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Longevity in space; Experiment on the life span of Paramecium cell clone in space
www.elsevier.nl/locate/asr
Adv. Space Res. Vol. 23, No. 12, pp. 2087-2090,1999 0 1999 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in&eat Britain 0273- 1177199 $20.00 + 0.00 PII: SO273-1177(99)00167-2
LONGEVITY IN SPACE; EXPERIMENT PARAMECIUM CELL CLONE IN SPACE
ON THE LIFE SPAN OF
Y. Mogami, N. Tokunaga, and S. A. Baba
Department of Biology, Ochanomizu University Otsuka 2-I-1, To&o 112-8610, Japan
ABSTRACT Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described. 01999 COSPAR. Published by Elsevier Science Ltd.
INTRODUCTION We proposed for the life science experiment onboard JEM (Japan Experimental Module) an investigation on the fluctuation of biological clock during a long stay in space. Time-dependent phenomena of biological activities result from or are themselves basic activities of organisms, which have been known to be affected at various levels of organization by the exposure to microgravity in space (Moore and Cogoli, 1996). Therefore, we might not anticipate that the biological clock is maintained under microgravity in space. If the effects are only marginally significant, they could not been clearly detected during a short stay in space. However, when accumulated after a long stay in space, they could appear as severe defects on the biological clock, which governs the long-termed activities such as development, growth, maturation, and aging. Among them, life span is the most interesting and also the most important time-related phenomena. WHY PARAMECIM? In order to gain an insight into the effect of long-term space flight on the life span, we proposed an experiment focusing on the life-time of the clone generation of the ciliates Paramecium, as a model experiment on the longevity in space. It was clearly demonstrated in Spacelah D-l that the population of Paramecium tetraurelia grew 3 to 4 times faster in microgravity than those of ground and onboard 1 x g-controls (Richoilley et al, 1986). In the life history of Paramecium, the total length of life span is defined not by clock time but by fission age (the number of cell divisions experienced after fertilization); about 500 and 250 divisions for P. caudatum and P. tetraurelia, respectively (Smith-Sonneborn, 1981).
2087
2088
Y Mogami et al.
Therefore, if the total fission age is strictly conserved in space, the faster the cell proliferates, the shorter the life span as measured by universal clock time.
R
7.
Hi
\
observation window
ling port
waste
Fig. 1. Schematic drawing of a closed-loop culture system.
Paramecium starts its life cycle by conjugation (mating), which is artificially induced by mixing cells of complementary mating types. During conjugation fertilization occurs by producing duplicated haploid nuclear gametes, exchanging one of the gametes between mating pairs and fusing of the exchanged gamete with the remaining one. Thus, each of a mating pair, through conjugation, becomes genetically identical; each cell with one half from its own haploid complement and one half from its partner’s The identical twins separate, and grow by successive cell divisions to a large population of clone cells. If the two individuals of the twin are isolated just after conjugation and cultivated separately in different environments, we can directly assess the environment effects on the genetically identical cell populations which begin the life cycle (clone generation) simultaneously. In our proposal, clone generation will be initiated in orbit and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 Xg-controls) will be precisely assessed in terms of fission age as compared to the clock time. For initiating clone generation, autogamy (self-mating; fusion of the duplicated haploid with each other in a single cell without pairing) in stead of conjugation would be available in some species, such as P. tetraurelia. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we have tested the methods of cell culture and of cell-density measurements, which will be available in closed environments under microgravity. The basic design of the experimental hardware and a preliminary result of the culture are described in this paper. DESIGN OF EXPERIMENTAL HARDWARE The minimum requirement for the experimental hardware is that it enables the counting of the cell divisions in the course of a long-lasting culture in a totally closed system. Figure 1 shows one of the basic designs of the hardware (closed-loop system). It has a closed-loop of a gas-impermeable tube,
Life Span of Purumecium
2089
in Space
which have been modified for special functions. The gas exchanger portion has a gas permeableThe observation window is used for the macroscopic membrane wall for oz/CGz exchange. observation for the measurement of the cell density of the population.
