Miniaturized platform for electroporation of cell monolayers

Goal

The goal of this project is to study of the effect of membrane polarization on the cell response to electroporation. In certain types of cells, like epithelial cell for instance, the composition of the cell membrane is organized into two distinct parts. The apical part is in contact with the exterior of the body whereas the basolateral part is in contact with other cells (see figure 1, left). From the experiment performed on planar cell membrane models, we expect the apical part of the MDCK cell membrane to be more resistant to pore formation. To test if this effect is also observed in MDCK cells, a simple miniaturized electroporation system is employed to study the influence of membrane polarization on sensitivity of cells to the applied electric field.

Motivation

A miniaturized system is employed (figure 1, right) where MDCK cells are grown as monolayer on two different substrates. Cells grown on a thin layer of hydrogel are expected to polarize, whereas those grown onto the bare electrode (indium-tin-oxide, ITO) are non-polarized. This allows us to compare the difference in response to electroporation for the two cases of membrane compartmentalization.

Figure 1. Schematic of the monolayer-based electroporation device

Left: Cells are grown in PDMS wells on two types of surfaces, an ITO electrode, or a layer of hydrogel made on the ITO, to induce or not their polarization (membrane organized in two distinct parts).

Figure 2: Immunofluorescence.

MDCK cell staining on hydrogel, for actin (phalloidin coupled to Texas Red) (left), E-cadherin (anti-E-cadherin and a secondary antibody coupled to FITC) (right) and nucleus (Hoechst, blue). 3D reconstruction of the cells and scheme illustrating cell polarization. Scale bar of 10 μm.

Results

Prior to the electroporation experiments, the cells are stained with a fluorescent dye, calcein AM. This dye remains in the cells until their membrane is permeabilized. Figure 3 demonstrates that (as one would expect) a higher applied potential results in the release of more fluorescent dye from the cells and thus a larger extend of electroporation.

Figure 3. Electroporation of MDCK cells cultured on hydrogel

Quantification of the calcein release caused by electroporation in MDCK cells for three different applied pulse amplitudes (8 pulses of 20 ms, treatment repeated every 5 min): 5 V (left), 7 V (middle) and 9 V (right). Striped lines in the graph indicate the decrease in fluorescence caused by photobleaching.

Contact persons

Dr. ir. Iris van Uitert (Alumni BIOS)

Dr. ir. Séverine Le Gac