Development of the Flight Hardware for the Experiment ... - Springer Link

Microgravity Sci. Technol. (2011) 23:243–248 DOI 10.1007/s12217-010-9182-0

ORIGINAL ARTICLE

Development of the Flight Hardware for the Experiment XENOPUS on the Kubik BIO4-Mission Eberhard R. Horn · Sybille Böser · Markus Franz · Martin Gabriel · Norbert Hiesgen · Ulrich Kübler · Massimiliano Porciani · Achim Schwarzwälder · Valfredo Zolesi

Received: 29 October 2009 / Accepted: 10 February 2010 / Published online: 24 February 2010 © Springer Science+Business Media B.V. 2010

Abstract The needs of developing aquatic animals depend on their age. For example, amphibian tadpole stages require regular food supply while embryos use their yolk as food source. Thus, life support systems have to be adapted to the different ages; an efficient control for water cleanness and steady food supply is mandatory for a safe flight in microgravity. A list of biological and technical requirements prompted the concept of the Dornier-Mini-System and the design for the Astrium SUPPLY Unit. These life support systems are connected with the Astrium miniaquarium that was used several times for the transport of small aquatic animals in space. Scientific experience from this concept was considered by Kayser Italia to design and develop a space suitable hardware. Its functionality was successfully demonstrated by the experiment XENOPUS that flew on the Soyuz TMA13/TMA12 mission in 2008. From 36 launched tadpoles, 35 returned back to Earth after the 12 days lasting space flight in physiologically stable conditions.

E. R. Horn (B) · M. Gabriel Gravitational Physiology at the Institute of Neurobiology, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Germany e-mail: [email protected] S. Böser Kiefersfelden, Germany M. Franz · N. Hiesgen · U. Kübler · A. Schwarzwälder EADS Astrium GmbH, Abt. TO53, Claude-Dornier-Strasse, 88090 Immenstaad, Germany M. Porciani · V. Zolesi Kayser Italia srl, Via di Popogna 501, 57128 Livorno, Italy

Keywords Aquatic animals · Life support · Osmotic pump · Space flight · Xenopus laevis

Introduction To use aquatic animals for space flight experiments, the flight hardware has to scope with biological requirements and with the technical feasibility. A strong biological requirement is that the hardware has to be adapted to various developmental stages because—in some sense—animals at the different stages of development have to be considered as different organisms with respect to their demands for survival. Embryos need for a certain period of their life no food supply; they take it from their yolk. For example, Xenopus laevis embryos can be exposed to a 10 days lasting space flight at a temperature of 20◦ C to 21◦ C with a survival rate of about 80% to 85% when they are not older then 3 days after fertilization at launch (Horn 2006). According to the stage table from Nieuwkoop and Faber (1967), they have reached at that time stage 40/41 when reared at 23◦ C. Clean spring water and permanent air-exchange by means of an air transparent membrane such as bioFolie25 as cover of the transport aquarium are sufficient (Horn and Sebastian 1999). Similar transport conditions are sufficient for young fish Oreochromis mossambicus that were launched at stages up to 16 (= 15 dpf at 20◦ C; cf. Anken et al. 1993) (Sebastian et al. 2001) or salamander embryos (Pleurodeles waltl) if launched at stages up to 25 (= 4 dpf at 18◦ C; cf. Gallien and Durocher 1957; Horn et al. 2009). In contrast, older animals need regular food supply. Space flights with older developmental stages are needed if age related sensitivities to altered gravity

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Table 1 Requirements for the development of a life support system for the transport of animals depending on regular food supply

After three successful experiments with Xenopus tadpoles up to stage 45 on the space missions STS-55, STS84 and Soyuz-Andromède (cf. Horn 2006), flights with more developed stages were required by the scientists

to find out whether the development of the vestibuloocular reflex is characterized by a critical period. This scientific goal per se required life support systems with a highly efficient control for water cleanness and steady food supply. A list of biological and technical requirements was proposed to the engineers that were considered as necessary for a successful flight (Table 1). This list prompted a concept that was called Dornier-MiniSystem (Sebastian et al. 1998). An important component of the Dornier-Mini-System was the miniaquarium developed for the transport of small aquatic animals in space and used successfully several times with Xenopus tadpoles, young fish and salamander tadpoles (Horn and Sebastian 1999; Sebastian et al. 2001; Horn et al. 2009). Transformation into reliable flight hardware went on in a stepwise manner. The basic technical and biological concept was adapted to the miniaquarium technique (Horn and Sebastian 1999). An intensive interdisciplinary cooperation between biologists and engineers was initiated because the technical development needed careful biological tests (Franz 2003; Franz et al. 2003). A survival system (Fig. 1) developed on the basis of biological and technical requirements (cf. Table 1) worked successfully in darkness made it independent of the integration of aquatic plants. Additional studies with this breadboard were dealing with the significance of denitrifying bacteria in this system as well as with the velocity of water flow that might affect tadpoles’ activity in case of microgravity exposure. Conditions could be defined to keep Xenopus tadpoles alive for 1 month. Based on the results of these bread-board tests, the design for the Astrium SUPPLY Unit was

