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A Novel Microgravity Simulator Applicable for Three-Dimensional Cell Culturing


Abstract and Figures

Random Positioning Machines (RPM) were introduced decades ago to simulate microgravity. Since then numerous experiments have been carried out to study its influence on biological samples. The machine is valued by the scientific community involved in space relevant topics as an excellent experimental tool to conduct pre-studies, for example, before sending samples into space. We have developed a novel version of the traditional RPM to broaden its operative range. This novel version has now become interesting to researchers who are working in the field of tissue engineering, particularly those interested in alternative methods for three-dimensional (3D) cell culturing. The main modifications concern the cell culture condition and the algorithm that controls the movement of the frames for the nullification of gravity. An incubator was integrated into the inner frame of the RPM allowing precise control over the cell culture environment. Furthermore, several feed-throughs now allow a permanent supply of gas like CO 2. All these modifications substantially improve conditions to culture cells; furthermore, the rewritten software responsible for controlling the movement of the frames enhances the quality of the generated microgravity. Cell culture experiments were carried out with human lymphocytes on the novel RPM model to compare the obtained response to the results gathered on an older well-established RPM as well as to data from space flights. The overall outcome of the tests validates this novel RPM for cell cultivation under simulated microgravity conditions.
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Microgravity Sci. Technol.
DOI 10.1007/s12217-014-9364-2
A Novel Microgravity Simulator Applicable
for Three-Dimensional Cell Culturing
Simon L. Wuest ·St´
ephane Richard ·
Isabelle Walther ·Reinhard Furrer ·
Roland Anderegg ·J¨
org Sekler ·Marcel Egli
Received: 8 July 2013 / Accepted: 26 March 2014
© Springer Science+Business Media Dordrecht 2014
Abstract Random Positioning Machines (RPM) were
introduced decades ago to simulate microgravity. Since then
numerous experiments have been carried out to study its
influence on biological samples. The machine is valued by
the scientific community involved in space relevant topics
as an excellent experimental tool to conduct pre-studies,
for example, before sending samples into space. We have
developed a novel version of the traditional RPM to broaden
its operative range. This novel version has now become
interesting to researchers who are working in the field of
tissue engineering, particularly those interested in alterna-
tive methods for three-dimensional (3D) cell culturing. The
main modifications concern the cell culture condition and
the algorithm that controls the movement of the frames for
the nullification of gravity. An incubator was integrated into
the inner frame of the RPM allowing precise control over
the cell culture environment. Furthermore, several feed-
throughs now allow a permanent supply of gas like CO2.
All these modifications substantially improve conditions to
culture cells; furthermore, the rewritten software responsi-
ble for controlling the movement of the frames enhances
the quality of the generated microgravity. Cell culture
S. L. Wuest ·R. Anderegg ·J. Sekler
Institute for Automation, University of Applied Science
Northwestern Switzerland, Windisch, Switzerland
S. L. Wuest ·S. Richard ·I. Walther ·M. Egli ()
CC Aerospace Biomedical Science and Technology, Lucerne
School of Engineering and Architecture,
Seestrasse 41, 6052 Hergiswil, Switzerland
R. Furrer
Institute of Mathematics, University of Zurich,
urich, Switzerland
experiments were carried out with human lymphocytes on
the novel RPM model to compare the obtained response to
the results gathered on an older well-established RPM as
well as to data from space flights. The overall outcome of
the tests validates this novel RPM for cell cultivation under
simulated microgravity conditions.
Keywords Random Positioning Machine ·Microgravity ·
Tissue engineering ·3D cell cuturing ·Kinematic
tTime [s]
IInner frame
OOuter frame
GGlobal frame
gI(t) Earth gravitation vector in the inner frame, at
the time point t[g]
gGEarth gravitation vector in the global frame
α(t) Rotation angle of the outer frame to the
global frame, at the time point t[rad]
α0Rotation angle of the outer frame to the
global frame, at the time point t=0 [rad]
β(t) Rotation angle of the inner frame to the outer
frame, at the time point t[rad]
β0Rotation angle of the inner frame to the outer
frame, at the time point t=0 [rad]
Gmean Mean gravity [g]
GX,mean ;GY,mean;GZ,mean Mean gravity in X-, Y-and
Z-direction (inner frame) [g]
gX,I,i ,g
Y,I,i ,g
Z,I,i Samples indicating the direction of
the earth gravity vector transposed to the
Microgravity Sci. Technol.
inner frame, in the local X-, Y-andZ-
directions, respectively [g]
pglobal (t)Arbitrary point in the global frame, at time
point t[m]
rlocal Arbitrary point in the local inner frame [m]
rX,loca l ,r
Y,local;rZ,local Coordinates in the X,Yand Z-
directions of an arbitrary point rlocal in the
local inner frame [m]
rDistance from the center of rotation [m]
ωαRotation velocity of the outer frame [rad/s]
ωβRotation velocity of the inner frame [rad/s]
ωRotation velocity if both frames rotate with
the same velocity ωα=ωβ=ω[rad/s]
Aglobal (t)Acceleration considered in the global frame,
at time point t[m/s2]
AX,global(t ),A
Y,global (t ), AZ,global(t ) Acceleration con-
sidered in the global frame in X,Y,andZ-
direction, respectively, at time point t[m/s2]
Amean,global Mean acceleration, considered in the global
frame [m/s2]
Amean,global Minimum expected mean acceleration
Amean,global Maximum expected mean acceleration
Alocal (t)Acceleration in the local inner frame, at time
point t[m/s2]
Amean,local Mean acceleration, considered in the local
frame [m/s2]
Regular 2-dimensional (2D) cell culture techniques repre-
sent a convenient method to study biological processes.
The use of this method has substantially improved our
understanding of cellular mechanisms over the last decades.
Tissue cells are cultured in plastic dishes, where they
anchor to the inner flat surface via the extracellular
matrix. Obviously, this conventional 2D approach does not
account for the third dimension. Cells naturally belong to a
3-dimensional (3D) organization forming cell clusters or
tissues in which mechanical, structural and chemical inter-
actions between cells and/or the extracellular matrix (ECM)
take place in all three dimensions.
Indeed, recent evidence reveals striking discrepancies in
the behavior of cells cultured in 2D or 3D (Anders et al.
2003;Weaveretal.1997;Wolfetal.2003). In fact, 3D cell
cultures reflect the in vivo situation more accurately than
2D cell cultures, and 3D culture allows reducing the gap
between the artificial cell culture in vitro and the physio-
logical situation. Therefore, researchers have been looking
for 3D cell culture strategies that enable more accurate
mimicking of tissue or organ structures and functions. To
do so, culture techniques have been constantly developed.
Single and co-culture techniques have been developed such
as cellular spheroids (Lin and Chang 2008) or polarized
epithelial cell culture (Shaw et al. 2004). Spheroidal cell
structures for example were introduced in tumor research
a long time ago (Santini and Rainaldi 1999;Ivascuand
Kubbies 2006; Friedrich et al. 2009).
