Stability of beating frequency in cardiac myocytes by their community effect measured by agarose microchamber chip
© Kojima et al; licensee BioMed Central Ltd. 2005
Received: 19 November 2004
Accepted: 31 May 2005
Published: 31 May 2005
To understand the contribution of community effect on the stability of beating frequency in cardiac myocyte cell groups, the stepwise network formation of cells as the reconstructive approach using the on-chip agarose microchamber cell microcultivation system with photo-thermal etching method was applied. In the system, the shapes of agarose microstructures were changed step by step with photo-thermal etching of agarose-layer of the chip using a 1064-nm infrared focused laser beam to increase the interaction of cardiac myocyte cells during cultivation. First, individual rat cardiac myocyte in each microstructure were cultivated under isolated condition, and then connected them one by one through newly-created microchannels by photo-thermal etching to compare the contribution of community size for the magnitude of beating stability of the cell groups. Though the isolated individual cells have 50% fluctuation of beating frequency, their stability increased as the number of connected cells increased. And finally when the number reached to eight cells, they stabilized around the 10% fluctuation, which was the same magnitude of the tissue model cultivated on the dish. The result indicates the importance of the community size of cells to stabilize their performance for making cell-network model for using cells for monitoring their functions like the tissue model.
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 . 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  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].
This paper reports the cell network size effect (community effect) for stabilizing their beating intervals using our on-chip single-cell-based cultivation assay with stepwise modification of micorcultivation chamber structures during cultivation.
The above results indicate two facts. First, for the single cell based research as the cell-network model, the consideration of community size of cell group is important for acquiring the reliable, stable data. Second, the community size required for reliable measurement like tissue model is not so large, i.e. nine cells were enough in this case. That might mean that smaller than we expected number of cells is required to produce the same community effect as in the tissue model. And therefore the potential for creating individual-cell-based cell-network model might be practical for reliable drug screening assay especially for human organ model. Moreover, it should be noted that we succeeded in the separating of two factors affecting the beating synchronization in this system, gap-junction connection and physical stretching. In other words, using this system we can measure the effect of gap-junction connections on synchronization clearly. If we could not remove the effect of physical stretching caused by the physical contact of neighbouring cells, we could hardly clarify the effect of chemicals to inhibit gap-junction connections. Because the cluster of cells still synchronized by their physical stretching even after gap-junction was inhibited (data not shown). From this viewpoint, our system might be most beneficial in drug screening.
In conclusion, we applied the 1064-nm photo-thermal etching method and made the on-chip agarose single cell microcultivation system for generating cardiac myocyte networks of different size, which is important for understanding the community effect of rhythm synchronization. Using the system, we for the first time observed the differences in the synchronization process of cardiac myocyte cells and their dependence on the community size. This system can potentially be used in the biological/medical fields for cultivating next generation of networks from individual cultured cells and measuring their properties.
Materials and Methods
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.
The spontaneous contraction rhythm of cultured cardiac myocytes was evaluated by a video-image recording method. Images of beating cardiac myocytes were recorded with a CCD camera through the use of a phase contrast microscope. The sizes (cross-sectional area of cell) of cardiac myocytes, which changed considerably with contraction, were also analyzed and recorded every 1/30 s by a personal computer with a video capture board and estimated their beating phenomenon by the change of their cross-sectional area sizes [6, 7].
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