Figure 1.
Schematic diagram of the cell–GUV electrofusion process.
Cells and GUVs are exposed to an alternative current (AC) field to induce cell alignment (chain-of-pearls-like structure), and are then pulsed with direct current (DC) voltage to create breaks in the contact region between the cell membrane and GUV surfaces. Representative images are shown to demonstrate the appearance of cell–GUVs that had been exposed to the AC field and DC pulse.
Figure 2.
Representative images of cultured HeLa cells containing fluorescent microbeads introduced by cell–GUV electrofusion.
(A) Microscopic images of cells that had been cultured for 1 (left column), 2 (middle column), or 5 (right column) days after the electrofusion process. The diameter of the beads used here was 0.2 µm. Phase contrast gray scale images were converted to red-scale. Scale bar = 50 µm. (B) Fluorescent beads with various diameters were introduced into HeLa cells by cell–GUV electrofusion. Left: Confocal microscopic images show the cross-section of the treated HeLa cells. These images show HeLa cells into which (from the top) no beads, or beads of 0.2 µm, 0.5 µm, 1 µm, and 2 µm diameter (green) had been introduced. The cytoplasm is shown in red and nuclei in blue, and merged images are shown in the right column. Scale bar = 10 µm. Right: Flow cytometric detection of microbeads introduced into HeLa cells. Single parameter histograms of the cell number versus log fluorescence intensity are shown. The histograms represent a total of 7,000–15,000 cells counted for each measurement.
Figure 3.
Biomaterials introduced into HeLa cells by cell–GUV electrofusion.
(A) Plasmid DNA encoding EGFP (pEGFP; CMV promoter, 4.7 kbp) was adopted as a reporter. pEGFP was introduced into HeLa cells and cells were then cultured for 1 or 5 days. From the top row, images obtained by fluorescent microscopy are shown for GUV-treated cells after culturing for 1 day, 5 days, cells lacking GUVs (1 day), or not exposed to the DC pulse (1 day). Phase contrast (left column), EGFP expression in cells (green, middle column), and merged images (phase contrast shown in red-scale; right column) are shown. Scale bar = 50 µm. (B) An artificially designed DNA nanostructure (origami) was also introduced into HeLa cells. The DNA origami structure was labeled with green fluorescent dye (by using FITC-conjugated oligonucleotides, 282 FITC molecules per single origami). Corresponding images of phase contrast (left column), DNA origami (green, middle column), and merged images (phase contrast shown in red-scale) were obtained by fluorescent microscopy immediately after cell–GUV electrofusion. Scale bar = 10 µm.
Figure 4.
Introducing multiple components of microbeads and plasmids into live cells by cell–GUV electrofusion.
GUVs including both the plasmid mCherry and fluorescent microbeads were prepared for electrofusion with HeLa cells. The treated cells were then cultured for 2 days. Confocal microscopic images show the cross-section of the treated HeLa cells into which (from the top) beads of 0.2 µm, 0.5 µm, and 1 µm diameter (green) had been introduced. The mCherry expression in cells is shown in red, and merged images are shown in the right column. Scale bar = 20 µm.