In that context, spheroids have been shown to better
represent characteristics of in vivo tumors. Furthermore,
they have also been developed to study angiogenesis
(Wenger et al. 2005; Wenger et al. 2004). Of all the strate-
gies to simulate 3D in vivo tissue growth, hydrogels have
emerged as an attractive option that provides an artifi-
cial environment for optimal 3D cell culture. Such gels
consist of collagen (Butcher and Nerem 2004), dextran
(Cadee et al. 2000; Cascone et al. 2001), Matrigel™
(Hughes et al. 2010) as well as other materials (Burdick
and Prestwich 2011; Eyrich et al. 2007;Hoetal.2010;
Masters et al. 2005). Those gels can be supplemented with
particular enzymes and growth factors (Kleinman et al.
1986; Kleinman et al. 1982). In addition to hydrogels, 3D
platforms have been developed such as the BioLevitator™
from Hamilton. There, cells loaded with magnetic beads are
cultured in 3D while exposed to a magnetic field.
In this paper, an alternative approach to 3D cell cultur-
ing by using the RPM is presented. The RPM (van Loon
2007; Borst and Van Loon 2009) is a 3-axis clinostat in
which the two axes are essentially rotated at constant speed.
Its working concept evolved from simple clinostats, which
were developed first in 1879 by Julius von Sachs, a botanist
who wanted to investigate gravitropism in plants (van Loon
2007). Through the particular movement generated by the
RPM, the weight vector is continually reoriented as in
traditional clinorotation, but with directional randomization.
The purpose of the original idea was to simulate weight-
lessness more accurately, but the concept can be applied to
generate 3D culture as well. Indeed, randomization of the
Earth gravity vector allows redistributing the gravity forces
constantly so that cells grow in a similar environment as
in organs/tissues (Kraft et al. 2000). Cells cultured under
those conditions will no longer sediment, therefore allow-
ing for omnidirectional cell growth. Supporting this idea,
a similar approach has already been successfully applied
using a rotating wall vessel bioreactor (Barrila et al. 2010).
Moreover, it has been recently shown that proliferating
endothelial cells cultured on the RPM start to grow as
multicellular spheroids. Later on, tubular structures were
formed by the spheroidal growing cell structure and a clear
increase of extracellular matrix proteins was measured as
well (Pietsch et al. 2011).
The evolution of the RPM reported here lies mainly
in the full integration of a CO2incubator directly on
the internal frame of the RPM. Such an upgrade allows
Microgravity Sci. Technol.
close control of the temperature without the need of a
completely air-conditioned room. In addition, CO2can con-
stantly be supplied to the cells. CO2regulation allows
maintaining a constant pH matching physiological condi-
tions (7.2–7.5) and makes additional buffering strategies
Material and Methods
Random Positioning Machine (RPM)
The RPM has a solid lightweight construction that sup-
ports two gimbal-mounted frames (Fig. 1). A commercially
available CO2incubator, slightly modified to fit the needs,
is attached to the inner frame, which provides a 14 liter
cell culture chamber. The chamber offers optimal culture
conditions, such as maintaining defined levels of CO2and
temperature. In addition, the chamber is equipped with
various sensors for monitoring crucial culturing parame-
ters. The movement of the rotating frames is driven by two
independently operated and controlled engines via timing
belts to avoid slippage. Complicated belt routing is avoided
by mounting one engine directly onto the outer frame. This
design ensures independent control of the frames move-
ment. The incubator motion is monitored through encoders
attached to the motors directly. 3D accelerometers fixed
to the inner frame are used for quality control of the
This version of the RPM is equipped with a rotational
feed-through for gas and liquids allowing, for example,
constant CO2supply to the incubator. Power supply for the
various devices on the inner and outer frame, as well as
time critical communication, are transmitted via slip ring
capsules. As an alternative route, non-time critical infor-
mation can also be transmitted via a WLAN. The WLAN
system offers the advantage of being able to monitor cru-
cial RPM data from a nearby office by using a standard web
browser. Having the incubator placed in the RPM instead of
placing a small (desktop) RPM inside an incubator brings
the advantage of a relatively large test chamber. At the same
time the confinement of the incubator chamber insures that
all samples are in a reasonable distance to the center of
rotation in order to avoid centrifugal forces. In addition
the incubator does not get contaminated through the RPM
machinery such as oil vapor or debris of wear.
The software designed to control the RPM functions is
run on a built-in industrial PC and is a LabVIEW based
application. Numerous functions have been implemented
into the operating software like an automatic start-stop func-
tion executable at predefined time/date or the option to
monitor all the crucial incubator parameters like tempera-
ture, gas composition and motion.
Biological Experiment
All chemicals and drugs used for conducting the mentioned
biological experiments were purchased from Sigma (Buchs,
Switzerland) unless otherwise stated.
Lymphocytes Culture
Peripheral blood (450 ml) was collected from healthy
donors and further processed for lymphocyte isolation as
previously described (Cogoli-Greuter et al. 1996; Cogoli-
Greuter et al. 1994; Gmunder et al. 1990; Pippia et al.
1996). In brief: a lymphocyte-enriched suspension was
prepared by gradient centrifugation using Lymphocyte
Separation Medium (PAA, LMS-1077, J15-004). A T lym-
phocyte enriched suspension was later obtained using a
Human T Cell Enrichment kit (RnD research, HTCC-25).
The cells were re-suspended in RPMI-1640 supplemented
with 40 mM HEPES, 5 mM sodium bicarbonate, gen-
tamycin (50 μl/ml), 4 mM L-Glutamine and 10 % fetal calf
serum (PAA A15-101). The cells were then transferred into
LYCIS containers (1 ml at a density of 1.5 million cells/ml)
(Chang et al. 2012) and mounted either at the rotating cen-
ter of the RPM or in a stationary laboratory incubator(equal
incubator as on the RPM). In the experiments described
here, the samples that were placed at the rotating center of
the RPM (within a radius of 10 cm) experienced a rota-
tion with an angular velocity of 40 deg/s and an ambient
temperature of 37 C. Cells on the RPM were exposed
to the RPM simulated microgravity for one hour prior to
lymphocyte activation by injection of concanavalin A /CD
28 mixture (10 μg/ml and 4 μg/ml final concentrations,
respectively; performed by a quick interruption of the
rotation, <1 min.). After activation, the cells remained
under their respective conditions (simulated microgravity or
stationary) for 22 h.
FACS Analysis
For the FACS analysis, T lymphocytes were stained at 4 C
using a CD-25 antibody (MACS, 130-091-024) and fixed
with 2 % paraformaldehyde according to the manufacture’s
instructions. Thereafter, the T lymphocyte populations were
investigated by a FACS analyzer (BD FACS Calibur,
Becton Dickinson). The CD-25 positive T lymphocytes
were considered as activated.
Statistical Analysis
Statistical variations were tested by applying the Wilcoxon
rank-sum Test (p values 0.05 were regarded as statis-
tically different). Data are reported as means ±standard
deviation (n =3).
Microgravity Sci. Technol.
Results and Discussion
Equalizing Gravitation Through Random Positioning
The basic idea followed, to generate simulated micrograv-
ity, is to distribute the Earth’s gravity vector in space evenly
over time by constantly reorienting the sample chamber
to random positions (van Loon 2007). Therefore the RPM
can be treated as a quasi-static machine (if the time frames
are chosen small enough). Kinematic effects greatly depend
on the position of the sample and the rotation velocity.
