ASTROBIOLOGY

Volume 13, Number 1, 2013 Review Article

ª Mary Ann Liebert, Inc.
DOI: 10.1089/ast.2012.0876

Ground-Based Facilities for Simulation of Microgravity:

Organism-Specific Recommendations for Their Use,
and Recommended Terminology

Raul Herranz,1 Ralf Anken,2,3 Johannes Boonstra,4 Markus Braun,5 Peter C.M. Christianen,6
Maarten de Geest,4 Jens Hauslage,2 Reinhard Hilbig,3 Richard J.A. Hill,7 Michael Lebert,8
F. Javier Medina,1 Nicole Vagt,5 Oliver Ullrich,9 Jack J.W.A. van Loon,10 and Ruth Hemmersbach2

Abstract

Research in microgravity is indispensable to disclose the impact of gravity on biological processes and organ-
isms. However, research in the near-Earth orbit is severely constrained by the limited number of flight oppor-
tunities. Ground-based simulators of microgravity are valuable tools for preparing spaceflight experiments, but
they also facilitate stand-alone studies and thus provide additional and cost-efficient platforms for gravitational
research. The various microgravity simulators that are frequently used by gravitational biologists are based on
different physical principles. This comparative study gives an overview of the most frequently used micro-
gravity simulators and demonstrates their individual capacities and limitations. The range of applicability of
the various ground-based microgravity simulators for biological specimens was carefully evaluated by using
organisms that have been studied extensively under the conditions of real microgravity in space. In addition,
current heterogeneous terminology is discussed critically, and recommendations are given for appropriate
selection of adequate simulators and consistent use of nomenclature. Key Words: 2-D clinostat—3-D clinostat—
Gravity—Magnetic levitation—Random positioning machine—Simulated microgravity—Space biology. Astro-
biology 13, 1–17.

1. Introduction tions are lacking. While the currently applied ‘‘-omics’’ tech-
nologies produce huge amounts of data, it remains unclear up

R esearch under the conditions of microgravity during

space missions has contributed greatly to our knowledge
of the impact of gravity on biological processes, gravity-
to now what exactly to look for. This strongly emphasizes the
need for ground-based facilities (GBFs) to define baselines
and enable thorough testing of the biological system to
sensing mechanisms, and gravity-mediated orientation of address gravity-related issues prior to space experiments.
organisms in their spatial environment. These processes, Since the introduction of the classical clinostat in 1879 by
however, are far from being fully understood. This is mainly Julius Sachs, a number of GBFs have been designed to sim-
due to the fact that access to flight opportunities is scarce, and ulate the condition of ‘‘weightlessness’’ or ‘‘free fall’’ in lab-
performing a sufficient number of experiments or even a oratories on Earth. Such simulators do not abolish the 1g
series of succeeding experiments has been realized only force of gravity but instead either randomize the direction of
sporadically. In addition, clear-cut distinctions between gravity with respect to the sample over time (omnilateral
gravity-related effects and stress responses to free-fall condi- stimulation—clinostat principle) or compensate the gravity

1
Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
2
German Aerospace Center (DLR), Institute of Aerospace Medicine, Cologne, Germany.
3
Zoological Institute, University of Stuttgart-Hohenheim, Stuttgart, Germany.
4
Department of Biology, Faculty of Science, University of Utrecht, Utrecht, the Netherlands.
5
Institute of Molecular Physiology and Biotechnology of Plants, University of Bonn, Bonn, Germany.
6
High Field Magnet Laboratory (HFML), Institute for Molecules and Materials, Radboud University, Nijmegen, the Netherlands.
7
School of Physics & Astronomy, University of Nottingham, Nottingham, UK.
8
Biology Department, Cell Biology, University of Erlangen, Erlangen, Germany.
9
Institute of Anatomy, Faculty of Medicine, University of Zurich, Zurich, Switzerland.
10
Dutch Experiment Support Center (DESC) @ ACTA, University of Amsterdam & VU University Amsterdam, Amsterdam; Department of
Oral Cell Biology, Research Institute MOVE, Amsterdam; European Space Agency (ESA), TEC-MMG, ESTEC, Noordwijk, the Netherlands.

1
2 HERRANZ ET AL.

force by creating a counteracting force (magnetic levitation). Further terms are ‘‘modeled microgravity’’ (e.g., Plett et al.,
On the ground, only drop towers are able to provide real 2004; Zayzafoon et al., 2004), ‘‘near-Earth free-fall orbit’’
free-fall conditions for a period of seconds. (Zayzafoon et al., 2004), ‘‘microweight simulator’’ (as a
In recent decades, numerous experiments have been per- synonym for the RPM; van Loon et al., 1999), ‘‘randomized
formed with different types of simulators and a great variety microgravity’’ (RPM; England et al., 2003), or ‘‘low shear
of organisms. Simulator experiments have provided excel- environment’’ (Hammond and Hammond, 2001; Nickerson
lent insight into a multitude of gravity-dependent phenom- et al., 2004). Other authors refrain from judging the accel-
ena. However, many researchers failed to compare results eration achieved with a simulation technique and therefore
from experiments in the ‘‘real’’ microgravity of a spaceflight use a wording that exactly describes the experimental
mission with experiments on simulators, with respect to the methods, such as ‘‘clinorotation’’ or ‘‘wall vessel rotation’’
sample used and to the parameters investigated, which (e.g., Anken et al., 2010; Brungs et al., 2011; Li et al., 2011).
represents the only way to unambiguously verify the suit- From the physical point of view, ‘‘Microgravity is the con-
ability of the facility for simulating microgravity conditions dition in which the absolute sum of all mass-dependent
of space. Without this direct comparison, it is difficult to accelerations does not exceed a certain small ‘noise’ level
conclude whether biological reactions or organismic re- (typically 10 - 5-10 - 6 times g)’’ (Albrecht-Buehler, 1992).
sponses are caused by the conditions of simulated micro- Weightlessness also has been described as ‘‘no mechanical
gravity or by any of the possible side effects of the simulation support of mass’’ (Briegleb, 1992) and as a ‘‘result from a net
technique. Each type of simulator has its specific artifacts, for sum of all forces present equaling zero, not from absence
example, centrifugal accelerations and vibrations in the case of gravity’’ (Klaus, 2001). The latter definition can be
of clinostats or differing magnetic susceptibility of cell com- misleading since in ‘‘microgravity’’ not all forces need to
ponents in the case of magnetic levitation, that may mask or be equal to zero. One still has, for example, capillary
distort the desired microgravity effect. Noncritical use of forces, hydrostatic pressure, and cell surface binding forces
simulators may easily result in a misinterpretation of re- (Albrecht-Buehler, 1991).
sponses to side effects as specific microgravity effects. The term ‘‘microgravity’’ (or ‘‘micro-g environment,’’ or
Comparing results from experiments in which samples were ‘‘lg’’) is frequently used as a synonym of ‘‘weightlessness’’
investigated on different simulators often revealed inconsis- and ‘‘zero-g,’’ which indicates that the g-forces are not
tent or even contradicting responses. Therefore, for each actually zero but just ‘‘very small.’’ ‘‘True’’ weightlessness,
simulator, the physical parameters and principles as well as for more than a few seconds at least, can only be achieved
their specific impact on the biological processes and objects in space. In practice, space experiments require a space
of different sizes need to be critically evaluated. The different vehicle (e.g., ISS, shuttle). Inside the vehicle, the quality of
simulators and different modes of operation of simulators weightlessness is influenced by so-called ‘‘g-jitters,’’ that is,
are not equally suited to simulate microgravity for all pro- vibrations caused by onboard machinery, movement of
cesses and organisms. astronauts, thruster operation, and so on. The ‘‘micro-
This comparative study gives an overview of the most gravity’’ level may range between * 10 - 3 to 10 - 6 g, de-
frequently used microgravity simulators and illustrates pending on the location within the spacecraft and the
their individual capacities and limitations. The range of frequency of vibration (Tryggvason et al., 2001; Penley
applicability of the various ground-based microgravity et al., 2002; Jules et al., 2004). In life-science experiments,
simulators for different biological specimens was carefully we are interested in the impact of mechanical stresses
evaluated by using organisms that have been studied generated through the force of gravity on the mass of the
extensively under the conditions of real microgravity in organism. In short, we are investigating the impact of
space. Since some papers suffer from inadequate descrip- weight on biological systems. As a consequence, the best
tions of how the simulators were operated and which description of the environment would be ‘‘near weight-
stimulations (i.e., accelerations and/or shearing forces) the lessness.’’ Terms such as ‘‘zero-g’’ or ‘‘weightlessness’’
samples were subjected to, current heterogeneous termi- should be avoided since we can never achieve such a level
nology is discussed critically, and recommendations are for more than a few seconds with space vehicles or
given for a proper selection of adequate simulators and ground-based simulations.
consistent use of terminology. In simulation experiments, the magnitude of the Earth
gravity vector cannot be changed—only its influence or effect
can be changed (Briegleb, 1992). In consequence, micro-
2. Simulated Microgravity
gravity cannot be achieved with a simulator. Rather, such a
First, we address nomenclatorial issues. Using experi- simulator may generate functional weightlessness from the
mental platforms such as two-dimensional (2-D) clinostats, perspective of the organism or cell. The term ‘‘functional near
rotating wall vessels (RWVs), random positioning machines weightlessness’’ may thus be applied when the physical en-
(RPMs), and so on, the authors mainly use the term ‘‘sim- vironment results in physical constraints that are (according
ulated microgravity’’ or ‘‘simulated weightlessness’’ to de- to Briegleb, 1992) below the known acceleration sensitivities
scribe the state of acceleration, which is assumed to be of relevant biological processes. Functional weightlessness
achieved with such machines. Sometimes, different phra- has also been described as ‘‘a state of relative motionlessness
seology is used for the same phenomenon/basic physical which is defined with respect to the simultaneous contribu-
principle. For instance, the 2-D clinostat has been defined as tions of gravity, centrifugation and Brownian motion acting
a tool to obtain a ‘‘vector-averaged gravity’’ (e.g., Sarkar on the suspended cell’’ (Klaus et al., 1998). By analogy, this
et al., 2000; Nakamura et al., 2003) or to provide the ‘‘nul- description is also regarded to be valid for multicellular or-
lification of the gravity stimulus’’ (Dedolph et al., 1967). ganisms.
SIMULATED MICROGRAVITY ON EARTH—REVIEW 3

