WO2003060114A2 - Procede de desintegration a l'aide de champs electriques alternatifs en continu, a modification brusque d'intensite - Google Patents

Procede de desintegration a l'aide de champs electriques alternatifs en continu, a modification brusque d'intensite Download PDF

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Publication number
WO2003060114A2
WO2003060114A2 PCT/EP2003/000128 EP0300128W WO03060114A2 WO 2003060114 A2 WO2003060114 A2 WO 2003060114A2 EP 0300128 W EP0300128 W EP 0300128W WO 03060114 A2 WO03060114 A2 WO 03060114A2
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WO
WIPO (PCT)
Prior art keywords
field strength
biological cell
cell material
substances
electric field
Prior art date
Application number
PCT/EP2003/000128
Other languages
German (de)
English (en)
Other versions
WO2003060114A3 (fr
Inventor
Frank Vitzthum
Jürgen Bernhagen
Herwig Brunner
Original Assignee
Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. filed Critical Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V.
Priority to AU2003215534A priority Critical patent/AU2003215534A1/en
Publication of WO2003060114A2 publication Critical patent/WO2003060114A2/fr
Publication of WO2003060114A3 publication Critical patent/WO2003060114A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms

Definitions

  • the present invention relates to a method for the disintegration of biological cell material for the extraction of cell contents or the introduction of active substances into biological cell material.
  • the cell walls and membranes have to be disrupted in order to be able to extract the cell contents from the cells in a suitable form.
  • disintegration can also be used to introduce active substances (xenobiotics) into biological cell material.
  • Disintegration is understood to mean a process that modifies the orderly state of the biological cell material or initiates or accompanies the modification.
  • Another non-mechanical, physical disintegration process is the use of electrical fields to disrupt biological cell material suspended in the liquid medium, for example (DE 197 52 961, PCT / DE98 / 02979, PCT / EP99 / 03047, PCT / EP99 / 03039 , DE 199 06 277, PCT / EP00 / 01201, WO97 / 08293, DE 37 33 927 AI, WO98 / 02399, WO98 / 54306).
  • pulsed electric fields with varying field strength or electric fields with constant electric field strength are used.
  • pulsed electric fields with pulse durations of a few seconds.
  • stronger fields and sufficient pulse duration especially with repeated treatment, there are permanent changes in the cell membrane, which leads to cell death, for example, in the treatment of living biological cells. This procedure is used as standard to kill microorganisms in food.
  • biological cell material consists of an electrically conductive content that is enclosed by an electrically insulating, polarizable cell membrane, the electrical charge carriers (ions) shift inside when an external electrical field is applied (polarization).
  • polarization the electrical charge carriers
  • local potential differences arise between the cell interior and the cell exterior, which lead to the cell membrane losing its semi-permeable and / or selectively permeable properties and cell contents subsequently escaping from the cell or active substances (xenobiotics) being able to penetrate into the cell interior.
  • the present invention is therefore based on the technical problem of providing a method for disintegrating biological cell material which enables the gentle extraction of cell contents or the introduction of active substances (xenobiotics) into biological cell material, the method being particularly suitable for use in simple, low-cost and space-saving electrical systems is suitable and completely or largely avoids the disadvantages mentioned above.
  • the process should be universally applicable for the disintegration of different types of biological cell material, in particular the process time for extracting and isolating the cell contents or for introducing xenobiotics should be significantly reduced. Furthermore, the efficiency of the disintegration process should be high and the cell constituents and / or the active ingredients to be introduced should be protected during its use.
  • the present invention solves this problem by providing a method for the disintegration of biological cell material, in particular for the extraction of cell contents and / or for the introduction of active substances into the biological cell material, the biological cell material having at least one electric field with con - is continuously exposed to alternating field strength with an abrupt change in the field strength and is thereby unlocked.
  • the method according to the invention is characterized in particular in that the abrupt change in field strength of at least one electrical field to which the biological cell material is exposed has a periodic rectangular or periodic triangular course. Abrupt changes in the electric field strength due to the rectangular or triangular vibrations particularly support the disintegration of the biological cell material. Abrupt and thus rapid changes in the electric field strength disintegrate the biological cell material particularly efficiently.
  • an electro-mechanical Maxwell stress advantageously occurs, which additionally effectively supports the disintegration of the biological cell material.
  • the abrupt change in field strength is generated by superimposing a periodic fundamental with higher-frequency periodic vibrations.
  • the abrupt change in field strength contains frequency components from 1 MHz to 10 GHz, preferably from 100 kHz to 1 MHz, particularly preferably from 200 kHz to 500 kHz.
  • the method according to the invention is further characterized in that the action of an electric field with a continuously alternating abrupt change in field strength as a function of Frequency, amplitude, duration of treatment and temperature lead to permanent and / or transient pores in the membrane of the biological cell material, wherein the pores can have different diameters.
  • the pores formed Through the pores formed, extracellular substances can get into the biological cell material and / or, conversely, intracellular cell contents can be released.
  • the release or infiltration of substances depends on the strength and type of interaction between these substances and / or between the substances and the biological cell material, and on the service life and the diameter of the pores formed.
