A microfluidics-based wound-healing assay for studying the effects of shear stresses, wound widths, and chemicals on the wound-healing process

Abstract

Collective cell migration plays important roles in various physiological processes. To investigate this collective cellular movement, various wound-healing assays have been developed. In these assays, a "wound" is created mechanically, chemically, optically, or electrically out of a cellular monolayer. Most of these assays are subject to drawbacks of run-to-run variations in wound size/shape and damages to cells/substrate. Moreover, in all these assays, cells are cultured in open, static (non-circulating) environments. In this study, we reported a microfluidics-based wound-healing assay by using the trypsin flow-focusing technique. Fibroblasts were first cultured inside this chip to a cellular monolayer. Then three parallel fluidic flows (containing normal medium and trypsin solution) were introduced into the channels, and cells exposed to protease trypsin were enzymatically detached from the surface. Wounds of three different widths were generated, and subsequent wound-healing processes were observed. This assay is capable of creating three or more wounds of different widths for investigating the effects of various physical and chemical stimuli on wound-healing speeds. The effects of shear stresses, wound widths, and β-lapachone (a wound healing-promoting chemical) on wound-healing speeds were studied. It was found that the wound-healing speed (total area healed per unit time) increased with increasing shear stress and wound width, but under a shear stress of 0.174 mPa the linear healing speed (percent area healed per unit time) was independent of the wound width. Also, the addition of β-lapachone up to 0.5 μM did not accelerate wound healing. This microfluidics-based assay can definitely help in understanding the mechanisms of the wound-healing process and developing new wound-healing therapies.

Figure 1

(a) 3D numerical simulation of trypsin concentration inside the microfluidic chip. The concentrations in the central and side inlets were 0.0214 mole/m³ and 0 mole/m³, respectively. Scale bar = 6 mm. (b) 1D concentration profiles along different widths of the microfluidic chip. Three colors represent x positions along three different widths, starting from the middle to the right, as shown in the left (blue: the 6-mm width; green: the 4.5-mm width; red: the 3-mm width). Scale bar = 6 mm. The dash lines indicate the regions where the concentrations of trypsin were close to the initial value of 0.0214 mole/m³.

Figure 2

Pictures of the wound-healing processes under a flow rate of 200 μL/hr and a β-lapachone concentration of 0.5 μM. Scale bar = 500 μm.

Figure 3

Wound-healing speeds under different wound widths and flow rates. The number of experiments N = 9, and the error bars represent the standard errors of the mean (SEM) (see Data analysis in Methods). Statistical analysis was performed. ns: no statistically significant difference (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 4

Wound-healing speeds under different wound widths and β-lapachone concentrations. The number of experiments N = 9, and the error bars represent the standard errors of the mean (SEM) (see Data analysis in Methods). Statistical analysis was performed. ns: no statistically significant difference (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 5

Design of the microfluidic chip. Scale bar = 6 mm.

Figure 6

Layer-by-layer structure of the microfluidic chip. (a): adaptors; (b), (e), and (h): 1-mm PMMA substrate; (c) and (f): 60-μm double-side tape; (d) and (g): 260-μm double-side tape.

Figure 7

Experimental procedure for creating wounds. Blue: washing buffer (PBS); Red: cell culture medium (DMEM + 10% CS); Green: 0.05% trypsin; Gray: cell.

Figure 8

ImageJ was used to draw the boundaries and calculated the area of the wound. Scale bar = 500 μm.