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Experiments with small animals in BIOLAB and EMCS on the international space station
~
Pergamon
www.elsevier.eom/locate/asr
Adv. SpaceRes. Vol.30, No. 4, pp. 809-814, 2002 © 2002COSPAR.Publishedby ElsevierScienceLtd.All rightsreserved Printedin GreatBritain 0273-1177/02 $22.00+ 0.00 PII: S0273-1177(02)00401-5
EXPERIMENTS WITH SMALL ANIMALS IN BIOLAB AND EMCS ON THE INTERNATIONAL SPACE STATION E. Brinckmann I and P. Schiller2
European Space Agency (ESA), ESTEC/MSM-GFB 1 and ESTEC/TOS-MMG 2 Postbus 299, NL-2200 AG Noordwijk, The Netherlands
ABSTRACT Two ESA facilities will be available for animal research and other biological experiments on the International Space Station: the European Modular Cultivation System (EMCS) in the US Lab "Destiny" and BIOLAB in the European "Columbus" Laboratory. Both facilities use standard Experiment Containers, mounted on two centrifuge rotors allowing either research in microgravity or acceleration studies with variable g-levels from 0.001 to 2.0xg. Standard interface plates provide each container with power and data lines, gas supply (controlled CO2, 02 concentration and relative humidity), and -for EMCS only- connectors to fresh and waste water reservoirs. The experiment hardware inside the containers will be developed by the user, but ESA conducted a feasibility study for several kinds of Experiment Support Equipment with potential use for research on small animals: design concepts for experiments with insects, with aquatic organisms like rotifers and nematodes, and with small aquatic animals (sea urchin larvae, tadpoles, fish youngsters) are described in detail in this presentation. Also ESA's initial steps to support experiments with rodents on the Space Station are presented. © 2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.
INTRODUCTION ESA's contribution to the International Space Station (ISS) for experiments with small animals will be the European Modular Cultivation System (EMCS) in the US Lab, and BIOLAB in the Columbus Laboratory. The design of these two facilities is based on experience with Biorack, ESA's precursor of the Space Station, flown six times on the Space Shuttle. The small animals used in Biorack experiments covered a wide range from unicellular organisms (Paramecium tetraurelia, Loxodes striatus) to nematodes (Caenorhabditis elegans), jellyfish (Aurelia aurita), sea urchin larvae (Sphaerechinus granularis), insects (Drosophila melanogaster, Carausius morosus eggs) and small vertebrates (Xenopus laevis embryos and tadpoles, fish youngsters of Oreochromis mossambicus). To accommodate these different organisms, Biorack offered two kinds of Experiment Containers (EC) with 65 ml and 345 ml volume. The biological samples were placed inside the experiment specific hardware, which was developed by the different investigators according to their specific needs. Several design concepts have been published in an overview by Briarty (1989) and by the experimenters themselves in the mission reports of D-1 (Longdon and David, 1987), IML-I (Mattok, 1995), IML-2 (Cogoli, 1996; Snyder 1997), S/MM-03, S/MM-05, and S/MM-06 (Perry, 1999). ESA's development concept for the Space Station experiments is similar to Biorack experiments: ESA provides the empty experiment container to the investigators, who are responsible for the development and testing of their experiment specific hardware. However, to avoid unnecessary parallel developments, ESA has studied in its Technology and Research Programme the technical feasibility of several designs, which could be used for experiments in BIOLAB and EMCS. Three of these studies are presented here for the support of experiments with aquatic organisms and insects.
