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CHEMICAL SENSORS, BIOSENSORS, AND BIOARRAYS
Chimia 53 (1999) 75-80
©Neue Schweizerische Chemische Gesellschaft
ISSN 0009-4293
75
CHIMIA 1999, 53, No.3
Microtechnology in Space Bioreactors
Isabelle Walther*a), Bart van der SChootb), Marc Boillatb), Otfried MOller
C),
and Augusto Cogolia)
Abstract. Space biology is a young and rapidly developing discipline comprising basic research and
biotechnology. In the next decades it will playa prominent role in the International Space Station (ISS).
Therefore, there is an increasing demand for sophisticated instrumentation to satisfy the requirements of the
future projects in space biology. Bioreactors will be needed to supply fresh living material (celis and tissues)
either to study still obscure basic biological mechanisms or to develop profitable bioprocesses which will take
advantage of the peculiar microgravity conditions. Since more than twenty years, the Space Biology Group of
the ETHZ is carrying out research projects in space (Space Shuttle/Spacelab, MIR Station, satellites, and
sounding rockets) that involve also the development of space-qualified instrumentation. In the last ten years
we have developed, in collaboration with Mecanex SA, Nyon, and the Institute of Microtechnology of the
University of Neuchatel, a space bioreactor for the continuous culture of yeast cells under controlled
conditions. Sensors, pH control, nutrients pump and fluid flowmeter are based on state-of-the-art silicon
technology. After two successful space flights, a further improved version is presently prepared for a flight in
the year 2000.
1. Introduction
Space biology has evolved from the scien-
tists' need to better understand the effects
of the space environment on living sys-
tems. The peculiarities of such an environ-
ment are a reduced gravity (i.e., Shuttle:
10-
2
-10-4
g),
the almost absence of con-
vection movements and cosmic radiation
(10 mrad/d in average). At the beginning
of human space exploration in the early
sixties, the investigations were oriented
essentially toward the health of the astro-
nauts, so that medical and physiological
experiments were predominant. Even if
this aspect is still very important, today's
investigators are increasingly interested
inbasic and applied research under micro-
gravity. Also, the upcoming of the ISS is
triggering the scientists to develop and
plan new instruments for long-term exper-
'Correspondence: Dr. I. Walther
a)
Space Biology Group ETH-Technopark
Technoparkstrasse 1
CH-8005 Zurich
Tel.: +41 14451280
Fax: +4114451271
E-Mail: walther@spacebiol.ethz.ch
b)
Seyonic SA
Rue du Puits-Godet 12
CH-2000 Neuchatel
(previously in the Institute of
Microtechnology)
0)
Institute of Anatomy University of Bern
Buhlstrasse 26
CH-3000 Bern
iments in space and for the recycling of
components essential for life, such as ox-
ygen, water, and air, from waste products.
Biotechnology in space will become a
daily process helping astronauts to live for
several months far away from Earth.
For twenty years, our group has been
active in this field focusing, first, on the
study of immune-system cells, in particu-
lar T lymphocytes, and, second, on the
development of 'space-qualified' (see be-
low) instruments to carry out studies on
single cells. We used 0 gas a tool to study
the mechanism of activation of T lym-
phocytes. This mechanism is extremely
complex and many of its steps still need
clarification [1][2]. Our first two space
experiments were performed in 1983
aboard the space shuttle Challenger [3]
and Columbia, respectively [1]. We man-
ufactured a tissue culture incubator that
had to comply with the peculiar constrai nts
of space flight.
During the past ten years, the develop-
ment of continuous cell culture systems
has also been an important aspect of our
activity. Our research was conducted in
orbit (Biokosmos, MIR, Spacelab, Space-
hab), in sounding rockets (MASER, MAX-
US) as well as on the ground in the centri-
fuge for hypergravity, and in clinostats
(rotating devices that randomize Earth-
gravity vector) for simulated microgravi-
ty. A summary of our experiments in space
is given in the Table.
The purpose of this article is to de-
scribe the four generations of the bioreac-
tor developed in collaboration with the
Swiss aerospace companies Contraves AG,
ZUrich, Mecanex SA and the Institute of
Microtechnology (IMT) of the University
of Neuchatel. We will also briefly present
the data of two flight experiments.
2. Space-Qualified Instrumentation
All equipment to be installed in space
laboratories must fulfil very strict require-
ments like crew safety, man-machine in-
teraction, energy consumption, volume,
electromagnetic contamination, and oth-
ers. For instance, safety requires the use of
non-offgassing, non-flammable, non-tox-
ic materials. Handling operations by the
crew shall be simple and not time-con-
suming. For biological samples, triple con-
tainment is necessary. On the other hand,
materials have to be biocompatible with
the biological specimen used.
