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An agar-based on-chip neural-cell-cultivation system for stepwise control of network pattern generation during cultivation
Sensors and Actuators B 99 (2004) 156–162
An agar-based on-chip neural-cell-cultivation system for stepwise control of network pattern generation during cultivation Yoshihiro Sugio, Kensuke Kojima, Hiroyuki Moriguchi, Kazunori Takahashi, Tomoyuki Kaneko, Kenji Yasuda∗ Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 16-723A, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan Received 24 June 2003; received in revised form 24 June 2003; accepted 25 June 2003
Abstract We have developed a new type of single-cell based on-chip cell-cultivation system with an agarose microchamber (AMC) array and a photo-thermal etching module for step-by-step topographical control of the network patterns of living neural cells during long-term cultivation. The advantages of this system are that (1) it can control positions and numbers of cells for cultivation by using agar-based microchambers, and (2) it can change the neural network complexity during cultivation by photo-thermal melting a portion of agar at the focal point of a 1064 nm infrared laser beam. This laser wavelength is permeable with respect to water and agarose, and it is only absorbed at the thin chromium layer on the chromium-coated glass slide surface at the bottom of the agarose layer. With adequate laser power, we can easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar layer without the complicated steps conventional microfabrication processes entail even during cultivation; we demonstrated that rat hippocampal cells in two adjacent chambers formed fiber connections through new connections between chambers after these had been photo-thermally fabricated. We also verified the fiber connection between those cells by using calcium-based fluorescent microscopy. These results indicate that this system can potentially be used for studying the complexity of neural network patterns for epigenetic memorization. © 2003 Elsevier B.V. All rights reserved. Keywords: On-chip culture system; Agar microchamber; Photo-thermal etching; Change of structure; Neural network pattern
1. Introduction Acquiring epigenetic information of life systems is important for understanding living systems. Especially in the field of neuroscience, one main focus of epigenetic studies is how information is controlled and recorded. Network connections and epigenetic information are assumed to be strongly linked. Numerous approaches and fabrications have been tested and applied to clarify the significance of network patterns [1–19]. Although these conventional microfabrication techniques provide structures with a fine spatial resolution, effective approaches to studying epigenetic information are still being sought; conventional techniques cannot change cell shapes easily during cell cultivation, which usually rules out fabricating them during cell cultivation. We therefore need to fabricate all the structures on the chip before we use it. Thus, we cannot measure the effect of the growth of network patterns that use the same neural cells.
We have developed a new single-cell-cultivation method and a system using agar microstructures based on photo-thermal etching [20,21]. With this method, we can change the structure of the microchambers even during cultivation. For example, this method can be used to control interactions among nerve cells during cultivation by adding channels between two adjacent microchambers one at a time. Comparing the changes in signals before and after the network shapes change helps understanding the significance of the spatial pattern of the neural network. In this paper, we report on the properties of our agar-microchamber system for creating hippocampal neural network patterns and on the results of network pattern growth after adding new tunnels during cultivation.
2. Materials and methods 2.1. Agar-microchamber cell-cultivation system
Abbreviations: EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride ∗ Corresponding author. Tel.: +81-3-5454-6749; fax: +81-3-5454-6749. E-mail address: [email protected] (K. Yasuda). 0925-4005/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-4005(03)00550-1
The system, as shown in Fig. 1, consists of the following four parts: an agar-microchamber (AMC) array chip, a cultivation dish with a nutrient-buffer-changing
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Fig. 1. Schematic drawing of the agar-microchamber cell-cultivation system. Cells are cultivated in individual microchambers molded on an agar plate on a glass slide. For phase-contrast/fluorescent microscopy, two different wavelengths (visible light for observation and a 1064 nm infrared laser for spot heating) were used simultaneously to observe single cells in the microchambers and to melt a portion of the agar in the heated area. Fresh medium was supplied into the chamber covering the microchamber array to provide nutrients for the cells in the microchambers. The system consists of the following four parts: (1) an agar-microchamber (AMC) array chip, (2) a cultivation dish with a nutrient-buffer-changing apparatus, (3) a visible/laser-light permeable cultivation container, and (4) a phase-contrast/fluorescent optical microscope with a 1064 nm Nd:YAG focused laser irradiation system.
