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Biology under microgravity conditions in Spacelab International Microgravity Laboratory 2 (IML-2)
,c.“lNAL
OF
Biotechnolo ELSEVIER
Journal
of Biotechnology
47 (1996) 67-70
Preface
Biology under microgravity conditions in Spacelab International Microgravity Laboratory 2 (IML-2) August0
Cogoli*
Space Biology Group, ETH Technopark, Technoparkstrasse Received
5 February
1996; accepted
The history of space biology can be divided in two phases. The first phase began with the experiments in the early rockets of the fifties and ended with the advent of Spacelab in the eighties. This period was characterised by the so-called ‘fishing experiments’ in which the behaviour of an organism was investigated in space without a specific working hypothesis. The rational to conduct such studies was the scientific curiosity to expose living systems to an environment never experienced throughout evolution and not reproducible on Earth. The main characteristics of this environment are microgravity and cosmic radiation. The first priority in life sciences at that time was the health and performance of humans aboard spaceships and not the study of biological mechanisms. All such experiments lacked proper controls, like on-board 1 x g centrifuges, and adequate analytical tools. The second phase started in 1985 with the first flight of BIORACK in Spacelab D-l. BIORACK is a multi-user facility developed in Europe by ESA and it is exclusively dedicated to the study of *Tel.: f41
1 4451270;
016%1656/96/$15.00
fax:
+41
0 1996 Elsevier
PII SOl68-1656(96)01413-7
1 4451271 Science
B.V. All rights
reserved
1, CH-8005 Zurich, Switzerland
7 February
1996
small organisms like bacteria, slime moulds, fungi, small plants and animals, and single plant and animal cells. Basic biology became a priority and it was the privilege of European scientists to be the firsts to make use of BIORACK. After D-l, BIORACK flew in Spacelab IML-1 in 1992, IML2 in 1994 and will fly in Spacelab three times between 1996-1998. BIORACK was followed by other biological multi-user facilities like BIOLABOR flown in Spacelab D-2 in 1993, NIZEMI flown in Spacelab IML-2 and BIOBOX, an automated incubation facility installed on Russian biosatellites. This phase is characterised by the transition from the ‘fishing experiments’ to investigations based on the study of molecular mechanisms at the cellular level. It is accompanied by studies on signal transduction on board sounding rockets delivering a few minutes of microgravity. This phase, that ended with the flight of BIORACK in IML-2 in 1994, permitted the collection of a large amount of data showing that even single cells undergo dramatic changes at 0 x g. A third and new phase is just at its beginning and will consist of experiments fitting within the goals defined recently by ESA and NASA. It will comprise the three flights of BIORACK in three
shuttle missions to the Russian space station MIR, investigations in sounding rockets and biosatellites and it will proceed with the use of BIOLAB aboard the International Space Station Alpha. There. the potential benefits of bioprocessing in space will be investigated in addition to basic research (see below). About 200 biological experiments with bacteria, slime moulds. fungi, protozoa, small plants, sea urchin and frog eggs, invertebrates, plant and mammalian cells have been conducted in space so far. Important findings in signal transduction at the cellular level, plant gravitropism, developmental biology have been obtained. In certain cases, investigations with single cells permitted the study of complex systems like the immune system with lymphocytes and the bone system with bone fragments or with osteoblasts. Most of the experiments hitherto conducted in space were dedicated to basic science. There were and there are speculations that microgravity may favour certain bioprocesses and lead to industrial pharmacological applications. The changes observed in the genetic expression and signal transduction of cells producing substances of pharmacological importance may suggest potential applications. However, it is questionable whether such technology may compete with the application of genetic engineering in ground laboratories. The data available are encouraging; however, not one application of commercial interest has been developed yet. In fact, due to the limited resources, it was not yet possible to conduct a systematic program of investigations on the potential of such technology. Only the advent of an international space station permanently attended by crews of specialists will allow to address this question. Great industrial interest is currently focused on the crystallisation of proteins (like receptors and hormones), a discipline more related to materials technology than to biology. Some data show that protein crystals grow with a more regular structure, and, therefore, are more suitable for X-ray structural analysis, than crystals grown at 1 x g. Despite the important results achieved, space biology is still a discipline not well known to the broad scientific community. The purpose of this volume of Journal of Biotechnology, dedicated to