There are two types of space instru-
ments used for biology: Multi-user facili-
ties and experiment-specific devices. A
multi-user facility, such as the Biorack
flown on several missions, consists of
freezer, incubators, cooler, and reference
centrifuges in which biological experi-
ments can be performed. They are mostly
built under supervision and financing of
the space agencies. Experiment-specific
CHEMICAL SENSORS, BIOSENSORS, AND BIOARRAYS
76
CHIMIA 1999. 53, NO.3
8T8-76, 3rd Shuttle to MIR
STS-107
ST8-81, 5th Shuttle to MIA
MIR MISSIon7
MIR MISSion 8
MIA MISSion 9
Kosmos Biosatellite 9
MASER 3
MASEA4
MAXU81
MAXU81b
MAXU82
StratospheriC balloon, program ODISSEA
StratospheriC balloon, program ODI8SEA
Table. Experiments Conducted in Space by the Space Biology Group of the ETHZ
Mission Year Experlmentlinstrument developed with Swiss aerospace industry
STS-8 1983 Incubator with human embryonic kidney cells
8T8-9, 8pacelab 1 1983 Incubator with human lymphocytes
STS-61-A, Dl 1985 2 experiments: lymphocyte cultures, lymphocytes from astronauts
8T8-40, 8pacelab SL8-1 1991 2 experiments: lymphocyte cultures, lymphocytes from astronauts
8T8-42, Spacelab IML-1 1992 Hybridoma cells, DCCS with hamster embryonic kidney cells (Contraves AG). Friend cells
STS-65, Spacelab IML-2 1994 3 experiments: lymphocyte activation and motion, bioreactor SSR I
(Mecanex SA, Univ. Neuchatel) wflh yeast cells
1996 Sioreactor 8SR I (Mecanex SA, Univ. Neuchatel) with yeast cells
2000 Sioreactor SSR II (Mecanex SA, Seyonic, Neuchatel), tentatively scheduled
1997 8tudy on preservation of mammalian cells
1988 Immunological 'skin-test' on cosmonauts with applicator
1989 Immunological 'skin-test' on cosmonauts With applicator
1990 Immunological 'skln-lest' on cosmonauts with applicator
1989 Test of the DCC8 (Contraves AG) with protoplasts
1989 Lymphocytes: mitogen binding, patching and capping
1990 Lymphocytes: cytoskeleton, mitogen binding, patching and capping
1991 Lymphocytes: motility, cytOSkeleton, mitogen binding, patching and capping, rocket failur
1992 Lymphocytes: cytoskeleton, mitogen binding, patching and capping
1995 Lymphocytes: motility, cytoskeleton, mitogen binding, patching and capping
1986 Cosmic radiation and lymphocyte activation, balloon failure
1987 Cosmic radiatron and lymphocyte activation
hardware is mostly developed for and
adapted to one specific biological experi-
ment. The best way for scientists to have
instruments optimally conceived for their
experiments is to participate in their de-
velopment and design. The use of silicon
microtechnology was a key element to
contend the extremely strict place restric-
tion imposed by the standard (Type II)
containers of the Biorack facility.
Here, we present a miniaturized and
controlled bioreactor as an example for
the utilization of microtechnology for space
instruments.
2.1. Dynamic Cell Culture System,
DCCS
With one exception (namely an auto-
mated device flown in Skylab in 1973), all
the early experiments performed in space
with single cells were so-called batch ex-
periments. A batch experiment is an ex-
periment in which the cells are cultivated
in a fixed amount of nutrient solution
(medium). As a consequence, such culti-
Fig. 2. SBR I: Miniaturized bioreactor with, on
its left, two syringes for the sampling and the
sample bottles. The cultivation chamber is on
the top of the bioreactor. The inspection win-
dow is located on the right upper side (w). The
sensors, which are integrated into the cham-
ber wall at the opposite of the window, are not
visible on this picture. The basis structure (bs)
contains the fresh and the used medium. Total
height: 8 em.
c
,
. ---_11
•
Fig. 1. Dynamic cell cultivation system DCCS. On the left is the container Type I (c).On the right
the DCCS with the two circular windows (arrow) over the cultivation chambers. The osmotic
pump was located inside of the metallic bloc (M).