10000
r
v
vvvvv
8000 -
6000 4000 2000 0 0
5
10
Days After
15
20
25
Inoculation
Fig. 2. Growth of cell population of P. tetraurelia in a closed-loop culture system. Total volume of Inner diameter of the the culture medium in the closed-loop used in this measurement was 3 ml. tubing of the loop was 2.2 mm on the average and silicone rubber of 0.5 mm thick was used for the wall of the gas exchanger. Cell density was monitored in an ‘optically sampled’ volume in the observation window of square quartz-tubing (2 x 2 x 20 mm inner dimensions) with slit-laser illumination of 0.2 mm thick. Inverted triangles indicate the time point of dilution at the stationary phase.
Cells are first introduced into a closed-loop through an access port (C) and confined to a closed-loop (B -+D-+E*B), where the medium is allowed to flow to facilitate gas exchange at the exchanger portion of the loop. When the cell population grows, through the log phase to the stationary phase, new medium is introduced to the loop (A-B) through a cell-discriminating filter. At the same time, an aliquot of the culture within the loop is pushed out to the sampling port (E-F-G), and the rest to the waste container (E-+F-+H). Sampled cells will be fixed by appropriate procedures and stored until recovery. The diluted cells in the loop will grow again to the stationary phase. Precise control of the dilution ratio would give the precise measure of the cell proliferation; i.e., the period necessary to regain the stationary phase after the dilution by l/2n might be considered as the mean clock time required for n divisions of each cell in the population. Cell density in the closed-loop is continuously monitored through the observation window. Since the density of Paramecium in culture is relatively low (up to ca. lo6 to IO7 cell/ml), it has been routinely measured by directly counting the cells in a given volume. In order to avoid the problem of direct sampling from the closed-loop system, a given volume in the culture is ‘optically’ sampled by illuminating the cell suspension through a side wall of observation window with a slit-laser of a known Dark-field images are obtained by observing the illuminated area through a TV beam thickness.
2090
Y. Mogami et al.
camera in the direction perpendicular to the slit illumination. Counting the cells in the dark-field images give a reliable measurement of the cell density of the practical range (Mogami et al, 1996). A preliminary result of the culture with the closed-loop system is shown in Figure 2. P. tetruurelia was grown in the closed-loop system with the culture medium of 4.1 mM KIQPO4, 4.1 mM Na2HP04, 4.1 mM sodium citrate, 1.5 mM CaC12,0.05% (w/v) Calory Mate (liquid type, Otsuka Pharmaceutical Co. Ltd., Osaka, Japan). In a totally closed condition, cell population grew to the stationary phase within 3 days after the inoculation of the cell. Growth of the cell population was initiated by dilution at various ratios and the density regained pre-dilution value before dilution. These facts confirm that the closedloop system developed here has the minimum specifications required for the space experiment. Basic models of experimental hardware will be improved in parallel with fine tuning of the experimental scenarios. Since paramecia, in this culture condition, are fed by bacteria grown in the medium, undigested particles secreted from the cytoproct sometimes make errors in cell counting. This will be avoided by replacing the culture medium with a non-bacterized one, which is currently in progress. For the short-term experiment at the early assembly phase of ISS, experimental scenarios of the longevity mission would be modified to allow an assessment of the fluctuation of immaturity period, which is much shorter, but still highly correlates with the length of the life span (Smith-Sonneborn, 1981). ACKNOWLEDGEMENTS This study was carried out as a part of “Ground Research Announcement for the Space Utilization” promoted by NASDA and Japan Space Forum. REFERENCES Mogami, Y., S.A. Baba, A. Ooki, M. Kasamatsu, N. Tokunaga, M. Takahashi, Y. Takagi, and I. Miwa, Fluctuation of Life Span in Space - Cell Culture and Counting in Closed Environment (in Japanese with abstract in English), Space UtilizationResearch, 13,27-29 (1996). Moore, D., and A. Cogoli, Gravitational and Space Biology, in Biological and Medical Research in Space. An Overview of Life Sciences Research in Microgravity, ESA, pp. 5-109, SpringerVerlag, Berlin Heidelberg (1996). Richoilley, G., R. Tixador, G. Gasset, J. Templier, and H. Planel, Preliminary Results of the “Paramecium” Experiment, Naturwissenschaften, 73,404-406 (1986). Smith-Sonneborn, J., Genetics and Aging in Protozoa, Znt.Rev. Cytol., 73, 3 19-354 (1981)
Adv. Space Res. Vol. 23, No. 12, pp. 2087-2090,1999 0 1999 COSPAR. Published by Elsevier Science Ltd. All rights reserved Printed in&eat Britain 0273- 1177199 $20.00 + 0.00 PII: SO273-1177(99)00167-2
LONGEVITY IN SPACE; EXPERIMENT PARAMECIUM CELL CLONE IN SPACE
ON THE LIFE SPAN OF
Y. Mogami, N. Tokunaga, and S. A. Baba
Department of Biology, Ochanomizu University Otsuka 2-I-1, To&o 112-8610, Japan
ABSTRACT Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described. 01999 COSPAR. Published by Elsevier Science Ltd.