To avoid confusion and provide a basis for acceptance, we 1-D or 2-D refers to whether the dimension of the rotated line
propose here to use the term ‘‘simulated microgravity’’ (in or the whole area is considered. Clinostats with two axes are
analogy to microgravity conditions in spaceflight). Figure called three-dimensional (3-D) clinostats and will be consid-
legends should explain clearly the GBF used and the main ered in the section ‘‘Random positioning machine.’’
parameters to reproduce the simulation (geometry of the The use of clinostats in plant research began with experi-
sample container, mode of operation and rotational speeds ments that rotated the object relatively slowly (1–10 rpm;
for RPM/clinostat, magnetic field intensity, and gradient in classical clinostat). Seedlings and small plants rotated slowly
each position for the magnet as well as residual accelerations in the 2-D clinostat axis did not exhibit any gravitropic re-
or effective radius). sponse. However, later on, morphological studies demon-
We propose that the term ‘‘microgravity’’ should be as- strated that slow clinorotation (1–2 rpm) induces
signed exclusively to those experiments that have been per- disturbances at the ultrastructural level, which were not
formed in an environment such as that of the International found under spaceflight conditions (Hensel and Sievers,
Space Station (ISS), satellites, sounding rockets, drop towers, 1980). These are indications that the slow rotation prevented
or aircraft during parabolic flight. Parabolic flights offer a a gravity-induced growth response but most likely also
special experimental scenario, as the microgravity phases are caused omnilateral mechanical stress in some sensitive plant
interrupted by phases of hyper-g accelerations, which tissues.
thereby demands careful control experiments in order to Briegleb introduced the concept of a fast rotating clinostat
discriminate both kinds of effects on the sample. Usage of the to achieve ‘‘functional weightlessness’’ for small objects,
term ‘‘microgravity’’ is independent of the actual acceleration mainly single cells (Briegleb, 1992). By fast and constant ro-
(real microgravity, i.e., *10 - 6 g, is not, in fact, achieved in tation it is assumed that sedimentation is prevented physi-
most of the environments listed above). The term ‘‘simulated cally by a continuous and constant change of the direction of
microgravity,’’ thus, should be used regarding experiments the gravity vector. The principles can be demonstrated by
performed in GBFs, in which the gravity level may be av- rotating particles in a small tube or cuvette vertically posi-
eraged to near zero with time but not neutralized. tioned in the horizontal axis (running through its geometric
center). In this arrangement, particles are forced to move on
3. Microgravity Simulators circular paths, whose diameters decrease with speed of ro-
tation and finally reach a state in which relative movements
Various GBFs with different physical concepts have been
with respect to gravity can be neglected. That is, a speed can
constructed to simulate microgravity on the ground. A de-
be achieved at which a cell rotates around its center together
scription and the mode of operations of the most well-used
with a small liquid boundary layer surrounding it (Klaus,
facilities in Europe is given. In principle, organisms of all
2001).
evolutionary levels can be used with these facilities: sessile
Under these conditions, biological samples no longer seem
organisms like plants or moving/swimming ones (e.g., small
to have the capacity to perceive gravity and, thus, experience
animals, protists, or bacteria) and cell cultures. Restrictions
simulated microgravity. Depending on the scientific ques-
are given by the maximum volume of exposure and the
tions and methodological demands, different kinds of
quality of simulation the experimenter wants to achieve. One
clinostats, besides the regular clinostats (Dedolph et al., 1967;
of the major problems that arises when comparing the dif-
van Loon et al., 1999; Hemmersbach et al., 2006), have been
ferent experimental designs and results is the lack of detailed
developed, which have enabled, for example, microscopic
technical descriptions of the operational modes (speed and
observation (clinostat microscope, Fig. 1A), online kinetic
direction of rotation) of the simulation facilities used in some
measurements (photomultiplier clinostat, Horn et al., 2011),
of the literature. In addition, details on the hardware (ma-
fixation during rotation (pipette clinostat, Fig. 1B), and de-
terial, dimensions, location within the GBF) are frequently
velopmental studies under submersed conditions (sub-
missing.
mersed clinostat, Hemmersbach et al., 2006; Brungs et al.,
In this study, the following simulation techniques were
2011) or even portable clinostats (Fig. 1C).
used for comparative studies with various biological model
systems:
3.2. Random positioning machine—two axes running
(1) 2-D clinostat with different speeds and directions
(2) Random positioning machine (RPM)
(3) Rotating wall vessel (RWV) Based on the hypothesis that the quality of simulation
(4) Diamagnetic levitation might be increased by rotating around two axes, especially
for larger objects, so-called 3-D clinostats have been devel-
oped in Japan and in the Netherlands (for review see van
3.1. Clinostats—one or two axes running
Loon, 2007). These 3-D systems have two independently
fast and constantly in one direction
rotating frames (Fig. 1D). The term ‘‘3-D clinostat’’ is ap-
A clinostat is a device in which samples are rotated to propriate as long as the device is running with constant
prevent the biological system from perceiving the gravita- speed and constant direction (simplest RPM mode of func-
tional acceleration vector. Different configurations exist with tion). However, both frames can also be operated with dif-
respect to the number of rotation axes, the speed, and the ferent speeds and different directions. In this case, the term
direction of rotation (Briegleb, 1992; Häder et al., 1995; Klaus ‘‘random positioning machine’’ (RPM) in combination with
et al., 1997; Klaus, 2001). Clinostats with one rotation axis, operational mode description should be used. These facilities
which runs perpendicular to the direction of the gravity vec- are characterized by the randomly changing rotation speed
tor, are called 1-D (seldom) or 2-D clinostats (more common). and direction (Hoson et al., 1997; Borst and van Loon, 2009).
4 HERRANZ ET AL.

FIG. 1. Images of the 2-D clinostat mi-

croscope (A) and the pipette 2-D clinostat
(B) hosted at DLR, Cologne, Germany; the
2-D clinostat (C) and the 3-D RPM (D)
hosted at DESC/ESA-ESTEC, Noordwijk,
the Netherlands; and the two magnetic
levitation facilities hosted at the HFML,
Nijmegen, the Netherlands, (E) and the
University of Nottingham, UK (F).