CHEMICAL SENSORS, BIOSENSORS, AND BIOARRAYS
77
CHIMIA 1999. 53,
No.3
outlet
glass base plate
t t
,,
glass membrane
with PROD EX funding. It is designed for
yeast-cell cultivation [7]. We chose yeast
cells because they are widely used in bio-
technology (i.e., beer and wine industry),
in basic and cancer research, and as a
model organism in molecular biology.
piezo disc
silicon pump body
+
into a Type lIfE B iorack contai ner that has
forvolume365 ml(size87 x 63 x 63 mm,
Fig.
2). This instrument was built in col-
laboration with Mecanex SA (Nyon, Swit-
zerland) and the Institute of Microtech-
nology of the University of Neuchatel
Fig. 3. Micropump. The piezo device is clearly visible as a disk on the right of the pump. The left
part of the structure contains the flow sensor with the electrical connector and the tube bringing
the medium to the culture chamber.
vation is very limited in time due to the
exhaustion of the nutrients and the accu-
mulation of waste products. To overcome
this problem, we decided in 1986 to devel-
op, in collaboration with Contraves AG
(Zurich, Switzerland), a totally automatic
cultivation instrument for animal cells with
continuous delivery of fresh medium, so
that the cells can grow for several days
without being starved. The development
of the instrument was funded by the PRO-
DEX program of the European Space
Agency (ESA). This program supports the
development of specific space hardware
in countries which have not their own
space agency such as Switzerland, Aus-
tria, Belgium and, since 1998, Hungary.
The first system we developed was called
Dynamic Cell Culture System (DCCS,
Fig.
J).
It is a completely closed system
consisting of three main parts: the pump,
housing, the pump and the culture cham-
ber [4]. The fresh-medium reservoir has a
capacity of 230~. The novelty of this
system was the self-powered osmotic pump
supplying the cells with fresh medium at a
continuousflowrateofl ~ h-'.TheDCCS
was designed to fit into one standard ESA
Type I container (81
x
40
x
20 mm). Its
biological performance was tested on the
Biokosmos 9 satellite (1989) with plant
protoplasts [5]. The protoplasts were cul-
tivated for 14 days in orbit. The results
showed that the DCCS worked well under
microgravity conditions. Aboard the IML-
I mission (STS-42, 1992) the DCCS was
used for the cultivation of hamster kidney
cells. These cells are interesting because
they produce tissue plasminogen activator
(t-PA). No difference in cell growth and t-
PA secretion was found between flight
and ground [6]. Though the DCCS, which
does not need any external power supply
or electronic controlling device, is suita-
ble for continuous cultivation in micro-
gravity, it has some limitations such as the
very small working volume of the culture
(200 ~), which does not allow the with-
drawal of a sample during experiment, and
the lack of control and on-line informa-
tion.
2.2. Space Bioreactor SBR
I:
Miniaturized anc:JControlled
Our next goal was to overcome the
limitations of the DCCS and to develop a
miniaturized bioreactor with sampling ca-
pability, pH control, gas exchange, con-
tinuous fresh-medium supply, and on-line
measurements. The real challenge was to
build a bioreactor with almost all the capa-
bilities of a commercial one (total volume,
electronics and mechanics included, of
about 1 m3for a I I bioreactor) but fitting
CHEMICAL SENSORS, BIOSENSORS, AND BIOARRAYS 78
CHIMIA 1999. 53, No.3
Current source
Furthermore, they have a very well-known
metabolism and are very sensitive to envi-
ronmental changes. The objective of the
experiment was, besides the technical chal-
lenge, to investigate the effect of mixing in
microgravity on biological parameters such
as growth rate and metabolism. As yeast
cells are non-motile it is necessary on
Earth to stir the cultivation medium to
avoid their sedimentation. In microgravi-
ty, they would not sediment, but the lack of
convection would favour the formation of
nutrient and waste gradients that could
affect cell growth. To avoid the formation
of such gradients, the bioreactor chamber
(3 ml) was equipped with a magnetic stir-
ring mechanism. A piezo-electric silicon
micropump of 20 x 20 x 2 mm size was
used for the delivery of fresh medium
(Fig.
3). The flow rate was variable be-
tween 200 and 1200 ~ per hour. The data
were collected on-line by means of a sen-
sor inserted directly into the cultivation
chamber. The chip size was 3.5 x 3.5 mm.
This sensor was an integration of:
i)
a pH-
ISFET (ion-sensitive field-effect transis-
tor) with Al
2
0
3
gate insulator (sensitivity
51 mV/pH),
ii)
a temperature-sensitive
diode in forward bias at 100
JlA,
and
iii)
thin-film platinum redox electrode [7].
The regulation of the pH was achieved by
coulometric generation of hydroxyl ions
at a titanium electrode in the bioreactor
(Fig.