INTRODUCTION We proposed for the life science experiment onboard JEM (Japan Experimental Module) an investigation on the fluctuation of biological clock during a long stay in space. Time-dependent phenomena of biological activities result from or are themselves basic activities of organisms, which have been known to be affected at various levels of organization by the exposure to microgravity in space (Moore and Cogoli, 1996). Therefore, we might not anticipate that the biological clock is maintained under microgravity in space. If the effects are only marginally significant, they could not been clearly detected during a short stay in space. However, when accumulated after a long stay in space, they could appear as severe defects on the biological clock, which governs the long-termed activities such as development, growth, maturation, and aging. Among them, life span is the most interesting and also the most important time-related phenomena. WHY PARAMECIM? In order to gain an insight into the effect of long-term space flight on the life span, we proposed an experiment focusing on the life-time of the clone generation of the ciliates Paramecium, as a model experiment on the longevity in space. It was clearly demonstrated in Spacelah D-l that the population of Paramecium tetraurelia grew 3 to 4 times faster in microgravity than those of ground and onboard 1 x g-controls (Richoilley et al, 1986). In the life history of Paramecium, the total length of life span is defined not by clock time but by fission age (the number of cell divisions experienced after fertilization); about 500 and 250 divisions for P. caudatum and P. tetraurelia, respectively (Smith-Sonneborn, 1981).
2087
2088
Y Mogami et al.
Therefore, if the total fission age is strictly conserved in space, the faster the cell proliferates, the shorter the life span as measured by universal clock time.
R
7.
Hi
\
observation window
ling port
waste
Fig. 1. Schematic drawing of a closed-loop culture system.
Paramecium starts its life cycle by conjugation (mating), which is artificially induced by mixing cells of complementary mating types. During conjugation fertilization occurs by producing duplicated haploid nuclear gametes, exchanging one of the gametes between mating pairs and fusing of the exchanged gamete with the remaining one. Thus, each of a mating pair, through conjugation, becomes genetically identical; each cell with one half from its own haploid complement and one half from its partner’s The identical twins separate, and grow by successive cell divisions to a large population of clone cells. If the two individuals of the twin are isolated just after conjugation and cultivated separately in different environments, we can directly assess the environment effects on the genetically identical cell populations which begin the life cycle (clone generation) simultaneously. In our proposal, clone generation will be initiated in orbit and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 Xg-controls) will be precisely assessed in terms of fission age as compared to the clock time. For initiating clone generation, autogamy (self-mating; fusion of the duplicated haploid with each other in a single cell without pairing) in stead of conjugation would be available in some species, such as P. tetraurelia. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we have tested the methods of cell culture and of cell-density measurements, which will be available in closed environments under microgravity. The basic design of the experimental hardware and a preliminary result of the culture are described in this paper. DESIGN OF EXPERIMENTAL HARDWARE The minimum requirement for the experimental hardware is that it enables the counting of the cell divisions in the course of a long-lasting culture in a totally closed system. Figure 1 shows one of the basic designs of the hardware (closed-loop system). It has a closed-loop of a gas-impermeable tube,
Life Span of Purumecium
2089
in Space
which have been modified for special functions. The gas exchanger portion has a gas permeableThe observation window is used for the macroscopic membrane wall for oz/CGz exchange. observation for the measurement of the cell density of the population.