Since the rotation velocity is small, kinematic effect such as
centrifugal forces are negligible. In this section the con-
cept of quasi-static random positioning is discussed in more
Gravity Vector Transposition to the Inner Frame
For the examination, it is important to distinguish between
the static global and the rotating local frame (coordinate
system) of the inner frame (Figs. 1and 2). The moving
frames of the RPM are mounted in the global frame, which
is fixed, and gravity acts always normal to the ground
(representing the global XY-plane). The local inner frame
represents the frame (coordinate system) of the sample and
is constantly reoriented with respect to the global frame. The
gravity vector of the global frame is transposed into the local
inner frame. This decomposition of the gravity vector to the
inner coordinate system (where the samples are mounted)
is computed, by employing two rotational matrixes. For this
operation the position of the outer and inner frames needs to
be known:
cos β(t)0sinβ(t)
sin β(t)0cosβ(t)
cos α(t)·sin β(t)
sin α(t)
cos α(t)·cos β(t)
10 0
0cosα(t)sin α(t)
0sinα(t)cos α(t)
Random Walk
To equalize gravity we employ a Random Walk algorithm
by which both frames of the RPM rotate at a constant
velocity, but the rotation direction is inverted at randomly
chosen time points. The velocity transition from forward
to backward rotation takes place at a constant rotational
Plotting the local gravity vector of the inner frames over
the course of time, it becomes clear that the vector tip travels
on a sphere with a radius of 1 g. However the distribu-
tion is not entirely even and concentrates at the two poles
lying at the rotational axis of the inner frame (local Y-axis;
Fig. 3). They become predominant after a short time. This
phenomenon can be explained by the rotation of the outer
frame. Every time the outer frame is in a vertical position,
with the rotation axis of the inner frame parallel to the grav-
ity, the local gravity vector is unavoidably pulled towards
one of the two poles. In general, the resulting picture is sym-
metrical. Figure 4illustrates the time course of how many
times the Earth’s gravity vector is pointing to an arbitrary
position after a given period of time (1, 6, 15, 30, 60 min
and 5 h).
Mean Gravity
As a quality measure for the distribution of orientation, we
use the mean gravity over time, defined as:
Gmean =GX,mea n2+GY,mean2+GZ,mean2
where the mean gravity values in the three directions are
defined as follows:
GX,mean =n
GY,mean =n
GZ,mean =n
The mean gravity is easy to compute and memory efficient,
since only the sum of all samples in X,Yand Z, as well as
the total number of samples, has to be stored. In Fig. 5the
mean gravity is plotted over time. The mean gravity falls
quickly below 0.1 g and stabilizes below 0.03 g within 2 h
of operation. With this concept two points lying opposite
each other (sign change) are compensated. Therefore the
two poles seen in Fig. 4also cancel themselves out. In Fig. 5
Microgravity Sci. Technol.
Fig. 1 Picture of the RPM construction. A CO2incubator is mounted
in the center of the gimbal framework. The frames are driven by two
independent precision motors
a temporal increase between approximately 0.5 and 1.5 h is
visible. This appears because the Random Walk algorithm
is based on random numbers and because the mean grav-
ity is computed through averaging. At the beginning of an
experiment little data contribute to the average, making it
sensitive to any deviations. With the elapsing experiment
time, the mean gravity becomes increasingly robust. This
temporal increase of the mean gravity thus depends on the
combination of the random numbers.
Numerical Illustration
Since the Random Walk algorithm depends on random
numbers, the outcome of two successive runs, are not iden-
tical. To demonstrate the reliability of the algorithm several
hundred runs of numerical simulations were performed.
Each of these simulations represents an experiment of sev-
eral hours. The resulting mean gravity at a defined time
were finally recorded and plotted as a histogram. As shown
in Fig. 6, all values stay below 0.03 g. The mean value of
these 500 samples is 0.0086 g ±0.0039 g (SD) for a rota-
tional velocity of 60 deg/sec and 0.0105 g ±0.0044 g (SD)
for 40 deg/sec. The histograms in Fig. 6are illustrating that
the Random Walk is reliably producing simulated micro-
gravity at both velocities. For particular angular velocities
and assuming piecewise constant accelerations, it is possi-
ble to show that the expected mean squared gravity vanishes
over time. The approach, based on a central limit theorem,
is mathematically quite involved and is outside the scope of
this article.
For slow rotations the quasi-static approach is sufficiently
valid and applies for all samples close enough to the cen-
ter of rotation. The acceleration caused by kinematics is,
however, much more difficult to deal with and depends
greatly on the sample position relative to the center of
rotation (Fig. 7).
The experienced acceleration of an arbitrary point can
be computed as follows: Any point
plocal in the inner
frame can be described with a vector
rlocal from the
center of rotation. The path of
plocal in the global frame, as
both frames rotate, is computed by employing two rotational
pglobal =G
pglobal (t ) =10 0
0cosα·t+α0)sin α·t+α0)
0sin α·t+α0)cos α·t+α0)
cos ωβ·t+β00sin ωβ·t+β0
01 0
sin ωβ·t+β00cos
·rX,loca l
By differentiating the position twice with respect to
time we get the accelerations AX,global (t),AY,global(t) and
AZ,global (t), acting in the direction of the global coordi-
nate systems axis X,Yand Z, respectively. From these three
accelerations the absolute acceleration magnitude Aglobal
can be computed.
Aglobal (t ) =AX,global (t )2+AY,global (t )2+AZ,gl obal (t)2
To simplify, we assume that both frames rotate with an
identical velocity, ωα=ωβ=ω.Thetermsα0and β0can
Fig. 2 Schematic of the RPMs
gimbal framework. The samples
(on the inner frame) are rotated
around two perpendicular axes
Microgravity Sci. Technol.
Fig. 3 Distribution of the local gravity vector over the course of time.
The blue line indicates the path of the gravity vector, as it would be
experienced by a sample at the center of rotation. The local gravity
vector frequently passes through two poles lying on the Y-axis (rota-
tion axis of inner frame). This becomes clearly visible already after
6min(top left). (Random Walk, Velocity: 60 deg/sec)
be set to zero (α0=β0=0), since they have no influence
on the mean and peak acceleration. This results in:
Aglobal (t)=ω2·r2
X,loca l 4.5+cos (2·ω·t)
Y,local +r2
Z,local 4.5cos (2·ω·t)
rX,loca l ·rY,local ·4·cos (ω·t)rX,local ·rZ,local
·sin (2·ω·t)+rY,local ·rZ,local ·4·sin (ω·t)
From this formula we can approximate the mean accelera-
Amean,global ω24.5·r2
X,loca l +r2
Y,local +4.5·r2
To illustrate the formula above, path and total acceleration
for three different points are shown in Fig. 7. All points
have the same radius length but acceleration is by far not the
Even though the kinematic acceleration depends on the
location, it is limited to a range given by the rotation velocity
and the distance from the center of rotation:
Amean,global ω2·r
Amean,global 3
Microgravity Sci. Technol.
