Robotic single cell electroporation

Transporting DNA and metabolites across the cell membrane is a fundamental mechanism to achieve a controlled, quantitative understanding of the complex processes occurring in the human body at the cellular level. However, the thin, cell membrane forms an effective barrier to foreign molecules and alternative means are required to coerce entry. One method capable of overcoming this barrier is single cell electroporation (SCE) via microcapillary, which can be applied to cells directly in culture, or tissue. SCE induces reversible pores in the membrane by applying an electric field at the cell surface. Membrane-impermeable molecules enter these pores by electrophoresis and diffusion. The tip of the microcapillary can be fabricated with micrometer size geometries allowing extraordinary cell selectivity and access to small cell features with sparing quantities of molecules. However, the technical complexity of SCE limits the use to highly trained operators. Operators must carefully position a microcapillary tip on cells only several micrometers in height and must perform the technique using conventional microscopy methods that lack depth-perception. Additionally, knowledge of the electrical characteristics of SCE influencing the rate and efficiency are required. These broad technical requirements and the fragile nature of thin cell structures limit the efficiency of manual throughput. Furthermore, the sequence of tasks have not been adequately achieved by automated efforts, thus the true potential of SCE has not been realized. In this thesis, a versatile system and methods are described for an infrastructure designed completely for automated SCE. The intent of the system is to abstract the technical challenges and exploit the accuracy and repeatability of automated instrumentation leaving only the focus of the experimental design to the operator. In addition, new milestones within automated cell manipulation have been achieved. The system described herein has the capability of fully automated SCE of 'thin' cell features less than 10 m in height. This achievement eliminates limitations imposed by many mammalian cell lines and provides a rapid, transmembrane transport method for a broad range of applications. The execution is demonstrated by inserting a combination of a fluorescing dye and a plasmid DNA with a reporter gene into NIH/3T3 fibroblasts.