3.3. Rotating wall vessel (Dijkstra et al., 2011), cell cultures (Babbick et al., 2007; Hammer
et al., 2009; Qian et al., 2009), a mouse (Liu et al., 2011), and
Rotating wall vessels (RWVs) or rotating bioreactors (Ro-
Drosophila melanogaster (Herranz et al., 2012; Hill et al., 2012).
tating Cell Culture System, initially developed by NASA)
Diamagnetic levitation requires a strong, spatially varying
have been designed for cell cultures (Schwarz et al., 1992) and
magnetic field, such as that produced by a Bitter solenoid or
aquatic organisms such as zebrafish eggs/embryos (Moor-
a superconducting solenoid magnet. For levitation of bio-
man et al., 1999; Li et al., 2011). The submersed version of the
logical material, a vertical bore magnet, in which the sole-
RWV used in this comparative study was designed and
noid axis is oriented vertically, is used. The bore of the
constructed at the German Aerospace Center (Brungs et al.,
magnet is a cylindrical hole, open at both ends, that passes
2011). Main components are a Plexiglas cylinder (diameter of
through the center of the solenoid. Inside the bore, the
10 cm) with a 5 cm wide central core, mounted on a hori-
magnetic field is strongest where the bore passes through the
zontal plane. A shaft is connected to a variable speed motor.
center of the solenoid. A diamagnetic material is one such as
water, many organic-based materials (e.g., oils, plastics), and
3.4. Diamagnetic levitation
biological materials that are repelled from a magnetic field.
In 1997, Andre Geim, along with researchers from the Inside the upper section of the vertical magnet bore, dia-
Universities of Nijmegen and Nottingham, succeeded in levi- magnetic material is repelled upward, away from the strong
tating a live frog in a high field magnet at the High Field field near the center; in the lower section, diamagnetic ma-
Magnet Laboratory (HFML), Nijmegen, using the diamagne- terial is repelled downward. The vertical repulsive force is
tism of the frog (Berry and Geim, 1997; Geim, 1998; Simon and proportional to the product of the magnitude of the magnetic
Geim, 2000); in the same year, Valles et al. at Brown University, field B and its vertical gradient vB/vz, and to the magnetic
USA, demonstrated diamagnetic levitation of frogs’ eggs susceptibility of the material. The field-gradient product
(Valles et al., 1997). These were followed by studies of levi- B · vB/vz varies continuously within the bore; it is zero
tating yeast (Coleman et al., 2007), swimming paramecia in exactly in the center of the solenoid (where the gradient of
gadolinium solution (Guevorkian and Valles, 2006b), E. coli the field is zero) and reaches a maximum near the top and
SIMULATED MICROGRAVITY ON EARTH—REVIEW 5

bottom of the solenoid (Fig. 2). If the product B · vB/vz is stresses induced by the pull of normal gravity (Valles et al.,
large enough, the diamagnetic force can support the weight 1997). There are, however, notable exceptions, for example,
of the diamagnetic material, allowing levitation. This occurs in experiments on seedling growth, which we discuss below.
when B · vB/vz equals ql0g/v, where q and v are, respec- The effective gravity acting on a diamagnetic body in the
tively, the density and volume magnetic susceptibility of the magnetic field is defined as the net force, that is, the sum of
material, l0 is the magnetic constant, and g is the gravita- the gravitational and magnetic forces, per unit mass. At a
tional acceleration. For example, the diamagnetic force on a levitation point, the effective gravity is zero, and the material
droplet of water counteracts exactly the weight of the droplet is weightless. For biological material in the magnetic field, it
where B · vB/vz = - 1361 T2/m, in the upper region of the is sometimes useful to calculate the effective gravity acting
bore. One can achieve stable levitation by this technique; a on water, especially in cases where the magnetic suscepti-
diamagnetic material can be made to levitate at a particular bility of all tissues of interest are similar to that of water.
point in space in stable equilibrium (Berry and Geim, 1997), Particularly useful as reference points are the places in the
with no mechanical means of support. The diamagnetic force magnetic field where the effective gravity on water is pre-
balancing the force of gravity on a levitating object acts cisely zero and 1g. These are commonly labeled as the 0g*
throughout its volume, just as the centrifugal force acts to and 1g* points, respectively (depending on the configuration
balance the force of gravity on a weightless body in Earth of the magnetic field, there can be more than one 0g* point,
orbit. In this respect, diamagnetic levitation is unlike flota- including ring-shaped or planar loci of such points). The
tion; a scuba diver who is neutrally buoyant under water will asterisk is used as a reminder that the labels refer to the
neither float nor sink, but the forces balancing gravity in this effective gravity on water, and also to indicate that there is a
case act only at the surface. The diver still feels ‘‘internal’’ strong magnetic field present. The magnitude and direction
stresses owing to the downward pull of the lead weight belt of the effective gravity varies continuously throughout the
and the upward-pulling force of the buoyancy jacket, for bore of the magnet, owing to the spatial variation of the
example. The aim of diamagnetic levitation is to reduce the magnetic field. Magnetic levitation therefore can be used to
gravitationally induced internal stresses to as near to zero as tune the effective gravity and provides a means by which to
possible in order to approach a weightless environment. create enhanced, reduced, or even inverted gravity (Heijna
Fortunately, the magnetic susceptibility of most soft biolog- et al., 2007; Micali et al., 2012). Depending on the experiment,
ical tissues differs from that of water by only 10% or less it may thus be useful to identify points in which the effective
(Schenck, 1992) and levitate under approximately the same gravity acting on water is the same as the gravity on the
conditions as water. Levitation can reduce the stresses within Moon (0.17g*) or Mars (0.38g*), in the upper region of
such tissues by an order of magnitude compared to the the bore or a hypergravity point (2g*) in the lower part of the

FIG. 2. An example of magnetic levitation experimental positions in relation to the intensity of the magnetic field (curve, left
axis) and the net effective force (curve, right axis) along the length of the magnetic bore. Color graphics available online at
www.liebertonline.com/ast
6 HERRANZ ET AL.

bore, for example (Fig. 2). The 0g* point (or points) is a gators who wish to use their particular model systems in space
mathematically determined location in the magnetic field, biology or those who need to corroborate their results with
where the forces on water balance exactly. Although the ef- previous literature. For instance, the microbiology community
fective gravity on water is a useful starting point in discus- would benefit greatly from a critical and updated review of the
sions of the internal stresses within the organism, a more related literature (Klaus et al., 1998; Beuls et al., 2009).
detailed discussion of the diamagnetic forces within the or-
ganism is often required to obtain a more accurate picture of
4.1. Paramecium and Euglena—unicellular
the residual forces within the organism. The label 0g* should
free-swimming cells
not be confused with the term ‘‘microgravity’’ (or ‘‘lg’’) used
to indicate the residual gravity ‘‘experienced’’ by an object in The protists Paramecium (ciliate) and Euglena (flagellate)
an orbiting spacecraft. The magnitude of the effective gravity show distinct gravity-guided modes of behavior in the form
typically increases by *0.01g per millimeter as one moves of negative gravitaxis (orientation against the direction of the
away from a 0g* point; the magnitude of the effective gravity gravity vector) and gravikinesis (regulation of the swimming
at the surface of a levitating water droplet, with volume speed with respect to the swimming direction) (Machemer
1 mL, for example, is typically 10 - 2g (Hill and Eaves, 2010). et al., 1991; Machemer and Bräucker, 1992; Lebert and Häder,
Note that, in cases where the susceptibility of a particular 1996; Hemmersbach and Häder, 1999; Hemmersbach et al.,
biological structure of interest is widely different from that of 1999; Häder et al., 2005a, 2005b, 2006, 2010; Hemmersbach
water (i.e., significantly different from the average suscepti- and Braun, 2007; Daiker et al., 2011). Studies in real micro-
bility of the bulk of the organism), it may not be useful to gravity have demonstrated the loss of these responses in a
discuss the results in terms of the effective gravity on water time frame of 1–2 min (Hemmersbach-Krause et al., 1993a,
at all. For example, in the case of levitation of Arabidopsis 1993b; Hemmersbach et al., 1996a, 1996b; Häder et al., 2005a).
seedlings, although the plant levitates under approximately These unicellular systems respond quickly to changes of
the same conditions as water, sedimentation of the starch- gravity conditions but also to further environmental stimuli
rich statoliths within the root-tip cells is not suppressed (as it such as mechanical disturbances, light, or temperature.
is in spaceflight), and the roots continue to bend down in the Furthermore, they can be immobilized in order to study their
direction of gravity. To suppress sedimentation of the sedimentation behavior. These are the main reasons why
statoliths, a field gradient product much larger than that Paramecium has been chosen as a model system for our
needed to levitate the plant is required. comparative studies. As all experimental platforms had been
One can anticipate that a strong magnetic field alone may equipped with in vivo observation by video recording, a
have significant effects on the behavior of living organisms; detailed computer analysis of the behavior of the exposed
for example, the 7 T field inside a modern MRI scanner can cells could be performed. Swimming cells were exposed in
make volunteers nauseous (Glover et al., 2007). In addition, round observation chambers (radius 15 mm, 0.5 mm depth).
strong magnetic fields are able to orient certain biomolecules In the 2-D clinostat, cells in the observation area of 1 mm
in solution, such as tubulin, actin, and DNA (Maret and radius were rotated at a speed of 60 rpm and thus experi-
Dransfeld, 1985). By performing careful control experiments enced a centrifugal force of 4 · 10 - 3g at the outer perimeter of
in different parts of the bore, it is possible to study and the observation area (6 · 10 - 2g at the perimeter of the
distinguish between effects of magnetically simulated chamber). Cuvettes were totally filled with fluid (culture
weightlessness and any other effects of the strong magnetic medium) and cells; gas bubbles were carefully avoided.
field. Experiments may be conducted simultaneously, under In the magnet, the unicellular systems were studied in
different effective gravities, in the same magnet; in this case, commercially available cuvettes [Hellma, Müllheim, Ger-
the magnitude of the magnetic field in the 1g* samples is many; 52 · 12.5 mm, 3.5 mm depth (Paramecium);
*30% larger than in the 0g* and 2g* samples. An additional 45 · 12.5 mm, 0.2 mm depth (Euglena)], owing to spatial re-
experiment exposing 1g* samples to the same field as in 0g* strictions within the magnet bore.
and 2g* may be performed by repeating the 1g* experiment Two-dimensional clinorotation [60 rpm; clinostat micro-
individually, using a lower solenoid current. scope, German Aerospace Center (DLR) Cologne] revealed
no change in the high linearity of the swimming paths of
Paramecium comparable to the swimming pattern in real
4. Biological Responses of Selected Organisms
microgravity. Exposure on the RPM, however, indicated an
Exposed to Simulated Microgravity
increase in directional turns. The random speed and random
In this study, a variety of organisms were investigated with direction mode on the RPM (Dutch Space, Leiden, the
different microgravity simulators (GBFs). The quality of simu- Netherlands) induced strong drifting and course corrections.
lation was assessed on the basis of comparison with results Euglena cells, three times smaller than Paramecium, even
from experiments under conditions of microgravity during drifted away passively, thereby making an analysis of the
spaceflight missions. To determine the optimal GBF and the swimming velocities and the degree of orientation impossi-
mode of operation, the threshold of gravisensitivity of the bi- ble. Two-dimensional clinorotation of immobilized cells
ological system should be known. However, this is not known (Paramecium) and glass beads resulted in a continuous cir-
in most cases and must be determined by space experiments cling, which demonstrated one-directional acceleration. The
with threshold acceleration profiles. The following examples speed of the clinostat determined the diameter of the circles
(performed in the framework of an ESA GBF project) might be and thus the relative movements of the particles. In contrast,
useful as a guide for choosing the proper microgravity simu- on the RPM, immobilized cells and beads were shown to
lator for a given biological system. Nevertheless, this list should bounce and move out of the rotation center (Fig. 3). These
not be considered exhaustive but some inspiration for investi- results demonstrate that samples on the RPM experience
SIMULATED MICROGRAVITY ON EARTH—REVIEW 7

