Comparison between direct and reverse electroporation of cells in situ: a simulation study

Abstract

The discovery of the human genome has unveiled new fields of genomics, transcriptomics, and proteomics, which has produced paradigm shifts on how to study disease mechanisms, wherein a current central focus is the understanding of how gene signatures and gene networks interact within cells. These gene function studies require manipulating genes either through activation or inhibition, which can be achieved by temporarily permeabilizing the cell membrane through transfection to delivercDNAorRNAi. An efficient transfection technique is electroporation, which applies an optimized electric pulse to permeabilize the cells of interest. When the molecules are applied on top of seeded cells, it is called "direct" transfection and when the nucleic acids are printed on the substrate and the cells are seeded on top of them, it is termed "reverse" transfection. Direct transfection has been successfully applied in previous studies, whereas reverse transfection has recently gained more attention in the context of high-throughput experiments. Despite the emerging importance, studies comparing the efficiency of the two methods are lacking. In this study, a model for electroporation of cells in situ is developed to address this deficiency. The results indicate that reverse transfection is less efficient than direct transfection. However, the model also predicts that by increasing the concentration of deliverable molecules by a factor of 2 or increasing the applied voltage by 20%, reverse transfection can be approximately as efficient as direct transfection.

Keywords: Electroporation; high‐throughput techniques; transfection efficiency.

Figure 1

Electroporation and uptake model that is implemented in this article.

Figure 2

(A) Geometry of an attached cell used in the modeling, including cell membrane, a; cytoplasm, b; nucleus, c; nucleus membrane, d; material below the cell, e; material covering the slides around the cell, f; buffer on top of the cell, g; and bottom electrode, h. (B) Geometry of adjacent cells in a multicellular layer.

Figure 3

(A) 2D axisymmetry model for a single spherical cell between two plate electrodes. The dashed‐dot line shows the symmetry axis. The upper and lower electrodes are shown by arrows in the figure. (B) A section of the 3D view of the model. The lines in the figure show the contours of electric potential. It can be seen that the lines are bent around the cell due to the presence of the cell.

Figure 4

(A) Induced transmembrane voltage and (B) membrane conductivity for a single cell attached to a conductive slide. The applied electric field was 400 V m⁻¹. The lines with the asterisks belong to the static case in which conductivity of the membrane is considered constant. The lines without asterisks correspond to the dynamic case in which conductivity of the membrane changes as a function of the ITV. The solid lines demonstrate the results for the apical side, while the dash‐dot lines show the results for the basal side of the cell. The ITV in the static case for a single cell is larger on the apical side. This results in a larger conductivity in the dynamic case, which, in turn, causes the reduction of ITV so that the ITV has almost the same maximum value for both the apical and basal sides.

Figure 5

ITV in static study for a single cell in (A) a real monolayer of the cells on the substrate and (B) a simplified model replacing all other cells with a layer that has characteristics of the cell membrane. The solid lines show the ITV for the apical side, and the dashed lines indicate ITV for the basal side of the cell. These results show that the actual model of a monolayer and the simplified model are within 95% of each other.

Figure 6

Study of the dynamic case for electroporation of endothelial cells within a monolayer, using a 10 msec pulse of 150 V m⁻¹. (A) Induced transmembrane voltage on the apical (solid line) and on the basal (dashed line) side of the membrane. The ITV is almost uniform along the membrane and slightly larger for the basal side compared with the apical (0.68 on basal and 0.67 on apical). (B) Resulting pore density on the apical (solid line) and basal (dashed line) sides of the membrane. Due to the larger ITV on the basal side during the pulse, the pore density is larger. The permeability related to diffusion has the same trend (not shown). (C) Shows the evolution of pore density at the highest point of the cell with time after the pulse ends. The pores reseal after approximately 10 sec, while ITV vanishes immediately after the pulse ends. (D) Permeability related to electrodiffusion for reverse electroporation at the apical (solid line) and the basal (dashed line) sides of the cell. The permeability of the basal membrane gets even larger for reverse electroporation. (E) and (F) show the uptake of the cell in direct and reverse electroporation, respectively. It is clear that direct electroporation is more effective. In (F), the dot‐dash line shows the uptake just due to the diffusion, and the solid line shows the uptake due to the electrodiffusion. Although the main factor of the uptake is diffusion, electrodiffusion has significant effect on the uptake in reverse electroporation.

Figure 7

Dynamic case study for electroporation of five adjacent endothelial cells in a multicellular layer or cluster. (A) Induced transmembrane voltage and (B) pore density of a 10 msec pulse of 150 V m⁻¹ at the apical (solid line) and the basal (dashed line) side of five adjacent cells on a spot. The vertical grids in (A) and (B) show the location of each cell, considering the middle cell on x = 0. The ITV and consequently pore density are not uniform along the membrane of each cell and very different on the adjacent cells. (C) and (D) display the uptake of different cells in the multicellular model for direct and reverse electroporation, respectively. The solid lines, dashed lines, and dotted lines are related to the cells on the edges, cells adjacent to the edge cells, and cells adjacent to the middle cell, respectively. It is clear that uptake of the cell in the middle is the highest, and uptake for the cells on the edges is the lowest.