10000
r
v
vvvvv
8000 -
6000 4000 2000 0 0
5
10
Days After
15
20
25
Inoculation
Fig. 2. Growth of cell population of P. tetraurelia in a closed-loop culture system. Total volume of Inner diameter of the the culture medium in the closed-loop used in this measurement was 3 ml. tubing of the loop was 2.2 mm on the average and silicone rubber of 0.5 mm thick was used for the wall of the gas exchanger. Cell density was monitored in an ‘optically sampled’ volume in the observation window of square quartz-tubing (2 x 2 x 20 mm inner dimensions) with slit-laser illumination of 0.2 mm thick. Inverted triangles indicate the time point of dilution at the stationary phase.
Cells are first introduced into a closed-loop through an access port (C) and confined to a closed-loop (B -+D-+E*B), where the medium is allowed to flow to facilitate gas exchange at the exchanger portion of the loop. When the cell population grows, through the log phase to the stationary phase, new medium is introduced to the loop (A-B) through a cell-discriminating filter. At the same time, an aliquot of the culture within the loop is pushed out to the sampling port (E-F-G), and the rest to the waste container (E-+F-+H). Sampled cells will be fixed by appropriate procedures and stored until recovery. The diluted cells in the loop will grow again to the stationary phase. Precise control of the dilution ratio would give the precise measure of the cell proliferation; i.e., the period necessary to regain the stationary phase after the dilution by l/2n might be considered as the mean clock time required for n divisions of each cell in the population. Cell density in the closed-loop is continuously monitored through the observation window. Since the density of Paramecium in culture is relatively low (up to ca. lo6 to IO7 cell/ml), it has been routinely measured by directly counting the cells in a given volume. In order to avoid the problem of direct sampling from the closed-loop system, a given volume in the culture is ‘optically’ sampled by illuminating the cell suspension through a side wall of observation window with a slit-laser of a known Dark-field images are obtained by observing the illuminated area through a TV beam thickness.
2090
Y. Mogami et al.
camera in the direction perpendicular to the slit illumination. Counting the cells in the dark-field images give a reliable measurement of the cell density of the practical range (Mogami et al, 1996). A preliminary result of the culture with the closed-loop system is shown in Figure 2. P. tetruurelia was grown in the closed-loop system with the culture medium of 4.1 mM KIQPO4, 4.1 mM Na2HP04, 4.1 mM sodium citrate, 1.5 mM CaC12,0.05% (w/v) Calory Mate (liquid type, Otsuka Pharmaceutical Co. Ltd., Osaka, Japan). In a totally closed condition, cell population grew to the stationary phase within 3 days after the inoculation of the cell. Growth of the cell population was initiated by dilution at various ratios and the density regained pre-dilution value before dilution. These facts confirm that the closedloop system developed here has the minimum specifications required for the space experiment. Basic models of experimental hardware will be improved in parallel with fine tuning of the experimental scenarios. Since paramecia, in this culture condition, are fed by bacteria grown in the medium, undigested particles secreted from the cytoproct sometimes make errors in cell counting. This will be avoided by replacing the culture medium with a non-bacterized one, which is currently in progress. For the short-term experiment at the early assembly phase of ISS, experimental scenarios of the longevity mission would be modified to allow an assessment of the fluctuation of immaturity period, which is much shorter, but still highly correlates with the length of the life span (Smith-Sonneborn, 1981). ACKNOWLEDGEMENTS This study was carried out as a part of “Ground Research Announcement for the Space Utilization” promoted by NASDA and Japan Space Forum. REFERENCES Mogami, Y., S.A. Baba, A. Ooki, M. Kasamatsu, N. Tokunaga, M. Takahashi, Y. Takagi, and I. Miwa, Fluctuation of Life Span in Space - Cell Culture and Counting in Closed Environment (in Japanese with abstract in English), Space UtilizationResearch, 13,27-29 (1996). Moore, D., and A. Cogoli, Gravitational and Space Biology, in Biological and Medical Research in Space. An Overview of Life Sciences Research in Microgravity, ESA, pp. 5-109, SpringerVerlag, Berlin Heidelberg (1996). Richoilley, G., R. Tixador, G. Gasset, J. Templier, and H. Planel, Preliminary Results of the “Paramecium” Experiment, Naturwissenschaften, 73,404-406 (1986). Smith-Sonneborn, J., Genetics and Aging in Protozoa, Znt.Rev. Cytol., 73, 3 19-354 (1981)