suitable model system for research on plant gravity sensing

and gravity-regulated growth responses (Limbach et al.,
2005; Braun and Limbach, 2006; Braun, 2007; Hemmersbach
and Braun, 2007). The apical actin cytoskeleton keeps the
statoliths, membrane-bound vacuoles filled with BaSO4
crystals, in a dynamically stable position close to tip by
precisely counteracting the apically directed force of gravity
(Braun and Wasteneys, 2000; Braun et al., 2002). Upon tilting
rhizoids, statoliths are displaced toward the physically lower
cell flank, where they initiate the gravitropic bending of the
cell tip back into the direction of gravity (positive gravi-
tropism). Consequently, under the conditions of micrograv-
ity during TEXUS, MAXUS, or space shuttle flights, statoliths
are rapidly displaced further away from the tip (Braun et al.,
FIG. 3. Paths of glass beads (2–10 lm) in water during 2002). After about 5 min, statoliths doubled their original
exposure on a fast rotating 2-D clinostat constantly running
distance from the cell tip.
with 60 rpm (A) and a RPM with randomly varied speed and
direction of the two frames (108–120 s - 1) (B). Notice the To test whether magnetic levitation has a similar effect on
scale bars indicating the strong drifting in the RPM in this statolith positioning as real microgravity, rhizoids were
experimental setup (S. Hoppe, personal communication). subjected to high-gradient magnetic fields (HGMF levitation)
with different magnetic field strengths (7–29 T) and field-
gradient products up to - 4160 T2/m at the HFML Nijme-
positive and negative acceleration forces induced by con-
gen. Optical components and mirrors were arranged and
tinuously changing the direction and speed of rotation.
inserted in the bore of the magnet such that rhizoids growing
Paramecium and Euglena were exposed to high-gradient
vertically and horizontally in a small agar-filled plastic
magnetic fields (HFML) with different magnetic field strengths
chamber (10 · 10 · 2 mm) could be observed within the dif-
(7–29 T) and field-gradient products up to - 4160 T2/m. These
ferent areas of magnetic field strength. The kinetics of the
experiments revealed a parallel and passive alignment of
movements of statoliths in several rhizoids was video re-
Paramecium to the static magnetic field, as a result of the
corded and analyzed. Statoliths subjected to high-gradient
magnetic alignment of anisotropic, structurally rigid compo-
magnetic fields did not show any significant displacement
nents of the cells, consistent with the results of Guevorkian and
away from the cell tip. Even field-gradient products up to
Valles (2006a). In contrast, Euglena demonstrated a perpendic-
- 4160 T2/m (at a magnetic field as high as 29 T) did not
ular orientation with respect to the applied field. The difference
cause any measurable displacement of the statoliths away
is caused by chloroplasts, as shown by the use of Astasia longa,
from the tip. In analogy with the results on the protists, also
a chloroplast-free Euglena, which did not show any magnetic
here due to the differences in densities and magnetic suscep-
alignment up to the highest magnetic fields used. Immobilized
tibilities of the BaSO4 statoliths and the surrounding water,
cells could not be levitated in the magnet for field-gradient
the levitation of the statoliths is expected to occur at higher
products up to - 4160 T2/m, as revealed by their sedimenta-
values of the field-gradient product than the levitation point
tion; due to the differences in densities and magnetic suscep-
of water. Given the q and v of BaSO4, levitation is expected at
tibility of the protists and the surrounding water, the
B · vB/vz = - 6250 T2/m, which is currently feasible in 33 T
suppression of sedimentation does not coincide with levitation
Florida-Bitter and 45 T Hybrid magnets (which, however,
of water but should occur at higher values of the field-gradient
have a smaller bore size, 32 mm). Besides the failure to pro-
product (e.g., - 7000 T2/m, estimated for Paramecium; Gue-
vide a simulated microgravity environment, no significant
vorkian and Valles, 2006b).
impact of strong magnetic fields was detected on the cell’s
We conclude that, for Paramecium and Euglena, the fast-
polar organization, integrity, and polarized growth.
rotating 2-D clinostat is a good simulator for microgravity,
In contrast to magnetic levitation, clinorotating rhizoids
whereas the RPM is less suited. In the current configuration,
exhibited statolith displacement that was very similar to the
the magnetic fields applied are not suited as a microgravity
one that was observed under the conditions of real micro-
simulation for these two species of protists as levitation was
gravity (Cai et al., 1997; Braun et al., 2002; Limbach et al.,
not achieved and there was a strong effect of the magnetic
2005). Chara rhizoids were studied on classical 2-D clinostats
field as such.
at 2–10 rpm (Cai et al., 1997), a fast-rotating 2-D clinostat
microscope at 60–90 rpm (DLR Cologne), a Japanese fast-
4.2. Rhizoids of characean green algae
rotating 3-D clinostat (Hoson et al., 1997), and a RPM (Dutch
The Chara rhizoid is one of the most extensively studied Space at DLR Cologne). Although clinorotation generally
plant cells in real microgravity. Experiments have been resulted in a clear basipetal displacement of statoliths, the
performed on board space shuttles (IML-2, SMM-05; Braun statoliths’ complex of rhizoids rotated on the classical
et al., 1996, 1999a, 1999b, 2002), during several sounding clinostats often appeared more dispersed than on fast
rocket flights (MAXUS and TEXUS; Buchen et al., 1991, 1993; clinostats or in real microgravity. After clinorotation, more
Braun, 2002; Braun et al., 2002) and parabolic plane flights dispersed statoliths sedimented more slowly, which resulted
(Limbach et al., 2005), in several types of clinostats (classical, in a delayed initiation of the gravitropic response. Obviously,
fast-rotating 2-D, 3-D clinostats), and in a RPM and a mag- rotation around two axes did not improve the quality of
netic levitator of the HFML. The transparency and the rela- microgravity simulation but reduced the volume in which
tively large size make this gravitropically tip-growing cell a specimens experience a good-quality microgravity simulation
8 HERRANZ ET AL.