apparatus, a permeable cultivation container, and a phasecontrast/fluorescent optical microscope with a 1064 nm Nd:YAG focused laser irradiation module, which is basically the same as the one we used in [21]. The temperature and humidity of the cultivation dish were kept constant (37 ◦ C, 100%) by circulating warm saturated vapor air containing 5% CO2 in the permeable cultivation container. The phase-contrast/fluorescent microscope (IX-70; with a phase-contrast objective lens, 40×, Olympus, Tokyo, Japan) was used to study the growth and division of the cells. Phase-contrast and fluorescent images were obtained simultaneously by using a charge-coupled device (CCD) camera (CS230, Olympus) or a cooled CCD camera (AQUA/ORCA C4742-98-24ER, Hamamatsu Photonics, K.K., Hamamatsu, Japan). The images of the cells were recorded with a VCR and analyzed using a video capture system on a personal computer. For photo-thermal etching, which is an area-specific melting of agar microchambers by heating a spot on the chromium layer, the 1064 nm Nd:YAG laser (T20-8S, Spectra Physics) was attached to the microscope. The maximum laser power at the focal point after passing through the 40× objective lens was 100 mW. 2.2. Grafting collagen molecules onto the surface of chromium-coated glass slides The agarose chip we used consisted of three layers on a 0.2 mm thick glass slide: agar, collagen, and chromium.
First, to fix the collagen on the surface of the 5 nm thick chromium-coated glass slide, we decorated an amino group on the surface of the glass slide and linked the amino group and the agarose by using EDC (PIERCE, IL, USA) as follows: The 5 nm thick chromium-coated glass slide was washed with detergent (Contaminon, WAKO Pure Chemical Co., Tokyo, Japan) for 15 min during ultrasound vibrations to remove the oil mist and dust. The slide was rinsed three times with water, washed with acetone (Wako), and dried in a 140 ◦ C oven for 30 min. Next, it was soaked in 1 M NaOHaq for 1 h (with ultrasound), rinsed three times with water, washed with acetone, and dried in a 140 ◦ C oven for 30 min. To create amino groups on the surface of the chromium layer, a drop of 30 l of 1.5% 3-(2-aminoethylaminopropyl) trimethoxysilane (LS-2480, Shinetsu Silicone, Nagano, Japan) solution was put onto the central area of the surface (a drop diameter is about 5 mm). The slide was then incubated overnight at room temperature (RT), after which it was soaked once in water and dried in a 140 ◦ C oven for 1 h. To link the collagen molecules and the amino groups on the glass slide, 30 l of EDC solution (10 mg/1 ml in 1.0 mM HClaq ) was put on the silane-coupled area, and the slide was incubated overnight at RT. In the morning, it was rinsed three times with water and dried at RT. Finally, 30 l of 150 g/ml collagen solution (Collagen type I-C (from pig skin), Nitta gelatin, Tokyo, Japan) was deposited onto the EDC-treated area, and the slide was kept at RT for 1 h, then rinsed with water, and dried at RT.
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Fig. 2. Schematic drawings of cultivation procedure. To control the number of cells and cell types in each chamber, cells are introduced into each chamber by using a micropipette and cultivated. Before/during cultivation, the AMCs are photo-thermally connected by microtunnels by using a focused infrared beam. For photo-thermal etching, a 1064 nm infrared laser beam was focused on the chromium layer on the glass slide. The focused beam was moved to the agar wall and a portion of agar at the spot-heated point melted and diffused into water. This eventually produced a tunnel connecting the microchambers.
