Electroporation

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Electroporation is a method of making cell membranes temporarily permeable (permeable) in order to smuggle macromolecules such as DNA or proteins into cells or tissue . Electroporation is often used in molecular biology to transfer nucleic acids into prokaryotic ( transformation ) and eukaryotic ( transfection ) cells . In the field of food and bioprocess engineering, electroporation can be used to improve mass transport processes or to inactivate microorganisms.

principle

Due to various effects , the cell membrane of cells located in the capacitor is permeabilized by an electric field , which is usually generated as a short pulse by the discharge current of a capacitor . It is controversial whether the cell membrane actually forms pores. It could depend on the field strength and duration of the pulses. For example, the conformation of membrane components can change. The constriction of membrane areas into vesicles is also observed , which can explain the import of macromolecules and organelles . The effect of electroporation was first described by Neumann in 1982. The temporary permeabilization leads to the release of intracellular components, induced by hydrostatic pressure differences ( turgor pressure ) and osmotic effects. In addition, substances from the external medium can be absorbed into the interior of the cell (dyes, DNA, ions). Electroporation is possible with all cell types, but since not all pores close again, cell viability decreases , possibly up to cell death. The transformation rate of bacteria with this method is higher than with the chemical transformation processes .

Electroporation can be used to kill microorganisms . Whether this method can be used industrially to sterilize various substances (e.g. water ) is still being discussed. The material to be treated is conveyed through a reaction space in which a pulsed electrical field is generated using one or more pairs of electrodes. The repetition rate of the pulses is adapted to the product flow. The required electrical field strength is usually in a range of 1 kV / cm for plant or animal cells or 10 to 40 kV / cm for microorganisms.

Procedure

Schematic representation of an electroporator with cuvette.
Cuvettes for electroporation with electrodes made of aluminum.

For the introduction of large molecules, e.g. B. plasmids , which cannot pass the cell membrane by themselves, are used an electroporator - a device that generates the electric field. The electroporator has space for a cuvette into which the cell suspension is pipetted . The cuvette has two electrodes .

The success rate of electroporation depends heavily on the purity of the plasmid solution; in particular, the solution must be free of salts . An impure solution can cause a small explosion during electroporation, killing the cells.

Tumor therapy

Electroporation can be used for ex vivo RNA transfection of immune cells such as dendritic cells or cytotoxic T cells. T cells can be equipped with a tumor antigen -specific T cell receptor or CAR (chimeric antigen receptor). The transfer of RNA , which codes for tumor antigens, into dendritic cells is also possible. By transferring the modified immune cells back into the patient, immune responses against the tumor should be induced. In contrast to stable transfection with DNA, RNA transfection offers a safer, transient alternative. The transfection process using electroporation is also relatively easy to implement in accordance with GMP ( good manufacturing practice ).

A new method of eliminating tumors, irreversible electroporation , is based on destroying the cells of the tissue with precisely localized electrical surges. The advantage over conventional surgery is that the intercellular matrix is spared, so that the original structure, e.g. B. of blood vessels, can be restored by the immigration of new cells.

Electric shock

In the event of accidents caused by electrical current , currents of several amperes through the body occur in the high voltage area . Very high electric field strengths occur on the cell membrane, which lead to the formation of pores. The pores can close again or enlarge and damage the cell irreversibly. Depending on the severity, this can even lead to amputation of body parts. An electric shock can affect all types of tissue.

literature

  • Bruce Alberts et al: Molecular Biology of the Cell, Fourth Edition. Taylor & Francis, 2002, ISBN 0-8153-4072-9 .
  • Ulrich Zimmermann: Electromanipulation of Cells. Crc Press, 1996, ISBN 0-8493-4476-X .

Individual evidence

  1. ↑ Shi J, Ma Y, Zhu J, Chen Y, Sun Y, Yao Y: A Review on Electroporation-Based Intracellular Delivery. . In: Molecules . 23, No. 11, 2018. doi : 10.3390 / molecules23113044 . PMID 30469344 . PMC 6278265 (free full text).
  2. ^ E. Neumann, M. Schaefer-Ridder, Y. Wang, PH Hofschneider: Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO Journal, Vol. 1 (7), 1982, pp. 841-845. PMID 6329708 ; PMC 553119 (free full text).
  3. ↑ CN Haas, D. Aturaliye: Semi-quantitative characterization of electroporation-assisted disinfection processes for inactivation of Giardia and Cryptosporidium. Journal of applied microbiology. Volume 86, Number 6, June 1999, pp. 899-905, ISSN  1364-5072 . PMID 10389240 .
  4. ↑ C. Liu, X. Xie, W. Zhao, N. Liu, PA Maraccini, LM Sassoubre, AB Boehm, Y. Cui: Conducting nanosponge electroporation for affordable and high-efficiency disinfection of bacteria and viruses in water. Nano letters. Volume 13, Number 9, September 2013, pp. 4288-4293, ISSN  1530-6992 . doi: 10.1021 / nl402053z . PMID 23987737 .
  5. ↑ LA Johnson, B. Heemskerk, DJ Powell Jr, CJ Cohen, RA Morgan, ME Dudley, PF Robbins, SA Rosenberg: Gene transfer of tumor-reactive TCR confers both high avidity and tumor reactivity to nonreactive peripheral blood mononuclear cells and tumor- infiltrating lymphocytes. J. Immunol. 177 (9), Nov 1, 2006, pp. 6548-6559.
  6. ↑ C. Krug, M. Wiesinger, H. Abken, B. Schuler-Thurner, G. Schuler, J. Dörrie, N. Schaft: A GMP-compliant protocol to expand and transfect cancer patient T cells with mRNA encoding a tumor specific chimeric antigen receptor. Cancer Immunol Immunother. 63 (10), Oct 2014, pp. 999-1008. doi: 10.1007 / s00262-014-1572-5 . Epub 2014 Jun 18.
  7. ↑ JA Kyte, G. Gaudernack: Immunogenic therapy of cancer with tumor-mRNA transfected dendritic cells. Cancer Immunol Immunother. 55 (11), Nov 2006, pp. 1432-1442. Epub 2006 Apr 13.
  8. ↑ S. Wilgenhof, J. Corthals, AM Van Nuffel, D. Benteyn, C. Heirman, A. Bonehill, K. Thielemans, B. Neyns: Long-term clinical outcome of melanoma patients treated with messenger RNA-electroporated dendritic cell therapy following complete resection of metastases. Cancer Immunol Immunother. 64 (3), Mar 2015, pp. 381–388. doi: 10.1007 / s00262-014-1642-8 . Epub 2014 Dec 30.
  9. ↑ Michael K. Stehling, Enric Günther, Boris Rubinsky: With electric shocks against cancer. Spectrum of science. April 2014, p. 40.
  10. ↑ DIN IEC / TS 60479-1 ( VDE V 0140-479-1): 2007-05 Effects of electric current on humans and livestock - Part 1: General aspects, Chap. 5.6, p. 26.