809
810
E. Brinckmann arld P. Schiller
A detailed description of both facilities, BIOLAB and EMCS, had been given elsewhere (Brinckmann, 1999a; Brinckmann, 1999b). In principle, both facilities have an incubator with two centrifuges each (diameter: 600 ram) allowing microgravity research or acceleration studies in the range of 0.001xg (or less in EMCS) to 2.0xg. The experiment containers (2x6 in BIOLAB, 2x4 in EMCS) are placed by the crew on the centrifuge platters, where they are connected to the Life Support System with a controlled atmosphere: 02 and CO2 concentration can be varied in a wide range, trace gases and CO2 can be removed, and the relative air humidity can be controlled, the latter in EMCS on an individual container level including a drying function with 30% r.h. Other than BIOLAB, the EMCS rotor has a fresh water and a waste reservoir in its centre. Illumination and video observations are also possible on the rotor: the EMCS camera has even a zoom optic to observe the entire interior of the EC with a resolution of 0.1 mm; "dark" observation in infrared light is also possible on the rotors of both facilities. Besides the upgraded environmental control, observation, data handling and other capabilities, the new EC's of BIOLAB and EMCS are larger than the Biorack containers, which had only a height of 20 mm with respect to the g-vector: the BIOLAB EC has a useful volume of 360 ml with 60 mm height, the EMCS EC allows specimens with a volume up to 580 ml and a height of 160 ~ These dimensions shall be sufficient for research on cell cultures and small organisms. For larger animals like rodents, a new animal research facility on the ISS has been envisaged by ESA and the Italian Space Agency (ASI). This concept will use animal models (mice) under very strict ethical rules to allow experiments in physiology and developmental biology, which are not possible with smaller organisms or cell and tissue cultures. EXPERIMENT CONTAINERS
The BIOLAB Experiment Container The standard Experiment Container (EC) provides a safe environment for the biological samples, isolating them from the external environment and vice-versa. The transparent cover allows illumination and video observation of the experiment (Figure 1); also non-transparent covers will be available. Each EC is equipped with a temperature and a pressure sensor, a power and analogue/digital data connector, a serial interface for data, commands and container identification, and filtered gas connections to the Life Support System; this interface plate connects the EC also thermally to the centrifuge platter. The bottom plate of the EC (with respect to the g-vector and opposite to the light source) can be. customised for automatic operations to be performed by the Handling Mechanism, e.g. with several septa to inject water, nutrients or fixative for experiment termination; tool functions (push/pull/rotate) are also possible with this robotic mechanism. The EC is internally 100 ram wide and leaves a volume of about 360 ml for the accommodation of experiment specific hardware (Figure 1).
Fig. 1. Experiment Containers for BIOLAB (left) and EMCS (right). The bottom plate is the interface to the centrifuge platter. Dimensions given are the useful volumes for experiment specific hardware. The observation direction in both containers is perpendicular to the g-vector through the cover top, the illumination is parallel to the g-vector.
Small Animal Facilities by ESA
811
Two places on the centrifuge rotor are foreseen for the accommodation of Advanced Experiment Containers (AEC) enabling the development of more sophisticated or larger, customised experiment hardware, e.g. a microscope or other equipment which may be needed to perform experiments on the rotating centrifuge; thus, it is possible to avoid stops of the centrifuge, otherwise required for manual or robotic operations. The interface of the AEC to BIOLAB is the same as for the standard Experiment Containers, but with an additional analogue NTSC video line. The outer dimensions of the AEC are 175x147x125 mm, height (with respect to the g-vector) x width x depth.
The EMCS Experiment Container In addition to power and data lines and inlet/outlet gas connectors, the EMCS EC has also quick-connects for water supply and back to a waste reservoir on the rotor. Nutrients for the organisms shall be added inside the EC to prevent contamination of the fresh water reservoir. The EC's are supplied with sensors for temperature and pressure monitoring. An internal volume of 580 ml (60x60x160 mm) is available for experiment hardware with its biological material, with the long axis in direction to the gravity vector (Figure 1). Other than BIOLAB, there is no Handling Mechanism in EMCS, but all experimental operations shall be done either manually by the crew or by automatic devices of the experiment specific hardware inside the EC. If an experiment is terminated by chemical fixation, single shot valves can be activated to seal permanently the air and water connections in the EC baseplate for safety reasons.
Fig. 2. Exploded view of the Automatic Culture System for Insects with two compartments for adults, one drum with six food trays and a heat fixation device. The compartments for the adults are covered by transparent windows for video observation (perpendicular to the g-vector in BIOLAB).