from a cylindrically shaped volume to a spherically shaped experimental designs (seedlings growing on a wet paper
volume of the same diameter of several millimeters de- containing Murashige and Skoog medium).
pending on the rotational speed. The higher the rotational Additionally, within the frame of the joint ESA-GBF pro-
speed, the higher the residual g-force, ergo the smaller the ject, magnetic levitation has been used as a device for
useful sample volume (van Loon, 2007). changing gravity conditions in plates/tubes in which seed-
In rhizoids that were mounted in the center of the axes lings were grown on agar-based medium. Interestingly, the
( – 1 mm) of a RPM running in the 2-D mode at 60 rpm, physical effect of levitation was not visually observed in the
statoliths also moved away from the tip, but in the real- roots of these seedlings, since root gravitropism was not
random mode of the RPM (2–11 and 30–60 rpm) the effect abolished in the conditions of our experiment, owing to the
was less pronounced; the statoliths moved more slowly and fact that the starch-based organelles, the gravisensors of
spread slightly more as compared to fast 2-D clinorotation Arabidopsis roots, were not levitated in the 1400 T2/m mag-
and real microgravity. Growth rates of Chara rhizoids were netic field applied to the plants, although the intracellular
not impaired by the clinostat and RPM treatment but rather water was levitated. However, under these conditions, in-
appeared to be slightly increased in some cases. tracellular water was unequivocally exposed to levitation
Sample housing used for clinorotation and magnetic levi- conditions. Studies on the cell proliferation rate, nucleolar
tation of Chara rhizoids was generally simple, consisting ultrastructure, and the levels of nucleolar proteins that were
mainly of an agar-filled chamber handmade of microscopic performed on samples grown in the 0g* position within the
slides covered with long cover glasses and sealed on all sides magnet (0g* at 16.5 T at the University of Nottingham facility,
with tape or of a macrolon chamber as was used extensively in UK) confirmed the decoupling of cell proliferation and cell
the space shuttle and parabolic plane flights (Braun et al., 2002). growth (Manzano et al., personal communication). Expression
In conclusion, for investigations on the statolith-based of the cyclin B1 gene, a marker of the G2/M transition in the
gravity sensing system of Chara rhizoids, the 2-D fast- cell cycle, evaluated by GUS staining on transgenic plants,
rotating clinostat represents an excellent simulator for mi- decreased in 0g* despite the increase of the cell proliferation
crogravity, whereas the 3-D clinostat, the RPM, and the rate. All these parameters showed the same alteration with
classical clinostat provide good simulators but are less suit- respect to the 1g control as in the RPM experiment and also,
able in the order listed. The high-gradient magnetic fields as when applicable, as in the space-grown samples (Fig. 4).
applied in this study (magnetic levitation) failed to provide Hypergravity conditions have been tested for the first time
proper simulated microgravity conditions, but with the use for these parameters in both the HFML magnet, at the sym-
of stronger magnets levitation of the statoliths in Chara rhi- metrical position of 0g*, which produces 2g*, and the Large
zoids should be feasible. Diameter Centrifuge (LDC, ESA-ESTEC, Noordwijk, the
Netherlands) used at 2g. The less evident observed alterations
might indicate that the stress produced is weaker than that
4.3. Arabidopsis thaliana—cell growth
under real (ISS) or simulated microgravity (RPM or 0g*).
and proliferation
Nevertheless, a decoupling between cell growth and prolif-
Early studies on plant biology in space did not use a single eration was also detected, however, in an opposite sense to
biological model. The list of plant species used in space ex- that one observed in microgravity (Manzano et al., 2012b).
periments until the first years of this century includes more On the RPM, as well as under the influence of the high
than 30 members, among which wheat, lentil, and several magnetic field (all positions within the magnet, including
Brassica species are the most repeated. However, in the last internal 1g* control), an inhibition of the auxin polar trans-
decades Arabidopsis thaliana has progressively become the port was observed in agreement with results previously
most frequently used biological model in all kinds of studies obtained in spaceflight (Ueda et al., 1999). This was visual-
on plant biology due to the advantages it offers in molecular ized in the root of transgenic seedlings by the staining pat-
biology, genetics, and developmental biology research. This tern of DR5-GUS, an artificial auxin responsive promoter
species was the first plant whose genome was fully se- coupled to a reporter gene. However, hypergravity per se
quenced. Consequently, A. thaliana has also quickly emerged (LDC, 2g) did not alter the auxin polar transport.
as the model of choice for space plant biology research. In addition to the work carried out with seedlings, solid
An essential topic in this research field consists of dis- in vitro cell cultures of Arabidopsis thaliana were used and ex-
cerning alterations in plant growth and development caused posed to the HFML magnet (maximum field of 16.5 T from 0g*
by the space environment and particularly by microgravity. to 2g*), the RPM (real random mode), and the LDC (2g, hy-
This can be addressed by investigating cellular processes pergravity), as a homogeneous proliferating cellular material
such as cell proliferation and growth that occur in meriste- with the use of agar-filled tubes/plates of the required size
matic tissues, where ‘‘meristematic competence’’ consists of (Manzano et al., 2012a). This material has been rarely used in
the strict coupling between the proliferative state of the cell real spaceflight experiments (Paul et al., 2012), but its use as
and its growing capability. In previous studies, carried out source material for altered gravity research has the purpose of
on the ISS, it was shown that the absence of gravity was testing whether cells not specialized in gravity perception re-
capable of modifying cell growth and proliferation rates in spond to gravity alteration. Such systems allow for the use of
the root meristem of seedlings after 4 days of growth. Similar molecular techniques that demand a large biomass of prolif-
disruption of this strict coupling was confirmed in a parallel erating cells, which is difficult to collect from seedlings.
experiment performed in a RPM by using the real random Therefore, the objectives of using GBFs with this material,
operation mode (Matı́a et al., 2010), demonstrating the suc- apart from the facility validation by comparison with space
cess of this microgravity simulation method for experiments experiments, included the preparation for the use of plant cell
on Arabidopsis. Experiments were performed with similar cultures in space, the comparison of the alterations observed in
SIMULATED MICROGRAVITY ON EARTH—REVIEW 9

FIG. 4. Ultrastructural images of the nucleolus of Arabidopsis root meristematic cells grown for 4 days under simulated
microgravity in a magnetic levitation instrument (upper part) and under real microgravity in the ISS (lower part). Nucleolar
parameters represent an accurate estimation of the rate of ribosome biogenesis and, consequently, of the level of protein
synthesis and cell growth. The nucleolus is smaller in both real and simulated microgravity compared to the corresponding
1g controls. Furthermore, the levels of the nucleolar protein nucleolin, an essential factor for pre-rRNA synthesis and
processing, estimated by ultrastructural immunogold procedure, were lower under both real and simulated microgravity
than in the respective 1g ground controls. Bars indicate 1 lm in each experiment. Data taken from Matı́a et al. (2010) and
Manzano et al. (personal communication). DFC, dense fibrillar component; GC, granular component; arrows indicate fibrillar
centers. N, nucleus; Nu, nucleolus.

cellular parameters in cell cultures with the results obtained on stress caused by the synergy between the altered gravity and
seedlings, and the extension of the analysis to new parameters the high magnetic field, both at the gene expression and
provided by high-throughput genomics and proteomics proteomic level (Manzano et al., 2012a; Herranz et al., 2013).
methods. Other laboratories are also involved in the use of In general, data obtained from cell cultures reveal alter-
these plant biological materials in altered gravity research, ations in cell growth and proliferation that could be com-
with application to other cellular and molecular processes pared to those found in seedlings, indicating that a
(Martzivanou et al., 2006; Barjaktarovic et al., 2009). significant part of the response to altered gravity does not
With the use of Agilent microarray-based technology to depend on specialized cells containing mechanoreceptors
detect global genome gene expression profiles, the results (Manzano et al., 2012a; Herranz et al., 2013). In cell cultures
from the RPM and the magnet were compared with Affy- we verified (i) the alteration of the relative proportion of cell
metrix microarray-based expression data obtained in real cycle phases (which probably leads to a change in the du-
microgravity (Paul et al., 2012). Although the gene ontologies ration of the cycle), (ii) the change in expression of numerous
affected are similar in all experiments (mainly stress-related genes acting as regulators in cell cycle checkpoints of phase
genes), the intensity of the gene expression changes and the transitions, and (iii) the deregulation of ribosome biogenesis
particular collection of genes affected are strongly dependent by means of the alteration of nucleolar structure and of
on the source material (seedlings, diffuse callus, confluent transcriptional and post-translational changes of key pro-
callus) and the duration of the treatment. teins of this process, such as nucleolin and fibrillarin.
Specifically, in the magnet experiment analyzed with ge- More recently, we used the pipette clinostat (Fig. 1B) for
nomic and proteomic methods, the effects of altered gravity exposing suspension plant cell cultures to fast clinorotation
were significantly masked by other magnetic field effects. In (60 rpm). Experiments have been done in which both asyn-
fact, the intensity of alterations caused by the gravitational chronous and synchronized samples were used to gain
stress was strengthened by an amplified environmental knowledge on the effects of clinorotation on cell cycle
10 HERRANZ ET AL.