2.3. Agar microchambers The agar-microchamber array chip is made up of 50 m square microchambers, called agarose microchambers (AMCs), that are molded by using a 50 m thick cube array cast of a thick photo resist, SU-8, microstructure as shown in Figs. 1 and 2. The fabrication procedure is basically the same as in our previous report [21]. The following process was used for the molding. First, a microchamber mold of the desired shape was created on the glass slide by using SU-8, and sol state 2% (w/v) agar (ISC BioExpress, GenePure LowMelt, melting temperature: 65 ◦ C) was spread on the 5 nm thick chromium-coated glass slide, the surface of which was decorated with collagen (Nitta gelatin, pH 9.0) as described above. The mold was then pressed onto the agar, and the agar was placed in a refrigerator at 4 ◦ C until it was set and hardened into gel. Finally, the mold was removed at room temperature, leaving 50 m high agar microchambers on the glass slide. The size of the agar microchambers precisely reflects the mold’s shape; possible sizes range from 2 to 100 m high and the resolution in the x–y plane is 1 m. For this experiment, we chose low-melting-point agar that melts at around 65 ◦ C. Using agar gel with such a low melting point is important for non-destructive photo-thermal etching, so the neighboring cells are not damaged by the heat from the microchambers. We also took care not to contaminate the agar, because agar itself does not absorb light with a 1064 nm wavelength, as long as there is no contamination such as dust. If we had not used contamination-free agar, melting might have occurred in an unexpected area. Next, the chip was placed in the cultivation dish filled with a cultivation medium (Fig. 1), and the individual cells were each put into a microchamber by using a micropipette (Fig. 2). The inner diameter of the tip of the micropipette is about 50 m, which is large enough to pass cells like
hippocampal cells (average size: 10 m in diameter). After the cells were set in the AMC, they were cultivated in the container. 2.4. Cell cultivation Rat hippocampi were obtained from 18 days old fetuses (E18) following a dissection protocol as previously described [22]. The isolated tissue was incubated in 0.25% trypsin (Sigma Chemical Co., St. Louis, MO) in Ca2+ - and Mg2+ -free Hank’s balanced salt solution (HBSS, Gibco) for 8 min at 37 ◦ C. After trypsination, the tissue was rinsed
Fig. 3. Photo-thermal etching during hippocampal-cell cultivation: (a) just after photo-thermal etching and after cells were introduced; (b) 5 days after starting cultivation; (c) photo-thermal etching to create a new tunnel between AMCs during cultivation (white arrow); (d) after another 5 days of cultivation in the AMC with microtunnels (10 DIV), the neurites connected cells through the newly added tunnel.
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in a 2 ml plating medium (Neurobasal medium with B27 supplement, Gibco) five times for 5 min each and mechanically dissociated with a fire-polished pipette into single cells. The cells were plated one by one into each agar microchamber with a pipette and incubated at 37 ◦ C with 5% CO2 at saturated humidity. 2.5. Fluorescence imaging of neural-cell excitation To measure the network linkage of cells, we used Ca2+ -sensitive fluorescence dye for monitoring as described in [23]. The cultured hippocampal neuron was loaded with 0.02 mM fluo-3/AM (Molecular Probes, Eugene, Oregon USA) dissolved into the cultivation buffer for 30 min at 37 ◦ C. The fluorescence image was recorded and analyzed with a cooled CCD camera system.
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3. Results and discussion 3.1. Rearranging the hippocampal neural network by photo-thermal etching during cell cultivation A 1064 nm photo-thermal etching is the area-specific melting of agar microchambers by spot heating using a focused laser beam and a thin layer made of a light-absorbing material such as chromium. Normally, agar and water do not absorb light with a 1064 nm wavelength. Thus, only the portion of agar touching the thin chromium layer melts at the beam spot of laser. The photo-thermal etching process is as follows (see [21] for details). First, we focused the 1064 nm infrared laser beam on the chromium layer while checking the position of the agar microchambers. Next, the focused beam was moved in the desired direction of the
Fig. 4. Three different types of AMC arrays for hippocampal-cells cultivation: the lengths of tunnels connecting the microchambers are 240 m (A), 80 m (B), and 30 m (C), respectively.