the results of the Spacelab IML-2 mission, is to present an insight into the technology, scientific approaches and possibilities of space biology, as well as of its limits and constraints, to a large readership in a journal of good international reputation. As suggested by Professor Fiechter, the Editor-in-Chief, we also decided to consider for publication papers describing experiments that were not completely successful or even failed. The intention is to give a first-hand description of the problems encountered in this young discipline. The authors were asked to describe their experimental approach, their technology and instrumentation, and to explain the reason of choosing the space environment for their study. Some of the common problems preventing a large community of scientists from conducting experiments in space are outlined here. First. the access to space is restricted. Only a small number of projects can be accommodated in a Spacelab flight. The consequence is that the statistical significance of the data is sometimes questionable and the reproducibility of important results is difficult to verify by independent teams. For instance, less than 20 experiments are hosted in each BIORACK flight. In addition, the number of flight opportunities in Spacelab. MIR, automated satellites and sounding rockets is very low compared to the number of investigations proposed. Second, the resources available in a space laboratory are very limited. Energy, weight and volume of the payload as well as crew time have to be shared among several users from different disciplines as material and fluid sciences. medicine and biology. The incubation temperatures usually available are 22°C and 37°C. While the last value is adequate for all mammalian cells, 22°C are often a compromise for ‘ambient temperature’. Freezing conditions are limited to - 10°C or 2O”C, different from standard preservation condition on ground of - 80°C and - 180°C. This means a significant restriction of manipulations, of analytical procedures (such as microscopic and biochemical determination), and of the controlled storage/stowage of biological samples in orbit. Another disturbing limitation is the so-called late access time, i.e. the latest time at which biological
A. Cogoli
/ Journal
of
Biotecilnologv
samples can be delivered for installation on board. This time ranges between 15 and 25 h before launch. Several living probes must undergo special treatment in order to be viable for the processing in orbit. The consequence is that the experimental protocols are less sophisticated and comprehensive than those of equivalent investigations on earth. Another important issue is that of the proper controls. There is a consensus today, that centrifuges providing 1 x g in space, in addition to synchronous controls on ground, are necessary to control all those environmental elements (as vibrations, accelerations, temperature fluctuations and, most important, cosmic radiation) typical of space flight. While BIORACK is fitted with a 1 x g centrifuge, most of the other experiments performed in other flights of the shuttle lack such control. Third, the safety of the astronauts requires severe acceptance criteria for instruments and biological materials on board. For example, the limits to off-gassing of toxic or bad-smelling gases and to electromagnetic contamination are extremely low, sharp edges must be avoided, and biological/chemical contamination from viruses and bacteria or biological fluids must be prevented by independent triple containment. Moreover, instruments shall not interfere (electrically or acoustically) with each other. Fourth, the time interval between the acceptance of a proposal and its flight is often several years. This was due to the delays of the development of the space shuttle (the first flight took place in 1981 instead of 1978) and to the loss of Challenger ten years ago. Nowadays, the interval between acceptance and flight of an investigation is approximately 2-3 years. The consequence was that the science proposed was obsolete at the time of flight. Requests for updates of flight protocols or of requirements during the preparation of the experiments were and are very difficult to obtain approval from the space agencies. Fifth, failures due to instrument malfunctions, break-down of resources, crew errors may even cause the total loss of an investigation prepared for years, often without an opportunity to re-fly. The considerations outlined above shall not, however, discourage those scientists who might be
47 (1996)
67- 70
69
interested to carry out experiments in space. The question is, why accept all these kind of hurdles to perform an investigation that may even fail when the resources in the ground laboratories permit the conducting of daily interesting studies on the most challenging questions of today’s biology? The answer is given by two main aspects that motivate the efforts and the patience of space biologists. One is the scientific curiosity to expose living systems to conditions (microgravity and cosmic radiation) never experienced before throughout evolution. The unexpected and important results of several experiments show that even very simple organisms display dramatic changes in microgravity. In this context, microgravity can be considered as a new tool to study certain biological mechanisms from a new perspective. For example, in the case of cell cultures, the transition from 1 x g to 0 x g changes the geometry of the system from two-dimensional to three-dimensional. Thus, cell-cell interactions and movements are completely changed with inevitable consequences in signal transduction as well as nutrients and waste concentrations around the cells. Since microgravity and cosmic radiation are not reproducible on earth, the only way to perform this research is to go to space. Simulations in devices like clinostats are a useful and necessary complement, but not a replacement for space. The other reason is the exploration of space. This includes trips to an earth orbit as well to the planets of the Solar system and. in the distant future, to other planetary systems. It is important that the adaptation of the physiological functions of humans and other mammals, as well as of other organisms like plants, invertebrates and microbes, are investigated and clarified. It was and it will be an irresistible drive of mankind to first explore all the continents of planet Earth and, later, any accessible site in the Universe as soon as the required technology becomes available. Space exploration also includes the search for extraterrestrial life. The study of terrestrial life out of the terrestrial environment will contribute to the identification and understanding of alien forms of life. The perspectives for space biology in the next ten years are the advent of the multi-user facility
