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Cell Locomotion in Archaeocyte-dominant Cell Population (ADCP) Primmorph Culture of Marine Sponge Hymeniacidon perlevis
CHINESE JOURNAL OF BIOTECHNOLOGY Volume 24, Issue 12, December 2008 Online English edition of the Chinese language journal RESEARCH PAPER
Cite this article as: Chin J Biotech, 2008, 24(12), 2133í2134.
Cell Locomotion in Archaeocyte-dominant Cell Population (ADCP) Primmorph Culture of Marine Sponge Hymeniacidon perlevis Xupeng Cao1, and Wei Zhang1, 2 1
Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2
Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide SA 5042, Australia
Abstract: The archaeocyte-dominant cell population (ADCP) primmorphs is a new sponge cell culture system, and is possible to develop into a continuous sponge cell line. However, the archaeocytes’ division, differentiation and development, as well as the cell interactions, have not yet been clear. Cell movement (cell locomotion) is the base of other cell interactions. We use real-time video cinemicrography technique to document the cell locomotion continuously up to 90 days in the ADCP cultures of marine sponge Hymeniacidon perlevis. Locomotion of three typical cell types, archaeocytes for mesohyl cells, pinacocytes for surface cells, and spicule-associated cells, were monitored and analyzed during the culture process including the inoculation of dissociated cells and the formation stage of functional primmorphs. We observed the unique particle transfer process between archaeocytes, the fluctuation spreading over cells and the production of silica spicules and the active transfer of spicules by sponge cells. A tentative model of material transfer and the coordinated locomotion of sponge cells were proposed. Keywords: Hymeniacidon perlevis; archaeocyte-dominant cell populations (ADCP); cell locomotion
Introduction Sponge is a well known natural products resource, and it contributes nearly 30% of bioactive compounds from the marine organisms within 10 years. But due to the “supply problem” of sponge biomass, only few have been commercialized. Sponge cell culture is a most promising approach to solve the supply problem but till now nobody has been successful in establishing a sponge cell line[1]. Wilson’s first observation of sponge cell aggregation set the foundation and prompted a rapid development of immunology and cell-sorting phenomena in the higher metazoans. However, the understanding of sponge cell interactions during aggregation process is still poor, which limits our study on the division, differentiation, and
development of sponge cells. This knowledge is also required to illustrate specific intercellular adhesion, the formation and maintenance of anatomical structures, and the coordination behaviors of sponge cells. Sponge cell locomotions in sponge tissues have been reported for a long time (reviewed by Simpson[2]). Bond used time-lapse techniques to observe continuous active movements in intact sponges, which resulted in the rearrangement of the anatomical structures[3]. Although the reaggregation of dissociated sponge cells is an important model for these observations, the opaque aggregates in the rotating and stirring culture system obstructed long time tracking cells locomotion by microscopes. Based on Müller’s[4] primmorphs culture method, archaeocyte-dominant cell populations (ADCP) primmorphs
Received: October 17, 2008; Accepted: November 25, 2008 Supported by: National High-tech R&D Program of China (863 Program) (Nos. 2001AA620404, 2006AA09Z435). Corresponding author: Wei Zhang. Tel: +86-411-84379069; Fax: +86-411-84379069; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
Xupeng Cao et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2133–2134
culture of Hymeniacidon perlevis (H. perlevis) was achieved by Zhang et al[5] after enriching archaeocytes with Ficoll gradient. Archaeocytes are believed to be totipotent cells with the capacity to differentiate into any other sponge cell types. Archaeocyte-dominant cell population primmorphs showed improved cell proliferation and spiculogenesis ability. Instead of shaking or stirring, static ADCP culture can produce anchored aggregations, which then spread and develop into functional tissues with spicules and canals in the form of thin sheets. One of the advantages of this technique is that the thin sheet cell aggregates are transparent and inner cells locomotion can be observed in almost the whole culture process.
1
Materials and methods
1.1 Sponge collection H. perlevis was collected at the tide area of Lingshui Bay (Dalian, China) several months before experiments. After washing away the dirt on the surface, sponges were cultured in a 150 L aquarium with sand-filtered natural sea water, 18°C–20°C. Hundred percentage of water was changed each week, and sponges were fed with golden algae (Isochrysis zhanjiangensis) until the concentration of 2×105 cells/mL was obtained. 1.2 ADCP primmorphs culture and cell locomotion capture Using the same protocol described in Cao et al’s early report[6], ADCP primmorphs culture were performed and video of cell locomotion were captured on a continuous-medium-replacement culture and observation platform (CMRP) and analyzed.