Fig. 4 Histograms of the orientation mapped as color code on an
imaginary sphere. The color of a particular spot on the sphere indi-
cates how often the gravity vector pointed at that direction. According
to the simulation, the orientation distribution is uneven, but symmetri-
cal and two poles become dominant after a short time (Random Walk,
Velocity: 60 deg/sec, fs=50 Hz)
Local Acceleration
The acceleration computed in the global frame (for a
specific time point) is not the acceleration experienced by
a point in the local frame. To be more precise, the absolute
value of the local and global acceleration is the same, but
not the direction:
Aglobal (t)=
Alocal (t)
Aglobal (t)=
Alocal (t)
Microgravity Sci. Technol.
Fig. 5 The mean gravity (upper track) as well as the mean gravity
of the components in the X-, Y-andZ- directions (lower track) plot-
ted over time. The temporal increase between 0.5 and 1.5 h appear
because the Random Walk algorithm is based on random numbers
and because the mean gravity is computed through averaging. With
the elapsing experiment time, the mean gravity becomes increasingly
robust to temporal deviations
To compute the local acceleration the global acceleration
has to be transformed by again employing the above rotation
Alocal (t)=0
Aglobal (t )
Alocal (t)=
cos ωβ·t+β00sin
sin ωβ·t+β00cos
10 0
(ωα·t+α0)sin (ωα·t+α0)
(ωα·t+α0)cos (ωα·t+α0)
Aglobal (t )
Again we can set the terms α0and β0to zero (α0=β0=0).
The rotation velocity shall be the same for both frames,
ωα=ωβ=ω. After substituting and simplifying we finally
Alocal (t )
rX,local ·cos(2·ω·t)3
2rZ,local ·sin(2·ω·t)
rX,local ·2·cos ·t) rY,local rZ,local ·2·sin ·t)
rX,local ·sin(2·ω·t)
2rZ,local ·cos(2·ω·t)+3
By computing the mean gravity, we first compute the mean
gravity for all three components in X,Yand Zand then we
Fig. 6 Histograms of 500
numerical simulations. Each
sample represents the mean
gravity at the center of rotation
after 5 h in operation. Left:
Random Walk, velocity:
60 deg/sec. Right: Random
Walk, velocity 40 deg/sec. The
histograms are illustrating that
the Random Walk is reliably
producing simulated
microgravity at both velocities
Microgravity Sci. Technol.
Fig. 7 Path and acceleration for three points with the same distance
from the center of rotation. Both (outer and inner) frames rotate at a
constant velocity with 60 deg/sec. The top row shows the path in blue.
The vector in magenta indicates the position of the specific point at
time t=0. On the bottom row, the global acceleration as a function
of time (in blue) and the corresponding mean (green)aswellasthe
minimum and maximum (red) is plotted. The resulting accelerations
depend greatly on the position, even though the distance to the center
of rotation is the same
Fig. 8 The mean global acceleration (Amean,gl obal) and peak global acceleration ( ˆ
Aglobal ) depending on the location (r=10 cm; ω=60 deg/sec)
Microgravity Sci. Technol.
compute the total mean gravity by using the vector addition.
If we do the same for the local acceleration we get:
Amean,local =ω2
1.5·rX,loca l
Amean,local =
=ω22.25 ·r2
X,loca l +r2
Y,local +2.25 ·r2
Acceleration Depending on the Position
As we have already seen above, the experienced accelera-
tion of a point depends strongly on the location. By iterating
through multiple points on a sphere and computing the mean
global acceleration (Amean,global )and the peak acceleration
Aglobal ), we get the results illustrated in Fig. 8. The radius
length ris constant and set to 10 cm. By visualizing the peak
acceleration, it becomes clear that the highest peak accel-
erations do not appear at the same locations as the highest
mean accelerations (Fig. 8). The points on the XZ-plane
(being perpendicular to the rotation axis of the inner frame)
experience the highest mean acceleration. The highest peak
accelerations appear on two planes parallel to the XZ-plane
and are approximately 0.38·raway from the XZ-plane. The
smallest accelerations appear on the two points lying on the
rotation axis of the inner frame (Y-axis).
Experimental Validation
T Lymphocytes purified from human peripheral blood
can be activated by Con A in vitro. The drug, a lectin
extracted from lentil seeds, exerts this effect by mimick-
ing the antigen-presenting process occurring during spe-
cific antigen-activation. The transmembrane protein CD25,
which is highly expressed on the surface of activated
Fig. 9 T lymphocyte activation by Con A/CD28 under 1 g and
simulated microgravity. Two healthy donors were tested
T lymphocytes, is used as a marker for the activation.
Several experiments have already shown that T lympho-
cytes reduce their activation substantially on exposure to
reduced gravity (either during space flights, sounding rocket
or RPM)(Cogoli-Greuter et al. 1994; Cogoli et al. 1984).
We use this effect to validate the quality of the algorithm
running on the novel RPM that generates simulated micro-
gravity for samples in the center of the rotating frames. By
comparing the activation values of samples under normal 1 g
conditions, simulated microgravity (created by the old and
established RPM as well as by the novel RPM), as well as
real space conditions, it appears that the simulated reduction
of the gravity field leads to a reduction of the T lymphocytes
activation by about 80–90 % (Fig. 9). These results are in
agreement with previous publications (Cogoli et al. 1984;
Gmunder et al. 1990; Cogoli-Greuter et al. 1994).
Here, we present a novel version of a RPM that is applicable
for experiments in the field of life science concerning micro-
gravity and 3D cell culturing. Our new machine applies
the well-established core principle of gravity nullification, a
concept that was introduced decades ago (van Loon 2007;
Borst and Van Loon 2009). In this novel RPM includes
features like stable environmental conditions (temperature,
CO2etc.) in the culture chamber as well as a constant sup-
ply of culture media or CO2to the samples, which creates
much better long-term cell culturing conditions than before.
A major objective of the novelRPM is to simulate micro-
gravity as accurately as possible. Thus, a major effort was
put into the theoretical concept of gravity nullification as
well as the conversion of that concept into the algorithm
controlling the movement of the frames. As we have shown
here, by applying the newly designed algorithm, the mean
gravity falls below 0.1 g within a few minutes and stabi-
lizes below 0.03 g thereafter within 2 h. The mathematical
analysis further demonstrated that samples placed anywhere
in the incubator experience low gravity levels. Despite the
difficulty to completely control kinematic accelerations,
they are limited to a given range, only depending on the rota-
tion velocity and the samples’ distance from the center of
rotation. These two parameters should therefore be kept as
low as possible. This novel RPM represents a valid tool for
simulating microgravity. This was confirmed by conduct-
ing experiments with human T lymphocytes. Their behavior
under microgravity (simulated on real space microgravity)
is well known and has been described in numerous pub-
lications (Cogoli et al. 1984; Cogoli-Greuter et al. 1994;
Gmunder et al. 1990). As illustrated in Fig. 9, exposing T
lymphocytes to reduced gravity leads to a severe reduction
of the activation. The figure further elucidates that there is
Microgravity Sci. Technol.
no difference between the old and well-established RPM
and our novel version of the RPM (Fig. 9). This indicates
that the algorithm of the novel RPM is comparable to the
one of the old RPM.