70
A. Cogoli / Journal of Biotechnology
BIOLAB for the space station. BIOLAB will be an assembly of instruments more sophisticated than BIORACK. It will offer the opportunity to conduct long-duration experiments as well as cycles of experiments over weeks and months, different from Spacelab experiments lasting no longer than 14 days. In addition, automated facilities will be developed for satellites and sounding rockets. The conditions of the experiments will be improved in terms of resources, analytical tools, reproducibility and access to space. In addition to basic science, the systematic search and screening for industrial bioprocesses will be promoted. This volume contains 23 articles describing investigations conducted aboard the Spacelab IML2 mission in July 1994 involving 30 laboratories in Europe, four in the USA and seven in Japan, and with the assistance of four space agencies (NASA in the USA, CNES in France, DARA in Germany and NASDA in Japan). Of the experiments described, 12 were conducted in BIORACK, five in NIZEMI, three with single cells in other facilities, and three in two electrophoresis apparatuses.
47 (1996) 67- 70
The technical specifications and the performance of BIORACK and NIZEMI are presented in two papers. Prof. Hider who is introducing the experiments in NIZEMI, helped me as a co-editor of the NIZEMI papers. The articles dedicated to bioprocessing by electrophoresis are introduced by Dr. Seaman. Finally, an important issue in space biology, namely the preservation of biological samples over days and weeks before they are used in experiments in space laboratories, is addressed by the last paper. In this, the data of a study conducted in five European laboratories on the preservation of viable probes of mammalian cells, plants, invertebrates and aquatic animals, in view of the activities planned on board the International Space Station Alpha in the next century, are presented. All articles have been peer reviewed by internationally known experts. I wish to thank Prof. Fiechter for the invitation to produce this volume, the colleagues who contributed with articles, the reviewers who took care of them, and my co-editor Prof. Hader.
OF
Biotechnolo ELSEVIER
Journal
of Biotechnology
47 (1996) 67-70
Preface
Biology under microgravity conditions in Spacelab International Microgravity Laboratory 2 (IML-2) August0
Cogoli*
Space Biology Group, ETH Technopark, Technoparkstrasse Received
5 February
1996; accepted
The history of space biology can be divided in two phases. The first phase began with the experiments in the early rockets of the fifties and ended with the advent of Spacelab in the eighties. This period was characterised by the so-called ‘fishing experiments’ in which the behaviour of an organism was investigated in space without a specific working hypothesis. The rational to conduct such studies was the scientific curiosity to expose living systems to an environment never experienced throughout evolution and not reproducible on Earth. The main characteristics of this environment are microgravity and cosmic radiation. The first priority in life sciences at that time was the health and performance of humans aboard spaceships and not the study of biological mechanisms. All such experiments lacked proper controls, like on-board 1 x g centrifuges, and adequate analytical tools. The second phase started in 1985 with the first flight of BIORACK in Spacelab D-l. BIORACK is a multi-user facility developed in Europe by ESA and it is exclusively dedicated to the study of *Tel.: f41
1 4451270;
016%1656/96/$15.00
fax:
+41
0 1996 Elsevier
PII SOl68-1656(96)01413-7
1 4451271 Science
B.V. All rights
reserved
1, CH-8005 Zurich, Switzerland
7 February
1996
small organisms like bacteria, slime moulds, fungi, small plants and animals, and single plant and animal cells. Basic biology became a priority and it was the privilege of European scientists to be the firsts to make use of BIORACK. After D-l, BIORACK flew in Spacelab IML-1 in 1992, IML2 in 1994 and will fly in Spacelab three times between 1996-1998. BIORACK was followed by other biological multi-user facilities like BIOLABOR flown in Spacelab D-2 in 1993, NIZEMI flown in Spacelab IML-2 and BIOBOX, an automated incubation facility installed on Russian biosatellites. This phase is characterised by the transition from the ‘fishing experiments’ to investigations based on the study of molecular mechanisms at the cellular level. It is accompanied by studies on signal transduction on board sounding rockets delivering a few minutes of microgravity. This phase, that ended with the flight of BIORACK in IML-2 in 1994, permitted the collection of a large amount of data showing that even single cells undergo dramatic changes at 0 x g. A third and new phase is just at its beginning and will consist of experiments fitting within the goals defined recently by ESA and NASA. It will comprise the three flights of BIORACK in three