regulation and progression. Preliminary results of these ex- perienced pseudo-weightless conditions. Flies were observed
periments suggest that the cell cycle duration was altered levitating within a few millimeters of the empirically deter-
under 2-D clinorotation, although in this experiment the 1g mined levitation point of water (Herranz et al., 2012; Hill
control was performed in the same container, without et al., 2012). Two additional groups of flies were exposed to
shaking, but otherwise with the same experimental setup pseudo-hypergravity conditions (in an arena enclosing the
(1 ml pipette) also reflected high differences in proliferation 2g* point) and normal gravity conditions (arena enclosing
rates. This indicates that the configuration of the 1g control the 1g* point) within the spatially varying field, and a fourth
experiments has to be carefully considered. arena was set up well away from the magnet (1g). The be-
We conclude that, for Arabidopsis seedlings and cell cul- havior of flies in the 0g* arena was found to be consistent
tures especially, magnetic levitation presents problems for with that of flies flown on the space shuttle Columbia and
use as a method of microgravity simulation, as it is necessary the subsequent mission to the ISS; a pronounced increase in
to separate the effects of altered effective gravity from other the frequency of locomotor activity and the walking speed of
effects of the strong magnetic field, which can be challenging. the flies was observed in the pseudo-weightless conditions of
The problem is more apparent in cell cultures and molecular levitation, compared to flies in the 1g* and 1g arenas (Hill
biology methods than in seedlings and cell biology tech- et al., 2012). Flies moved more slowly in the 2g* arena com-
niques (i.e., ultrastructural analyses). So far, the results with pared to flies in the 1g* and 1g arenas. Comparison of flies in
respect to Arabidopsis on the RPM are totally homologous to the 1g* arena with flies in the 1g arena revealed no additional
those obtained in real microgravity. Specifically for cell cul- effects of the strong magnetic field on behavior, up to 16.5 T.
tures, it is mandatory to expose experimental and control All the experiments in the magnet and the 1g control located
samples to the same environment, including temperature, away from the magnet were performed simultaneously,
shaking, and magnetic and inertial forces, parameters often under the same conditions of atmospheric pressure, tem-
difficult to control in GBFs. perature, humidity, lighting, and with the same batch of flies
(Fig. 5).
Similar effects have been observed in the RPM/LDC fa-
4.4. Drosophila behavior and gene expression
cilities, in which flies were constrained to move within a
Drosophila is used extensively as a model animal system in Biorack Type-I container, of the type employed on board the
space research due to its small size, short life cycle, and the ISS. In this case, however, mechanical forces and vibration
wealth of genetic literature support. Additionally, their life- produced during the sudden changes in rotation and speed
support requirements are relatively undemanding, requiring linked to the real random mode disturb Drosophila behavior
little or no human intervention once in orbit. As part of this similarly to the effect detected in paramecia, making a video-
GBF comparison project, we have studied the behavioral re- based analysis of the walking behavior impossible.
sponse of the flies to simulated microgravity conditions and the The RPM/LDC experiments showed that the influence of
effect of simulated microgravity on the gene expression profile. microgravity on the motility of imagoes varied between
The behavioral changes of the flies in microgravity con- different Drosophila strains. Furthermore, for a particular
ditions have been studied previously in experiments on strain, the influence of microgravity was found to be de-
board the space shuttle Columbia, STS-65 IML-2 mission pendent on the age and gender of the flies and closely related
(Benguria et al., 1996) and later as part of the Cervantes with environmental conditions (Serrano et al., 2010; Serrano
mission to the ISS (de Juan et al., 2007). Video recordings of et al., 2012). Consequently, not only the facility and envi-
the flies in space were analyzed and compared with re- ronmental condition but also the biological state (i.e., age,
cordings made of flies on the ground. The analysis showed gender, strain) of the organisms should be carefully consid-
that, in containers small enough to prevent flight, flies move ered in the experiment design.
more often and walk longer distances in microgravity com- The global gene expression profile has been observed to be
pared to flies in 1g. Since it was not possible to implement a affected in space, but different studies have found diverse
1g control for behavioral comparisons on board these par- intensity effects, from being nearly insignificant to affecting
ticular spaceflight missions, all the influences of microgravity thousands of genes. Our own real microgravity data belong
on behavior were identified by comparison with a 1g control to the latter case, suggesting a major disturbance of the
experiment implemented on the ground. Therefore, we global gene expression profile during Drosophila metamor-
cannot be certain that the behavior of the flies in space was phosis in microgravity as detected by microarray analyses
sensitive to the spaceflight preparations made immediately (Herranz et al., 2010). Our expectation is that these differ-
before launch, introducing some doubt about the root cause ences rely on different suboptimal environmental conditions
of the anomalous behavior observed in space. under previous space experiments.
To address these uncertainties, we performed experiments Experiments on the response of the transcriptome have
using ground-based devices for hypergravity and simula- been performed in different GBFs: real random RPM, levita-
tions of microgravity; the LDC (2g), RPM (real random mode tion in a superconducting magnet (0g*), and hypergravity in
at DESC-ESTEC), and magnetic levitation in a super- the LDC and in the magnet (2g and 2g*), replicating previous
conducting magnet at the University of Nottingham (Hill experiments in real microgravity (ISS) (Herranz et al., 2010,
et al., 2012) were used to clarify the contribution of gravity to 2012). The analysis indicated firstly that the RPM real random
motility and the aging processes. mode produces a very similar gene expression profile to the
In experiments in which magnetic levitation was used, the one obtained in microgravity; 90% of genes found altered in
walking paths of flies confined to a 25 mm diameter, 10 mm RPM experiments are also observed in ISS samples. Secondly,
tall cylindrical ‘‘arena’’ enclosing the stable levitation point of 10g hypergravity produces a less intense and opposite effect;
water (0g*) were analyzed. Within this arena, the flies ex- similar genes are altered in 10g hypergravity, but the genes
SIMULATED MICROGRAVITY ON EARTH—REVIEW 11

FIG. 5. Comparison in the motility of flies exposed to simulated and real microgravity. (A) Motility in the magnet (sim-
ulated microgravity and hypergravity) expressed as global activity in cm2/10 min. Activity is almost 3 · in unloaded
conditions and 0.5 · in 2g* simulation. Average activity of 20 periods of 0.5 min is indicated with the bars (statistical
significance *p < 0.05 has been calculated with the Bonferroni–multiple ANOVA method). (B) Magnetic levitation data (Hill
et al., 2012) shows the same trend as real spaceflight mission data obtained from the IML2 shuttle (Benguria et al., 1996) and
the AGEing Cervantes mission (de Juan et al., 2007) experiments as resumed in part (B); motility increased from 3 to 9 times
under real microgravity conditions depending on Drosophila selected strains exposed [SL, short life strain, comparable to wild
type; GA, altered gravity strain; LL, long life strains; y, young ( < 2-week-old imagoes); m, mature].