  • the pores formed by the action of the continuously alternating electric field need not necessarily have a size similar to that of the substances to be extracted or introduced.
  • the disintegrated membrane of the biological cell material is disintegrated to a high degree by the action of the continuously alternating electric field, so that the cell membrane is irreversible and in particular completely is constantly dissolved and the cell contents are completely released into the medium.
  • the biological cell material is in a liquid or gel-like medium which is characterized in its physicochemical property as an electrolyte and which is electrically conductive.
  • the medium preferably has a specific electrical resistance of 0.1 ⁇ S / cm to 1 ⁇ S / cm, preferably 0.1 mS / cm to 100 mS / cm, particularly preferably 1 mS / cm to 10 mS / cm.
  • the medium is gaseous or essentially solid.
  • the medium is characterized in that it is not an electrolyte.
  • the biological cell material is brought into contact with substances before or during the disassembly which support the digestion and / or the cell Stabilize substances and / or stabilize the active substances to be introduced and / or introduced into the biological cell material.
  • Substances with these functions are, for example, detergents, proteins, organic solvents, reducing agents, complexing agents or chaotropic substances.
  • the detergents are preferably sodium dodecyl sulfate (SDS), Tween 20 or Triton X100.
  • the proteins are preferably enzymes from the group of hydrolases, particularly preferably proteases or lipases.
  • the organic solvents are preferably dimethyl sulfoxide (DMSO), glycerol, ethanol or phenol.
  • the reducing agents are preferably ⁇ -mercaptoethanol, diethotholeol, lipoic acid or glutathione.
  • the complexing agents are preferably ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • the chaotropic substances are preferably urea and / or guanidium salts.
  • these substances are used in particular by acting on the biological cell material in such a way that the cell content is released more easily and quickly from inside the biological cell material.
  • these substances diffuse through the electrical treatment of pores in the membrane into the interior of the cell, break down proteins, for example, or specifically cut DNA molecules into smaller fragments. The resulting proteins or DNA fragments can then diffuse freely through pores to the outside.
  • a further mode of action of the substances is that the transient or permanent pores in the membrane of biological cell material that are created by the electrical treatment according to the invention are attacked by the substances. This can lead to an enlargement of the pores, which facilitates the passage of cell contents from the cell interior or the introduction of active substances (xenobiotics) from the outside into the cell interior.
  • the substances used lead to the breakdown of the cell membrane or cell wall of the biological cell material.
  • the biological cell material is exposed to at least one continuously alternating electrical field, in order to achieve these desired effects, lower concentrations of the abovementioned substances which come into contact with the biological cell material are necessary in comparison with the prior art. This advantageously prevents the substances used from reacting with the cell contents to be extracted or with the active substances to be introduced.
  • the voltage on which the continuously alternating field strength is based is selected in its time-varying course in such a way that the current flow it inscribes is symmetrical with respect to its zero crossing. This means that no net load is transported across the field over the course of time. This reduces electrochemical processes on the electrodes of the apparatus used for disintegration in a particularly advantageous manner and minimizes the contamination of the medium with electrolysis products.
  • the cell contents preferably leave the cell interior of the biological cell material due to diffusion and / or due to osmotic processes. Active ingredients (xenobiotics) are preferably introduced using the same mechanisms.
  • the time course of the continuously alternating field strength is characterized in that a net charge is transported in a directional manner over the field on average.
  • a net charge is transported in a directional manner over the field on average.
  • the current flow described is asymmetrical with respect to its zero crossing over time.
  • the directional transport of a net charge is achieved by superimposing at least one electric field with a constant electric field strength and a constant inscribed electric current on an electric field with a continuously alternating field strength with a symmetrical current profile.
  • At least two electrical fields with parallel field vectors are superimposed.
  • a two-dimensional arrangement is essentially assumed, with at least two overlapping electric fields in one variant having field vectors running perpendicularly or approximately perpendicularly to one another.
  • the biological cell material in the medium can be exposed to an electrical field with continuously alternating field strengths, for example to enable disintegration, and at the same time to an electrical field running perpendicular or approximately perpendicular thereto, which has a constant field strength that iontophoretic introduction of active substances enables or the electrophoretic separation of the extracted cell contents according to their electrical-physical properties (for example, isoelectric focusing, zone electrophoresis, pulse field electrophoresis).
  • the at least two overlapping electrical fields have field vectors which run at any angle to one another.
  • essentially a three-dimensional arrangement is used, the at least two superimposed electrical fields in space have perpendicular or approximately perpendicular field vectors.
  • the amplitude of the continuously alternating field strength is slowly or continuously changed over time.
  • the frequency of the periodic fundamental oscillation of the continuously alternating field strength is slowly or continuously changed over time.
