7):1161– can accentuate the altered gravity response of Drosophila transcriptome. 1165. Mol Ecol 19(19):4255–4264. Vandenburgh H, Chromiak J, Shansky J, Del Tatto M, Lemaire J. 1999. Space Hoson T, Kamisaka S, Masuda Y, Yamashita M, Buchen B. 1997. Evaluation of travel directly induces skeletal muscle atrophy. FASEB J 13(9):1031– the three-dimensional clinostat as a simulator of weightlessness. Planta 1038. 203(Suppl):S187–S197. Wagner EB, Fulford-Jones TR. 2006. Sensorimotor investigations for the Infanger M, Kossmehl P, Shakibaei M, Bauer J, Kossmehl-Zorn S, Cogoli A, Mars Gravity Biosatellite: A rotating spacecraft for partial gravity Curcio F, Oksche A, Wehland M, Kreutz R, Paul M, Grimm D. 2006. research. Brain Res 1091(1):75–78. Simulated weightlessness changes the cytoskeleton and extracellular Wagner EB, Granzella NP, Saito H, Newman DJ, Young LR, Bouxsein ML. matrix proteins in papillary thyroid carcinoma cells. Cell Tissue Res 2010. Partial weight suspension: A novel murine model for investigating 324(2):267–277. adaptation to reduced musculoskeletal loading. J Appl Physiol Kordi M, Kluge N, Kloeckner M, Russomano T. 2012. Gender influence on 109(2):350–357. the performance of chest compressions in simulated hypogravity and Watson JV, Chambers SH, Smith PJ. 1987. A pragmatic approach to the analysis microgravity. Aviat Space Environ Med 83(7):643–648. of DNA histograms with a definable G1 peak. Cytometry 8(1):1–8. Lewis RS. 2001. Calcium signaling mechanisms in T lymphocytes. Annu Rev Wickman LA, Luna B. 1996. Locomotion while load-carrying in reduced Immunol 19:497–521. gravities. Aviat Space Environ Med 67(10):940–946. Louisy F, Guezennec CY, Guell A. 1994. Leg vein hemodynamics during Yip DK, Auersperg N. 1972. The dye-exclusion test for cell viability: Persistence bedrests simulating lunar trip. J Gravit Physiol 1(1):P100–P101. of differential staining following fixation. In Vitro 7(5):323–329. 1190 Biotechnology and Bioengineering, Vol. 111, No. 6, June, 2014

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Published: Jan 22, 2014

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