Study of a Robotic Cell Injection Technology for Single Cell Transfection

Abstract

Microinjection is a widely used technique in biological research, particularly for delivering DNA, RNA, and proteins into single cells. Most cell injections are currently operated manually, which requires experienced operators to accomplish the task. Automation can improve the efficiency and accuracy of this cell manipulation process. Compared with many existing microinjection technologies that are primarily designed for relatively large-scaled biological samples, the technology investigated in this study will enable automated injection into cells with diameters less than 25 μm, which is the typical size of many human cells. This thesis presents the study of an automated quantitative microinjection technology with high productivity for small cells (10–25 μm) from the three following aspects.

First, a specially designed cell manipulation platform that uses microfluidic chip technology is proposed. This manipulation platform, also referred to as cell holder, is used to automatically locate cells and handle a large amount of cells in a 1D array to accomplish the injection of small individual cells. The irregular morphology of human cells makes them considerably difficult to recognize and locate. The developed cell holder can trap cells in a semicircular cell channel, allowing the cells to be easily and accurately located. This specially designed cell holder allows the development of a highly efficient automated cell injection system for small human cells.

Second, an automated cell injection system is developed with the microfluidic cell holder. The developed system utilizes a bent micropipette for cell injection and a novel cell holder design to simplify the injection process in a single-axis motion control. Compared with numerous existing injection systems that handle relatively large cells (e.g., >50 μm), the proposed system can work efficiently on human cells with smaller sizes (e.g., <25 μm). Moreover, the developed cell injection system can achieve higher throughput and efficiency compared with other injection systems for small adherent cells. These advantages have been experimentally demonstrated in human cell injections.

Third, a quantitative microinjection technology that can deliver precise amounts of materials into cells is produced using the developed cell injection system. The amount of injected materials is precisely controlled by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and those of water droplets with a known injection amount of dye in oil. This method is evaluated by injecting a synthetically modified mRNA that encodes for green fluorescence proteins and injecting a cocktail of plasmids that encode for green and red fluorescence proteins into human foreskin fibroblast cells. Experimental results demonstrate that the resulting green fluorescence intensity or green/red fluorescence intensity ratio can be well correlated with the amount of genetic material injected into the cells.

In summary, the proposed automated cell injection technology can achieve quantitative single-cell transfection for small human cells with high efficiency. This single-cell transfection via the developed quantitative microinjection technique will be particularly useful for applications where cell transfection is challenging and genetic modification of selected cells is desired.