WO1991018103A1 - Method and device for making living cells permeable - Google Patents

Method and device for making living cells permeable Download PDF

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Publication number
WO1991018103A1
WO1991018103A1 PCT/BE1991/000030 BE9100030W WO9118103A1 WO 1991018103 A1 WO1991018103 A1 WO 1991018103A1 BE 9100030 W BE9100030 W BE 9100030W WO 9118103 A1 WO9118103 A1 WO 9118103A1
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WIPO (PCT)
Prior art keywords
discharge
cells
duration
short
pulse
Prior art date
Application number
PCT/BE1991/000030
Other languages
French (fr)
Inventor
Christian Servais
Joël LOUETTE
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Scientific Equipment Design & Development S.C.
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Application filed by Scientific Equipment Design & Development S.C. filed Critical Scientific Equipment Design & Development S.C.
Publication of WO1991018103A1 publication Critical patent/WO1991018103A1/en

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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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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

Definitions

  • the present invention relates to this field and relates more particularly to a process for permeabilization of living cell membranes as well as to a device for implementing this process.
  • State of the art relates to this field and relates more particularly to a process for permeabilization of living cell membranes as well as to a device for implementing this process.
  • US Pat. No. 4,663,292 (WONG et al.) Describes a system capable of supplying trains of square pulses of high voltage and of short duration, identical, and of applying them to a suspension containing cells and biological macromo ⁇ molecules such as genes. Where appropriate, the application of the discharge train is preceded by the application to the suspension of a non-uniform high-frequency electric field, which cannot be assimilated to a pulse train and which is known per se. There is no direct contact between the discharge electrode and the suspension, according to the technique described in this document. Purpose of the invention
  • the present invention aims to provide a process for permeabilization of cell membranes which does not have the drawbacks of the state of the art and which makes it possible to improve the performance of manipulations by increasing the rate of pierced cells and / or cell survival. and / or the entry of foreign molecules into cells.
  • Another object of the present invention consists in providing a device allowing the implementation of the method. Essential elements of the invention
  • a suspension containing the cells to be made permeable as well as the molecules intended to be housed inside the cells successively at least one short-term electric discharge generating in the suspension an electric field of high value and then at least one electric discharge of longer duration and generating in the suspen ⁇ sion an electric field of lower value.
  • the short-duration discharge consists of an exponentially decreasing pulse, in particular obtained by the discharge of a capacitor. It can also consist of one or more pulses of the rectangular type.
  • the values of the electric field generated by the short-term discharge can reach 400 to 25,000 V / cm depending on the duration of the pulse and the type of cells to be treated, the duration being of the order of 10 ⁇ s to 10 s .
  • the characteristics of the short-term discharge are selected so that the electric field generated by it is understood:
  • the long-term discharge can advantageously consist of an essentially continuous voltage, for example supplied by a low-voltage generator.
  • the electric field generated can be of the order of 50 to 1000 V / cm, depending on the duration of the pulse and the type of cells to be treated.
  • the voltage is generally applied for a cumulative time of the order of 1 ms to 1 s.
  • the long duration pulse is possibly an expo ⁇ nential decay pulse.
  • the first discharge and / or the second discharge may consist of a train of waves.
  • provision can also be made to swap the polarity of the potential difference across the solution during discharge.
  • the reversal of polarity during the long-term discharge can cause back and forth movements of the molecule, for example DNA, to be introduced into the cells, which increases the chances of penetration. in the cell.