4). The counter electrode contained
a chlorinated silver wire in a potassium
chloride gel. The counter electrode was
separated from the chamber by a cation-
selective membrane. This type of pH con-
trol allows to avoid the use of concentrated
NaOH. The pH sensor measured the level
between pH 2 and pH 9 with an accuracy
of
±
0.05 pH units. Its only disadvantage
was its limited capacity due to the con-
sumption of both the silver anode and the
KCI
in the counter electrode. The sam-
pling port consists of a silicone-rubber
septum and the gas exchange occurs by
passive diffusion through seven
Silastic®
membranes. This bioreactor flew in 1994
and in 1996 aboard the shuttle missions
STS-65 and STS-76. For the second flight,
a flow sensor was implemented to im-
prove the regulation of medium delivery.
In fact, if the flow measured by the sensor
was not nominal, the current delivered to
the pump was adapted accordingly. This
sensor was also measuring the pressure in
the cultivation chamber. This measure-
ment allowed detecting possible blockage
of the outlet leading to an increase of
pressure in the chamber. A new, improved
version of the bioreactor is currently under
development with
Mecanex
and
Seyonic
SA,
Neuchatel, a spin-off company of the
KCI gel
Ag
120 144 168 192
96
Time [h]
Stirred
72
48
24
pH
Unstirred
9.0
pH
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0 024 48
72
96
120
144 168 192
Time [h]
Titanium cover
Medium with cells
00
9.0
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
o
2 H20 +2e- ~ 2 OW +H2
,I
Ag+Cr
-7
AgCI+e-
Bioreactor \ Counter electrode
Nafion membrane
Fig.5.
pH values for both ground bioreactors.
Inthe stirred one, the sampling times are very well
visible (drops). About
1
ml of fresh medium at pH
2.5
is delivered to the chamber (pH
4.5)
to
replace the sampling volume, a pH drop is resulting especially because no compensation of pH
is effected during sample withdrawal (no electrical connection of the bioreactor).
Fig.
4. Principle of the electrochemical pH regulation.
K+ions are tormed inthe counter electrode,
they pass through the Nation membrane to the bioreactor chamber and combined with the
hydroxyl ions to form KOH.
CHEMICAL SENSORS, BIOSENSORS, AND BIOARRAYS
79
CHIMIA 1999, 53.
No.3
--~ •• 00
--"--Glu
• EtOH
stirred 10
9
8
7
6
5
4
3
.0.0,
C· "[] 2
1
o
Ground
unstirred
.0.0
C· ,
I
I
.
I
.
result we can conclude that both physico-
chemical environment and microgravity
have an effect on the bud location.
3.2. Cultivation in Batch Chamber
Additionally to the bioreactors that
could not be placed on the 1
g
reference
Dilution rate [h·
l]
Bioreactors
Flight
unstirred stirred
•
". ..
,
,
.0/0
r \
-
•••
C
N
In
0>
(V)
....
~~~
c:i
000
00
160
140
120
100
80
60
40
20
o
Fig. 7. Scanning electron micrograph of yeast cells with budding scars (arrows). Magnification
5000
x.
Fig. 6. Optical density at 610 nm, glucose and ethanol concentration in the bioreactors' samples.
This diagram shows the evolution of the culture parameters (growth, glucose consumption, and
ethanol production) during a continuous cultivation at increasing dilution rates. Mean values of
dual measurements of a single sample are shown.
tors than in the stirred ones, we tend to
assume that the last reason is relevant.
Nevertheless, the fact that the percentage
of randomly positioned scars is higher in
both cultivation conditions in space than
on Earth indicates that microgravity has
indeed an effect on the cells. From this
3. Results
As an example, in this section, we
present the performance and part of the
scientific results of the second flight of the
SBR I in STS-76. The experiment consist-
ed of two elements, namely cultivation of
yeast cells in the bioreactor and, as a
control, in batch chambers.
3.1. Cultivation in the Bioreactor
From the technical point of view, the
second flight of SBR I was a success. The
sensors worked according to the expecta-
tions and the pH was regulated correctly
(Fig. 5). It was not a surprise to observe
higher fluctuations of the pH under un-
stirred conditions in microgravity as well
as on Earth. In fact, to obtain the correct
pH at the sensor level (side of the cham-
ber), more hydroxyl and K+ions have to be
produced at the source (cover of the cham-
ber and compensation electrode) when
mixing is achieved only by diffusion and
not
by
stirring. Moreover, in microgravi-
ty, no convection movements are occur-
ring which leads to a larger fluctuation
amplitude of the pH values.