Fig. 1 Flow diagram (left) and breadboard design (right) used during the course of the development of the Astrium SUPPLY Unit. The syringes fixed at the Astrium miniaquaria were used

(1) to inject food suspension into the system and (2) to take water samples to measure regularly the nitrate and nitrite concentration as well as the pH (from Franz 2003)

Biological requirements

Technical requirements

Exposure of old stages Suitable food Dosage of food

Increase of 0 g-exposure durations Working under 0 g Fully automatic system → no crew time needed No pulsed or strong water currents Adjustable to many animal species Modular system

Appropriate water quality No plants are allowed Optimal growth of animals

are investigated. This question is closely linked to the demonstration of a critical period during development. Critical periods are typical for most sensory systems including vision (cf. Hubel and Wiesel 1970), hearing, feeling, olfaction, or gustation. During these periods of life animals are very sensitive to deprivation from environmental stimuli. The experimental demonstration of a critical period is only possible if animals at different periods of their life are exposed to sensory deprivation. For the sense of gravity that controls body, head and eye posture, weightlessness during space flight is the respective deprivation. Safety regulations require a specific number of containments; limited availability of crew time favours automated hardware. The EADS-Astrium SUPPLY Unit


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Fig. 2 Flow diagram (left) and design of the Astrium SUPPLY unit (right) (from Franz 2003). By means of appropriate fittings, an exhausted Supply unit can be exchanged extending the period of keeping biological samples within the bioreactor alive

developed (Fig. 2). From the technical point of view, an exhausted unit can be exchanged by a new one increasing life duration of the life support system.

Design and development of SM and FM XNP Hardware used for the experiment XENOPUS (XNP) on the Soyuz TMA13 flight in 2008 were performed by KAYSER ITALIA, Livorno/Italy. The basic components of the flight module (FM-XNP) were the Xenopus Units (XU) and the Xenopus Container (XC) that was composed of the base plate and the cover

(Fig. 3). Eight XUs could be integrated into one XC. Each XU had its own peristaltic pump; the osmotic pump was integrated within the aquarium. Tests on a Science Module (SM-XNP) led to significant improvements for the Flight Module (FM-XNP). The FM-XC as well as the SM-XC Mockup had an available air volume of 1.8 l or L. Calculations of the air volume needed by 24 tadpoles during a period of 14 days were done on the basis of data about oxygen consumption published by Territo and Burggren (1998). During the necessary 2-week period in the closed container, a group of 24 stage 45 tadpoles needs a total air volume of 172.14 ml, while a group of 24

Fig. 3 XENOPUS Unit from the Science Module SM (left) showing the peristaltic pump and the tubing for water flow, integration of the tadpole protector at the left side of the osmotic pump (middle), and the Flight Module FM immediately before

its closure, showing the base plate of the XENOPUS Container (XC) with the eight XUs and the electronic device for the control of water circulation by means of the peristaltic pumps, and the XC cover

The Kayser Italia XENOPUS Flight Hardware

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Table 2 Parameters tested on the Science Module SM-XNP for final handling of Flight Module FM-XNP

Parameters tested on the Science Module (SM-XNP) Critical parameters Handling and pre-selection of tadpoles Area of air-exchange (bioFolie25) Concentration of food in osmotic pump Tadpole Protector Multiple use of XU supplies Cleaning of experiment equipment Depressurization (p > 300 mb) Autoclavation of food suspension

stage 54 tadpoles needs 1721.40 ml. Based on the preEST observations and these calculations, the number of tadpoles for the EST were put to five per XU for stage48 and four per XU for stage-51 animals. A total number of 10 tests were performed with the SM-XNP containing eight SM-XUs with the goal to keep tadpoles alive in the system active for 15 days. Five supporting tests each with a group of 8 EADSAstrium miniaquaria completed the pre-flight tests. The number of active tadpoles in each XU was checked once every day, starting with the day of SM-XNP activation. It became clear that some parameters were critical; others were less critical (Table 2). Handling and pre-selection of tadpoles included daily exchange of water during the last 4 days before loading of the SM XUs; for the experiment EVIAN spring water was used. The tadpoles were fed with a suspension of nettle powder; its concentration should not exceed 1 g nettle powder in 40 ml water. A so-called tadpole protector was mandatory to avoid trapping of tadpoles in corners between the osmotic pump and the narrow sides of the aquarium. Bacterial contamination was significantly reduced if new tubes were used. Use of antibiotics can be recommended because analyses of water from XUs and nettle powder suspension after rearing of tadpoles for 3–7 days in the closed XUs revealed a reduction in the number of non-denitrifying bacteria strains. Autoclavation of food was disadvantageous as indicated by high rates of tadpole losses after living 10 days in the system. Depressurization has to be limited to a level of not more than p = 300 mb (Fig. 4). Less critical parameters are listed in Table 2.