  • the process of the present invention makes it possible to transform cell strains which we had not yet managed to transform until now. Experiments on human lymphocytes and on certain yeast strains have been particularly conclusive.
  • the penetration rate can be further increased by successively carrying out several steps in accordance with the invention.
  • the device which allows the implementation of the method of the invention comprises at least one high voltage source and one low voltage source, the voltage released by one and the other being alternately placed at the terminals of electrodes adapted to a well known per se intended to contain the suspension.
  • the high voltage source can advantageously consist of a capacitor associated with a charging circuit; it can also be a high voltage generator.
  • the low voltage source can for example be a low voltage generator, or a capacitor associated with a charging circuit.
  • the voltage and the time constant of the capacitor (s) are programmable.
  • the method and the device of the invention are particularly suitable for permeabilization of cell walls in order to introduce molecules such as DNA, proteins, antibodies, drugs or other chemicals.
  • Another application lies in the extraction of molecules, in particular DNA, from cells made permeable. This technique makes it possible in particular to obtain "clean" DNA, greatly simplifying the purification steps later.
  • FIG. 1 is a schematic representation of an embodiment of a device for permeability of living cells according to the invention.
  • This comprises a capacitor 1 associated with a charging circuit 3.
  • a switching device 2 (of the relay, thyristor, GTO or assimilated) puts it in contact with an electroporation dish 7 containing, in suspension, living cells and biological macromolecules.
  • the first discharge is then applied.
  • the device 2 switches to the open position and another switching device 4, of the same type, then brings the low-voltage generator 5 into contact with the electroporation cuvette.
  • the second discharge can then take place. Its characteristics (shape, amplitude, duration) are also determined by the control circuit 11.
  • the switching device 4 At the end of the second discharge, the switching device 4 returns to the open state and the device of the invention is ready for a new electroporation.
  • a shunting device 9 controlled by the control circuit 11 is placed in parallel on the electrolytic basin 7 to allow the determination of the discharge time constant of the capacitor 1.
  • the device of the invention does not include a condenser, nor a charging circuit.
  • a high voltage generator generates the first discharge, of short duration, which is then of the rectangular type.
  • the switching device 2 remains permanently closed during each discharge of the rectangular type.
  • the second discharge may also be of decreasing exponential type, in which case the low voltage generator will be replaced by a circuit assembly. load and capacitor, the latter's charge value also being determined by the control circuit.
  • the values of the various parameters are adjustable and programmable.
  • a capacitor can be replaced by several capacitors of different capacities selected by the control circuit 11 mounted in parallel.
  • the invention is described in more detail below using examples of cell permeabilization carried out using a device according to the invention.
  • Example 1 Permeabilization of rat pituitary cells Cligne GH. GC. GH3b6. GH4,
  • the cells are prepared as for a conventional electropora ⁇ tion. They are concentrated at 4 million cells / 800 ⁇ l, in the usual culture medium for these cells. DNA is added, from 3 ⁇ g to 100 ⁇ g depending on the plasmid or the DNA fragment used. 800 ⁇ l of the mixture are placed in a conventional 4 mm electroporation cuvette. A first discharge of 300 V (750 V / cm) is applied, with a shunt resistance of 74 ⁇ and a capacitor of 40 ⁇ F. Then apply a second discharge of 100 V (250 V / cm) with a shunt resistance of 132 ⁇ and a capacitor of 1800 ⁇ F. The cells are immediately transferred to fresh culture medium. By the enzymatic measurement method "CAT", a response 10 to 100 times greater is obtained compared to the state of the art.
  • Example 2 By the enzymatic measurement method "CAT", a response 10 to 100 times greater is obtained compared to the state of the art.
  • ÎO 11 transformants / ⁇ g DNA that is to say up to 10 times more than according to the state of the art.
  • 10 * to 10 ⁇ transformants / ⁇ g DNA are obtained, i.e. up to