shuttle missions to the Russian space station MIR, investigations in sounding rockets and biosatellites and it will proceed with the use of BIOLAB aboard the International Space Station Alpha. There. the potential benefits of bioprocessing in space will be investigated in addition to basic research (see below). About 200 biological experiments with bacteria, slime moulds. fungi, protozoa, small plants, sea urchin and frog eggs, invertebrates, plant and mammalian cells have been conducted in space so far. Important findings in signal transduction at the cellular level, plant gravitropism, developmental biology have been obtained. In certain cases, investigations with single cells permitted the study of complex systems like the immune system with lymphocytes and the bone system with bone fragments or with osteoblasts. Most of the experiments hitherto conducted in space were dedicated to basic science. There were and there are speculations that microgravity may favour certain bioprocesses and lead to industrial pharmacological applications. The changes observed in the genetic expression and signal transduction of cells producing substances of pharmacological importance may suggest potential applications. However, it is questionable whether such technology may compete with the application of genetic engineering in ground laboratories. The data available are encouraging; however, not one application of commercial interest has been developed yet. In fact, due to the limited resources, it was not yet possible to conduct a systematic program of investigations on the potential of such technology. Only the advent of an international space station permanently attended by crews of specialists will allow to address this question. Great industrial interest is currently focused on the crystallisation of proteins (like receptors and hormones), a discipline more related to materials technology than to biology. Some data show that protein crystals grow with a more regular structure, and, therefore, are more suitable for X-ray structural analysis, than crystals grown at 1 x g. Despite the important results achieved, space biology is still a discipline not well known to the broad scientific community. The purpose of this volume of Journal of Biotechnology, dedicated to
the results of the Spacelab IML-2 mission, is to present an insight into the technology, scientific approaches and possibilities of space biology, as well as of its limits and constraints, to a large readership in a journal of good international reputation. As suggested by Professor Fiechter, the Editor-in-Chief, we also decided to consider for publication papers describing experiments that were not completely successful or even failed. The intention is to give a first-hand description of the problems encountered in this young discipline. The authors were asked to describe their experimental approach, their technology and instrumentation, and to explain the reason of choosing the space environment for their study. Some of the common problems preventing a large community of scientists from conducting experiments in space are outlined here. First. the access to space is restricted. Only a small number of projects can be accommodated in a Spacelab flight. The consequence is that the statistical significance of the data is sometimes questionable and the reproducibility of important results is difficult to verify by independent teams. For instance, less than 20 experiments are hosted in each BIORACK flight. In addition, the number of flight opportunities in Spacelab. MIR, automated satellites and sounding rockets is very low compared to the number of investigations proposed. Second, the resources available in a space laboratory are very limited. Energy, weight and volume of the payload as well as crew time have to be shared among several users from different disciplines as material and fluid sciences. medicine and biology. The incubation temperatures usually available are 22°C and 37°C. While the last value is adequate for all mammalian cells, 22°C are often a compromise for ‘ambient temperature’. Freezing conditions are limited to - 10°C or 2O”C, different from standard preservation condition on ground of - 80°C and - 180°C. This means a significant restriction of manipulations, of analytical procedures (such as microscopic and biochemical determination), and of the controlled storage/stowage of biological samples in orbit. Another disturbing limitation is the so-called late access time, i.e. the latest time at which biological
A. Cogoli
/ Journal
of
Biotecilnologv
samples can be delivered for installation on board. This time ranges between 15 and 25 h before launch. Several living probes must undergo special treatment in order to be viable for the processing in orbit. The consequence is that the experimental protocols are less sophisticated and comprehensive than those of equivalent investigations on earth. Another important issue is that of the proper controls. There is a consensus today, that centrifuges providing 1 x g in space, in addition to synchronous controls on ground, are necessary to control all those environmental elements (as vibrations, accelerations, temperature fluctuations and, most important, cosmic radiation) typical of space flight. While BIORACK is fitted with a 1 x g centrifuge, most of the other experiments performed in other flights of the shuttle lack such control. Third, the safety of the astronauts requires severe acceptance criteria for instruments and biological materials on board. For example, the limits to off-gassing of toxic or bad-smelling gases and to electromagnetic contamination are extremely