Collective cell migration of fibroblasts is affected by horizontal vibration of the cell culture dish

Abstract

Regulating the collective migration of cells is an important issue in bioengineering. Enhancing or suppressing cell migration and controlling the migration direction is useful for various physiological phenomena such as wound healing. Several methods of migration regulation based on different mechanical stimuli have been reported. While vibrational stimuli, such as sound waves, show promise for regulating migration, the effect of the vibration direction on collective cell migration has not been studied in depth. Therefore, we fabricated a vibrating system that can apply horizontal vibration to a cell culture dish. Here, we evaluated the effect of the vibration direction on the collective migration of fibroblasts in a wound model comprising two culture areas separated by a gap. Results showed that the vibration direction affects the cell migration distance: vibration orthogonal to the gap enhances the collective cell migration distance while vibration parallel to the gap suppresses it. Results also showed that conditions leading to enhanced migration distance were also associated with elevated glucose consumption. Furthermore, under conditions promoting cell migration, the cell nuclei become elongated and oriented orthogonal to the gap. In contrast, under conditions that reduce the migration distance, cell nuclei were oriented to the direction parallel to the gap.

Keywords: cell migration; fibroblast; mechanotransduction; vibrational stimulation; wound healing.

FIGURE 1

The fabricated system applies horizontal vibration to the cells: (A‐B) Overview of the system. (C) Enlarged view of the dish holder and coordinate system. The red arrows indicate the direction of the vibration measurements conducted for system characterization

FIGURE 2

Outline of the collective cell migration experiment: (A) Experimental procedure and (B) initial gap between the two rectangular cell areas

FIGURE 3

Characteristics of the fabricated system: (A) Relationship between the applied voltage and the vibration amplitude at x₁ = 0 under an applied frequency of 11.2 kHz and amplitude of the applied voltage was 0 to 5 V. (B) Vibration amplitude distribution of the dish holder along the x₁, x₂, and x₃ axes when the frequency and amplitude of the applied voltage were 11.2 kHz and 2.5 V, respectively. (C) Temperature of the culture medium in the dish vibrated by the fabricated system for 24 h in a humidified 5% CO₂ incubator at 37°C when the frequency of the applied voltage was 11.2 kHz and the voltage amplitude was 2 or 4 V

FIGURE 4

Migration distance results: (A‐B) Migration distances when vibrational stimulation was applied (A) orthogonal to the gap and (B) parallel to the gap. (C) Cell migration over 24 h under three conditions; no vibration (referred to as “non‐vibrating”), vibration orthogonal to the gap with a voltage amplitude of 2 V (“2 V/Orthogonal”), vibration parallel to the gap with a voltage amplitude of 4 V (“4 V/Parallel”). The dashed and solid white lines represent the edges of the cell areas before and after migration. Data are presented as mean ± SD, **p < 0.01, n = 3

FIGURE 5

Normalized glucose consumption for 24 h under three conditions; no vibration (referred to as “non‐vibrating”), vibration orthogonal to the gap with a voltage amplitude of 2 V (“2 V/Orthogonal”), vibration parallel to the gap with a voltage amplitude of 4 V (“4 V/Parallel”). Data are presented as mean ± SD, *p < 0.05, n = 3

FIGURE 6

Effect of vibration on cell nuclei orientation: (A) Definition of the angle of a cell nucleus. (B) Nuclear staining image. (C) Orientation angle distributions of the cell nuclei under three conditions. (D) Aspect ratios of cell nuclei; no vibration (referred to as “non‐vibrating”), vibration orthogonal to the gap with a voltage amplitude of 2 V (“2 V/Orthogonal”), vibration parallel to the gap with a voltage amplitude of 4 V (“4 V/Parallel”). n = 300