Abstract

A method and a device for making cell membranes permeable using electrical discharges (electroporation), wherein at least one short electrical discharge is applied to a suspension containing cells to be made permeable and molecules to be accommodated therein, in order to generate in said suspension a high-value electric field, whereafter at least one longer electrical discharge is applied thereto to generate a lower-value electric field therein.

Description

PROCÉDÉ ET DISPOSITIF DE PERMÉABILISATION DE CELLULES VIVANTES Objet de 1/inventionMETHOD AND DEVICE FOR PERMEATING LIVE CELLS Object of 1 / invention
Le transfert de matériel génétique nouveau ainsi que de protéines étrangères ou autres molécules ou macro- molécules dans des cellules vivantes a gagné en importance depuis les derniers développements en biochimie et notamment en génétique.The transfer of new genetic material as well as foreign proteins or other molecules or macro-molecules into living cells has gained importance since the last developments in biochemistry and in particular in genetics.
La présente invention est relative à ce domaine et concerne plus particulièrement un procédé de perméabilisation des membranes de cellules vivantes ainsi qu'un dispositif pour la mise en oeuvre de ce procédé. Etat de la techniqueThe present invention relates to this field and relates more particularly to a process for permeabilization of living cell membranes as well as to a device for implementing this process. State of the art
Parmi les différentes méthodes destinées à rendre les membranes cellulaires perméables, de façon à permettre l'introduction de "corps étrangers" on connait notamment les techniques de perméabilisation par chocs électriques, couram¬ ment dénommées "électroporation".Among the various methods intended to make the cell membranes permeable, so as to allow the introduction of "foreign bodies", the techniques for permeabilization by electric shocks, commonly known as "electroporation", are known.
L'article "Transformation of bacteria by électro¬ poration" de B.M. Chassy et al., Trends in Biotechnology, vol. 6 , n° 12, décembre 1988, pages 303-309 décrit les prin¬ cipes de base utilisés dans les méthodes classiques d'élec¬ troporation: il mentionne des expériences de transformation par électroporation qui ont été réalisées antérieurement sur des cellules eucaryotiques et procaryotiques. On utilise habituellement une impulsion ou une série d'impulsions identiques, le plus souvent de haut vol¬ tage (en règle générale, des impulsions générant un champ électrique de 2 à 12 kV/c ) . Les décharges appliquées aux cellules sont soit une décharge unique, soit un train d'ondes identiques de type exponentiel décroissant ou carré par exemple.The article "Transformation of bacteria by electroporation" by BM Chassy et al., Trends in Biotechnology, vol. 6, No. 12, December 1988, pages 303-309 describes the basic principles used in conventional methods of electroporation: he mentions experiments of transformation by electroporation which have been carried out previously on eukaryotic and prokaryotic cells . Usually an identical pulse or series of pulses is used, most often of high vol¬ tage (as a general rule, pulses generating an electric field of 2 to 12 kV / c). The discharges applied to cells are either a single discharge or a train of identical waves of decreasing or square exponential type for example.
Une illustration d'une technique d'électroporation de ce type, appliquée à des cellules eucaryotiques est donnée dans l'article "Enhancer-dependent expression of human immu- noglobulin gènes introduced into mouse pre-B lymphocytes by électroporation" de H. POTTER et al., Proc. Natl. Acad. Sci -An illustration of an electroporation technique of this type, applied to eukaryotic cells is given in the article "Enhancer-dependent expression of human immunoglobulin genes introduced into mouse pre-B lymphocytes by electroporation" by H. POTTER and al., Proc. Natl. Acad. Sci -
USA, Vol. 81, pages 7161-7165, Novembre 1984, qui décrit une méthode dans laquelle l'application d'un choc électrique de 2 à 4 kV dans une suspension contenant de l'ADN clone dans un vecteur plasmidien et des cellules urines a permis d'ob¬ tenir la transfection de gènes dans ces cellules.USA, Vol. 81, pages 7161-7165, November 1984, which describes a method in which the application of an electric shock of 2 to 4 kV in a suspension containing DNA cloned into a plasmid vector and urine cells has made it possible to Obtain the transfection of genes in these cells.
Par l'article "Electric shock-mediated transfection of cells", Biochem. J. (1988) 251, 427-434, de David J. Winterbourne et al., il est connu d'appliquer des impulsions de durée variable à une solution contenant les cellules à percer ainsi que de l'ADN à faire pénétrer dans lesdites cellules. On y mentionne notamment des impulsions du type rectangulaire créant un champ électrique de l'ordre de 2,5 à 3 kV/cm. Il y a également été montré une corrélation entre la durée des impulsions et l'efficacité de l'opération.By the article "Electric shock-mediated transfection of cells", Biochem. J. (1988) 251, 427-434, from David J. Winterbourne et al., It is known to apply pulses of variable duration to a solution containing the cells to be pierced as well as DNA to be penetrated into said cells. Mention is made in particular of pulses of the rectangular type creating an electric field of the order of 2.5 to 3 kV / cm. There has also been shown a correlation between the duration of the pulses and the effectiveness of the operation.