MULTI-GENERATION CULTIVATION SYSTEM FOR INSECTS To study the embryological development of insects under Space conditions, the prototype of an automated culture system for multi-generation experiments has been tested on ground with Drosophila melanogaster(Marco et al. 1999). Figure 2 shows the main parts of this system in an exploded view: two transparent chambers contain the adults which can feed on the food trays and lay eggs on the food surface. Once the food tray is full with embryos, the drum is rotated and the embryos may either develop in the other adult chamber for a second generation, or they are preserved by heat shock (50°C) for later analysis on ground. New food trays can easily be replaced by the crew in a glovebox, where the experiment container can be opened in a safe environment. A
812
E. Brinckmannand P. Schiller
photoelectric sensor in the adult chambers is used for monitoring of the activity of the flies in addition to the video observation provided by BIOLAB or EMCS. One of the adult chambers contains also a receptacle compartment, which can be opened to fix a population chemically with ethanol. All functions of this insect cultivation system are handled by a microcontroller, which fits together with it into the BIOLAB or the EMCS container. The microcontroller can be commanded from ground and transmits science and status data to the experimenter on ground, thus allowing telescience operations. AUTOMATED CULTURE SYSTEMS FOR AQUATIC SPECIES
Aquatic Animals Cultivation System For studies on small aquatic species like sea urchin larvae, jellyfish ephyrae, tadpoles, fish youngsters, a miniaquarium system has been developed, based on results with a similar design on a tadpole experiment on STS-84 (Sebastian et al., 1998; Sebastian and Horn, 1998; Horn and Sebastian, 1999); in this experiment, the tadpoles survived in the aquaria a period of several weeks in perfect condition. The connection module (Figure 3) is the interface between the EC baseplate and the aquarium: fresh and waste water can be supplied to/from the EMCS reservoirs on the rotor; with BIOLAB, the Handling Mechanism has to inject liquids through a special septum port (not shown). The connection module can accommodate 6/4/3/2/1 mini-aquaria with water flowing through a volume of 30/45/60/90/180 ml each. By different lay-outs of the interconnecting tubes, the aquaria can be supplied e.g. in a parallel or serial mode, also the use of a EC specific pump module or filter/nutrient module is possible, replacing one or more aquaria on the connection module. Each mini-aquarium is covered by a biofoil membrane, supporting gas exchange of the medium to the atmosphere in the EC, which is conditioned by the Life Support System. This transparent foil allows also the visual control on the small animals by the video observation system of the facility. A ground test with sea urchin larvae proved the excellent biocompatibility of this system: swimming behaviour and development was normal during 14 days even without any water exchange.
Fig. 3. Breadboard model of the Aquatic Animals Cultivation System. The connection module is attached to the breadboard adapter plate, simulating the actual interface for gas and liquids to the EMCS container. Up to 6 miniaquaria can be installed on the connection module, connected to each other by tubes below the liquid inlet/outlet pods. The white dots on each side of the connection module are lateral pressure pads allowing a quick and safe attachment of the mini-aquaria.
Small Animal Facilities by ESA
813
Automated Culture System for Small Aquatic Species Several small organisms can thrive unfavourable conditions by forming special live forms, e.g. the "Dauerlarvae" of nematodes, or by complete desiccation, e.g. common with rotifers, until the improved environment, e.g. the addition of water or nutrients, terminates the dormant phase and re-establishes normal life. These organisms should be good candidates for experiments on the ISS, where periods of several days for transport to and from the ISS are expected: the transportation phase will most likely not be sensed in this inactive, dormant state. The Automated Culture System for Small Aquatic Species, shown schematically in Figure 4, was designed for experiments in EMCS with rotifers and nematodes there, a microcontroller would handle the liquid distribution system by opening the respective "curtains" which are separating the different compartments. The drying mode of the Life Support System in EMCS can provide the required relative humidity of 30%. A similar aquatic culture system shall be possible in BIOLAB, but mainly with manual functions due to the smaller EC size. Medium Inlet/Outlet C'1"3 t ' ~
/~"~/Separating
.....................
i D ying
Filter 40 ~'n
Gas Dryer Tube
211
1
i
Filter 80 ~
......i / '
7 ................................................
Drying Chamber
I
/ J
/
Fig. 4. Schematic drawing of the Automated Culture System for Small Aquatic Species (Rotifers), containing in total 4 of the depicted culture & drying chamber assemblies. After injection of dry animals through a septum into the outermost culture chamber (volume: 3 ml), animals can be cultivated until they lay eggs. These eggs (size 90x El 50 pro) can be separated from the adults (size 400x El 100 pm) by opening the separating curtain and flushing them through the 80 pm filter to the next culture chamber for 2nd/3rd/4th generation. The adults can be dried for conservation: after opening the drying curtain, the main liquid is absorbed by the sponge; the remaining humidity is removed by means of a dry air stream (30% rh) flowing through the semi-permeable gas dryer tube.
