Living Green Paramecia as Micro-Particle Transporting Automata
Paper:
Platform for Two-Dimensional Cellular Automata Models Implemented by Living Cells of Electrically Controlled Green Paramecia Designed for Transport of Micro-Particles Kohei Otsuka and Tomonori Kawano The University of Kitakyushu 1-1 Hibikino, Wakamatsu-ku, Kitakyushu 808-0135, Japan E-mail:
[email protected] [Received August 31, 2012; accepted September 2, 2013]
Microscopic traffic flow models are a class of scientific models of vehicular traffic dynamics. Here, we attempted to establish an experimental platform for mimicking microscopic traffic flow models at microscopic dimensions. We achieved this, by monitoring the flow of micro-sized particles transported by the motile cells of living microorganisms. Some researchers have described the cells of protozoan species as “swimming neurons” or “swimming sensory cells” applicable to biological micro-electro-mechanical systems or micro-biorobotics. Therefore these cells, in a controlled environment, may form a good model system for bio-implementable cellular automata for traffic simulation. The living cells of the Paramecium species including those of green paramecia (Paramecium bursaria), actively migrate towards a negatively charged electrode when exposed to an electric field. This type of cellular movement is known as galvanotaxis. P. bursaria was chosen as a model organism since the ideal micro-vehicles required for micro-particle transport must have a particular particle packing capacity within the cells. The present study establishes that the movement of cells with or without the loadΦ, 9.75 μ m) can be controlled ing of microspheres (Φ on a two-dimensional plane under strict electrical controls. Lastly, implementation of microchips equipped with optimally sized micro-flow channels that allow the single-cell traffic of swimming P. bursaria was proposed for further studies and mathematical modeling.
computational problem. Nagel and Schreckenberg [2] introduced a cellular automata model for the microscopic modeling of traffic movement, and extensions of this model presume that traffic demands (vehicles) are particles without route. Each vehicle simulated by cellular automata models must decide when to turn left, right, continue straight ahead, stop, or give way [3]. Microscopic traffic flow models are a class of scientific models of vehicular traffic dynamics. In the present study, we attempted to establish an experimental platform for mimicking traffic flow models at microscopic size by monitoring the flow of micro-particles transported by the motile cells of living microorganisms. Generally, the concepts of traffic or transportation often accompany the phenomena of flow stagnation or jamming. Accordingly, we can handle these jamming phenomena as a system of interacting particles, such as vehicles, pedestrians, ants, and Internet packets, driven far from equilibrium [4]. By considering all the above particles in various transportation processes as “self-driven particles (SDPs),” we are able to treat various transportation phenomena, universally governed by the physics of complex systems [4]. These models must have the properties of being solvable. Note that we can approximate the behavior of particles in complex systems by not only numerical simulations but also by experimental simulations. By considering passengers as SDPs freely traveling by boarding on cars, buses and trains, Tomoeda [4] has described a mathematical model for the passenger transport system built on analytical rule-based models, and has introduced the real-time railway network simulation tool.
Keywords: bio-automata, bio-MEMS, biorobotics, paramecium bursaria
1.2. Swimming Cells as Micro-Biorobots In one of our previous articles, some living organisms, cells or bio-signaling molecules are listed and classified as natural sequential machines (namely, the Mealy or Moore machines) or finite state automata [5]. Defining the actions (states and transition functions) of these natural automata clarified the similarity between the computational data processing and cellular decision-making processes. This study reports an attempt to implement an electrically controllable bio-automata model using green paramecia (Paramecium bursaria).