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agar microchambers, and the agar at the heated point melted and diffused into water. Eventually, a tunnel connecting the adjacent microchambers formed. To verify our photo-thermal etching method, we fabricated additional microchannels by using photo-thermal etching during hippocampal-cell cultivation 4 days after beginning the process. As shown in Fig. 3a, tunnels formed to connect the AMCs (see white arrows in Fig. 3a). After the tunnels formed, the neural-cell cultivation started. After cultivation for 5 days, the neurite connections were formed completely (white arrows in Fig. 3b). Then, a new tunnel was created to make new connections between the neighboring chambers by using the infrared laser (white arrow in Fig. 3c). Here, no cells and no neurites appeared to have been directly damaged by the irradiation. After 5 days of cultivation, a new fiber connection was observed in the new microchannel, as shown in the Fig. 3d. This suggests that we can photo-thermally etch microchannels without damaging the cells in the AMCs. 3.2. Observing hippocampal neurite outgrowth in the AMC network Fig. 4 shows the three AMC-array types. The lengths of their tunnels connecting the microchambers are 240, 80 and 30 m, respectively. The AMC in Fig. 4C has a round shape to strengthen the agar-walls, which is different from the other AMCs as shown in Fig. 4A and B. By using these AMC chambers, we checked the tunnel-length dependence of neural network connections generation. After 11 days of cultivation, the cell connection stabilized as shown in Fig. 5. In this condition, we measured the number of tunnels connecting chambers from whole tunnels on the chips and summarized them in Table 1. The results show that the difference in connection efficiency was only reduced by 15% even when the tunnel length increased eight times. It indicates that the tunnel length was not the determinant factor for the network formation, which is
Fig. 5. Micrographs of cells after 11 days of cultivation.
Table 1 Tunnel-length dependence of neural network connection generation
Tunnel width (m) Tunnel length (m) Average neurite thickness (m) S.D. Success rate of connection (%)
Chamber A
Chamber B
Chamber C
15 240 1.94
15 80 5.33
15 30 3.82
3.56 57.1 (16/24)
2.08 59.6 (34/57)
0.59 45.8 (11/24)
Fig. 6. Ca2+ oscillation of hippocampal cells in four AMCs.
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Fig. 7. Time–course change of Ca fluorescent images of neural cells. Lines A, B and C indicate the relative change of fluorescent image in cells A, B and C in Fig. 6.
important for the practical application of topological control of pathways. 3.3. Confirming neurite connections in the microtunnels connecting AMCs As shown in phase-contrast micrograph in Fig. 6, hippocampal cells were cultivated in four AMCs, which were connected by microtunnels. After flou-3 staining, we observed a synchronization of Ca2+ oscillation. The fluorescent micrographs (Fig. 6a–i) show the time course of Ca2+ oscillation. The graph shown in Fig. 7 indicates the change in time-course intensities of the cells in chambers A–C. These results indicate that the cells in chamber A and B were synchronized. Thus, this result may reflect the ability of the neurites to rearrange themselves to promote a network pattern formation and information connection.
4. Conclusion We have developed an agar-microchamber neural-cellcultivation system that can be used in combination with photo-thermal etching to change the shape of agarmicrochamber connections even during cell cultivation. The results of the hippocampal cell culture experiment demonstrated that the photo-thermal etching method can be used during cultivation. We believe this system can potentially be used for the next stage of single-cell-based neural-cell network cultivation and measurements including real-time control of network patterns during cell cultivation.
Acknowledgements This study was financially supported by the Japan Science and Technology Corporation and by Grants-in-Aids for Science Research from the Ministry of Education, Science and Culture of Japan.
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Biographies Y. Sugio is a graduate student at Yasuda Lab. He studies single-cell-based neural network analysis using a new on-chip analysis system. K. Kojima is a graduate student at Yasuda Lab. He studies single-cell-based neural network analysis and microfabrication techniques for neural cells using a new on-chip analysis system. H. Moriguchi is a graduate student at Yasuda Lab. He studies single-cell-based post-genome analysis using a new on-chip analysis system. K. Takahashi is a graduate student at Yasuda Lab. His subject is making nano/microfabrications for single-cell based cellomics studies. T. Kaneko is a Research Associate at Yasuda Lab. His subject is single-cell-based cultivation and analysis of ‘community effect’ using a new single-cell based on-chip analysis system. K. Yasuda is Associate Professor in The University of Tokyo. He studies Biophysics specializing in muscle contractile mechanism in the skeletal muscle using micromanipulation techniques and cellomics using microfabrications. He also studies the basics and applications of acoustic radiation force for biological analyses in microchambers.