EXPERIMENTS WITH RODENTS To close the gap between cellular biology and human physiology, ESA's Microgravity Programme Board initiated a Phase A/B study on an animal holding facility for long term experiments with mice, which should be complementary to the facilities of NASA, NASDA and CSA already planned for the ISS. In close co-operation with a similar approach by the Italian Space Agency (ASI), this study will investigate the need of a reference centrifuge, the possible increase of volume capacity for 6 or 12 mice, advanced analytical capabilities and the provision for birthing and multi-generation research. The Phase A/B study will include a number of technical issues such as requirements on biological air filters, incorporation of a glovebox, administration of drugs, microlitre-analyses, and possible in-flight tomography. The scientific focus of the facility will be related to the use of animals for pharmacological research, for research in post natal developmental biology and for multi-generation research. The duration of this study will be one year to baseline the system specifications and to define the Phase C/D development and cost. This shall allow a further decision about the animal research facility on ESA's Ministerial Conference in 2001.
814
E. Brinckmann and P. Schiller
FUTURE PERSPECTIVES With the described designs for experiment specific hardware and the new rodent facility, ESA likes to encourage scientists to submit proposals for animal research in Space, particularly on the International Space Station (ISS), which will be the main research platform in the future. Annual international Life Science Research Announcements (LSRA) will call for experiment proposals on the ISS (up to 3 months experiment duration) and possibly also on a limited number of Shuttle flights with up to 16 days for experimentation. These opportunities will allow progress in Space Life Sciences research in both, basic and applied disciplines. REFERENCES Briarty, L.G., Biology in Microgravity. A Guide for Experimenters. ESA TM-02, ed. B. Kaldeich, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1989). Brinckmann, E., Spaceflight Opportunities on the ISS for Plant Research - the ESA Perspective. Adv. SpaceRes.,24, 779-788 (1999a). Brinckmann, E., ESA-Built Hardware: MCS and BIOLAB. In: Wilson, A. (ed.) Proceedings of the 2~ Eur. Syrup. on the Utilisation of the ISS, ESA SP-433, pp. 433-440, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999b). Cogoli, A. (ed.), Biology under Microgravity Conditions in Spacelab IML-2, J. Biotechnol. 47, 65-403 (1996). Horn, E. and Sebastian, C., A Comparison of Normal Vestibulo-Ocular Reflex Development Under Gravity and in the Absence of Gravity. In: Perry, M. (ed.), Biorack on Spacehab: Experiments on Shuttle to Mir Missions 03, 05 & 06, pp. 127-138, ESA SP-1222, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999). Longdon, N, and V. David (eds.), Biology on Spacelab D1, ESA SP-1091, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1987). Mattok, C. (ed.), Biorack on Spacelab IML-1, ESA SP-1162, ESA Publications Division, ESTEC, Noordwijk, The Nethedands (1995). Marco, R., Dfaz, C., Bengnrfa, A., Mateos, J., de Juan, E., Drosophila melanogaster, a key arthropod model in the study of the evolutionary long term adaptation of multicellular organisms to the space environment. In: Wilson, A. (ed.) Proceedings of the 2~ Eur. Syrup. on the Utilisation of the ISS, ESA SP-433, pp. 433-440, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999). Perry, M. (ed.), Biorack on Spacehab: Experiments on Shuttle to Mir Missions 03, 05 & 06, ESA SP-1222, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999). Sebastian, C., Pfau, K., Horn E., An Age-Dependent Sensitivity of the Roll-Induced Vestibuloocular Roflex to Hypergravity Exposure of Several Days in an Amphibian (Xenopus laevis). Acta Astronautica, 42, 419 (1998). Sebastian, C. and Horn, E., The Minimum Duration of Microgravity Experience During Spaceflight which Affects the Development of the Roll Induced Vestibuloocular Reflex in an Amphibian. Neuroscience Letters 253,171 (1998). Snyder, R. S. (ed.), Second International Microgravity Laboratory (IML-2) Final Report, NASA Reference Publication 1405, Marshall Space Flight Center, MSFC, Alabama (1997).
