Electrophoretic Nanoinjection: An Alternative to Microinjection for Living Cells

Image Credit: shuttersotck.com/SergeyNivens

Several different types of foreign molecules such as antibodies, plasmid DNA and functionalized fluorescent probes are required to be introduced into cells in various techniques involved in gene therapy² and fluorescence microscopy¹. These foreign molecules could be introduced to image the substrate- or fluorescent probe-tagged antigens or markers in either a live or a fixed dead cell. Techniques such as electroporation, lipofection and microinjection are most often used in the case of live cells. As impressive as these traditional methods may sound, either the viability of cells is reduced or the cell cycle can have up to a 50% disruption rate, when the foreign molecules are introduced into the cells³. To deliver these molecules into the cytoplasm, the molecules need to cross the cell membrane, which is composed of a phospholipid bilayer. One of the most commonly used methods to deliver the molecules involves microinjection, which is a process capable of delivering microliter volumes of liquids and dissolved molecules.  

The microinjection method involves injection of the molecules dissolved in a liquid using a glass capillary with a very fine tip measuring only 0.5 to 1 mm in diameter, while using the micromanipulators to direct the tip towards the target site³. The successful delivery of the molecules without harming the cells depends upon the skills of the operator, volume of the injection and the cell type. Lack of accurate positioning feedback from the micropipette, difficulty in the estimation of the adherent cells and multiple injection attempts are the main reasons for the shortcomings of the microinjection³. The diameter of the pipette tip relative to the cell size, the technology that provides accurate feedback regarding the approach, penetration, and the injection process seem to be the determining factors for cell’s viability.

Researchers at the University of Bielefeld’s Department of Physics’ Biomolecular Photonics recently demonstrated a novel method published in Nature Scientific Reports on their successful delivery of foreign molecules without causing harm to the cells by using a nanopipette, measuring only 100 nm in diameter.¹ Simon Hennig’s team injected a cell impermeable fluorophore-labelled Dextran into a total of 239 human bone osteosarcoma (U2OS) cells ³. The fluorescent Dextran - Alexa Fluor 647 (DAF) was injected using the piezo-actuated approach, where the electrodes placed in the cell medium sense the change in the ionic current with the approaching pipet tip towards the target site. This detected change in the ionic current serves as a constant and real time feedback mechanism for monitoring the pipette tip using a computer software. Pre-warmed phosphate buffer silane (PBS) is used instead of the culture media throughout the injection process in order to achieve a more precise ionic current³. After confirming the successful fluorescent signal of the cell using a red exciation light, the pipette tip is finally retracted and directed towards a new cell for DAF injection. A wide field imaging system analysis is then used to determine the cell survival and proliferation rate of the DAF injected cells.

To determine the safe voltage and duration of the current applied to deliver these fluorescent molecules, the survival rates of the cells after 24 h were assessed at voltages of 0.5 volts (V) and 1 V at both 1 min and 5 min durations of the current applied³. The results revealed that the cell survival was best when the electric current was applied for 1 min, in both voltages of 0.5 V or 1 V. Therefore, to avoid the detrimental factor of duration of the applied electric current on cell survival, all further injections were carried out with a 1 minute duration of the applied electric current.  

Hennig’s research team compared the effect of the diameter (D) of the pipette tip on cell survival by using two different sized pipette tips for injection; one with a 100 nm D, resembling the nanopipette, and the other with 500 nm D, which resembles a traditional micropipette. When the same voltage was applied for 1 minute and observed for cell viability following a 24 hour period, cells injected with the nanopipette exhibited a 92% survival rate, whereas the cells injected with the micropipette only showed a 40% survival rate³.

The mitotic cell division images from the time lapse series, which can be identified by the doubling of fluorescent cells, was obtained using the wide field fluorescence imaging of the DAF injected cells. These images revealed that applying the voltages of 0.5 V – 1 V for a duration of 1 minute did not affect the proliferation rate of the cells nor cell health, even after administering the DAF into the nucleus³. This research therefore shows that injecting the molecules into living cells using a nanopipette with electrophoretic delivery will greatly improve cell survival rates as compared to the traditional injection methods. The nanopipette injection method described in this study also serves as an exceptional alternative to micropipette injections when working with living cells.

References:

1. "Nanoinjection Increases Survival Rate of Cells." Phys.org. 01 Mar. 2017. Web. https://phys.org/news/2017-03-nanoinjection-survival-cells.html.
2. "Transfection." Promega Corporation. Web. https://www.promega.com/resources/product-guides-and-selectors/protocols-and-applications-guide/transfection/.
3. Matthias Simonis et al, Survival rate of eukaryotic cells following electrophoretic nanoinjection, Scientific Reports (2017).