  • the method according to the invention is further characterized in that the biological cell material is exposed to the electrical fields for a treatment period of 1 ms to 48 h, preferably from 1 s to 1 h, particularly preferably from 10 s to 30 min.
  • the biological cell material to be disintegrated is replaced by an electrical one Field heated with continuously alternating field strength.
  • the biological cell material to be disintegrated is tempered in the medium such that there is no measurable increase in temperature due to the action of the electric field.
  • sensitive cell constituents to be extracted or active substances to be introduced are particularly protected against degradation (for example by hydrolysis, oxidation, denaturation).
  • the biological cell material is tempered in such a way that a temperature of -196 ° C. to 200 ° C., in particular from -20 ° C. to 95 ° C., preferably from 4 ° C. to, results from the action of the electric field 80 ° C, particularly preferably from 40 ° C to 79 ° C.
  • a temperature of -196 ° C. to 200 ° C. in particular from -20 ° C. to 95 ° C., preferably from 4 ° C. to, results from the action of the electric field 80 ° C, particularly preferably from 40 ° C to 79 ° C.
  • an increase in the temperature level compared to room temperature can directly promote the extraction of cell contents or the introduction of active substances into the cell interior, without bringing the biological cell material into contact with additional substances.
  • it can be achieved by vigorous cooling that the continuous Nuely alternating fields with abrupt changes in field strength, electro-mechanically acting Maxwell stress are particularly effective in supporting the disintegration of the biological cell material.
  • the method according to the invention is characterized in that the pH of a preferably liquid or gel-like medium in which the biological cell material is suspended is pH -0.3 to pH 14.2, preferably pH 2 to pH 12, particularly preferred pH 6 to pH 9.
  • the acidification of the medium with, for example, 2 normal acids effectively supports the disintegration of the biological cell material.
  • process conditions can be created in detail that optimize with the aim of an automated, possibly miniaturized and routine use of the disintegration process according to the invention enable.
  • cell constituents are understood to mean all substances which can be extracted from the interior of the biological cell material by means of the method according to the invention, in particular nucleic acids, proteins, carbohydrates or other extractable molecules and structures from the cell interior.
  • active substances and “xenobiotics” are understood to mean all substances which can be introduced into the interior of the biological cell material by means of the method according to the invention, in particular nucleic acids, proteins, carbohydrates or other chemical molecules with a biological effect.
  • the expression “continuously alternating” is understood to mean a periodic process over time, with more than ten, preferably more than a hundred, particularly preferably more than a thousand, changes in consecutive succession in amplitude, period duration and phase position.
  • 1A to F show preferred exemplary embodiments of the electrical fields used according to the invention.
  • 1A and 1B exemplarily show the rectangular or triangular field strength profiles according to the invention in the medium.
  • 1 C and 1 D show quasi-rectangular or quasi-triangular field strength profiles, in FIGS. 1 E and 1 F the amplitudes of the abrupt changes in field strength are shown by way of example, which change in field strength profiles according to FIGS. 1 C and 1 D result.
  • FIG. 2 shows schematically the potential curves along the extent of an individual cell of the biological cell material, on the left, when treated with the method according to the invention, on the right, when treating according to the prior art.
  • biological cell material is exposed to continuously alternating fields with an abrupt change in field strength in an electrically conductive, liquid medium.
  • 1A, 1B, 1C and 1D show the embodiments of the continuously alternating electric fields used according to the invention. Five different approaches are followed, with in A) the biological cell material is continuously exposed to alternating fields with a rectangular shape, in
  • the conductivity factor (r " ( ⁇ m)) is a function of the cell radius (r z ), the electrical cell membrane thickness (d ZM ), the specific conductivities of the membrane ( ⁇ m ), the cell interior ( ⁇ j . ) And the external solution ( ⁇ ⁇ ). Part 2 )
  • substances such as detergents, proteins, organic solvents, reducing agents, complexing agents and / or chaotropic substances are introduced either individually or in combination and / or the pH in the medium is shifted away from the neutral point and / or becomes the temperature of the medium varies.
  • the intrinsic membrane potential difference ( ⁇ -, i.e. the total potential difference across the membrane of the biological cell material including that of the natural membrane ran potential and the surface membrane potential, from equation 2.
  • ⁇ ⁇ p m When ⁇ ⁇ p m reaches a critical value ⁇ c , which is usually 1 V, the lipid layer of the biological cell material breaks down electrically, forming pores in the cell membrane. Since the effective electrical thickness of the membrane of biological cell material is usually in the range of 5-10 nm, ⁇ c corresponds to an electrical field in the cell membrane of over 1 MV / cm.
  • continuously alternating fields are used, a continuous low-frequency fundamental oscillation being overlaid with higher-frequency oscillations.
  • This exemplary embodiment corresponds to example 1, but, according to the invention, high electrical field strengths are additionally used to disintegrate the biological cell material. In this way, rapid changes in voltage are generated in order to achieve high short-term changes in electrical field strength.
  • FIG. 2 shows the treatment according to the invention of a biological cell (1) whose plasma membrane is represented by two concentric circles: a) In the treatment according to the invention, a potential curve develops over the cross-section of the cell, the induced membrane potential ( ⁇ ⁇ p nc ⁇ ) being significantly greater than in the disintegration methods according to the prior art.