An agar-based on-chip neural-cell-cultivation system for stepwise control of network pattern generation during cultivation Yoshihiro Sugio, Kensuke Kojima, Hiroyuki Moriguchi, Kazunori Takahashi, Tomoyuki Kaneko, Kenji Yasuda∗ Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 16-723A, 3-8-1 Komaba, Meguro, Tokyo 153-8902, Japan Received 24 June 2003; received in revised form 24 June 2003; accepted 25 June 2003
Abstract We have developed a new type of single-cell based on-chip cell-cultivation system with an agarose microchamber (AMC) array and a photo-thermal etching module for step-by-step topographical control of the network patterns of living neural cells during long-term cultivation. The advantages of this system are that (1) it can control positions and numbers of cells for cultivation by using agar-based microchambers, and (2) it can change the neural network complexity during cultivation by photo-thermal melting a portion of agar at the focal point of a 1064 nm infrared laser beam. This laser wavelength is permeable with respect to water and agarose, and it is only absorbed at the thin chromium layer on the chromium-coated glass slide surface at the bottom of the agarose layer. With adequate laser power, we can easily fabricate narrow tunnel-shaped channels between the microchambers at the bottom of the agar layer without the complicated steps conventional microfabrication processes entail even during cultivation; we demonstrated that rat hippocampal cells in two adjacent chambers formed fiber connections through new connections between chambers after these had been photo-thermally fabricated. We also verified the fiber connection between those cells by using calcium-based fluorescent microscopy. These results indicate that this system can potentially be used for studying the complexity of neural network patterns for epigenetic memorization. © 2003 Elsevier B.V. All rights reserved. Keywords: On-chip culture system; Agar microchamber; Photo-thermal etching; Change of structure; Neural network pattern
1. Introduction Acquiring epigenetic information of life systems is important for understanding living systems. Especially in the field of neuroscience, one main focus of epigenetic studies is how information is controlled and recorded. Network connections and epigenetic information are assumed to be strongly linked. Numerous approaches and fabrications have been tested and applied to clarify the significance of network patterns [1–19]. Although these conventional microfabrication techniques provide structures with a fine spatial resolution, effective approaches to studying epigenetic information are still being sought; conventional techniques cannot change cell shapes easily during cell cultivation, which usually rules out fabricating them during cell cultivation. We therefore need to fabricate all the structures on the chip before we use it. Thus, we cannot measure the effect of the growth of network patterns that use the same neural cells.
We have developed a new single-cell-cultivation method and a system using agar microstructures based on photo-thermal etching [20,21]. With this method, we can change the structure of the microchambers even during cultivation. For example, this method can be used to control interactions among nerve cells during cultivation by adding channels between two adjacent microchambers one at a time. Comparing the changes in signals before and after the network shapes change helps understanding the significance of the spatial pattern of the neural network. In this paper, we report on the properties of our agar-microchamber system for creating hippocampal neural network patterns and on the results of network pattern growth after adding new tunnels during cultivation.
2. Materials and methods 2.1. Agar-microchamber cell-cultivation system
Abbreviations: EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride ∗ Corresponding author. Tel.: +81-3-5454-6749; fax: +81-3-5454-6749. E-mail address: [email protected] (K. Yasuda). 0925-4005/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0925-4005(03)00550-1
The system, as shown in Fig. 1, consists of the following four parts: an agar-microchamber (AMC) array chip, a cultivation dish with a nutrient-buffer-changing
Y. Sugio et al. / Sensors and Actuators B 99 (2004) 156–162
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Fig. 1. Schematic drawing of the agar-microchamber cell-cultivation system. Cells are cultivated in individual microchambers molded on an agar plate on a glass slide. For phase-contrast/fluorescent microscopy, two different wavelengths (visible light for observation and a 1064 nm infrared laser for spot heating) were used simultaneously to observe single cells in the microchambers and to melt a portion of the agar in the heated area. Fresh medium was supplied into the chamber covering the microchamber array to provide nutrients for the cells in the microchambers. The system consists of the following four parts: (1) an agar-microchamber (AMC) array chip, (2) a cultivation dish with a nutrient-buffer-changing apparatus, (3) a visible/laser-light permeable cultivation container, and (4) a phase-contrast/fluorescent optical microscope with a 1064 nm Nd:YAG focused laser irradiation system.