Pergamon
www.elsevier.eom/locate/asr
Adv. SpaceRes. Vol.30, No. 4, pp. 809-814, 2002 © 2002COSPAR.Publishedby ElsevierScienceLtd.All rightsreserved Printedin GreatBritain 0273-1177/02 $22.00+ 0.00 PII: S0273-1177(02)00401-5
EXPERIMENTS WITH SMALL ANIMALS IN BIOLAB AND EMCS ON THE INTERNATIONAL SPACE STATION E. Brinckmann I and P. Schiller2
European Space Agency (ESA), ESTEC/MSM-GFB 1 and ESTEC/TOS-MMG 2 Postbus 299, NL-2200 AG Noordwijk, The Netherlands
ABSTRACT Two ESA facilities will be available for animal research and other biological experiments on the International Space Station: the European Modular Cultivation System (EMCS) in the US Lab "Destiny" and BIOLAB in the European "Columbus" Laboratory. Both facilities use standard Experiment Containers, mounted on two centrifuge rotors allowing either research in microgravity or acceleration studies with variable g-levels from 0.001 to 2.0xg. Standard interface plates provide each container with power and data lines, gas supply (controlled CO2, 02 concentration and relative humidity), and -for EMCS only- connectors to fresh and waste water reservoirs. The experiment hardware inside the containers will be developed by the user, but ESA conducted a feasibility study for several kinds of Experiment Support Equipment with potential use for research on small animals: design concepts for experiments with insects, with aquatic organisms like rotifers and nematodes, and with small aquatic animals (sea urchin larvae, tadpoles, fish youngsters) are described in detail in this presentation. Also ESA's initial steps to support experiments with rodents on the Space Station are presented. © 2002 COSPAR. Published by Elsevier Science Ltd. All rights reserved.
INTRODUCTION ESA's contribution to the International Space Station (ISS) for experiments with small animals will be the European Modular Cultivation System (EMCS) in the US Lab, and BIOLAB in the Columbus Laboratory. The design of these two facilities is based on experience with Biorack, ESA's precursor of the Space Station, flown six times on the Space Shuttle. The small animals used in Biorack experiments covered a wide range from unicellular organisms (Paramecium tetraurelia, Loxodes striatus) to nematodes (Caenorhabditis elegans), jellyfish (Aurelia aurita), sea urchin larvae (Sphaerechinus granularis), insects (Drosophila melanogaster, Carausius morosus eggs) and small vertebrates (Xenopus laevis embryos and tadpoles, fish youngsters of Oreochromis mossambicus). To accommodate these different organisms, Biorack offered two kinds of Experiment Containers (EC) with 65 ml and 345 ml volume. The biological samples were placed inside the experiment specific hardware, which was developed by the different investigators according to their specific needs. Several design concepts have been published in an overview by Briarty (1989) and by the experimenters themselves in the mission reports of D-1 (Longdon and David, 1987), IML-I (Mattok, 1995), IML-2 (Cogoli, 1996; Snyder 1997), S/MM-03, S/MM-05, and S/MM-06 (Perry, 1999). ESA's development concept for the Space Station experiments is similar to Biorack experiments: ESA provides the empty experiment container to the investigators, who are responsible for the development and testing of their experiment specific hardware. However, to avoid unnecessary parallel developments, ESA has studied in its Technology and Research Programme the technical feasibility of several designs, which could be used for experiments in BIOLAB and EMCS. Three of these studies are presented here for the support of experiments with aquatic organisms and insects.