Abstract

L'invention concerne un procédé de désintégration de matériau cellulaire biologique pour extraire des substances cellulaires ou transférer des principes actifs dans le matériau cellulaire biologique à l'aide de champs alternatifs en continu, à modification brusque d'intensité.
PCT/EP2003/000128 2002-01-15 2003-01-09 Procede de desintegration a l'aide de champs electriques alternatifs en continu, a modification brusque d'intensite WO2003060114A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003215534A AU2003215534A1 (en) 2002-01-15 2003-01-09 Disruption method by means of continuously alternating electric fields with abrupt field strength changes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE10201174.5 2002-01-15
DE2002101174 DE10201174A1 (de) 2002-01-15 2002-01-15 Desintegrationsverfahren mittels kontinuierlich alternierender elektrischer Felder mit abrupter Feldstärkeänderung

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WO2003060114A2 true WO2003060114A2 (fr) 2003-07-24
WO2003060114A3 WO2003060114A3 (fr) 2003-12-31

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DE (1) DE10201174A1 (fr)
WO (1) WO2003060114A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE202011004177U1 (de) * 2011-03-18 2012-06-25 Hugo Vogelsang Maschinenbau Gmbh Vorrichtung zur elektrischen Desintegration
WO2014195343A1 (fr) * 2013-06-04 2014-12-11 Hugo Vogelsang Maschinenbau Gmbh Dispositif de désintégration électrique de groupes de cellules

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999062592A1 (fr) * 1998-06-03 1999-12-09 Genetronics, Inc. TECHNIQUE D'ELECTROPORATION A FLUX TRAVERSANT POUR THERAPIE GENIQUE $i(EX VIVO)
US6043066A (en) * 1997-09-04 2000-03-28 Mangano; Joseph A. Cell separation using electric fields
WO2000049173A2 (fr) * 1999-02-15 2000-08-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Procede et systeme de support d'echantillon pour la separation et l'enrichissement de substances in situ
US6117660A (en) * 1997-06-10 2000-09-12 Cytopulse Sciences, Inc. Method and apparatus for treating materials with electrical fields having varying orientations

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117660A (en) * 1997-06-10 2000-09-12 Cytopulse Sciences, Inc. Method and apparatus for treating materials with electrical fields having varying orientations
US6043066A (en) * 1997-09-04 2000-03-28 Mangano; Joseph A. Cell separation using electric fields
WO1999062592A1 (fr) * 1998-06-03 1999-12-09 Genetronics, Inc. TECHNIQUE D'ELECTROPORATION A FLUX TRAVERSANT POUR THERAPIE GENIQUE $i(EX VIVO)
WO2000049173A2 (fr) * 1999-02-15 2000-08-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Procede et systeme de support d'echantillon pour la separation et l'enrichissement de substances in situ

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE202011004177U1 (de) * 2011-03-18 2012-06-25 Hugo Vogelsang Maschinenbau Gmbh Vorrichtung zur elektrischen Desintegration
WO2014195343A1 (fr) * 2013-06-04 2014-12-11 Hugo Vogelsang Maschinenbau Gmbh Dispositif de désintégration électrique de groupes de cellules

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DE10201174A1 (de) 2003-07-31
AU2003215534A1 (en) 2003-07-30
WO2003060114A3 (fr) 2003-12-31

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