apparatus, a permeable cultivation container, and a phasecontrast/fluorescent optical microscope with a 1064 nm Nd:YAG focused laser irradiation module, which is basically the same as the one we used in [21]. The temperature and humidity of the cultivation dish were kept constant (37 ◦ C, 100%) by circulating warm saturated vapor air containing 5% CO2 in the permeable cultivation container. The phase-contrast/fluorescent microscope (IX-70; with a phase-contrast objective lens, 40×, Olympus, Tokyo, Japan) was used to study the growth and division of the cells. Phase-contrast and fluorescent images were obtained simultaneously by using a charge-coupled device (CCD) camera (CS230, Olympus) or a cooled CCD camera (AQUA/ORCA C4742-98-24ER, Hamamatsu Photonics, K.K., Hamamatsu, Japan). The images of the cells were recorded with a VCR and analyzed using a video capture system on a personal computer. For photo-thermal etching, which is an area-specific melting of agar microchambers by heating a spot on the chromium layer, the 1064 nm Nd:YAG laser (T20-8S, Spectra Physics) was attached to the microscope. The maximum laser power at the focal point after passing through the 40× objective lens was 100 mW. 2.2. Grafting collagen molecules onto the surface of chromium-coated glass slides The agarose chip we used consisted of three layers on a 0.2 mm thick glass slide: agar, collagen, and chromium.
First, to fix the collagen on the surface of the 5 nm thick chromium-coated glass slide, we decorated an amino group on the surface of the glass slide and linked the amino group and the agarose by using EDC (PIERCE, IL, USA) as follows: The 5 nm thick chromium-coated glass slide was washed with detergent (Contaminon, WAKO Pure Chemical Co., Tokyo, Japan) for 15 min during ultrasound vibrations to remove the oil mist and dust. The slide was rinsed three times with water, washed with acetone (Wako), and dried in a 140 ◦ C oven for 30 min. Next, it was soaked in 1 M NaOHaq for 1 h (with ultrasound), rinsed three times with water, washed with acetone, and dried in a 140 ◦ C oven for 30 min. To create amino groups on the surface of the chromium layer, a drop of 30 l of 1.5% 3-(2-aminoethylaminopropyl) trimethoxysilane (LS-2480, Shinetsu Silicone, Nagano, Japan) solution was put onto the central area of the surface (a drop diameter is about 5 mm). The slide was then incubated overnight at room temperature (RT), after which it was soaked once in water and dried in a 140 ◦ C oven for 1 h. To link the collagen molecules and the amino groups on the glass slide, 30 l of EDC solution (10 mg/1 ml in 1.0 mM HClaq ) was put on the silane-coupled area, and the slide was incubated overnight at RT. In the morning, it was rinsed three times with water and dried at RT. Finally, 30 l of 150 g/ml collagen solution (Collagen type I-C (from pig skin), Nitta gelatin, Tokyo, Japan) was deposited onto the EDC-treated area, and the slide was kept at RT for 1 h, then rinsed with water, and dried at RT.
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Fig. 2. Schematic drawings of cultivation procedure. To control the number of cells and cell types in each chamber, cells are introduced into each chamber by using a micropipette and cultivated. Before/during cultivation, the AMCs are photo-thermally connected by microtunnels by using a focused infrared beam. For photo-thermal etching, a 1064 nm infrared laser beam was focused on the chromium layer on the glass slide. The focused beam was moved to the agar wall and a portion of agar at the spot-heated point melted and diffused into water. This eventually produced a tunnel connecting the microchambers.