micro-
1. Introduction 1.1. Traffic Flow and Automata The automata theory pertains to the mathematical investigation of abstract machines called automata, commonly studied in theoretical computer science [1]. Automata enable researchers to properly solve a range of Vol.18 No.1, 2014
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To biological researchers, paramecia are very commonly used laboratory tools for cell biological and environmental studies [6]. Paramecium cells are currently considered model systems for studying symbiosis [7] and cellular signal transduction mechanisms [8]. Since signal perception, processing and induced biochemical reactions all occur within these unicellular organisms, some researchers have described Paramecium cells as “swimming sensory cells” or “swimming neurons” [9]. Over a century ago, paramecia were found to exhibit responses to electric stimuli since they align with an electric field or voltage gradient and swim toward the negatively charged electrode if the electric field is sufficiently strong [10]. This type of cellular movement is known as galvanotaxis. The aforementioned studies indicated the technical possibility for finely geared neuronal controls and the engineering of unicellular ciliate micro-machineries under applied electric stimulus. In fact, the galvanotactic responsiveness observed in Paramecium species (particularly P. caudatum) has attracted the attention of researchers in the fields of microrobotics, biorobotics, or Bio-MEMS (biological microelectro-mechanical systems), in order to develop electrically controlled micro-machinery [11–13]. The living cells used as ideal micro-machines for micro-particle transport must be equipped with a certain capacity for particle loading. P. caudatum, one of the most intensively studied galvanotactically controllable species [8–13], lacks such characteristics suggesting that it should be replaced with a related species with greater particle-loading capacity. Our previous study revealed that an electric stimulus applied to P. bursaria, the green species, is converted to a galvanotactic cellular movement with the involvement of the T-type calcium channels on the plasma membrane [14]. Within its cytosol, a single P. bursaria cell naturally harbors several hundred endosymbiotic green algal cells that are morphologically and genetically similar to those of Chlorella species [15]. Recent studies have demonstrated that the majority of symbiotic algae inside the host cells can be forcibly replaced [16], with various artificial particles such as plastic microspheres [17, 18]. Notably, in addition to inert particles, several living organisms such as photosynthetic and non-photosynthetic bacteria [7] or non-symbiotic algae can be introduced. For micro-biorobotic demonstration, it is tempting to exploit the above-mentioned characteristics of P. bursaria, namely, particle internalization and swimming capability. In the micro-biorobotic approach, Aonuma et al. [14] have quantified the galvanotactic migration of P. bursaria in an open top bath. Using P. bursaria, Furukawa et al. [17] demonstrated for the first time that the electrically driven transportation of two distinct types of small particles differed in size, namely nanosized particles and micro-sized particles (microspheres) in the mono-directional galvanotactic system in microcapillaries. The present work demonstrates that the movement of cells (vehicle) with or without microspheres (passengers) 4
Fig. 1. Replacement of symbiotic algae inside the cells of P. bursaria with artificial microspheres. (a) Schematic diagram showing the excretion and uptake of symbiotic algae or microspheres by the cells of P. bursaria. (b) A single cell of wild-type P. bursaria harboring green algae as symbionts. (c) Alga-free apo-symbiotic ciliates prepared after treatment with a herbicide. (d) The conditioned cells after internalization of plastic microsheres diameter, 9.75 μ m.
loaded could be controlled on a two-dimensional (2D) plane under strict electrical controls. This work aims to develop an experimental model for cellular automata employing living cells.
2. Materials and Methods 2.1. Paramecium Cell Line P. bursaria strain INA-1 (Fig. 1(b); syngen 1, mating type I) was originally collected from the known ecological studying point INA on the Ongagawa River (Kama City, Fukuoka Prefecture, Japan) [19]. Since the cell line was established after single cell isolation, all the cells in the culture were clones that share identical genetic background. INA-1 ciliate cells were propagated and maintained in a modified culture medium as previously reported [20]. Briefly, the culture medium was prepared with a yeastextract-based nutrition tablet (single EBIOS tablet/L; Asahi Food and Healthcare, Tokyo, Japan). The culture medium was renewed at 2-week intervals. A single nutrition tablet (250 mg) contained 94.2% (w/w) dried yeast homogenates and 5.5% (w/w) carbohydrates. The bacterized nutrition medium was prepared by inoculating the medium with the food bacterium Klebsiella pneumoniae, 1 day prior to the subculture of the ciliate cells. The ciliate culture was initiated with a cell density of approximately 10-20 cells/mL and propagated to the confluent level (over 1000 cells/mL) under a light cycle of 12 h light and 12 h dark with approximately 3000 lux under fluorescent natural-white light at 23◦ C.