2
Results and discussion
2.1 Overall locomotion of archaeocytes, pinacocytes, and aggregates We found that the development process of H. perlevis ADCP primmorphs can be defined as three stages. The first 12 hours after inoculation is the formation stage in which
dissociated cells settled down, reaggregated, and formed cell clusters. The second stage is the predeveloping stage that starts from 1 to 3 days in which cell clusters keep fusing and get attached to the substratum. In the early second stage, cell aggregates often undergo amoeboid locomotion as a whole. Sponge cells begin to spread, grow, and differentiate inside aggregates. The third stage is the development stage, during which cells proliferate and the complex inner structures are formed accompanying with the spiculogenesis. After 20 days to one month of fast development, aggregates transformed to functional tissues with steady size and biomass. Cells locomotion in all stages was recorded. Overall description of locomotion of archaeocytes, pinacocytes, and aggregates as a whole is listed in Tab.. We found that pinacocytes and archaeocytes have different locomotion modes during different stages. In early stages, pinacocytes form the basal layer. It appears to be the preparation for aggregates move. Soon the linkage is constructed between the basal and surface layer of aggregate. However, only very slow and local locomotions were observed from either of the bodies. The locomotion of aggregates started from the pinacocytes where the linkage has been established. Leys[7] reported the electrical recording from a glass sponge. In the embryo, one ectoderm cell has two options, epidermal or neural development, which is governed by signals. So it is possible that signals can be transferred far and quickly through the dynamic network of pinacocytes, may include even the electrical signals. We observed that the archaeocytes actively migrate after they come into inner of aggregates and move to the pinacocytes layer vigorously. We also found that archaeocytes could migrate by following certain traces. Inside of aggregates were full of cells but no central putrescence happened even for those that were more than 5 mm in diameter. This may be due to the nonstop migration of archaeocytes by which materials were exchanged between inner and environment. For example, some particles were found to be transferred between cells.
Tab. Locomotion of different cells types and aggregates in three stages of aggregates forming and developing Stage
Pinacocytes
Archaeocytes
I
Fast locomotion in random directions with intensive shape changes. Speed: 0.15 to 0.2 Pm/s
Round-ball like, aggregating fast and no obviously active locomotion in small clusters
II
III
In leading margins, speed of cells between 0.03 to 0.06 Pm/s; speed for single pinacocytes up to 0.2 Pm/s before re-suspending Forming pinacocytes sheets and cells oscillating locally
Choanocytes No obvious active locomotion and weakly combining to aggregates Speed: <0.01ȝm/s
Locomotion in clusters, circling inside of aggregates Speed: 0.020.2 Pm/s
Cannot be identified
As above
Cannot be identified
Aggregates Oscillating in small area with the aid of pinacocytes Locomotion as a whole, average locomotion speed of the center below 0.03 Pm/s and finally stopped Relatively steady as a whole with edges movement
Xupeng Cao et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2133–2134
2.2 Locomotion of spicule-associated cells The appearance of spicules is an important sign of the development and differentiation. Müller et al[8] proposed two steps for spicules synthesis, intracellular synthesis and extracellular growth. Therefore, the growth and positioning of spicules are closely related to cells locomotion. Due to the limit of resolution, we were not able to track the newly born spicules that were within 0.5 ȝm diameters in the video. But numerous cells that are possibly involved in spicules growth and positioning were monitored. 2.3 Spread of aggregates and front line regeneration Like a breeze over the calm surface of a pond, a kind of fluctuation spreading over cells inside aggregates was also recorded. Waves of fluctuations transferred across the view filed at the speed of 6 ȝm/s and lasted 1 minute to 8 minutes, which is long enough to exclude the possibility of random environment disturbance. No fixed interval or cycle of waves was found, but if continuous waves appeared, intervals were roughly less than 15 minutes. It was also found that the original routes of archaeocytes were not disturbed by waves. There have already been some reports on contracts in cellular sponges[8], such as waves of contraction traveling in sponge tissues at speeds between 4 to 100 Pm/s in different parts. The waves observed here may have some relation to the contraction of sponges. 2.4 Coordination of different sponge cells in primmorphs migration It is reasonable to assume that both cell signaling and particles have been transferred within the aggregates during the highly coordinated cell development and migration process. Cells are stimulated with wound, pressure, or starvation response specifically by undergoing corresponding locomotion, and some signal molecules could be released to surroundings nearby the trace of locomotion or directly to the other cells. It is obvious that cell migration increases the cell-cell communication. It seems that there could exist a very comprehensive extracellular or/and intracellular signal transduction network. The coordinating characteristics of sponge cells locomotion may be explained by the mechanism of “swarm intelligence,” which is the property of a system whereby the collective behaviors of (unsophisticated) agents interacting locally with their environment cause coherent functional global patterns to emerge[9]. A sponge cells aggregate can be treated as a swarm of sponge cells,
and furthermore, sponge tissue can be treated as the swarm of swarms of sponge cells.
