Fig 1.
The droplet actuation device and cells transfected by the W/O droplet electroporation method.
(A), (B) Two pieces of conductive tape were set parallel on a single cuvette or 8 wells in a line for 96-well plastic microwell plates. The droplet continued to bounce between the edges of the two electrodes. One was the ground electrode, and the other was the high-voltage electrode. Images of droplets bouncing between the anode and cathode in each well are shown. Many cells in droplets can be transfected simultaneously. (C) The upper row : Bright-field (BF), fluorescence, and merge images of HEK293 cells 24 hours after transfection by W/O droplet electroporation. Cells were examined at 24 hours after transfection to evaluate the expression of fluorescent protein (Venus). Scale bars, 100 μm. The lower row : BF, fluorescence, and merge images of HEK293 cells 24 hours after transfection by lipofection. (D) Variation of cell viability with time of droplet actuation determined by trypan blue staining. All experiments were performed at least twice.
Fig 2.
Screening of suitable conditions for W/O droplet electroporation.
(A) The pair of pin electrodes for single well of 96multi-well plate. (B) Merge image between bright-field (BF) and fluorescence images of HEK cells 1, 4, 7, and 12 days after W/O droplet electroporation. All scale bars, 30 μm. (C) HEK293 cells transfected with Venus fluorescent plasmid 1, 6, and 9 days after W/O droplet electroporation. Transfection efficiency was compared with 5-minute application time. (D) HEK293 cells transfected with Venus plasmid with application times of 1, 5, and 10 minutes 6 and 9 days after electroporation.
Fig 3.
Fibroblast, SCN neural cells and HEK293 cells transfected Venus plasmid by W/O droplet electroporation.
(A) Bright field, fluorescence and merge images of fibroblast cells from a subject aged 81 years old one day after electroporation. (B) 7 days after W/O droplet electroporation. (C) Bright field, fluorescence and merge images of SCN neural cells three days after W/O droplet electroporation. SCN cells had been differentiated to neural cells by incubation at higher culture temperature [26]. (D) Twelve days after W/O droplet electroporation. All scale bars (A-D), 30 μm. (E) Bright field, fluorescence and merge images of HEK293 cells colonies were picked up and cultured for one month after W/O droplet electroporation. (F) Bright field, fluorescence and merge images of HEK293 cells colonies were cultured though frozen stock for a total of more than two months after W/O droplet electroporation. scale bars (D-E), 100 μm.
Fig 4.
Double and stable transfected cells by W/O droplet electroporation.
(A), (B) Transgene plasmid DNAs of two kinds of fluorescent protein: green fluorescent protein (GFP) and Tag-RFP (red fluorescent protein), were successfully double transfected into HEK293 cells by W/O droplet electroporation for 3 minutes. Bright-field image and fluorescent GFP or RFP image of HEK293 cells (A) 5 days (71% of cells maintained GFP and 71% of cells maintained TagRFP signal) and (B) 8 days (41% of cells maintained GFP and 37% of cells maintained TagRFP signal) after W/O droplet electroporation. Scale bars, 30 μm. (C) Transgene plasmid DNAs of Venus and mCherry (red FP) were successfully double transfected into hippocampus primary neural cell lines by W/O droplet electroporation for 5 minutes. Scale bars, 30 μm. (D), (E) Bright-field image and fluorescent image of Venus and mCherry expression in human fibroblast cells (D) 2 days and (E) 10 days after W/O droplet electroporation. Scale bars, 30 μm.
Fig 5.
Image of the parallel W/O droplet electroporation electrode for the 8-well string of disposable 96-well plates.
(A) Conductive electrodes were produced and set on an 8-well string in 96-well microwell plates for W/O droplet electroporation. (B), (C) Venus plasmid was transfected into some HEK293 cells using 8-well W/O droplet electroporation electrodes (Nepa Gene) for 96-well plates at 1.8–2.2 kV. Venus fluorescence signals were observed after incubation in 5 of 8 wells for 4 and 7 days following electroporation. Scale bars, 20 μm.