that are up-regulated in simulated microgravity are down- other hand, the RPM offers a relatively good simulation
regulated in hypergravity and vice versa. Thirdly, the most method (similar effect of increased motility was observed);
profound effects were very closely related to suboptimal en- but in this case, we suggest that magnetic levitation should
vironmental conditions (Herranz et al., 2010). In the case of be used preferentially since the magnet allows the behavioral
magnetic levitation, although some global-scale effects were effect of microgravity on the flies to be observed, unaffected
found, the 1g* control (samples exposed to 16.5 Tesla but by the random inertial movements induced by the RPM.
without field gradient) also showed many of these effects,
making it challenging to isolate the microgravity effects from
4.5. Fish behavior
‘‘simulation side effects’’ (Herranz et al., 2012), that is, other
effects of the strong magnetic field besides that of levitation. Fishes are ideal model organisms to investigate the per-
Our investigations included analysis of the impact of formance of the vertebrate vestibular system in correlation
population-related and environmental factors such as tem- with features of the inner ear (especially of the ear stones, the
perature, oxygen concentration, and magnetic field; many of otoliths), as these structures are highly conserved among all
the environmental factors studied are related to confinement vertebrates. Concerning the vestibular fitness in different
imposed by the Biorack Type I container conditions (Herranz gravitational environments, the observation of specific ab-
et al., 2007, 2009). The pooled analysis of all gene expression errant behavioral responses (i.e., kinetoses such as spinning
experiments suggested a synergic effect of environmental movements and looping responses) is an excellent method.
factors in the microgravity response. The response to mi- Experiments in which a RWV was used and drop-tower
crogravity is not consistent but depends strongly on the experiments at various g-levels were carried out on larval
original state of the organism and relates to stress responses, cichlid fish Oreochromic mossambicus and zebrafish Danio rerio.
in particular a mixture of biotic, abiotic, and defense re- The animals did not show any altered behavior on the RWV,
sponses. We speculate that an explanation for this finding such as aberrant spinning movements and looping responses.
might have an evolutionary background; organisms have At high speeds, they were centrifuged away; at lower speeds,
evolved groups of genes and stress pathways to control the the fish behaved completely normally. In the drop-tower ex-
adaptation to multiple stresses they have encountered tem- periments, at first, the stationary platform of the drop-tower
porarily during their evolution. Gravity has remained con- capsule (10 - 6g) was used, and subsequently, a centrifuge
stant on Earth since life appeared on our planet, but within the drop capsule was employed to achieve a series of
microgravity has never been experienced and, thus, a specific 10 distinct levels of reduced gravity. Under 10 - 6g, five types
response could not have evolved. of behavior were observed: normal swimming, normal rest-
We conclude that, for Drosophila gene expression arrays, ing, kinetotic behavior (namely spinning movements), looping
the RPM is a good simulator for real microgravity, as it re- responses, and zigzag movements.
vealed very similar results. In the experiments in which Experiments designed to deprive fish of the gravity stim-
magnetic levitation was used, effects of the strong magnetic ulus with a RPM (Dutch Space) and magnetic levitation were
field besides that of levitation were observed, making it not carried out for the following reasons. The maximum
challenging to identify the effects of simulated microgravity. velocity of the RPM (20 rpm) was not sufficient to yield a
For behavioral studies (fast detection of the g-vector), on the disorientation of animals that remained in the center of an
12 HERRANZ ET AL.

approximately 8 · 4 · 4 cm cubic chamber. We intend to use a when clinorotation covers a comparably brief period of de-
modified RPM with 60 rpm in a future experiment. Regard- velopment of late-staged cichlids (Li et al., 2011). Interest-
ing magnetic levitation, the number of animals needed for ingly, wall vessel rotation yielded larger than normal otoliths
statistical analyses could not be accommodated in the hold- in early-staged zebrafish Danio rerio, but otoliths of cichlid
ing chamber for technical reasons. fish were not affected.
Most animals that display spinning behavior under 10 - 6g We conclude that an RPM, as it has been available (Dutch
had highly asymmetric utricular otoliths as compared with Space), is of no use concerning developmental studies, as
normally behaving individuals, and the ratio of animals long as the speed of rotation is too low to disorient the ani-
swimming kinetotically increased with decreasing environ- mals. It may be used, however, with very early larval stages
mental gravity. (respective experiments have not been undertaken yet). We
We also found a clear correlation between otolith asym- also conclude that a RWV only can be used regarding ana-
metry and the level of gravity that induced spinning move- lyses on otolith growth/developmental issues when animals
ments: the higher the otolith asymmetry, the higher was the have not yet reached a stage when they can swim freely. It
threshold of gravity-inducing kinetosis, for example, 3.48% also depends on the species (mouth-breeding like the cichlid
otolith asymmetry in animals spinning at 0.3g and 1.12% at versus egg-laying like the zebrafish) whether a RWV is able
0.015g. Comparative analyses in which zebrafish were used to provide a good simulation of microgravity. Extreme care
were not carried out, as we wanted first to get a picture as has to be taken when considering the use of a RWV. A 2-D
thorough as possible of the cichlids’ behavior and associated clinostat is well suited for carrying out experiments on de-
issues at altered gravity. veloping fish (all stages). Yet it is not fully understood under
We conclude that the RWV is not suited for behavioral which circumstances (developmental stage of animals, de-
studies on fish, as free swimming late-larval stages are not velopmental period analyzed) clinorotation causes similar
affected by a rotation at low speed. At high speeds, the RWV effects as real microgravity in spaceflights.
acts like a centrifuge. Moreover, the RPM used in this study
is also not suitable for behavioral studies as long as the speed
4.7. Mammalian cell cultures (adherent)
of rotation is too low to disorient the animals. The drop-
tower is a fine instrument that provides real microgravity During previous studies in real microgravity, it was found
for the analysis of behavior at ‘‘high quality microgravity’’ that epidermal growth factor (EGF)-induced signal trans-
(10 - 6g) and at distinct g-levels below 1g (centrifuge active). duction is sensitive to microgravity. Studies during sounding
rocket missions (real microgravity) demonstrated that EGF-
induced early gene expression of c-fos and c-jun was de-
4.6. Fish otolith gravisensing
creased under microgravity conditions. Moreover, cells
Studies in which zebrafish were used were carried out by showed increased cell rounding under microgravity condi-
Chinese colleagues in close cooperation with us, who used a tions. Cell rounding is largely determined by the actin fila-
RWV (Li et al., 2011). As discussed above concerning studies ment system. The relative F-actin content was shown to
on behavior, the maximum speed of the RPM used (20 rpm) increase during microgravity conditions. Therefore, it can be
is not regarded as sufficient to provide stimulus deprivation said that real microgravity changes gene expression and cell
in fish. It is therefore to be expected that otolith growth will morphology in A431 cells (de Groot et al., 1991; Rijken et al.,
not be affected by RPM treatment. 1991; Boonstra, 1999).
Regarding magnetic levitation (HFML, Nijmegen, the The EGF-induced early gene expression and cell morphol-
Netherlands), it is well known from previous studies under ogy changes were also studied on the fast rotating 2-D clinostat
hypergravity that several days of altered gravity are required (CCM, Nuenen, the Netherlands) at 60 rpm, and similar results
to yield statistically relevant effects on otolith growth. For were obtained under simulated microgravity conditions as
technical reasons, runs that last days cannot be executed with compared to real microgravity (Boonstra, 1999). Similar ob-
this Bitter magnet but are possible with the use of a super- servations were also done with cells in suspension, such as
conducting magnet, such as the ones used for levitation ex- lymphocytes (Cogoli, 1992). This rounding of A431 cells was
periments at the University of Nottingham, UK. Hence, only further studied in simulated microgravity with an RPM, used
experimental results from the RWV (designed and con- at random speed, random direction, and random interval. The
structed at DLR) were compared with real microgravity data. maximum random speed was set as 360s - 1. It has been
In the course of an earlier space experiment, late-larval demonstrated that exposure of the cells in the RPM resulted in
cichlids (vestibular system operational) were subjected to a transient process of cell rounding and subsequent reattach-
microgravity during the FOTON-M3 spaceflight mission. ment and flattening of the cells. The cell rounding was ac-
Animals of another batch were subsequently clinorotated in a companied by an increased cortical actin cytoskeleton,
submersed fast-rotating clinostat with one axis of rotation (2- decreased actin stress fibers, and disappearance of focal adhe-
D clinostat, 60 rpm; designed and constructed at DLR). After sions (Moes et al., 2010). Similar results were also obtained in
the experiments, animals had reached a free-swimming stage. non-transformed mouse fibroblasts. Magnetic levitation in-
Animals that were grown under spaceflight conditions duced similar changes in the actin morphology of A431 cells
exhibited significantly larger than normal otoliths (both la- and mouse fibroblast that were also described in real micro-
pilli and sagittae, involved in sensing gravity and the hearing gravity. A transient process of cell rounding and renewed
process, respectively). Clinorotation resulted in larger than spreading was observed in time, illustrated by a changing actin
1g sagittae. However, no effect on lapilli was obtained. cytoskeleton and variation in the presence of focal adhesions.
Earlier studies had shown that clinorotation resulted in lar- However, further experiments demonstrated that the magnetic
ger than 1g otoliths when early-staged animals are used or fields may induce similar changes in actin morphology under
SIMULATED MICROGRAVITY ON EARTH—REVIEW 13