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Fig. 2. Devices for 2D cell migration controls. (a) Top views of the devices. Arrows, distances between electrodes (5 mm). (b) Electronic circuit design employed. Electrodes were prepared with Cu-based conductive tape. Applied voltage ranged between 1.5 and 6 V. Eight capacitors (4.7 nF) were inserted to the circuit when required. Fig. 3. Conventional (1D) migration of galvanotactically stimulated cells of P. bursaria.
2.2. Conditioned Cells and Microspheres Conditioned P. bursaria cells, which are ready for the loading of micro-particles (employed as model passengers) and are the model vehicles to be driven under the microscope, were prepared by the forced removal of algal symbionts from the hosting ciliates as described by Tanaka et al. [16]. Briefly, the green cells were incubated in the presence of 0.1 μ M paraquat for 5 days under light (approximately 3000 lux at minimum). Then, a single ciliate lacking algae was separated under a microscope and the cell line of apo-symbiotic paramecia derived from this single cell was propagated in the culture medium inoculated with food bacteria as described above. The resultant white paramecium cells were ready for particle feeding as illustrated in Fig. 1. Micro-particles (polystyrene microspheres; Φ, 9.75 μ m), which mimicked the passengers, were obtained from Bangs Laboratories, Inc. (Fishers, IN, USA) and used for particle internalization by the conditioned cells. 2.3. Galvanotactic Cell Migration Using a 2DGalvanotactic Device An electrode in an electrochemical cell (battery) is referred to as either an anode or a cathode. The anode is defined as the electrode where electrons leave the battery (cell) and the cathode as the electrode where electrons Vol.18 No.1, 2014
enter the battery (cell). There was confusion in defining the polarity of galvanotactic migration of living cells since each of paired electrodes used for galvanotaxis assays can be considered bipolar electrode that functions as the anode or cathode of one cell (battery) and the cathode or anode, respectively, of another (culturing medium). Therefore, in this article, we avoided using the terms anode and cathode, but instead, labeled the electrodes placed on the galvanotactic apparatus either negatively or positively charged. An apparatus with electric circuits was newly designed for a demonstration with 2D-galvanotaxis (Fig. 2). Paramecium cells with and without internalized microparticles were placed on a depletion glass slide equipped with 4 individual, radially arranged Cu-based electrodes (Cu-conductive tapes). This apparatus enabled us to attract the cells at any randomly and transiently positioned negatively or positively charged area on the slide simply through switching and channeling of the electric circuit. Applied voltage ranged between 1.5 and 6.0 V. The duration of single electric stimulus was 15 s. Switching the electrode polarity was sequentially repeated several times.
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Fig. 4. Repeated galvanotactic migration of particle-loaded P. bursaria cells on the 2D plane, demonstrated on the newly designed device.
3. Results and Discussion 3.1. Preliminary Demonstration Using MonoDirectional (1D) Galvanotactic Device Using a conventional 1D galvanotactic device described by Aonuma et al. [14], both wild-type and conditioned P. bursaria cells were shown to be attracted by the negatively charged electrode (1.5 V, distance 7 cm, 10 min), confirming the galvanotactic property of these ciliate cells (see the data for wild type cells, Fig. 3). 3.2. 2D-Galvanotactic Model Unlike in 1D galvanotaxis, electric control for 2D galvanotaxis required newly designed circuits with complexity as shown in Fig. 2. Similar to the 1D model, forced mobilization of both the wild-type green cells and conditioned white cells was successfully achieved in the newly designed 2D-model system (preliminary data, not shown). Here, the conditioned white P. bursaria cells were used as the vehicle model in the microscopic environment while the polystyrene micro-particles (Φ, 9.75 μ m) were used as the models for passengers. As expected, the particle-loaded cells of pre-conditioned P. bursaria accumulated around the randomly and transiently positioned negatively charged electrode in assessments using four independent polarity-controlled Cu-based electrodes (Fig. 4). Once the accumulation of living cells was observed at one spot, the polarity of the cell-attracting electrode was reversed from negative to positive and the newly created negatively charged electrode was used for further attrac6
Fig. 5. Statistic analysis of galvanotactic migration of particle-loaded P. bursaria cells. (a) Diagram showing the experimental design. (b) Changes in population of P. bursaria cells around the 4 electrodes observed after electrical stimulation.