809
810
E. Brinckmann arld P. Schiller
A detailed description of both facilities, BIOLAB and EMCS, had been given elsewhere (Brinckmann, 1999a; Brinckmann, 1999b). In principle, both facilities have an incubator with two centrifuges each (diameter: 600 ram) allowing microgravity research or acceleration studies in the range of 0.001xg (or less in EMCS) to 2.0xg. The experiment containers (2x6 in BIOLAB, 2x4 in EMCS) are placed by the crew on the centrifuge platters, where they are connected to the Life Support System with a controlled atmosphere: 02 and CO2 concentration can be varied in a wide range, trace gases and CO2 can be removed, and the relative air humidity can be controlled, the latter in EMCS on an individual container level including a drying function with 30% r.h. Other than BIOLAB, the EMCS rotor has a fresh water and a waste reservoir in its centre. Illumination and video observations are also possible on the rotor: the EMCS camera has even a zoom optic to observe the entire interior of the EC with a resolution of 0.1 mm; "dark" observation in infrared light is also possible on the rotors of both facilities. Besides the upgraded environmental control, observation, data handling and other capabilities, the new EC's of BIOLAB and EMCS are larger than the Biorack containers, which had only a height of 20 mm with respect to the g-vector: the BIOLAB EC has a useful volume of 360 ml with 60 mm height, the EMCS EC allows specimens with a volume up to 580 ml and a height of 160 ~ These dimensions shall be sufficient for research on cell cultures and small organisms. For larger animals like rodents, a new animal research facility on the ISS has been envisaged by ESA and the Italian Space Agency (ASI). This concept will use animal models (mice) under very strict ethical rules to allow experiments in physiology and developmental biology, which are not possible with smaller organisms or cell and tissue cultures. EXPERIMENT CONTAINERS
The BIOLAB Experiment Container The standard Experiment Container (EC) provides a safe environment for the biological samples, isolating them from the external environment and vice-versa. The transparent cover allows illumination and video observation of the experiment (Figure 1); also non-transparent covers will be available. Each EC is equipped with a temperature and a pressure sensor, a power and analogue/digital data connector, a serial interface for data, commands and container identification, and filtered gas connections to the Life Support System; this interface plate connects the EC also thermally to the centrifuge platter. The bottom plate of the EC (with respect to the g-vector and opposite to the light source) can be. customised for automatic operations to be performed by the Handling Mechanism, e.g. with several septa to inject water, nutrients or fixative for experiment termination; tool functions (push/pull/rotate) are also possible with this robotic mechanism. The EC is internally 100 ram wide and leaves a volume of about 360 ml for the accommodation of experiment specific hardware (Figure 1).
Fig. 1. Experiment Containers for BIOLAB (left) and EMCS (right). The bottom plate is the interface to the centrifuge platter. Dimensions given are the useful volumes for experiment specific hardware. The observation direction in both containers is perpendicular to the g-vector through the cover top, the illumination is parallel to the g-vector.
Small Animal Facilities by ESA
811
Two places on the centrifuge rotor are foreseen for the accommodation of Advanced Experiment Containers (AEC) enabling the development of more sophisticated or larger, customised experiment hardware, e.g. a microscope or other equipment which may be needed to perform experiments on the rotating centrifuge; thus, it is possible to avoid stops of the centrifuge, otherwise required for manual or robotic operations. The interface of the AEC to BIOLAB is the same as for the standard Experiment Containers, but with an additional analogue NTSC video line. The outer dimensions of the AEC are 175x147x125 mm, height (with respect to the g-vector) x width x depth.
The EMCS Experiment Container In addition to power and data lines and inlet/outlet gas connectors, the EMCS EC has also quick-connects for water supply and back to a waste reservoir on the rotor. Nutrients for the organisms shall be added inside the EC to prevent contamination of the fresh water reservoir. The EC's are supplied with sensors for temperature and pressure monitoring. An internal volume of 580 ml (60x60x160 mm) is available for experiment hardware with its biological material, with the long axis in direction to the gravity vector (Figure 1). Other than BIOLAB, there is no Handling Mechanism in EMCS, but all experimental operations shall be done either manually by the crew or by automatic devices of the experiment specific hardware inside the EC. If an experiment is terminated by chemical fixation, single shot valves can be activated to seal permanently the air and water connections in the EC baseplate for safety reasons.
Fig. 2. Exploded view of the Automatic Culture System for Insects with two compartments for adults, one drum with six food trays and a heat fixation device. The compartments for the adults are covered by transparent windows for video observation (perpendicular to the g-vector in BIOLAB).