However, the novel version of the RPM is not only an
interesting tool for microgravity-related biological studies.
Long-term cultivation of cells under low gravity condi-
tions offers new aspects for tissue engineering. By adding
the described features, long-term cultivation of cells under
simulated microgravity is now possible for the first time
and is of a comparable quality to regular (stationary) cell
cultures. Permanent exchange of culture media can be pre-
cisely controlled by the novel RPM, as well as the addition
of particular drugs to the cell cultures at a particular time of
day for a defined period of time. Built in log files record any
action of the system allowing a retrospective analysis of the
experiment. Our former studies have already demonstrated
that long-term cultivation of endothelial cells under sim-
ulated microgravity conditions leads to spheroidal growth
behavior, including vascularization (Pietsch et al. 2011).
Thus, this novel version of the RPM offers an ideal platform
for studies such as tumor growth.
It will be interesting in future studies to show how other
cell types respond to cultivation under reduced gravity or
under mechanical unloading. It is likely that this culture
method bears new options and possibilities for cell culturing
in general, allowing tissue to grow that shows unexpected
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... 3D clinostats can be large machines that do not fit in an incubator (Hoson et al., 1997); Wuest et al., 2014;Ikeda et al., 2016). The 3D clinostat designed by Wuest et al. (2014) overcame this problem by integrating a small incubator into the inner frame of the 3D clinostat. ...
... 3D clinostats can be large machines that do not fit in an incubator (Hoson et al., 1997); Wuest et al., 2014;Ikeda et al., 2016). The 3D clinostat designed by Wuest et al. (2014) overcame this problem by integrating a small incubator into the inner frame of the 3D clinostat. The inexpensive 3D clinostat described in our study is similar to the 3D clinostat developed by Kim et al. (2017) and the RPM 2.0 (Yuri), which have a small enough footprint to fit within a standard cell culture incubator. ...
... The base design of our 3D clinostat with two rotating frames, a sample stage and a stand is the same as previously developed machines (Hoson et al., 1997;Van Loon 2007;Wuest et al., 2014;Kim et al., 2017); however, most 3D clinostats in the literature are made of metal. The structure of our new 3D clinostat is made of PLA plastic and designed to be 3D printed, as is the SciSpinner (CosE). ...
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2D and 3D Clinostats are used to simulate microgravity on Earth. These machines continuously alter the sample’s orientation, so the acceleration vector changes faster than the biological endpoint being monitored. Two commercially available microgravity simulators are the Rotary Cell Culture System (Synthecon Inc.), which is a 2D clinostat, and the RPM 2.0 (Yuri), which is a 3D clinostat that can operate as a random positioning machine or in constant frame velocity mode. We have developed an inexpensive 3D clinostat that can be 3D printed and assembled easily. To determine the optimal combination of inner (I) and outer (O) frame velocities to simulate microgravity, two factors were considered: the time-averaged magnitude and the distribution of the acceleration vector. A computer model was developed to predict the acceleration vector for combinations of frame velocities between 0.125 revolutions per minute (rpm) and 4 rpm, and a combination of I = 1.5 rpm and O = 3.875 rpm was predicted to produce the best microgravity simulation. Two other frame velocity combinations were also used in further tests: I = 0.75 rpm and O = 3.625 rpm, and I = 2 rpm and O = 1.125 rpm. By operating the RPM 2.0 in constant velocity mode at these three velocity combinations, the RPM 2.0 algorithm data confirmed that these operating conditions simulated microgravity. Mycobacterium marinum was selected for biological comparison experiments as this bacterium can grow as a biofilm or a planktonic culture. Biofilm experiments revealed that the RPM 2.0 and the 3D clinostat with I = 1.5 rpm and O = 3.825 rpm produced similar structures in attached biofilm, and similar changes in transcriptome for the bacteria in suspension compared to the normal gravity transcriptome. Operating the 3D clinostat at I = 2 rpm and O = 1.125 rpm, and the Synthecon 2D clinostat in simulated microgravity orientation at 25 rpm resulted in the same decreased planktonic growth and increased rifampicin survival compared to normal gravity. This study validates the inexpensive 3D clinostat and demonstrates the importance of testing the operating conditions of lab-developed clinostats with biological experiments.
... A miniaturized RPM with a maximum size of 50 cm  50 cm  50 cm allows the experiment to be done under a regular cell culture incubator [76]. Wuest et al. fitted the commercially available CO2 incubator onto the frames, in which temperature and other culture parameters are maintained and monitored through the incubator [77]. This device is called Random Position Incubator (RPI) [78]. ...
... This device is called Random Position Incubator (RPI) [78]. Moreover, due to the suitable gas supply, this device also found application in 3-D tissue culture similar to RWV [77]. In RPI, frames are rotated at a constant speed, and the direction is changed at random time points [78]. ...
Full-text available
Gravity plays an important role in the development of life on earth. The effect of gravity on living organisms can be investigated by controlling the magnitude of gravity. Most reduced gravity experiments are conducted on the Lower Earth Orbit (LEO) in the International Space Station (ISS). However, running experiments in ISS face challenges such as high cost, extreme condition, lack of direct accessibility, and long waiting period. Therefore, researchers have developed various ground-based devices and methods to perform reduced gravity experiments. However, the advantage of space conditions for developing new drugs, vaccines, and chemical applications requires more attention and new research. Advancements in conventional methods and the development of new methods are necessary to fulfil these demands. The advantages of Lab-on-a-Chip (LOC) devices make them an attractive option for simulating microgravity. This paper briefly reviews the advancement of LOC technologies for simulating microgravity in an earth-based laboratory.
... В качестве модельных клеток нами была выбрана линия мегакариобластных клеток (MEG-01) [38]. В качестве моделирования условий микрогравитации был выбран прибор RPM, который валидирован и широко используется в различных лабораториях разных стран [12,31,39]. ...