tion of the cells in a different area. By repeating this process at 15 s intervals, step-wise migration of the mass of cells on the 2D plane was facilitated (Fig. 4). Figure 5 summarizes the quantitation of the cell migration performed on the 2D galvanotactic cell mobilization device. Unfortunately, cell migration hardly continued following repeated polarity changes largely due to the loss of intact cells sensitive to successive electric stimuli (Fig. 5(b)). The loss of cells could be attributed to rapid and drastic changes in voltage during polarity switching events, since additional insertion of capacitors in the circuits significantly lowered both the level of transient increases in voltage and the extent of induced cell death (Fig. 6). Milder electric polarity shock at the cellattracting electrodes is favorable for maintaining the repeatedly migrating cellular population under the continuous galvanotactic controls (Fig. 6).
3.3. Conclusion and Perspectives In this work, we attempted to establish an experimental platform for simulating microscopic traffic flows at microscopic size by employing micro-particles as the model
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[6]
[7]
[8] [9]
[10] [11] [12]
[13]
[14]
Fig. 6. Acute changes in voltage due to switching procedure.
[15] [16]
for passengers and apo-symbiotically conditioned P. bursaria cells as electrically driven model vehicles. We illustrated that the movement of the conditioned P. bursaria cells with and/or without loading of plastic microspheres (9.75 μ m in diameter) can be reproducibly controlled on the 2D plane under strict electrical controls. For future research and mathematical modeling, we recommend implementation of microchips equipped with optimally-sized micro-flow channels (traffic routes) to allow the single-cell traffic of swimming P. bursaria.
[17]
[18] [19]
[20]
Acknowledgements This work was supported by a grant from the Regional Innovation Strategy Support Program implemented by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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References: [1] Y. Tomita and T. Yokomori, “Automata and formal language,” Morikita Publ. Tokyo, 1992. [2] K. Nagel and M. Schreckenberg, “A cellular automaton model for freeway traffic,” J. Phys. I France, Vol.2, No.12, pp. 2221-2229, 1992. [3] J. Sanchez-Medina, E. Medina-Machin, M. Diaz-Cabrera, M. J. Galan-Moreno, and E. Rubio-Royo, “Overtaking and giving way: Design and validation of a lightweight extended cellular automata urban traffic simulator,” Proc. Intell. Transportation Systems (ITSC), 2012 15th Int. IEEE Conf., pp. 746751 (10.1109/ITSC.2012.6338736). [4] A. Tomoeda, “Cellular automaton modeling of passenger transport systems,” In: Infrastructure Design, Signalling and Security in Railway, P. Xavier (Ed.), pp. 255-274, 2012. [5] T. Kawano, F. Bouteau, and S. Mancuso, “Finding and defining the natural automata acting in living plants: Towards the synthetic bi-
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Otsuka, K. and Kawano, T.
Name: Kohei Otsuka
Affiliation: Techno Ryowa Ltd.
Address: 2-26-20, Minami-Otsuka, Toshima-ku, 170-0005 Tokyo, Japan
Brief Biographical History: 2011- Graduate School of Environmental Engineering, The University of Kitakyushu 2013- Techno Ryowa Ltd.
Main Works:
• “On-chip platform for biological cellular automata models using swimming paramecium cells,” ICIC Express Letters, Part B: Applications (in press), 2014.
Membership in Academic Societies:
• Society of Chemical Engineering of Japan
Name: Tomonori Kawano
Affiliation: Associate Professor, Faculty of Environmental Engineering, The University of Kitakyushu
Address: 1-1 Hibikino, Wakamatsu-ku, Kitakyushu 808-0135, Japan
Brief Biographical History: 2000- Post-Doctoral Scientist, Institut National de la Recherche Agronomique (INRA) 2001- Research Associate, Graduate School of Science, Hiroshima University 2003- Associate Professor, Graduate School of Environmental Engineering, The University of Kitakyushu
Main Works:
• “Run-length encoding graphic rules, molecular biologically editable designs, and steganographical on-image numeric data embedment for DNA-based cryptographical coding system,” Communicative and Integrative Biology, Vol.6, e23478.
Membership in Academic Societies:
• Information Processing Society of Japan (IPSJ) • Society of Plant Signaling and Behavior
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