MULTI-GENERATION CULTIVATION SYSTEM FOR INSECTS To study the embryological development of insects under Space conditions, the prototype of an automated culture system for multi-generation experiments has been tested on ground with Drosophila melanogaster(Marco et al. 1999). Figure 2 shows the main parts of this system in an exploded view: two transparent chambers contain the adults which can feed on the food trays and lay eggs on the food surface. Once the food tray is full with embryos, the drum is rotated and the embryos may either develop in the other adult chamber for a second generation, or they are preserved by heat shock (50°C) for later analysis on ground. New food trays can easily be replaced by the crew in a glovebox, where the experiment container can be opened in a safe environment. A
812
E. Brinckmannand P. Schiller
photoelectric sensor in the adult chambers is used for monitoring of the activity of the flies in addition to the video observation provided by BIOLAB or EMCS. One of the adult chambers contains also a receptacle compartment, which can be opened to fix a population chemically with ethanol. All functions of this insect cultivation system are handled by a microcontroller, which fits together with it into the BIOLAB or the EMCS container. The microcontroller can be commanded from ground and transmits science and status data to the experimenter on ground, thus allowing telescience operations. AUTOMATED CULTURE SYSTEMS FOR AQUATIC SPECIES
Aquatic Animals Cultivation System For studies on small aquatic species like sea urchin larvae, jellyfish ephyrae, tadpoles, fish youngsters, a miniaquarium system has been developed, based on results with a similar design on a tadpole experiment on STS-84 (Sebastian et al., 1998; Sebastian and Horn, 1998; Horn and Sebastian, 1999); in this experiment, the tadpoles survived in the aquaria a period of several weeks in perfect condition. The connection module (Figure 3) is the interface between the EC baseplate and the aquarium: fresh and waste water can be supplied to/from the EMCS reservoirs on the rotor; with BIOLAB, the Handling Mechanism has to inject liquids through a special septum port (not shown). The connection module can accommodate 6/4/3/2/1 mini-aquaria with water flowing through a volume of 30/45/60/90/180 ml each. By different lay-outs of the interconnecting tubes, the aquaria can be supplied e.g. in a parallel or serial mode, also the use of a EC specific pump module or filter/nutrient module is possible, replacing one or more aquaria on the connection module. Each mini-aquarium is covered by a biofoil membrane, supporting gas exchange of the medium to the atmosphere in the EC, which is conditioned by the Life Support System. This transparent foil allows also the visual control on the small animals by the video observation system of the facility. A ground test with sea urchin larvae proved the excellent biocompatibility of this system: swimming behaviour and development was normal during 14 days even without any water exchange.
Fig. 3. Breadboard model of the Aquatic Animals Cultivation System. The connection module is attached to the breadboard adapter plate, simulating the actual interface for gas and liquids to the EMCS container. Up to 6 miniaquaria can be installed on the connection module, connected to each other by tubes below the liquid inlet/outlet pods. The white dots on each side of the connection module are lateral pressure pads allowing a quick and safe attachment of the mini-aquaria.
Small Animal Facilities by ESA
813
Automated Culture System for Small Aquatic Species Several small organisms can thrive unfavourable conditions by forming special live forms, e.g. the "Dauerlarvae" of nematodes, or by complete desiccation, e.g. common with rotifers, until the improved environment, e.g. the addition of water or nutrients, terminates the dormant phase and re-establishes normal life. These organisms should be good candidates for experiments on the ISS, where periods of several days for transport to and from the ISS are expected: the transportation phase will most likely not be sensed in this inactive, dormant state. The Automated Culture System for Small Aquatic Species, shown schematically in Figure 4, was designed for experiments in EMCS with rotifers and nematodes there, a microcontroller would handle the liquid distribution system by opening the respective "curtains" which are separating the different compartments. The drying mode of the Life Support System in EMCS can provide the required relative humidity of 30%. A similar aquatic culture system shall be possible in BIOLAB, but mainly with manual functions due to the smaller EC size. Medium Inlet/Outlet C'1"3 t ' ~
/~"~/Separating
.....................
i D ying
Filter 40 ~'n
Gas Dryer Tube
211
1
i
Filter 80 ~
......i / '
7 ................................................
Drying Chamber
I
/ J
/
Fig. 4. Schematic drawing of the Automated Culture System for Small Aquatic Species (Rotifers), containing in total 4 of the depicted culture & drying chamber assemblies. After injection of dry animals through a septum into the outermost culture chamber (volume: 3 ml), animals can be cultivated until they lay eggs. These eggs (size 90x El 50 pro) can be separated from the adults (size 400x El 100 pm) by opening the separating curtain and flushing them through the 80 pm filter to the next culture chamber for 2nd/3rd/4th generation. The adults can be dried for conservation: after opening the drying curtain, the main liquid is absorbed by the sponge; the remaining humidity is removed by means of a dry air stream (30% rh) flowing through the semi-permeable gas dryer tube.
