High-throughput and real-time study of single cell electroporation using microfluidics: Effects of medium osmolarity

Abstract

Electroporation has been widely accepted as an important tool for the delivery of exogenous molecules into cells. Previous mechanistic studies have been carried out by observing either the average behavior from a large population of cells or the response from a small number of single cells. In this study, we demonstrated a novel microfluidic method with high throughput (up to 30 Hz) for real-time studies of single cell electroporation events. Electroporation occurred when cells flowed through a section of a microfluidic channel defined by special geometry. A CCD camera was used to monitor the response of cells starting from the onset of the electroporation. We studied the swelling of Chinese hamster ovary cells and the rupture of cell membrane during electroporation using this technique. We applied buffers with different osmolarities to investigate the effects of medium osmolarity, based on results from a population of single cells. We were able to establish the distributions of the rates of swelling and membrane rupture in the cell population. We also explored establishing the correlation between the property (the cell diameter) and the behavior (the swelling rate) of single cells. Our results indicated that the processes of swelling and rupture occurred more rapidly in the hypotonic or hypertonic buffers than in the isotonic buffer. Statistical analysis did not reveal strong linear correlation between the cell size and the swelling rate. These proof-of-concept studies reveal the potential of applying microfluidics to study electroporation of a cell population at single cell level in real time with high throughput. The limitations associated with this approach were also addressed.

Detection of Kinase Translocation Using Microfluidic Electroporative Flow Cytometry

“…We have observed the rapid release of intracellular contents during electroporation in similar devices with alternating wide and narrow sections in our previous work 16,18,25 . Such release is due to a combination of outward diffusion and electrophoretic motion of intracellular molecules when the plasma membrane is compromised by the electrical field.…”

“…A similar field intensity distribution in the channel was modeled in our previous work 25 . We have shown that when the overall voltage is in the right range, electroporation exclusively occurs in the narrow section since the field intensity in the wide sections are too weak to compromise the cell membrane 16,18 . The field in the reservoirs is negligible due to the large size of the volume in there.…”

“…To incorporate electroporation into the device, two platinum electrodes connected to a DC power supply were inserted in the sample and outlet reservoirs to establish an electrical field across the horizontal channel. A new microfluidic electroporation technique based on constant DC voltage was applied here 16,18 . The horizontal channel was composed of two wide sections (W 1 = 300 μm) and one narrow section (W 2 = 30 μm) with the depth of the whole channel being uniform (Figure 1a).…”

“…More complete release might be attainable by the optimization of the electroporation conditions other than the electrical parameters (e.g. osmolarity of the electroporation medium) 16 .…”

“…During electroporation the electrical field opens up pores in the cell membrane. Such pores allow the release of intracellular materials into the surrounding solution [14][15][16] . In this study, we found that the amount of SykEGFP left in the cells after electroporation was related to whether or not translocation of Syk to the plasma membrane occurred.…”