Development of reliable cell-based assay is important for high-speed, low cost drug screening. However, the conventional method using cells are still unstable and thus are still under trial to make reliable cell models showing the same extent of reliability as tissue/organ models. As heart is one of the most important organs for toxicology in drug screening, the properties of heart cells are examined and reported strenuously. For example, it has been reported that one beating cell can influence the rate of a neighbor with which it makes contact, and that a group of heart cells in culture, beating synchronously with a rapid rhythm, can act as pacemaker for a contiguous cell sheet from earlier tissue culture studies of cardiac myocyte cells [1]. Although these former results predicted that the importance of a rapidly beating region of tissue acts as pacemaker for a slower one and examined how the synchronization process of two isolated beating cardiac myocytes [2] and that the importance of the communication of each cells in the cell-network, the community size effect could not be measured successfully using the conventional cultivation method on the culture dish plate. As means of attaining the spatial arrangement of cardiac myocytes even during cultivation, we have developed a new single-cell based cultivation method and a system using agarose microstructures, based on 1064-nm photo-thermal etching [3-5]. Using this system, we measured the time course of synchronization process of adjacent two beating cardiac myocyte cells connected by 2-μm-width pathways, and found the synchronization of two cells occurred 90 min after their first physical contact [6,7].

The schematic drawing of the on-chip single-cell-based cultivation assay is illustrated on Figure Figure1.1. Our system consists of three parts: temperature-controlled cell cultivation part, in which single cardiac myocytes are arranged in each microchambers of agarose cell cultivation chip; photo-thermal etching system to fabricate both the microchambers and microchannels by melting of a porting of the 5-μm-thick agarose layer by the spot heating of a 1064-nm infrared focused laser beam; and image acquire/analysis system. Figure Figure22 summarizes the chip design and cell cultivation procedure on the chip. In this example, we arranged nine 30-μm-diameter microchambers on the chip and the adjacent chambers are connected by the microchannels, which are fabricated during cultivation by the photo-thermal etching. As the 1064-nm laser beam is not absorbed by either water or agarose, it melts a portion of the agarose on the chromium thin layer because only the chromium layer absorbs the beam. Using this non-contact etching, we can easily make microstructures such as holes and channels within only a few minutes without using any cast molding process. The melting of agarose by laser occurred as follows (see Figure Figure2).2). We have focused the 1064-nm infrared laser beam on the agarose layer on the glass slide to melt the agarose at the focal point and on the light pathway until the shape of the hole for cells formed (Figure (Figure2a).2a). When the focused beam was moved parallel to the chip surface, a portion of agarose around the focal spot of laser melted and diffused into water (Figure (Figure2b).2b). After the heated spot had been moved, a channel was created at the bottom of the agarose layer connecting the two adjacent holes (Figure (Figure2c).2c). A microscope observation confirmed that the melting had occurred, and then either the heating was continued until the spot size reached the desired one, or the heating position was shifted to achieve the desired shape. Individual cardiac myocytes were cultivated in each hole of the agarose microchambers on the chip as shown in Figure Figure2d.2d. In our method we added micro channels one by one to connect neighbouring cardiac myocytes in adjacent microchambers during their cultivation. To improve the attachment of the cells to the bottom of the microchambers, collagen-type I (Nitta gelatin, Osaka, Japan) was coated on the chromium layer of the chip before coating of agarose layer (Figure (Figure33).

Ventricular myocytes were isolated from 1- to 3-day-old neonatal Wistar rats as described earlier [6,7]. Hearts were excised from rats anaesthetized with ethyl ether and transferred to phosphate buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4) containing 0.9 mM CaCl2 and 0.5 mM MgCl2. after which ventricles were separated and minced into small fragments. Tissue fragments were further dissociated by incubating them twice with PBS containing 0.25% collagenase (Wako, Osaka, Japan) for 30 minutes at 37°C. The cell suspensions were transferred to a cell culture medium (DMEM [Invitrogen Corp., Carlsbad, CA USA] supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml Streptomycin) at 4°C. The cells were filtered through a 40-μm nylon mesh and were centrifuged at 180 g for 5 minutes at room temperature. The cell pellet was re-suspended in a HEPES buffer (20 mM HEPES, 110 mM NaCl, 1 mM NaH₂PO₄, 5 mM glucose, 5 mM KCl, and 1 mM MgSO₄, pH 7.4). The cardiac myocytes present in the suspension were separated from other cells (i.e., fibroblasts and endothelial cells) by the density centrifugation method. The cell suspension was then layered onto 40.5% Percoll (Amersham Biosciences, Uppsala, Sweden) diluted in the HEPES buffer, which had previously been layered on 58.5% Percoll diluted in the buffer. The cell suspension was then centrifuged at 2200 g for 30 minutes at room temperature. Cardiac myocytes were retrieved from the interface of the 40.5% and 58.5% Percoll concentrations. Retrieved cells were then re-suspended in the cell culture medium. The 5-μl of the suspension, which was diluted to achieve a final concentration of 3.0 × 10⁵ cells/ml, was plated into the chip and each cardiac myocyte was picked up by a micropipette and manually introduced into each microchamber in the chip. Then, it was incubated on a cell-cultivation microscope system at 37°C in a humidified atmosphere of 95% air and 5% CO₂. It should be noted that, because the microchamber sidewalls were made of agarose, the cells could not easily pass over the chambers. A phase-contrast microscope was used both to measure the contraction rhythm (i.e. beating frequency) of the cardiac myocytes, and to record the shape of cell network in microchambers.