1g conditions. Therefore, it is required to confirm that responses the findings of the ESA GBF project. Table 1 could serve as a
are induced by the simulation of microgravity and not caused guide for scientists preparing space experiments or who wish
by magnetic field side effects. Exposure of mouse fibroblasts to do stand-alone experiments under altered gravity condi-
C3H10T1/2 to magnetic fields with or without levitation was tions. The quality of microgravity simulation strongly de-
shown to result in similar cytoskeletal changes. This indicates pends on numerous factors, such as sample size, type of
that the use of magnetic levitation for microgravity simulation tissue and cells, and so on, which are discussed in this paper,
may not be a suitable method to study gravisensing of as well as the reaction time and the threshold of gravity
mammalian cells. However, quantitative measurements of sensing of the biological process studied.
cytoskeletal polymers need to be done to fully understand the Table 1 reveals that the 2-D clinostat especially running in
impact of the magnetic field per se and of simulated micro- the fast rotating mode provides a good simulation of micro-
gravity and their effect on the cytoskeleton. gravity and can be recommended for most biological organ-
We conclude that both the fast rotating clinostat and the isms studied. The RPM was also found to be a suitable
RPM are valuable tools for simulating microgravity condi- simulator for some larger organisms as was shown for
tions in adherent mammalian cells. In contrast, the magnetic Drosophila and Arabidopsis. In studies of Drosophila behavior,
field used for levitation has significant effects in these cells, magnetic levitation was particularly successful at reproducing
especially on the cytoskeleton; therefore, magnetic levitation is the effects of real microgravity and obtained better results
not applicable to simulate microgravity in these adherent cells. than the RPM. In the other studies, magnetic levitation was
found to be of limited use as a microgravity simulator, owing
4.8. Mammalian cell cultures (suspension) to the difficulty in separating the effects of levitation from
other effects of the strong magnetic field on the organism.
Expression of the cell cycle regulatory protein p21Waf1/Cip1
The fast-rotating clinostat and the RPM are based on the
in human Jurkat T cells and phagocytosis and oxidative burst
principle of changing the direction of gravity with respect to
reaction in rat N8383 macrophages were used as a model
the sample mounted in the rotation axis. If the biological
system for non-adherent/semi-adherent cells. After treat-
sensing process is slow enough, the system will no longer
ment of Jurkat T cells with PMA, p21Waf1/Cip1 protein ex-
respond to the omnilateral stimulation. However, if the
pression was reduced 4.2-fold after 15 min in 1g but was
sensing process is fast and sensitive, the system might be
enhanced 1.6-fold after 15 min during clinorotation (2-D test-
continuously mechanically stimulated and might respond
tube clinostat, 60 rpm, pipette inner radius 1.5 mm, maximal
with typical stress reactions or even cell death (van Loon,
residual acceleration 6 · 10 - 3g, 37C) (Thiel et al., 2012). In
2007). Randomization of the gravity vector requires time;
addition to the protein expression, mRNA transcription
therefore, only processes that require a certain lag-time phase
levels were analyzed by real-time PCR. After 2 h of clino-
can be studied. Since the randomization time is dependent on
rotation, p21Waf1/Cip1 mRNA was decreased, whereas we
the rotational speed, different processes require different
detected increased levels after 2 h under magnetic levitation
rotational speeds, depending on their intrinsic lag-time phase.
at 0g* position (HFML, Nijmegen, the Netherlands). In gen-
For this reason, clinostats or RPMs cannot properly simulate
eral, we observed severely degraded RNA in all magnetic
microgravity for relatively fast molecular and cellular
levitation experiments in which Jurkat T cells were used.
Primary CD4-T cells from human donors did not survive the
magnetic levitation experiment but were viable before Table 1. Biological Responses in Microgravity
transfer into the magnet. Phagocytosis-mediated reactive Simulators (GBFs) in Comparison
oxygen species (ROS) production by N8383 cells was re- to Real Microgravity
duced by clinorotation, while no significant influence of
Object 2-D Clinostat RPM Levitation
clinorotation on the vitality of the cells was detected. A re-
duction in ROS was also observed in real microgravity Paramecium ++ - -
during parabolic flights. Interestingly, the amount of reduc- Euglena ++ - -
tion in ROS was directly influenced by the speed of clinor- Chara ++ + -*
otation. This result clearly shows that comparative studies by Arabidopsis
changing the speed of the clinostat are necessary to provide  Cell proliferation/growth n.a. ++ +*
 Gene expression n.a. + -
similar conditions as in microgravity. According to the
clinostat experiments, endpoint measurements were per- Drosophila
 Behavior n.a. + ++
formed also in the magnetic levitation setup. No significant  Gene expression n.a. ++ +
influence of magnetic levitation on phagocytotic activity or Fish
ROS production could be detected.  Behavior n.a. n.a. n.a.
Therefore, we conclude that the fast rotating clinostat is a  Development + n.a. n.a.
valuable tool to simulate microgravity for suspension cell Mammalian
cultures. In contrast, the magnetic levitation setup as used in  Adherent cells + + -
this study failed to reproduce the results obtained by clin-  Cells in suspension + n.a. -
orotation and from real microgravity conditions.
For further details see corresponding sections.
Symbols indicate that biological response to simulation is identical
5. Conclusions and Recommendations ( + + ), similar ( + ), or different ( - ) to those of real microgravity
experiments. n.a. means not applicable or data not available from
To anticipate which simulator might be the most appro- spaceflights.
priate GBF for a given biological system to provide a good *With the available field-gradient, levitation of statoliths was not
simulation of microgravity, the following table summarizes achieved.
14 HERRANZ ET AL.

processes. Increasing the speed of rotation strongly increases approaches (ESA contract 4200022650).’’ Nevertheless, pri-
the quality of the simulated microgravity and can even pro- mary research reviewed in this manuscript has been possible
vide near weightlessness (Briegleb, 1992). However, fast ro- by grants from the Spanish Space Program in the ‘‘Plan
tation also strongly reduces the area along the rotation axis in Nacional de Investigacion Cientifica y Desarrollo Tecnologico’’
which the omnilateral stimulation prevents effectively gravity AYA2009-07792-E to R.H., AYA2009-07952 and AYA2010-
sensing. Further away from the rotation axis, centrifugal 11834-E to F.J.M., German Space Administration grants on
forces dominate over the randomization effect. Therefore, fast behalf of Bundesministerium für Wirtschaft und Technologie
clinorotation provides simulated near weightlessness for only 50WB0527 to R.H., 50WB0815 to M.B., 50WB0828 to M.L.,
small samples that are positioned along the rotation axis. For 50WB0921 to O.U., and Dutch Space Research Organization
RPM experiments in which a relatively large liquid volume is NWO-ALW-SRON grant MG-057 to J.J.W.A.vL. Magnetic
used, one should note liquid movement and shear forces levitation at the High Field Magnet Laboratory in Nijmegen
within the volume (Leguy et al., 2011). The current study was granted by EuroMagNET II under the EU contract n
confirms that the RPM seems to be the system of choice for 228043 and by the Stichting voor Fundamenteel Onderzoek
Arabidopsis (Hoson et al., 1992, 1997; Kraft et al., 2000). der Materie (FOM), financially supported by the Nederlandse
We considered magnetic levitation to be an interesting new Organisatie voor Wetenschappelijk Onderzoek (NWO). Re-
technology for microgravity simulation with which to over- search using the University of Nottingham’s superconducting
come problems such as lag phase of perception due to its magnet was supported by grants from the UK Engineering
molecular scale effects and continuous (not time-averaged like and Physical Sciences Research Council (EPSRC), Nos. GR/
in the case of clinostat or RPM) presence of the magnetic field S83005/01 and EP/G037647/1. R.J.A.H acknowledges EPSRC
gradient (Beaugnon and Tournier, 1991a, 1991b). Conse- for support under a Research Fellowship EP/I004599/1 and
quently, no lag-time phase is required, and organism response C-DIP grant EP/J005452/1.
time to microgravity is not an issue under magnetic levitation.
Results of our studies on Drosophila melanogaster (Hill et al., Disclosure Statement
2012), in which the effect of real microgravity on the behavior
of flies was successfully reproduced by levitation, suggest that No competing financial interests exist.
levitation may be suited to study behavioral responses to
microgravity in small animals; further studies on other or- Abbreviations
ganisms are required to assess this possibility. Our other 2-D, two-dimensional; 3-D, three-dimensional; DLR, Ger-
studies in which levitation was used showed limitations of man Aerospace Center; EGF, epidermal growth factor; GBF,
this technique for investigating wider effects of microgravity ground-based facility; HFML, High Field Magnet Labora-
on the organism and the need of further investigations on the tory; ISS, International Space Station; LDC, Large Diameter
effects of strong magnetic fields on biological systems. Centrifuge; ROS, reactive oxygen species; RPM, random
These considerations clearly show that the experimenter positioning machine; RWV, rotating wall vessel.
has to carefully balance and consider the different aspects of
the various microgravity simulators and operation modes.
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