Spatial control of cell position is essential for numerous studies in tissue engineering, such as generation of linearly patterned muscle tissues, radially patterned liver lobule tissues, and circularly formed anal sphincter. This study proposes a novel method capable of patterning cells in a certain shape on a cell cultivation substrate. The concept is to pattern cells along the nodal position of resonance vibration on a cell cultivation substrate. Note that there are numerous resonance vibrations with different nodal patterns. In this study, we constructed a cell cultivation device consisting of a metallic cell cultivation substrate that can be excited to generate resonance vibration using a piezoelectric ceramic disk glued to the back of the substrate. In this study, we generated resonance vibration with a cross-circle nodal pattern. The cultivation substrate was a ø40 mm × 1 mm stainless steel plate, and the resonance frequency of the vibration was 21.4 kHz. Using the device, we conducted cell patterning experiments employing calf chondrocytes using various vibration amplitudes (1.0, 1.5, and 2.0 µmp-p). Cells were cultured for 2 hours. After the experiment, the cell density distribution on the substrate was measured by staining viable cells with calcein. Additionally, we confirmed the viability of the patterned cells after exposure to acoustic pressure produced by the resonance vibration by counting the number of cells attached to the substrate. Viable cells were successfully patterned along the nodal position when the vibration amplitude was 1.5 µmp-p. The number of cells attached to the substrate was 99.2% of that without vibration. Conversely, when the amplitude was 1.0 µmp-p, the cells were not patterned. The acoustic pressure produced by 1.0-µmp-p vibration was not large enough to move cells. Similarly, the cells could not be patterned when the amplitude was 2.0 µmp-p. In this case, flow of the culture medium induced by the high acoustic pressure probably prevented the cells from patterning. In conclusion, cells can be patterned along the nodal position of resonance vibration generated with an appropriate vibration amplitude. We believe that this method has great potential for use in tissue engineering.
2016 Japanese Society for Medical and Biological Engineering