From the biological point of view, as
also observed in the previous flight [8], the
yeast cells grew well in microgravity. Their
metabolism was comparable to that on
Earth as they consumed glucose and pro-
duced alcohol in similar ways (Fig. 6).
Morphologically, no differences were ob-
served between flight and ground sam-
pies. Fig. 7 presents a transmission micro-
graph where the bud scars are visible.
Normally, the bud scars left on the
mother cell after separation of the daugh-
ter cell are located bi polarl y. We observed
that the specific bipolar bud-scar position-
ing was altered under microgravity condi-
tions. Interestingly enough, the percent-
age of cells with bud scars located ran-
domly is much higher in space than on
Earth when comparing the same cultiva-
tion conditions (Fig. 8). This might be due
to: i) a higher mutation rate, the location
being genetically defined [9], ii) the dys-
function of the cytoskeleton in space, the
cytoskeleton playing an important role in
the choice of the bud location [10], and/or
iii)
a disturbed metabolism because cells
under starvation 'forget' where the bud
has to be located [11]. With respect to the
fact that the percentage of randomly locat-
ed scars is higher in the unstirred bioreac-
IMT of the University of Neuchatel. It is
scheduled to fly in a new instrument, called
Biopack, in 2000 in the shuttle flight STS-
107.
CHEMICAL SENSORS, BIOSENSORS, AND BIOARRAYS
80
CHIMIA 1999, 53, No.3
5
10
F unstirred F stirred G unstirred G stirred
15
particular, will be of primary importance
in the next decades. The trend to miniatur-
ization and automation will be a techno-
logical challenge that will probably also
favour spin-offs for Earth applications.
Several types of bioreactors will be re-
quired: small-sized reactors (3-50 ml) of
the type describe here for basic research
and pilot bioprocesses, medium-sized re-
actors (11) for established bioprocesses,
and large bioreactors (100
I)
for closed
ecological life-support systems to support
long-duration human life in space. Thus,
stable and reliable chemical and biologi-
cal sensors for the measurement of param-
eters such as glucose consumption, con-
centration of dissolved O
2
and CO
2,
me-
tabolite production (i.e., alcohol, lactate)
as well as environmental control systems
(pH, temperature, waste management) sat-
isfying the requirements of manned space
missions, will be needed. Depending on
the specific purpose, both optical and elec-
trochemical sensors might be of interest.
The most important thing is that they cope
with the space requirements for safety
(i.e., non-flammable, non-corrosive), pow-
erconsumption (normally only few Watts
available per experiment), and that their
size is compatible with the restricted vol-
ume available.
Batch chambers
7
OD61Onm
8
o
20
%
25
Budding position Random vs. Bipolar
Fig. 8. Percentage of the randomly located bud scars vs. the normally bipolar positioning. The
values were obtained counting 500 cells per sample; the standard deviation was calculated on
the four samples available per bioreactor.
F:
Flight, G: Ground.
6
centrifuge onboard due to their size, small
batch chambers were incubated for 32 h at
22° in static position in the flight (0
g),
on
ground (I
g)
and in the centrifuge in the
flight (lg). After this time, the chambers
were frozen to stop the growth of the cells.
The analyses were performed after the
flight. Optical density (aD), glucose con-
sumption and ethanol production were
measured. As expected, the glucose was
fully consumed and ethanol was produced
in every culture with no noticeable differ-
ence. At the aD level, a difference was
observed between the cultures grown at 1
g
and at 0
g.
Both 1
g
cultures (on board
and static on ground) had a lower aD than
the 0
g
culture
(Fig.
9). One could, thus,
conclude that microgravity has a positive
effect on the growth of the cells. However,
experiments performed on the ground with
agitation demonstrated that the cells grew
4. Conclusions and Outlook
The application of silicon microtech-
nology for the development of instrumen-
tation for space laboratories, the ISS in
also better than in a static position. There-
fore, the growth difference observed in the
batch cultures is rather caused by a nega-
tive effect of the sedimentation of the cells
during centrifugation than by a positive
microgravity effect. Static or centrifuge
conditions are certainly not particularly
favourable due to the accumulation of
waste and the formation of gradients. This
leads to the conclusion that for certain
types of cells, at least for those not used to
grow in static conditions, the centrifuge
might not furnish the ideal control condi-
tions.
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Fig. 9. Optical density in
the batch chamber after
32 h growth. The stand-
ard deviation was calcu-
lated on duplicate meas-
urements on quadrupli-
cates per position. F:
Flight, G: Ground.
FOg
F 19
G
19
4
5