EXPERIMENTS WITH RODENTS To close the gap between cellular biology and human physiology, ESA's Microgravity Programme Board initiated a Phase A/B study on an animal holding facility for long term experiments with mice, which should be complementary to the facilities of NASA, NASDA and CSA already planned for the ISS. In close co-operation with a similar approach by the Italian Space Agency (ASI), this study will investigate the need of a reference centrifuge, the possible increase of volume capacity for 6 or 12 mice, advanced analytical capabilities and the provision for birthing and multi-generation research. The Phase A/B study will include a number of technical issues such as requirements on biological air filters, incorporation of a glovebox, administration of drugs, microlitre-analyses, and possible in-flight tomography. The scientific focus of the facility will be related to the use of animals for pharmacological research, for research in post natal developmental biology and for multi-generation research. The duration of this study will be one year to baseline the system specifications and to define the Phase C/D development and cost. This shall allow a further decision about the animal research facility on ESA's Ministerial Conference in 2001.
814
E. Brinckmann and P. Schiller
FUTURE PERSPECTIVES With the described designs for experiment specific hardware and the new rodent facility, ESA likes to encourage scientists to submit proposals for animal research in Space, particularly on the International Space Station (ISS), which will be the main research platform in the future. Annual international Life Science Research Announcements (LSRA) will call for experiment proposals on the ISS (up to 3 months experiment duration) and possibly also on a limited number of Shuttle flights with up to 16 days for experimentation. These opportunities will allow progress in Space Life Sciences research in both, basic and applied disciplines. REFERENCES Briarty, L.G., Biology in Microgravity. A Guide for Experimenters. ESA TM-02, ed. B. Kaldeich, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1989). Brinckmann, E., Spaceflight Opportunities on the ISS for Plant Research - the ESA Perspective. Adv. SpaceRes.,24, 779-788 (1999a). Brinckmann, E., ESA-Built Hardware: MCS and BIOLAB. In: Wilson, A. (ed.) Proceedings of the 2~ Eur. Syrup. on the Utilisation of the ISS, ESA SP-433, pp. 433-440, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999b). Cogoli, A. (ed.), Biology under Microgravity Conditions in Spacelab IML-2, J. Biotechnol. 47, 65-403 (1996). Horn, E. and Sebastian, C., A Comparison of Normal Vestibulo-Ocular Reflex Development Under Gravity and in the Absence of Gravity. In: Perry, M. (ed.), Biorack on Spacehab: Experiments on Shuttle to Mir Missions 03, 05 & 06, pp. 127-138, ESA SP-1222, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999). Longdon, N, and V. David (eds.), Biology on Spacelab D1, ESA SP-1091, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1987). Mattok, C. (ed.), Biorack on Spacelab IML-1, ESA SP-1162, ESA Publications Division, ESTEC, Noordwijk, The Nethedands (1995). Marco, R., Dfaz, C., Bengnrfa, A., Mateos, J., de Juan, E., Drosophila melanogaster, a key arthropod model in the study of the evolutionary long term adaptation of multicellular organisms to the space environment. In: Wilson, A. (ed.) Proceedings of the 2~ Eur. Syrup. on the Utilisation of the ISS, ESA SP-433, pp. 433-440, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999). Perry, M. (ed.), Biorack on Spacehab: Experiments on Shuttle to Mir Missions 03, 05 & 06, ESA SP-1222, ESA Publications Division, ESTEC, Noordwijk, The Netherlands (1999). Sebastian, C., Pfau, K., Horn E., An Age-Dependent Sensitivity of the Roll-Induced Vestibuloocular Roflex to Hypergravity Exposure of Several Days in an Amphibian (Xenopus laevis). Acta Astronautica, 42, 419 (1998). Sebastian, C. and Horn, E., The Minimum Duration of Microgravity Experience During Spaceflight which Affects the Development of the Roll Induced Vestibuloocular Reflex in an Amphibian. Neuroscience Letters 253,171 (1998). Snyder, R. S. (ed.), Second International Microgravity Laboratory (IML-2) Final Report, NASA Reference Publication 1405, Marshall Space Flight Center, MSFC, Alabama (1997).