Актуальность. Исследования, выполненные в невесомости, показали, что космический полет вызывает серьезные физиологические изменения в живом организме. В клетках млекопитающих микрогравитация способна индуцировать и модулировать протекание таких ключевых процессов, как апоптоз, пролиферация, миграция и адгезия. Несмотря на возросший интерес к космической биологии и медицине, исследования клеточного цикла в условиях микрогравитации остаются спорными. Цель исследования – изучение экспрессии циклинов клеточного цикла клеток мегакариобластного лейкоза человека при воздействии моделированной микрогравитации. Методика. Для экспериментов клетки мегакариобластного лейкоза человека (MEG-01) высевали в культуральные флаконы. Условия микрогравитации моделировали с использованием прибора случайного позиционирования (RPM – Random Positioning Machine). Клетки размещали в центре платформы прибора случайного позиционирования (группы RPM) и сравнивали со статической контрольной группой (1g). Анализ экспрессии циклинов клеточного цикла проводили методом вестерн блота и на проточном цитофлоуриметре. Результаты. Результаты исследований показывают, что под воздействием микрогравитации клетки мегакариобластного лейкоза человека MEG-01 демонстрируют сопоставимые уровни экспрессии циклина D и E при сравнении с контрольной группой. Однако, уровни циклинов A и B повышались в течение первых 96 ч. В дальнейшем, количество этих циклинов снижалось к 168 ч в сравнении с предыдущей временной точкой и контрольной группой. Заключение. Таким образом, на основе полученных данных можно сделать заключение, что клетки MEG-01, подверженные RPM-моделированной микрогравитации успешно входят в клеточный цикл и завершают синтетическую фазу, но останавливаются в фазе G2 и не способны завершить митоз. Однако в более поздние сроки (168 ч) клетки MEG-01 успешно адаптируются к условиям невесомости. Результаты согласуются с экспериментальными данными, полученными при исследовании различных типов клеток при различных способах моделирования микрогравитации. Дальнейшие исследования влияния гравитации на клеточные реакции мегакариоцитов помогут понять патогенез заболеваний человека, приобретенных в экстремальных условиях. Background. Studies of weightlessness have shown that space flight causes serious physiological changes in a body. In mammalian cells, microgravity is able to induce and modulate key processes such as apoptosis, proliferation, migration, and adhesion. Despite growing interest to space biology and medicine, reports of cell cycle in microgravity remain controversial. Aim. This paper analyzes the expression of cell cycle cyclins in human megakaryoblastic leukemia cells exposed to simulated microgravity. Methods. Human megakaryoblastic leukemia (MEG-01) cells were seeded in culture flasks. Microgravity conditions were simulated using a Random Positioning Machine (RPM). Cells were placed at the center of the platform of the RPM (RPM group) and compared with a static control group (1 g). Cell cycle cyclin expression was analyzed by Western blotting and with a flow cytometer. Results. In the conditions of microgravity, MEG-01 showed comparable expression levels of cyclins E and D vs. the control group. However, concentrations of cyclins A and B increased during the first 96 h. Subsequently, concentrations of these cyclins decreased by 168 h compared to the previous time point and the control group. Conclusion. This study allowed a conclusion that MEG-01 cells exposed to RPM-modeled microgravity start proliferating and successfully finish the synthetic phase but stop in the G2 phase being unable to complete mitosis. However, at a later time (168 h), MEG-01 cells successfully adapt to the weightlessness conditions. In addition, the results are also consistent with reports of experiments on various cells under different conditions of simulated microgravity. Further studies of the effect of gravity on responses of megakaryocytes will provide insight into pathophysiology of human diseases acquired in extreme conditions.
... Current technology allows for the production and implementation of the ZPG effect, which has been extensively employed to study the physiological and behavioral reactions Life 2023, 13, 407 2 of 12 of higher plants, microbes, and animal tissues and organs to the ZPG effect [12][13][14][15][16][17][18]. For instance, a random positioning device that can continuously alter its orientation randomly with respect to an experiment's gravity vector and produce outcomes similar to those of real microgravity when the direction changes occur more quickly than the body's reaction time to gravity [13,19]. ...
Full-text available
Zero and partial gravities (ZPG) increase cardiovascular risk, while the corresponding theoretical foundation remains uncertain. In the article, the ZPG were generated through a rotating frame with two degrees of freedom in combination with the random walk algorithm. A precise 3D geometric configuration of the cardiovascular system was developed, and the Navier-Stokes laminar flow and solid mechanics were used as governing equations for blood flow and the surrounding tissue in the cardiovascular system. The ZPG were designed into governing equations through the volume force term. The computational fluid dynamics’ (CFD) simulations in combination with proper boundary conditions were carried out to investigate the influences of ZPG on the distribution of blood flow velocity, pressure, and shear stress in the cardiovascular system. The findings show that as simulated gravity gradually decreases from 0.7 g to 0.5 g to 0.3 g to 0 g, as opposed to normal gravity of 1 g, the maximum values of blood flow velocity, pressure, and shear stress on the walls of the aorta and its ramification significantly increase, which would lead to cardiovascular diseases. The research will lay a theoretical foundation for the comprehension of the ZPG effect on cardiovascular risk and the development of effective prevention and control measures under the circumstance of ZPG.
... Association in Vatican City in 2013) and a group from Switzerland published a comparable design [19]. ...
... Therefore, it is essential to establish an acceptable model to simulate microgravity on the ground. Currently, microgravity simulators specifically used for cell research principally include the suspension induced by strong magnetic fields, two-dimension and three-dimensional clinostats, rotating wall vessels (RWV) and random positioning machines (RPM) [18,[20][21][22]. Among them, the RPMs have been considered to be an efficient tool to construct the condition of simulated microgravity [22][23][24]. ...
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With the increasing application of nanomaterials in aerospace technology, the long-term space exposure to nanomaterials especially in the space full of radiation coupled with microgravity condition has aroused great health concerns of the astronauts. However, few studies have been conducted to assess these effects, which are crucial for seeking the possible intervention strategy. Herein, using a random positioning machine (RPM) to simulate microgravity, we investigated the behaviors of cells under simulated microgravity and also evaluated the possible toxicity of titanium dioxide nanoparticles (TiO2 NPs), a multifunctional nanomaterial with potential application in aerospace. Pulmonary epithelial cells A549 were exposed to normal gravity (1 g) and simulated gravity (~10-3 g), respectively. The results showed that simulated microgravity had no significant effect on the viability of A549 cells as compared with normal gravity within 48 h. The effects of TiO2 NPs exposure on cell viability and apoptosis were marginal with only a slightly decrease in cell viability and a subtle increase in apoptosis rate observed at a high concentration of TiO2 NPs (100 μg/mL). However, it was observed that the exposure to simulated microgravity could obviously reduce A549 cell migration compared with normal gravity. The disruption of F-actin network and the deactivation of FAK (Tyr397) might be responsible for the impaired mobility of simulated microgravity-exposed A549 cells. TiO2 NPs exposure inhibited cell migration under two different gravity conditions, but to different degrees, with a milder inhibition under simulated microgravity. Meanwhile, it was found that A549 cells internalized more TiO2 NPs under normal gravity than simulated microgravity, which may account for the lower cytotoxicity and the lighter inhibition of cell migration induced by the same exposure concentration of TiO2 NPs under simulated microgravity at least partially. Our study has provided some tentative information on the effects of TiO2 NPs exposure on cell behaviors under simulated microgravity.
... The two axes were each driven by electrical engines, which operated independently from each other. Both engines were controlled by custom-made software [Wuest et al., 2014) running on a laptop. The platform was rotated with constant velocity, set to 60°/s, but the rotation direction was inverted at random time points. ...
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Understanding how stem cells adapt to space flight conditions is fundamental for human space missions and extraterrestrial settlement. We analyzed gene expression in boundary cap neural crest stem cells (BC), which are attractive for regenerative medicine by their ability to promote proliferation and survival of co-cultured and co-implanted cells. BC were launched to space (space exposed cells) (SEC), on board sounding rocket MASER 14 as free-floating neurospheres or in bioprinted scaffold. For comparison, BC were placed in a random positioning machine (RPM) to simulate microgravity on earth (RPM cells) or were cultured under control conditions in the laboratory. Using Next-Generation RNA sequencing and data post-processing, we discovered that SEC upregulated genes related to proliferation and survival, whereas RPM cells upregulated genes associated with differentiation and inflammation. Thus, i) space flight provides unique conditions with distinctly different effects on the properties of BC compared to earth controls, and ii) the space flight exposure induces post-flight properties that reinforce the utility of BC for regenerative medicine and tissue engineering. This article is protected by copyright. All rights reserved.