• • • • •

• •

Less critical parameters Amount of food in aquarium at XU integration Use of denitrifying bacteria Pretreatment of water with oxygen Use of antibiotica Number of tadpoles in each XU Natural or artificial fertilization Depressurization (p < 300 mb)

Pre-selection of tadpoles in three steps during the 3 days before integration into the FM-XUs or SMXUs, using always fresh EVIAN spring water; Enrichment of water with medical oxygen for 15 min, immediately before filling of the FM-XUs or SM-XUs, Cleaning of all new tubes by alcohol, ultrasound (15 min), H2 O2 , dist. water; Concentration of nettle powder suspension 1 g nettle powder in 40 ml EVIAN water; Osmotic pumps, type 2ML1, that deliver nettle suspension at a rate of 10 μl/h, filled with 1 ml nettle powder suspension and 1 ml antibiotic solution (penicillin/streptomycin from Biochrom AG, Berlin) 120 mg charcoal per aquarium, freely floating; 680– 700 mg Zeolith in each waste control. Spaces between osmotic pump and bioFolie25 were closed by a biocompatible foam to avoid trapping of the animals

The EST schedule followed the time as planned for the mission with transport of animals from Ulm to Zürich on July 15, 2008, and activation of the experiment

XENOPUS-EST, Zürich, July 15–31, 2008 Based on the tests with SM-XNP and supporting tests with the Astrium miniaquaria, conditions for the Experiment Sequence Test (EST) were fixed to •

Four stage-50 or five stage-47 tadpoles in each aquarium;

Fig. 4 Depressurization test with tadpoles. At the beginning of the test, embryos were at stages 25–28; at termination of the test, stages ranged from 46 to 48. D indicates periods of depressurization for 3 to 4 h. Experiments were performed as a control for the STS-84 experiment TADPOLE in 1997


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Table 3 Survival rate after the Experiment Sequence Test (EST) performed in Zürich/CH and for a period of 4 days as planned for the first set of rVOR recordings after the mission FM

FM

FM spare

SM

FM

FM

FM spare

SM

Location

FM-XC

FM-XC

SM-XC

FM-XC

FM-XC

2

2

2

2

2

Laboratory incubator 1

SM-XC

Number of containments Animal stages Depressurization 19.07.2008 EST Start/ Zürich

Laboratory incubator 1

50

50 Yes

50 Yes

50

47

47 Yes

47 Yes

47

8 4–4

8 4–4

8 4–4

16 4–4–4–4

10 5–5

10 5–5

10 5–5

20 5–5–5–5

8 (8) 4–4 7 (8) 7 7 7

8 (8) 4–4

15 (16) 4–4–4–3 15 (16) 15 14 14

10 (10) 5–5 10 (10) 10 8 8

5 (10) 5–0

31.07.2008 EST End/ Zürich 31.07.2008 in Ulm 01.08.2008 02.08.2008 04.08.2008

4 (8) 4–0 11 (16) 11 11 11

9 (10) 5–4 14 (20) 14 14 12

2

13 (20) 4–0–4–5 11 (20) 10 10 10

Italicized numbers indicate total number of the respective group; numbers below, separated by slashes indicate the number of alive tadpoles in each XU FM XENOPUS Flight Module, SM XENOPUS Science Module, FM-XC XENOPUS Flight container

after the relevant safety tests including leakage test and depressurization on July 19, 2008. The experiment was deactivated in the morning of July 31, 2008; the containers were opened 12 h thereafter. Special attention was given to the survival rate. The overall survival rate immediately after opening of the aquaria was 35 out of 40 (= 87.5%) for the stage50 tadpoles and 37 out of 50 (= 74.0%) for the stage47 tadpoles. After transportation of the animals from Zürich to the PI’s laboratory in Ulm, the survival rate decreased to 82.5% for the stage-50 group and to 70.0% for the stage-47 tadpoles (Table 3). These rates were in the range of those obtained from earlier missions with Xenopus tadpoles (cf. Horn 2006). During the following 4 days, the number of animal losses was low.

Ground control survived. This successful cooperation between scientists and space companies from Germany and Italy should be used to continue with the development of the modular Dornier Mini-System for longer missions with an additional option for inflight video recordings. Acknowledgements The Miniaquarium of the Dornier-MiniSystem was developed by Astrium under DLR contract; the SUPPLY unit was part of an Astrium R&D project. Design and development of SM-XNP and FM-XNP Hardware was done by Kayser Italia under ESA contract. Horn was supported by DLR, grant 50WB0630. Permissions for the experiments with tadpoles were given by Regierungspräsidium Tübingen/D, no. 796/Ulm, and by Veterinäramt/Kanton Zürich, CH, no. 3916.

References The Experiment XENOPUS on TMA13 On the basis of this EST, ESA gave the permission for a flight of the experiment XENOPUS on Soyuz TMA13. The FM-XNP hardware including 8 XNP Units with 36 tadpoles and 1 XNP Container as second containment used during the EST was integrated in Soyuz TMA13 for the flight to the International Space Station ISS on October 11, 2008, in Bajkonur/Kazahkstan. Soyuz was launched on October 12, 2008, at 07:01 GMT. Landing took place on October 24, 2008, at 03:37 GMT. Thirtyfive out of 36 tadpoles launched in FM-XNP returned to Earth in excellent physiological conditions after a 12days lasting space flight; 64 out of 72 tadpoles from the

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