Impedimetric melanoma invasion assay device using a simple paper membrane and stencil-printed electrode on PMMA substrate

Abstract

The transwell assay is currently the most popular approach to studying cellular invasion due to its ease of use and readout, and the possibility for quantitative measurements. However, it only allows end-point measurements without the possibility for real-time tracking of the dynamics of cell movement during an invasion. Moreover, it requires cell labeling, and construction of customized devices is hampered by the commercial standard membrane inserts, only available in certain designs. Recently, paper has been used as a scaffold for three-dimensional (3D) cell cultures. Because of its microfibrous structure and easy handling, it could be a versatile alternative as a membrane insert in customized devices. Here, we develop a low-cost real-time invasion assay device using paper as an alternative membrane insert. The device was designed for two-electrode impedance measurements and fabricated using CNC micromilling. It also comprised a disposable low-cost stencil-printed working electrode on a poly(methyl methacrylate) substrate below the membrane and glassy carbon counter electrode above the membrane inserted in a specially designed lid. Thus, the impedance measurements during cell invasion addressed the entire membrane. We demonstrated the function of the device by monitoring the invasion of B16 melanoma 4A5 cells from a mouse using insulin growth factor-1 as the chemoattractant. The cell invasion on paper was visualized using scanning electron microscopy and confocal microscopy with Z-stack 3D imaging. Melanoma cell invasion could be observed within 7 h after the chemoattractant treatment, which was faster than the conventional assay and less likely to be influenced by cell proliferation.

Keywords

• 3D cell culture
• Paper-based cell culture
• Stencil-printed electrode
• Impedance monitoring
• Invasion assay
• Melanoma