4
Acknowledgments
The authors thank financial supports from the European Commission (Project: SILICON BIOTECHNOLOGY). We are grateful for Mr Yuanling Liu to construct the video capture system and Dr. Zhao’an Chen’s discussion. The authors thank the reviewers for the constructive comments for improving this article.
REFERENCES [1]
Sipkema D, Osinga R, Schatton W, et al. Large-scale production of pharmaceuticals by marine sponges: Sea, cell, or synthesis? Biotechnol Bioeng, 2005, 90: 201222.
[2]
Simpson TL. The cell biology of sponges. New York: Springer-Verlag, 1984.
[3]
Bond C. Continuous cell movements rearrange anatomical structures in intact sponges. J Exp Zool, 1992, 263: 284302.
[4]
Custodio MR, Prokic I, Steffen R, et al. Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death. Mech Ageing Dev, 1998, 105: 4559.
[5]
Zhang XY, Cao XP, Zhang W, et al. Primmorphs from archaeocytes-dominant cell population of the sponge Hymeniacidon perleve: Improved cell proliferation and spiculogenesis. Biotechnol Bioeng, 2003, 84: 583590.
[6]
Cao XP, Fu WT, Yu XJ, et al. Dynamics of spicule production in the marine sponge Hymeniacidon perlevis during in vitro cell culture and seasonal development in the field. Cell Tissue Res, 2007, 329: 595608
[7]
Leys SP, Mackie GO. Electrical recording from a glass sponge. Nature, 1997, 387: 2930.
[8]
Müller WE, Belikov SI, Tremel W, et al. Siliceous spicules in marine demosponges (example Suberites domuncula). Micron, 2005, 37: 107120.
[9]
Hoffmeyer J. The swarming body// Rauch PI, Carr GF (Eds.). The Fifth Congress of the International Association for Semiotic Studies. Berlin/New York: Mouton de Gruyter, 1994, 937940.
Cite this article as: Chin J Biotech, 2008, 24(12), 2133í2134.
Cell Locomotion in Archaeocyte-dominant Cell Population (ADCP) Primmorph Culture of Marine Sponge Hymeniacidon perlevis Xupeng Cao1, and Wei Zhang1, 2 1
Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China
2
Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide SA 5042, Australia
Abstract: The archaeocyte-dominant cell population (ADCP) primmorphs is a new sponge cell culture system, and is possible to develop into a continuous sponge cell line. However, the archaeocytes’ division, differentiation and development, as well as the cell interactions, have not yet been clear. Cell movement (cell locomotion) is the base of other cell interactions. We use real-time video cinemicrography technique to document the cell locomotion continuously up to 90 days in the ADCP cultures of marine sponge Hymeniacidon perlevis. Locomotion of three typical cell types, archaeocytes for mesohyl cells, pinacocytes for surface cells, and spicule-associated cells, were monitored and analyzed during the culture process including the inoculation of dissociated cells and the formation stage of functional primmorphs. We observed the unique particle transfer process between archaeocytes, the fluctuation spreading over cells and the production of silica spicules and the active transfer of spicules by sponge cells. A tentative model of material transfer and the coordinated locomotion of sponge cells were proposed. Keywords: Hymeniacidon perlevis; archaeocyte-dominant cell populations (ADCP); cell locomotion
Introduction Sponge is a well known natural products resource, and it contributes nearly 30% of bioactive compounds from the marine organisms within 10 years. But due to the “supply problem” of sponge biomass, only few have been commercialized. Sponge cell culture is a most promising approach to solve the supply problem but till now nobody has been successful in establishing a sponge cell line[1]. Wilson’s first observation of sponge cell aggregation set the foundation and prompted a rapid development of immunology and cell-sorting phenomena in the higher metazoans. However, the understanding of sponge cell interactions during aggregation process is still poor, which limits our study on the division, differentiation, and
development of sponge cells. This knowledge is also required to illustrate specific intercellular adhesion, the formation and maintenance of anatomical structures, and the coordination behaviors of sponge cells. Sponge cell locomotions in sponge tissues have been reported for a long time (reviewed by Simpson[2]). Bond used time-lapse techniques to observe continuous active movements in intact sponges, which resulted in the rearrangement of the anatomical structures[3]. Although the reaggregation of dissociated sponge cells is an important model for these observations, the opaque aggregates in the rotating and stirring culture system obstructed long time tracking cells locomotion by microscopes. Based on Müller’s[4] primmorphs culture method, archaeocyte-dominant cell populations (ADCP) primmorphs
Received: October 17, 2008; Accepted: November 25, 2008 Supported by: National High-tech R&D Program of China (863 Program) (Nos. 2001AA620404, 2006AA09Z435). Corresponding author: Wei Zhang. Tel: +86-411-84379069; Fax: +86-411-84379069; E-mail: [email protected] Copyright © 2008, Institute of Microbiology, Chinese Academy of Sciences and Chinese Society for Microbiology. Published by Elsevier BV. All rights reserved.
