Dielectrophoresis Prototypic Polystyrene Particle Synchronization toward Alive Keratinocyte Cells for Rapid Chronic Wound Healing

Abstract

Diabetes patients are at risk of having chronic wounds, which would take months to years to resolve naturally. Chronic wounds can be countered using the electrical stimulation technique (EST) by dielectrophoresis (DEP), which is label-free, highly sensitive, and selective for particle trajectory. In this study, we focus on the validation of polystyrene particles of 3.2 and 4.8 μm to predict the behavior of keratinocytes to estimate their crossover frequency (f_XO) using the DEP force (F_DEP) for particle manipulation. MyDEP is a piece of java-based stand-alone software used to consider the dielectric particle response to AC electric fields and analyzes the electrical properties of biological cells. The prototypic 3.2 and 4.8 μm polystyrene particles have f_XO values from MyDEP of 425.02 and 275.37 kHz, respectively. Fibroblast cells were also subjected to numerical analysis because the interaction of keratinocytes and fibroblast cells is essential for wound healing. Consequently, the predicted f_XO from the MyDEP plot for keratinocyte and fibroblast cells are 510.53 and 28.10 MHz, respectively. The finite element method (FEM) is utilized to compute the electric field intensity and particle trajectory based on DEP and drag forces. Moreover, the particle trajectories are quantified in a high and low conductive medium. To justify the simulation, further DEP experiments are carried out by applying a non-uniform electric field to a mixture of different sizes of polystyrene particles and keratinocyte cells, and these results are well agreed. The alive keratinocyte cells exhibit N_DEP force in a highly conductive medium from 100 kHz to 25 MHz. 2D/3D motion analysis software (DIPP-MotionV) can also perform image analysis of keratinocyte cells and evaluate the average speed, acceleration, and trajectory position. The resultant N_DEP force can align the keratinocyte cells in the wound site upon suitable applied frequency. Thus, MyDEP estimates the Clausius-Mossotti factors (CMF), FEM computes the cell trajectory, and the experimental results of prototypic polystyrene particles are well correlated and provide an optimistic response towards keratinocyte cells for rapid wound healing applications.

Keywords: Clausius–Mossotti factor; dielectrophoresis; electrical stimulation technique; fibroblast; keratinocyte.

Figure 1

Geometry of the single-shell model: (a) keratinocyte cell and (b) fibroblast cell. An asterisk (*) indicating complex permittivity.

Figure 2

Flowchart with governing equations and solver for each interface.

Figure 3

Tapered microelectrode geometry with specifications: (a) 2D geometry of a tapered microelectrode device; (b) illustration of a particle trajectory sequence in a tapered microelectrode.

Figure 4

Overview of the dielectrophoresis (DEP) framework: (a) pictorial view of the experimental setup; (b) fabricated tapered electrode; (c) top view of the tapered microelectrode; (d) region of interest (ROI)—particle in a tapered electrode.

Figure 5

Clausius–Mossotti factors (CMF) of polystyrene particles with sizes of 3.2 and 4.8 µm.

Figure 6

CMF result for epidermal layer keratinocyte and dermal layer fibroblast cells in low conductive medium.

Figure 7

CMF plot for keratinocyte and fibroblast cells in dulbecco’s modified eagle medium/nutrient mixture F-12 (DMEM/F-12) and dulbecco’s modified eagle medium (DMEM) in high conductive medium.

Figure 8

Performance analysis from the MyDEP simulation of polystyrene particles, epidermal layer keratinocyte, and dermal layer fibroblast cells.

Figure 9

At stationary study: (a) spectral change in electrical field in the tapered microelectrode with contour plot; (b) the enlarged view of the high field intensity spot at the edges of the microelectrode.

Figure 10

Particle trajectories at (a) the initial stage at 0 s with an initial particle velocity of 0 m/s, (b) 140 kHz, 20 s, 3.2 and 4.8 µm—P_DEP, (c) 275.012 kHz, 20 s, 3.2 µm—P_DEP, 4.8 µm—f_XO, (d) 350 kHz, 20 s, 3.2 µm—P_DEP, 4.8 µm—N_DEP, (e) 425 kHz, 20 s, 3.2 µm—f_XO, 4.8 µm—N_DEP, and (f) 1 MHz, 20 s, 3.2 and 4.8 µm—N_DEP. The color gradient of the trajectory line indicates the instantaneous particle velocity u ranging from 0 m/s (white) to 0.16 m/s (dark red). The contour and intensity of the electric field are indicated by the background blue color.

Figure 11

Particle trajectories in low conductive medium at (a) the initial stage at 0 with an initial particle velocity of 0 m/s, (b) 15 MHz, 20 s, keratinocyte and fibroblast—P_DEP, (c) 28.07 MHz, 20 s, keratinocyte—P_DEP, fibroblast—f_XO, (d) 270 MHz, 20 s, fibroblast—P_DEP, keratinocyte—N_DEP, (e) 510.1 MHz, 20 s, fibroblast—f_XO, keratinocyte—N_DEP, and (f) 600 MHz, 20 s, keratinocyte and fibroblast—N_DEP. The color gradient of the trajectory line indicates the instantaneous particle velocity u ranging from 0 m/s (white) to 0.6 m/s (dark red). The contour and intensity of the electric field are indicated by the background blue color.

Figure 12

Particle trajectory in high conductive medium at (a) the initial stage at 0 with an initial particle velocity of 0 m/s and (b) 15 and 600 MHz, 20 s, keratinocyte and fibroblast—N_DEP. The color gradient of the trajectory line indicates the instantaneous particle velocity u ranging from 0 m/s (white) to 46 m/s (dark red). The contour and intensity of the electric field are indicated by the background blue color.

Figure 13

Fluorescence microscopy observations of 3.2 and 4.8 µm polystyrene particles in deionized water conductive medium for effective F_DEP: (a) 4× and (b) 10× magnifications of microelectrode with polystyrene particles; (c) initial 0 V_PP at 200 kHz; (d) final 10 V_PP–P_DEP force at 200 kHz; (e) initial 0 V_PP at 350 kHz; (f) final 10 V_PP–intermediate frequency at 350 kHz; (g) initial 0 V_PP at 460 kHz; (h) final 10 V_PP–N_DEP force at 460 kHz.

Figure 14

Fluorescence microscopy observations of keratinocyte cells in DMEM/F-12 conductive medium for effective F_DEP: (a) initial 0 V_PP at 300 kHz; (b) final 10 V_PP–N_DEP at 300 kHz; (c) initial 0 V_PP at 800 kHz; (d) final 10 V_PP–N_DEP at 800 kHz; (e) initial 0 V_PP at 15 MHz; (f) final 10 V_PP–N_DEP force at 15 MHz.