Summary Ana Valero

In this thesis the results of the development of microfluidic cell trap devices for single cell electroporation are described, which are to be used for gene transfection. The performance of two types of Lab-on-a-Chip trapping devices was tested using beads and cells, whereas the functionality for single cell electroporation of these chips was verified by means of gene transfection studies.

In order to be able to design microfluidic cell trapping devices for single cell electroporation (SCE), the fundamentals of the electrical permeabilization of cell membranes were discussed, as well as the advantages of using microdevices (chips) for SCE. The current status on micro-electroporation devices for analysis, transfection or pasteurization of biological cells is reviewed: most of the reported devices and concepts focus on the electroporation process itself, and do, however, not incorporate integrated separation and detection processes (analysis methods). The field of single-cell content analysis, where the cell content is released by electroporation, is promising, in particular when it is done in chips since it is difficult to accomplish this in a well-controlled way in larger structures. Clearly, microfluidic devices as a platform for electroporation fulfill the necessities to perform single cell electroporation and analysis, and are therefore important ‘tools’ to get more insight in the fundamentals of the electroporation process.

Trapping of cells by means of integrated microstructures, i.e. “mechanical filters”, was identified as the best method to trap single cells in chips due to the simplicity of this method. The design and fabrication of silicon-glass chips containing two different micromachined trapping filters are shown. For both devices cells were trapped at the sites using two different methods (pinched injection and sheath flow), and the trapping performance of both methods was tested with beads (cell suspension model) and HL60 cells.

The analysis of apoptosis in HL60 cells was studied in these microfluidic cell trap devices. The silicon-glass chip enabled the immobilization of HL60 cells and real time monitoring of the apoptotic process. Induction of apoptosis of the cells was addressed in two ways: electric field mediated (i.e. electroporation) or chemically. Fluorescent dyes (FLICA and PI) were used to discriminate between viable, apoptotic and necrotic cells. This work presented the first results to analyse programmed cell death dynamics using a chip and a first step towards an integrated chip for high-throughput drug screening on a single cellular level.

Single cell electroporation was carried out in chips containing mechanical trapping sites and integrated electrodes. The electrodes were positioned in such a way that each trapped cell could be addressed individually. Electropermeabilization experiments were performed on two cell lines, viz. HL60 and C2C12 cells, using a membrane-impermeable stain (PI) and a plasmid encoding green fluorescent protein (GFP). The experiments performed with the cell membrane integrity marker (PI) showed that single cell electroporation was achieved. Moreover, only the cell that was electrically addressed with the electroporation signal showed red fluorescence (PI uptake), cells located at neighbouring traps did not. For both cell lines, the electrical parameters, voltage (or E-field) and pulse length needed for cell membrane permeabilization of individual cells were studied in detail in these chips. Furthermore, for the first it was shown that microfluidic cell trap devices can be used for successful and efficient gene transfection.

Electrical detection is another method to detect single cell electroporation. In this method the current flowing through the cell membrane is measured. Since electroporation of a cell results in a resistance change of the membrane, membrane permeation can be detected by characteristic ‘jumps’ in current that correspond to drops in cell resistance. An electrical model was developed to verify whether it is possible to detect these characteristic current variations in the chips used in this thesis. The model showed that it is not trivial to electrically detect single cell electroporation with the current measurement set-up. Solutions to optimize the electrical detection of electroporation are presented, e.g. reduction of the leak resistance around the cell and of the electrodes’ impedance.

Pulse electric field (PEF) electroporation can be used for the inactivation of microorganisms (e.g. pasteurization of foods). Two different microdevices were used to study PEF electroporation of yeast cells: a single cell trapping device and a flow-through electroporation device. The electroporation process was followed using fluorescent DNA stain PI. It was found that electroporation of yeast cells is a progressive process.

Finally, the microfluidic single cell trapping devices were used successfully for studying gene transfection into stem cells by electroporation. After electro-poration, green fluorescent-erk1 fusion protein was expressed in mouse myoblastic cells (C2C12) and in human mesenchymal stem cells (hMSCs) and subsequently the dynamics of the introduced protein were followed in real time. The long-term survival and responsiveness of the cells to external stimuli demonstrated the bio-compatibility of the microdevice. This study clearly showed that single cell electroporation in microfluidic trapping devices is a powerful tool for personalized gene therapy, tissue engineering and regenerative medicine. Due to the limited availability of adult stem cells, the use of microdevices for SCE makes these chips extremely important and attractive for stem cell research.