... 2021, 22, 6331 2 of 12 on these simulated microgravity platforms. Current procedures for biological studies primarily use relatively large vessels to carry out experiments (e.g., T-25 and T-75 cell culture flasks) [12][13][14][15][16] (Figure 1) or custom-made cell culture vessels [17][18][19]. Comparatively, these larger vessels require a copious amount of cells and cell culture media which is cost intensive, uses large amounts of single use plasticware, and limits biological studies that necessitate smaller amounts of media, for example the analysis of cytokine secretion profiles. Larger vessels also limit the number of experimental replicates (low throughput) to be run concurrently which ultimately requires more time for experimental completion with sufficient statistical relevance and will eliminate batch-to-batch discrepancies between samples. ...
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As the number of manned space flights increase, studies on the effects of microgravity on the human body are becoming more important. Due to the high expense and complexity of sending samples into space, simulated microgravity platforms have become a popular way to study these effects on earth. In addition, simulated microgravity has recently drawn the attention of regenerative medicine by increasing cell differentiation capability. These platforms come with many advantages as well as limitations. A main limitation for usage of these platforms is the lack of high- throughput capability due to the use of large cell culture vessels. Therefore, there is a requirement for microvessels for microgravity platforms that limit waste and increase throughput. In this work, a microvessel for commercial cell culture plates was designed. Four 3D printable (polycarbonate (PC), polylactic acid (PLA) and resin) and castable (polydimethylsiloxane (PDMS)) materials were assessed for biocompatibility with adherent and suspension cell types. PDMS was found to be the most suitable material for microvessel fabrication, long-term cell viability and proliferation. It also allows for efficient gas exchange, has no effect on cell culture media pH and does not induce hypoxic conditions. Overall, the designed microvessel can be used on simulated microgravity platforms as a method for long-term high-throughput biomedical studies.
... The random positioning machine (RPM), also referred to as 3D-clinostat, mimics microgravity conditions by rotating samples around two axes through which the gravitational acceleration on the samples changes direction ceaselessly. Averaged over time, the residual acceleration at the intersection of the two rotation axes where the samples are placed reaches near-zero mathematically [8]. The RPM has been extensively used to study the effects of microgravity on biological or physical processes [9,10]. ...
Full-text available
We introduce a new benchtop microgravity simulator (MGS) that is scalable and easy to use. Its working principle is similar to that of random positioning machines (RPM), commonly used in research laboratories and regarded as one of the gold standards for simulating microgravity. The improvement of the MGS concerns mainly the algorithms controlling the movements of the samples and the design that, for the first time, guarantees equal treatment of all the culture flasks undergoing simulated microgravity. Qualification and validation tests of the new device were conducted with human bone marrow stem cells (bMSC) and mouse skeletal muscle myoblasts (C2C12). bMSC were cultured for 4 days on the MGS and the RPM in parallel. In the presence of osteogenic medium, an overexpression of osteogenic markers was detected in the samples from both devices. Similarly, C2C12 cells were maintained for 4 days on the MGS and the rotating wall vessel (RWV) device, another widely used microgravity simulator. Significant downregulation of myogenesis markers was observed in gravitationally unloaded cells. Therefore, similar results can be obtained regardless of the used simulated microgravity devices, namely MGS, RPM, or RWV. The newly developed MGS device thus offers easy and reliable long-term cell culture possibilities under simulated microgravity conditions. Currently, upgrades are in progress to allow real-time monitoring of the culture media and liquids exchange while running. This is of particular interest for long-term cultivation, needed for tissue engineering applications. Tissue grown under real or simulated microgravity has specific features, such as growth in three-dimensions (3D). Growth in weightlessness conditions fosters mechanical, structural, and chemical interactions between cells and the extracellular matrix in any direction.
The random positioning machine (RPM), which continuously changes the gravity direction acting on a subject, can provide the simulated microgravity and planet hypogravity environments to the bioreactor on Earth. In this study, a theoretical analysis of the microgravity and planet hypogravity fields generated by the RPM motion was attempted. The simulated microgravity fields around the subject were quantitatively analyzed by the newly defined degree of gravity dispersion (DGD) parameter, and the simulated planet hypogravity fields were analyzed by the gravity ratio between the RPM simulation and a real planet, corresponding to the normalized DGD. The motion of the gravity vector tip (GVT) on an imaginary sphere attached to a rotational subject was traced and the cause of the GVT trajectory repetitions, which occur in certain combinations of constant (C) angular velocities of the inner and outer frames in the RPM, was identified. A countermeasure for the trajectory repetition was also developed. The linear sawtooth (LS) and parabolic sawtooth (PS) time-varying angular velocity profiles for the outer rotational frame were suggested to prevent concentration of the GVT trajectory, and their effectiveness was numerically verified using an in-house program. Furthermore, appropriate RPM operating conditions for simulating the hypogravity fields were proposed for the Moon and Mars. The mathematical theory presented for the first time in this study can be extended as an important theoretical background in research on the bioreactor, which can be applied to enhanced three-dimensional cell culturing conditions using the RPM.
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Transactivation of immediate early genes, especially targets of the Rel/NFκB pathway, is disrupted in T cells activated in microgravity. This study tested the hypothesis that transcription of immediate early genes is inhibited in T cells activated in μg. Immunosuppression during spaceflight is a major barrier to safe, long-term human space habitation and travel. The goals of these experiments were to prove that μg was the cause of impaired T cell activation during spaceflight, as well as understand the mechanisms controlling early T cell activation. T cells from four human donors were stimulated with Con A and anti-CD28 on board the ISS. An on-board centrifuge was used to generate a 1g simultaneous control to isolate the effects of μg from other variables of spaceflight. Microarray expression analysis after 1.5 h of activation demonstrated that μg- and 1g-activated T cells had distinct patterns of global gene expression and identified 47 genes that were significantly, differentially down-regulated in μg. Importantly, several key immediate early genes were inhibited in μg. In particular, transactivation of Rel/NF-κB, CREB, and SRF gene targets were down-regulated. Expression of cREL gene targets were significantly inhibited, and transcription of cREL itself was reduced significantly in μg and upon anti-CD3/anti-CD28 stimulation in simulated μg. Analysis of gene connectivity indicated that the TNF pathway is a major early downstream effector pathway inhibited in μg and may lead to ineffective proinflammatory host defenses against infectious pathogens during spaceflight. Results from these experiments indicate that μg was the causative factor for impaired T cell activation during spaceflight by inhibiting transactivation of key immediate early genes.