low, sharp edges must be avoided, and biological/chemical contamination from viruses and bacteria or biological fluids must be prevented by independent triple containment. Moreover, instruments shall not interfere (electrically or acoustically) with each other. Fourth, the time interval between the acceptance of a proposal and its flight is often several years. This was due to the delays of the development of the space shuttle (the first flight took place in 1981 instead of 1978) and to the loss of Challenger ten years ago. Nowadays, the interval between acceptance and flight of an investigation is approximately 2-3 years. The consequence was that the science proposed was obsolete at the time of flight. Requests for updates of flight protocols or of requirements during the preparation of the experiments were and are very difficult to obtain approval from the space agencies. Fifth, failures due to instrument malfunctions, break-down of resources, crew errors may even cause the total loss of an investigation prepared for years, often without an opportunity to re-fly. The considerations outlined above shall not, however, discourage those scientists who might be
47 (1996)
67- 70
69
interested to carry out experiments in space. The question is, why accept all these kind of hurdles to perform an investigation that may even fail when the resources in the ground laboratories permit the conducting of daily interesting studies on the most challenging questions of today’s biology? The answer is given by two main aspects that motivate the efforts and the patience of space biologists. One is the scientific curiosity to expose living systems to conditions (microgravity and cosmic radiation) never experienced before throughout evolution. The unexpected and important results of several experiments show that even very simple organisms display dramatic changes in microgravity. In this context, microgravity can be considered as a new tool to study certain biological mechanisms from a new perspective. For example, in the case of cell cultures, the transition from 1 x g to 0 x g changes the geometry of the system from two-dimensional to three-dimensional. Thus, cell-cell interactions and movements are completely changed with inevitable consequences in signal transduction as well as nutrients and waste concentrations around the cells. Since microgravity and cosmic radiation are not reproducible on earth, the only way to perform this research is to go to space. Simulations in devices like clinostats are a useful and necessary complement, but not a replacement for space. The other reason is the exploration of space. This includes trips to an earth orbit as well to the planets of the Solar system and. in the distant future, to other planetary systems. It is important that the adaptation of the physiological functions of humans and other mammals, as well as of other organisms like plants, invertebrates and microbes, are investigated and clarified. It was and it will be an irresistible drive of mankind to first explore all the continents of planet Earth and, later, any accessible site in the Universe as soon as the required technology becomes available. Space exploration also includes the search for extraterrestrial life. The study of terrestrial life out of the terrestrial environment will contribute to the identification and understanding of alien forms of life. The perspectives for space biology in the next ten years are the advent of the multi-user facility
70
A. Cogoli / Journal of Biotechnology
BIOLAB for the space station. BIOLAB will be an assembly of instruments more sophisticated than BIORACK. It will offer the opportunity to conduct long-duration experiments as well as cycles of experiments over weeks and months, different from Spacelab experiments lasting no longer than 14 days. In addition, automated facilities will be developed for satellites and sounding rockets. The conditions of the experiments will be improved in terms of resources, analytical tools, reproducibility and access to space. In addition to basic science, the systematic search and screening for industrial bioprocesses will be promoted. This volume contains 23 articles describing investigations conducted aboard the Spacelab IML2 mission in July 1994 involving 30 laboratories in Europe, four in the USA and seven in Japan, and with the assistance of four space agencies (NASA in the USA, CNES in France, DARA in Germany and NASDA in Japan). Of the experiments described, 12 were conducted in BIORACK, five in NIZEMI, three with single cells in other facilities, and three in two electrophoresis apparatuses.
47 (1996) 67- 70
The technical specifications and the performance of BIORACK and NIZEMI are presented in two papers. Prof. Hider who is introducing the experiments in NIZEMI, helped me as a co-editor of the NIZEMI papers. The articles dedicated to bioprocessing by electrophoresis are introduced by Dr. Seaman. Finally, an important issue in space biology, namely the preservation of biological samples over days and weeks before they are used in experiments in space laboratories, is addressed by the last paper. In this, the data of a study conducted in five European laboratories on the preservation of viable probes of mammalian cells, plants, invertebrates and aquatic animals, in view of the activities planned on board the International Space Station Alpha in the next century, are presented. All articles have been peer reviewed by internationally known experts. I wish to thank Prof. Fiechter for the invitation to produce this volume, the colleagues who contributed with articles, the reviewers who took care of them, and my co-editor Prof. Hader.