Xupeng Cao et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2133–2134
culture of Hymeniacidon perlevis (H. perlevis) was achieved by Zhang et al[5] after enriching archaeocytes with Ficoll gradient. Archaeocytes are believed to be totipotent cells with the capacity to differentiate into any other sponge cell types. Archaeocyte-dominant cell population primmorphs showed improved cell proliferation and spiculogenesis ability. Instead of shaking or stirring, static ADCP culture can produce anchored aggregations, which then spread and develop into functional tissues with spicules and canals in the form of thin sheets. One of the advantages of this technique is that the thin sheet cell aggregates are transparent and inner cells locomotion can be observed in almost the whole culture process.
1
Materials and methods
1.1 Sponge collection H. perlevis was collected at the tide area of Lingshui Bay (Dalian, China) several months before experiments. After washing away the dirt on the surface, sponges were cultured in a 150 L aquarium with sand-filtered natural sea water, 18°C–20°C. Hundred percentage of water was changed each week, and sponges were fed with golden algae (Isochrysis zhanjiangensis) until the concentration of 2×105 cells/mL was obtained. 1.2 ADCP primmorphs culture and cell locomotion capture Using the same protocol described in Cao et al’s early report[6], ADCP primmorphs culture were performed and video of cell locomotion were captured on a continuous-medium-replacement culture and observation platform (CMRP) and analyzed.
2
Results and discussion
2.1 Overall locomotion of archaeocytes, pinacocytes, and aggregates We found that the development process of H. perlevis ADCP primmorphs can be defined as three stages. The first 12 hours after inoculation is the formation stage in which
dissociated cells settled down, reaggregated, and formed cell clusters. The second stage is the predeveloping stage that starts from 1 to 3 days in which cell clusters keep fusing and get attached to the substratum. In the early second stage, cell aggregates often undergo amoeboid locomotion as a whole. Sponge cells begin to spread, grow, and differentiate inside aggregates. The third stage is the development stage, during which cells proliferate and the complex inner structures are formed accompanying with the spiculogenesis. After 20 days to one month of fast development, aggregates transformed to functional tissues with steady size and biomass. Cells locomotion in all stages was recorded. Overall description of locomotion of archaeocytes, pinacocytes, and aggregates as a whole is listed in Tab.. We found that pinacocytes and archaeocytes have different locomotion modes during different stages. In early stages, pinacocytes form the basal layer. It appears to be the preparation for aggregates move. Soon the linkage is constructed between the basal and surface layer of aggregate. However, only very slow and local locomotions were observed from either of the bodies. The locomotion of aggregates started from the pinacocytes where the linkage has been established. Leys[7] reported the electrical recording from a glass sponge. In the embryo, one ectoderm cell has two options, epidermal or neural development, which is governed by signals. So it is possible that signals can be transferred far and quickly through the dynamic network of pinacocytes, may include even the electrical signals. We observed that the archaeocytes actively migrate after they come into inner of aggregates and move to the pinacocytes layer vigorously. We also found that archaeocytes could migrate by following certain traces. Inside of aggregates were full of cells but no central putrescence happened even for those that were more than 5 mm in diameter. This may be due to the nonstop migration of archaeocytes by which materials were exchanged between inner and environment. For example, some particles were found to be transferred between cells.
