Electrode microchamber for noninvasive perturbation of mammalian cells with nanosecond pulsed electric fields

Nanosecond pulsed electric fields can pass through the external membrane of biological cells and disturb fast-responding intracellular structures and processes. To enable real-time imaging and investigation of these phenomena, a microchamber with integral electrodes and optical path for observing individual cells exposed to ultrashort electric pulses was designed and fabricated utilizing photolithographic and microelectronic methods. SU-8 photoresist was patterned to form straight sidewalls from 10 to 30 /spl mu/m in height, with gold film deposited on the top and sidewalls for conductive, nonreactive electrodes and a uniform electric field. Channel dimensions (10-30 /spl mu/m/spl times/100 /spl mu/m/spl times/12000 /spl mu/m) are suitable for observations of mammalian cells during nanosecond, megavolt-per-meter pulsed electric field exposure. Experimental studies utilizing the electrode microchamber include live-cell imaging of nanoelectropulse-induced intracellular calcium bursts and membrane phospholipid translocation.

[1]  J Teissié,et al.  In vitro and in vivo electric field-mediated permeabilization, gene transfer, and expression. , 2004, Methods.

[2]  Jian Zhang,et al.  Polymerization optimization of SU-8 photoresist and its applications in microfluidic systems and MEMS , 2001 .

[3]  Laura Marcu,et al.  Pulse generators for pulsed electric field exposure of biological cells and tissues , 2003 .

[4]  M. Berridge,et al.  Calcium signalling: dynamics, homeostasis and remodelling , 2003, Nature reviews. Molecular cell biology.

[5]  Warren W. Flack,et al.  The optimization and characterization of ultra-thick photoresist films , 1998 .

[6]  E. Neumann,et al.  Membrane electroporation and direct gene transfer , 1992 .

[7]  S. Simasko,et al.  Recruitment of Calcium from Intracellular Stores Does Not Occur during the Expression of Large Spontaneous Calcium Oscillations in GH3 Cells and Lactotropic Cells in Primary Culture , 2000, Neuroendocrinology.

[8]  K. Schoenbach,et al.  Nanosecond pulsed electric field (nsPEF) effects on cells and tissues: apoptosis induction and tumor growth inhibition , 2001, PPPS-2001 Pulsed Power Plasma Science 2001. 28th IEEE International Conference on Plasma Science and 13th IEEE International Pulsed Power Conference. Digest of Papers (Cat. No.01CH37251).

[9]  Laura Marcu,et al.  Ultrashort pulsed electric fields induce membrane phospholipid translocation and caspase activation: differential sensitivities of Jurkat T lymphoblasts and rat glioma C6 cells , 2003 .

[10]  E. Neumann,et al.  Gene transfer into mouse lyoma cells by electroporation in high electric fields. , 1982, The EMBO journal.

[11]  Warren W. Flack,et al.  Optimization and characterization of ultrathick photoresist films , 1998, Advanced Lithography.

[12]  Ulrich Zimmermann,et al.  Electromanipulation of cells , 1996 .

[13]  U. Zimmermann,et al.  Electrotransfection of anchorage-dependent mammalian cells. , 2003, Experimental cell research.

[14]  J Teissié,et al.  Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse. , 1999, Biophysical journal.

[15]  J Teissié,et al.  Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes. , 1998, Biophysical journal.

[16]  U. Zimmermann,et al.  Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration , 2001, The Journal of Membrane Biology.

[17]  K. Schoenbach,et al.  Intracellular effect of ultrashort electrical pulses , 2001, Bioelectromagnetics.

[18]  J Teissié,et al.  Control by electrical parameters of short- and long-term cell death resulting from electropermeabilization of Chinese hamster ovary cells. , 1995, Biochimica et biophysica acta.

[19]  J. Gehl,et al.  Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. , 2003, Acta physiologica Scandinavica.

[20]  Laura Marcu,et al.  Nanosecond pulsed electric fields perturb membrane phospholipids in T lymphoblasts , 2004, FEBS letters.

[21]  J. Teissié,et al.  Optimized conditions for electrotransformation of bacteria are related to the extent of electropermeabilization. , 1991, Biochimica et biophysica acta.

[22]  Laura Marcu,et al.  Calcium bursts induced by nanosecond electric pulses. , 2003, Biochemical and biophysical research communications.

[23]  E Neumann,et al.  Control by pulse parameters of electric field-mediated gene transfer in mammalian cells. , 1994, Biophysical journal.

[24]  Peter Lipp,et al.  Mitochondria are morphologically and functionally heterogeneous within cells , 2002, The EMBO journal.

[25]  A. Marks Intracellular calcium-release channels: regulators of cell life and death. , 1997, The American journal of physiology.