2.3. Agar microchambers The agar-microchamber array chip is made up of 50 m square microchambers, called agarose microchambers (AMCs), that are molded by using a 50 m thick cube array cast of a thick photo resist, SU-8, microstructure as shown in Figs. 1 and 2. The fabrication procedure is basically the same as in our previous report [21]. The following process was used for the molding. First, a microchamber mold of the desired shape was created on the glass slide by using SU-8, and sol state 2% (w/v) agar (ISC BioExpress, GenePure LowMelt, melting temperature: 65 ◦ C) was spread on the 5 nm thick chromium-coated glass slide, the surface of which was decorated with collagen (Nitta gelatin, pH 9.0) as described above. The mold was then pressed onto the agar, and the agar was placed in a refrigerator at 4 ◦ C until it was set and hardened into gel. Finally, the mold was removed at room temperature, leaving 50 m high agar microchambers on the glass slide. The size of the agar microchambers precisely reflects the mold’s shape; possible sizes range from 2 to 100 m high and the resolution in the x–y plane is 1 m. For this experiment, we chose low-melting-point agar that melts at around 65 ◦ C. Using agar gel with such a low melting point is important for non-destructive photo-thermal etching, so the neighboring cells are not damaged by the heat from the microchambers. We also took care not to contaminate the agar, because agar itself does not absorb light with a 1064 nm wavelength, as long as there is no contamination such as dust. If we had not used contamination-free agar, melting might have occurred in an unexpected area. Next, the chip was placed in the cultivation dish filled with a cultivation medium (Fig. 1), and the individual cells were each put into a microchamber by using a micropipette (Fig. 2). The inner diameter of the tip of the micropipette is about 50 m, which is large enough to pass cells like
hippocampal cells (average size: 10 m in diameter). After the cells were set in the AMC, they were cultivated in the container. 2.4. Cell cultivation Rat hippocampi were obtained from 18 days old fetuses (E18) following a dissection protocol as previously described [22]. The isolated tissue was incubated in 0.25% trypsin (Sigma Chemical Co., St. Louis, MO) in Ca2+ - and Mg2+ -free Hank’s balanced salt solution (HBSS, Gibco) for 8 min at 37 ◦ C. After trypsination, the tissue was rinsed
Fig. 3. Photo-thermal etching during hippocampal-cell cultivation: (a) just after photo-thermal etching and after cells were introduced; (b) 5 days after starting cultivation; (c) photo-thermal etching to create a new tunnel between AMCs during cultivation (white arrow); (d) after another 5 days of cultivation in the AMC with microtunnels (10 DIV), the neurites connected cells through the newly added tunnel.
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in a 2 ml plating medium (Neurobasal medium with B27 supplement, Gibco) five times for 5 min each and mechanically dissociated with a fire-polished pipette into single cells. The cells were plated one by one into each agar microchamber with a pipette and incubated at 37 ◦ C with 5% CO2 at saturated humidity. 2.5. Fluorescence imaging of neural-cell excitation To measure the network linkage of cells, we used Ca2+ -sensitive fluorescence dye for monitoring as described in [23]. The cultured hippocampal neuron was loaded with 0.02 mM fluo-3/AM (Molecular Probes, Eugene, Oregon USA) dissolved into the cultivation buffer for 30 min at 37 ◦ C. The fluorescence image was recorded and analyzed with a cooled CCD camera system.
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3. Results and discussion 3.1. Rearranging the hippocampal neural network by photo-thermal etching during cell cultivation A 1064 nm photo-thermal etching is the area-specific melting of agar microchambers by spot heating using a focused laser beam and a thin layer made of a light-absorbing material such as chromium. Normally, agar and water do not absorb light with a 1064 nm wavelength. Thus, only the portion of agar touching the thin chromium layer melts at the beam spot of laser. The photo-thermal etching process is as follows (see [21] for details). First, we focused the 1064 nm infrared laser beam on the chromium layer while checking the position of the agar microchambers. Next, the focused beam was moved in the desired direction of the
Fig. 4. Three different types of AMC arrays for hippocampal-cells cultivation: the lengths of tunnels connecting the microchambers are 240 m (A), 80 m (B), and 30 m (C), respectively.