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A Random Positioning Machine (RPM) is a laboratory instrument to provide continuous random change in orientation relative to the gravity vector of an accommodated (biological) experiment. The use of the RPM can generate effects comparable to the effects of true microgravity when the changes in direction are faster than the object’s response time to gravity. Thus, relatively responsive living objects, like plants but also other systems, are excellent candidates to be studied on RPMs. In this paper the working principle, technology and control modes will be explained and an overview of the previously used and available experiment systems will be presented. Current and future developments like a microscope facility or fluid handling systems on the RPM and the option to provide partial gravity control modes simulating for instance Mars or Moon gravity will be discussed.
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Appropriately simulating the three-dimensional (3D) environment in which tissues normally develop and function is crucial for engineering in vitro models that can be used for the meaningful dissection of host-pathogen interactions. This Review highlights how the rotating wall vessel bioreactor has been used to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observed in vivo. The application of these models to the study of infectious diseases is discussed.
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Numerous cell types require a surface for attachment to grow and proliferate. Certain cells, particularly primary and stem cells, necessitate the use of specialized growth matrices along with specific culture media conditions to maintain the cells in an undifferentiated state. A gelatinous protein mixture derived from mouse tumor cells and commercialized as Matrigel is commonly used as a basement membrane matrix for stem cells because it retains the stem cells in an undifferentiated state. However, Matrigel is not a well-defined matrix, and therefore can produce a source of variability in experimental results. In this study, we present an in-depth proteomic analysis of Matrigel using a dynamic iterative exclusion method coupled with fractionation protocols that involve ammonium sulfate precipitation, size exclusion chromatography, and one-dimensional SDS-PAGE. The ability to identify the low mass and abundance components of Matrigel illustrates the utility of this method for the analysis of the extracellular matrix, as well as the complexity of the matrix itself.
We have studied the extractability of type IV collagen, laminin, and heparan sulfate proteoglycan from EHS tumor tissue growth in normal and lathyritic animals. Laminin and heparan sulfate proteoglycan were readily extracted with chaotropic solvents from both normal and lathyritic tissue. The collagenous component was only solubilized from lathyritic tissue in the presence of a reducing agent. These results indicate that lysine-derived cross-links and disulfide bonds stabilize the collagenous component in the matrix but not the laminin or the heparan sulfate proteoglycan. The majority of the collagen present in the extracts had a native triple helix based upon the pattern of peptides resistant to pepsin digestion and visualization in the electron microscope by the rotary shadow technique. This protein was composed of chains (Mr 185000 and 170000) identical in migration to the chains of newly synthesized type IV procollagen. This finding confirms earlier work that indicates that the biosynthetic form, type IV procollagen, is incorporated as such in the basement membrane matrix. Material with smaller chains (Mr 160000 and 140000) appeared on storage in acetic acid solutions. These results indicate that the lower molecular weight collagen in acid extracts of basement membrane arises artifactually due to an endogenous acid-active protease.
Human peripheral blood lymphocytes and monocytes were activated with concanavalin A with or without exogenous recombinant interleukin 1 (IL-1) alone or IL-1 + interleukin 2 (IL-2) under microgravity conditions to test the hypothesis that lack of production of IL-1 by monocytes is the cause of the near total loss of activation observed earlier on several Spacelab flights. The 60 min failure of the on-board 1 × g reference centrifuge at the time of the addition of the activator renders the in-flight data at 1 × g unreliable. However, the data from a previous experiment on SLS-1 show that there is no difference between the results from the in-flight 1 × g centrifuge and 1 × g on ground. The comparison between the data of the cultures at 0 × g in space and of the synchronous control at 1 × g on ground show that exogenous IL-1 and IL-2 do not prevent the loss of activity (measured as the mitotic index) at 0 × g; production of interferon-γ, however, is partially restored. In contrast to a previous experiment in space, the production of IL-1 is not inhibited.
The first experiments using machines and instruments to manipulate gravity and thus learn about its impact to this force onto living systems were performed by Sir Thomas Andrew Knight in 1806, exactly two centuries ago. What have we learned from these experiments and in particular what have we learned about the use of instruments to reveal the impact of gravity and rotation on plants and other living systems? In this essay I want to go into the use of instruments in gravity related research with emphases on the Random Positioning Machine, RPM. Going from water wheel via clinostat to RPM, we will address the usefulness and possible working principles of these hypergravity and mostly called microgravity, or better, micro-weight simulation techniques.
The mitogenic activation of human lymphocytes resuspended in vitro is dramatically reduced in microgravity. As cell-cell contacts are one of the elements essential for activation, the behaviour of human leukocytes (mainly lymphocytes and monocytes as accessory cells) in the presence of the mitogen concanavalin A was studied in the centrifuge microscope NIZEMI at 0 × g. Aggregates (formed by intercellular bindings of membrane glycoproteins via the tetravalent α-glucoside ligand concanavalin A) were found at 0 × g as well as at 1 × g already 12 h after the addition of the mitogen. In general, the aggregates observed at 0 × g after an incubation time of 46 and 78 h were smaller than the corresponding aggregates in the ground control. The findings are of primary importance since they confirm the indirect evidence we had from earlier Spacelab experiments and demonstrate that cell-cell contacts are occurring also in microgravity. In addition, single cells in 0 × g show a significant higher locomotion velocity than the cells at 1 × g. The fact that the locomotion capability is not decreased during the 78-h incubation with concanavalin A provides further evidence that the cells are not proceeding through the cell cycle.
The human cell lines FTC-133 and CGTH W-1, both derived from patients with thyroid cancer, assemble to form different types of spheroids when cultured on a random positioning machine. In order to obtain a possible explanation for their distinguishable aggregation behaviour under equal culturing conditions, we evaluated a proteomic analysis emphasising cytoskeletal and membrane-associated proteins. For this analysis, we treated the cells by ultrasound, which freed up some of the proteins into the supernatant but left some attached to the cell fragments. Both types of proteins were further separated by free-flow IEF and SDS gel electrophoresis until their identity was determined by MS. The MS data revealed differences between the two cell lines with regard to various structural proteins such as vimentin, tubulins and actin. Interestingly, integrin α-5 chains, myosin-10 and filamin B were only found in FTC-133 cells, while collagen was only detected in CGTH W-1 cells. These analyses suggest that FTC-133 cells express surface proteins that bind fibronectin, strengthening the three-dimensional cell cohesion.
Hyaluronic acid (HA), an immunoneutral polysaccharide that is ubiquitous in the human body, is crucial for many cellular and tissue functions and has been in clinical use for over thirty years. When chemically modified, HA can be transformed into many physical forms-viscoelastic solutions, soft or stiff hydrogels, electrospun fibers, non-woven meshes, macroporous and fibrillar sponges, flexible sheets, and nanoparticulate fluids-for use in a range of preclinical and clinical settings. Many of these forms are derived from the chemical crosslinking of pendant reactive groups by addition/condensation chemistry or by radical polymerization. Clinical products for cell therapy and regenerative medicine require crosslinking chemistry that is compatible with the encapsulation of cells and injection into tissues. Moreover, an injectable clinical biomaterial must meet marketing, regulatory, and financial constraints to provide affordable products that can be approved, deployed to the clinic, and used by physicians. Many HA-derived hydrogels meet these criteria, and can deliver cells and therapeutic agents for tissue repair and regeneration. This progress report covers both basic concepts and recent advances in the development of HA-based hydrogels for biomedical applications.