Tab. Locomotion of different cells types and aggregates in three stages of aggregates forming and developing Stage
Pinacocytes
Archaeocytes
I
Fast locomotion in random directions with intensive shape changes. Speed: 0.15 to 0.2 Pm/s
Round-ball like, aggregating fast and no obviously active locomotion in small clusters
II
III
In leading margins, speed of cells between 0.03 to 0.06 Pm/s; speed for single pinacocytes up to 0.2 Pm/s before re-suspending Forming pinacocytes sheets and cells oscillating locally
Choanocytes No obvious active locomotion and weakly combining to aggregates Speed: <0.01ȝm/s
Locomotion in clusters, circling inside of aggregates Speed: 0.020.2 Pm/s
Cannot be identified
As above
Cannot be identified
Aggregates Oscillating in small area with the aid of pinacocytes Locomotion as a whole, average locomotion speed of the center below 0.03 Pm/s and finally stopped Relatively steady as a whole with edges movement
Xupeng Cao et al. / Chinese Journal of Biotechnology, 2008, 24(12): 2133–2134
2.2 Locomotion of spicule-associated cells The appearance of spicules is an important sign of the development and differentiation. Müller et al[8] proposed two steps for spicules synthesis, intracellular synthesis and extracellular growth. Therefore, the growth and positioning of spicules are closely related to cells locomotion. Due to the limit of resolution, we were not able to track the newly born spicules that were within 0.5 ȝm diameters in the video. But numerous cells that are possibly involved in spicules growth and positioning were monitored. 2.3 Spread of aggregates and front line regeneration Like a breeze over the calm surface of a pond, a kind of fluctuation spreading over cells inside aggregates was also recorded. Waves of fluctuations transferred across the view filed at the speed of 6 ȝm/s and lasted 1 minute to 8 minutes, which is long enough to exclude the possibility of random environment disturbance. No fixed interval or cycle of waves was found, but if continuous waves appeared, intervals were roughly less than 15 minutes. It was also found that the original routes of archaeocytes were not disturbed by waves. There have already been some reports on contracts in cellular sponges[8], such as waves of contraction traveling in sponge tissues at speeds between 4 to 100 Pm/s in different parts. The waves observed here may have some relation to the contraction of sponges. 2.4 Coordination of different sponge cells in primmorphs migration It is reasonable to assume that both cell signaling and particles have been transferred within the aggregates during the highly coordinated cell development and migration process. Cells are stimulated with wound, pressure, or starvation response specifically by undergoing corresponding locomotion, and some signal molecules could be released to surroundings nearby the trace of locomotion or directly to the other cells. It is obvious that cell migration increases the cell-cell communication. It seems that there could exist a very comprehensive extracellular or/and intracellular signal transduction network. The coordinating characteristics of sponge cells locomotion may be explained by the mechanism of “swarm intelligence,” which is the property of a system whereby the collective behaviors of (unsophisticated) agents interacting locally with their environment cause coherent functional global patterns to emerge[9]. A sponge cells aggregate can be treated as a swarm of sponge cells,
and furthermore, sponge tissue can be treated as the swarm of swarms of sponge cells.
4
Acknowledgments
The authors thank financial supports from the European Commission (Project: SILICON BIOTECHNOLOGY). We are grateful for Mr Yuanling Liu to construct the video capture system and Dr. Zhao’an Chen’s discussion. The authors thank the reviewers for the constructive comments for improving this article.
REFERENCES [1]
Sipkema D, Osinga R, Schatton W, et al. Large-scale production of pharmaceuticals by marine sponges: Sea, cell, or synthesis? Biotechnol Bioeng, 2005, 90: 201222.
[2]
Simpson TL. The cell biology of sponges. New York: Springer-Verlag, 1984.
[3]
Bond C. Continuous cell movements rearrange anatomical structures in intact sponges. J Exp Zool, 1992, 263: 284302.
[4]
Custodio MR, Prokic I, Steffen R, et al. Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death. Mech Ageing Dev, 1998, 105: 4559.
[5]
Zhang XY, Cao XP, Zhang W, et al. Primmorphs from archaeocytes-dominant cell population of the sponge Hymeniacidon perleve: Improved cell proliferation and spiculogenesis. Biotechnol Bioeng, 2003, 84: 583590.
[6]
Cao XP, Fu WT, Yu XJ, et al. Dynamics of spicule production in the marine sponge Hymeniacidon perlevis during in vitro cell culture and seasonal development in the field. Cell Tissue Res, 2007, 329: 595608
[7]
Leys SP, Mackie GO. Electrical recording from a glass sponge. Nature, 1997, 387: 2930.
[8]
Müller WE, Belikov SI, Tremel W, et al. Siliceous spicules in marine demosponges (example Suberites domuncula). Micron, 2005, 37: 107120.
[9]
Hoffmeyer J. The swarming body// Rauch PI, Carr GF (Eds.). The Fifth Congress of the International Association for Semiotic Studies. Berlin/New York: Mouton de Gruyter, 1994, 937940.