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agar microchambers, and the agar at the heated point melted and diffused into water. Eventually, a tunnel connecting the adjacent microchambers formed. To verify our photo-thermal etching method, we fabricated additional microchannels by using photo-thermal etching during hippocampal-cell cultivation 4 days after beginning the process. As shown in Fig. 3a, tunnels formed to connect the AMCs (see white arrows in Fig. 3a). After the tunnels formed, the neural-cell cultivation started. After cultivation for 5 days, the neurite connections were formed completely (white arrows in Fig. 3b). Then, a new tunnel was created to make new connections between the neighboring chambers by using the infrared laser (white arrow in Fig. 3c). Here, no cells and no neurites appeared to have been directly damaged by the irradiation. After 5 days of cultivation, a new fiber connection was observed in the new microchannel, as shown in the Fig. 3d. This suggests that we can photo-thermally etch microchannels without damaging the cells in the AMCs. 3.2. Observing hippocampal neurite outgrowth in the AMC network Fig. 4 shows the three AMC-array types. The lengths of their tunnels connecting the microchambers are 240, 80 and 30 m, respectively. The AMC in Fig. 4C has a round shape to strengthen the agar-walls, which is different from the other AMCs as shown in Fig. 4A and B. By using these AMC chambers, we checked the tunnel-length dependence of neural network connections generation. After 11 days of cultivation, the cell connection stabilized as shown in Fig. 5. In this condition, we measured the number of tunnels connecting chambers from whole tunnels on the chips and summarized them in Table 1. The results show that the difference in connection efficiency was only reduced by 15% even when the tunnel length increased eight times. It indicates that the tunnel length was not the determinant factor for the network formation, which is
Fig. 5. Micrographs of cells after 11 days of cultivation.
Table 1 Tunnel-length dependence of neural network connection generation
Tunnel width (m) Tunnel length (m) Average neurite thickness (m) S.D. Success rate of connection (%)
Chamber A
Chamber B
Chamber C
15 240 1.94
15 80 5.33
15 30 3.82
3.56 57.1 (16/24)
2.08 59.6 (34/57)
0.59 45.8 (11/24)
Fig. 6. Ca2+ oscillation of hippocampal cells in four AMCs.
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Fig. 7. Time–course change of Ca fluorescent images of neural cells. Lines A, B and C indicate the relative change of fluorescent image in cells A, B and C in Fig. 6.
important for the practical application of topological control of pathways. 3.3. Confirming neurite connections in the microtunnels connecting AMCs As shown in phase-contrast micrograph in Fig. 6, hippocampal cells were cultivated in four AMCs, which were connected by microtunnels. After flou-3 staining, we observed a synchronization of Ca2+ oscillation. The fluorescent micrographs (Fig. 6a–i) show the time course of Ca2+ oscillation. The graph shown in Fig. 7 indicates the change in time-course intensities of the cells in chambers A–C. These results indicate that the cells in chamber A and B were synchronized. Thus, this result may reflect the ability of the neurites to rearrange themselves to promote a network pattern formation and information connection.
4. Conclusion We have developed an agar-microchamber neural-cellcultivation system that can be used in combination with photo-thermal etching to change the shape of agarmicrochamber connections even during cell cultivation. The results of the hippocampal cell culture experiment demonstrated that the photo-thermal etching method can be used during cultivation. We believe this system can potentially be used for the next stage of single-cell-based neural-cell network cultivation and measurements including real-time control of network patterns during cell cultivation.
Acknowledgements This study was financially supported by the Japan Science and Technology Corporation and by Grants-in-Aids for Science Research from the Ministry of Education, Science and Culture of Japan.
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Biographies Y. Sugio is a graduate student at Yasuda Lab. He studies single-cell-based neural network analysis using a new on-chip analysis system. K. Kojima is a graduate student at Yasuda Lab. He studies single-cell-based neural network analysis and microfabrication techniques for neural cells using a new on-chip analysis system. H. Moriguchi is a graduate student at Yasuda Lab. He studies single-cell-based post-genome analysis using a new on-chip analysis system. K. Takahashi is a graduate student at Yasuda Lab. His subject is making nano/microfabrications for single-cell based cellomics studies. T. Kaneko is a Research Associate at Yasuda Lab. His subject is single-cell-based cultivation and analysis of ‘community effect’ using a new single-cell based on-chip analysis system. K. Yasuda is Associate Professor in The University of Tokyo. He studies Biophysics specializing in muscle contractile mechanism in the skeletal muscle using micromanipulation techniques and cellomics using microfabrications. He also studies the basics and applications of acoustic radiation force for biological analyses in microchambers.