Toutefois, l'application d'un champ électrique entraine une dissipation d'énergie importante dans la solu¬ tion qui a pour résultat une mortalité élevée des cellules. L'article "High efficiency transformation of E. coli by high voltage électroporation", Nucleic Acids Research (1988), volume 16, nβ 13, 6127-6144, de William J. Dower et al. et le brevet US-A-4 910 140 mentionnent des essais faisant appel à une électroporation à l'aide d'impul¬ sions de courte durée. On y atteint des résultats satisfai¬ sants avec des champs électriques allant jusqu'à 12,5 kV/c et 16,7 kV/cm et une corrélation entre la valeur du champ électrique et la durée des impulsions est également établie.However, the application of an electric field results in significant energy dissipation in the solution which results in high cell mortality. The article "High efficiency transformation of E. coli by high voltage electroporation", Nucleic Acids Research (1988), volume 16, n β 13, 6127-6144, by William J. Dower et al. and US Pat. No. 4,910,140 mention tests using electroporation using short duration pulses. Satisfactory results are achieved with electric fields up to 12.5 kV / c and 16.7 kV / cm and a correlation between the value of the electric field and the duration of the pulses is also established.
Bien que l'énergie dissipée dans la solution soit réduite, l'efficacité de l'opération est limitée par une mortalité des cellules due à l'éclatement suite à des chocs électriques trop importants.Although the energy dissipated in the solution is reduced, the efficiency of the operation is limited by cell mortality due to bursting following shocks electrical too large.
Le brevet US-A-4 663 292 (WONG et al.) décrit un système apte à fournir des trains d'impulsions carrées de haut voltage et de courte durée, identiques, et à les appli- quer à une suspension contenant des cellules et des macromo¬ lécules biologiques telles que des gènes. Le cas échéant, l'application du train de décharges est précédée par l'appli¬ cation à la suspension d'un champ électrique non uniforme de haute fréquence, non assimilable à un train d'impulsions et connue en soi par ailleurs. Il n'y a pas de contact direct entre l'électrode de décharge et la suspension, selon la technique décrite dans ce document. But de l'inventionUS Pat. No. 4,663,292 (WONG et al.) Describes a system capable of supplying trains of square pulses of high voltage and of short duration, identical, and of applying them to a suspension containing cells and biological macromo¬ molecules such as genes. Where appropriate, the application of the discharge train is preceded by the application to the suspension of a non-uniform high-frequency electric field, which cannot be assimilated to a pulse train and which is known per se. There is no direct contact between the discharge electrode and the suspension, according to the technique described in this document. Purpose of the invention
La présente invention vise à fournir un procédé de perméabilisation des membranes cellulaires qui ne présente pas les inconvénients de l'état de la technique et qui permet d'améliorer le rendement des manipulations en augmentant le taux de cellules percées et/ou la survie des cellules et/ou l'entrée des molécules étrangères dans les cellules. Un autre but de la présente invention consiste à fournir un dispositif permettant la mise en oeuvre du procédé. Eléments essentiels de l'inventionThe present invention aims to provide a process for permeabilization of cell membranes which does not have the drawbacks of the state of the art and which makes it possible to improve the performance of manipulations by increasing the rate of pierced cells and / or cell survival. and / or the entry of foreign molecules into cells. Another object of the present invention consists in providing a device allowing the implementation of the method. Essential elements of the invention
Conformément à la présente invention, on applique à une suspension contenant les cellules à rendre perméables ainsi que les molécules destinées à être logées à l'intérieur des cellules, successivement au moins une décharge électrique de courte durée engendrant dans la suspension un champ élec¬ trique de valeur élevée et ensuite au moins une décharge électrique de plus longue durée et engendrant dans la suspen¬ sion un champ électrique de valeur moins élevée.In accordance with the present invention, there is applied to a suspension containing the cells to be made permeable as well as the molecules intended to be housed inside the cells, successively at least one short-term electric discharge generating in the suspension an electric field of high value and then at least one electric discharge of longer duration and generating in the suspen¬ sion an electric field of lower value.
On a constaté que cette façon de procéder permet de réduire le taux de mortalité des cellules tout en augmen¬ tant le taux de pénétration de celles-ci. II est bien entendu que les deux étapes de l'inven¬ tion doivent se suivre dans un laps de temps très court. En effet, plus le laps de temps séparant les deux étapes est important, plus le taux de mortalité des cellules est élevé et plus le taux de transformation est faible. On a constaté qu'un temps d'environ 10 secondes constitue un seuil à ne pas dépasser.It has been found that this procedure makes it possible to reduce the rate of cell death while increasing the rate of penetration of the latter. It is understood that the two stages of the invention must follow each other in a very short period of time. The longer the time between the two stages, the higher the cell mortality rate and the lower the transformation rate. It has been found that a time of approximately 10 seconds constitutes a threshold not to be exceeded.
Avantageusement, la décharge de courte durée consiste en une impulsion à décroissance exponentielle, notamment obtenue par la décharge d'un condensateur. Elle peut également consister en une ou plusieurs impulsions du type rectangulaire.Advantageously, the short-duration discharge consists of an exponentially decreasing pulse, in particular obtained by the discharge of a capacitor. It can also consist of one or more pulses of the rectangular type.
Les valeurs du champ électrique engendré par la décharge de courte durée peuvent atteindre 400 à 25000 V/cm en fonction de la durée de l'impulsion et du type de cellules à traiter, la durée étant de l'ordre de 10 μs à 10 s.The values of the electric field generated by the short-term discharge can reach 400 to 25,000 V / cm depending on the duration of the pulse and the type of cells to be treated, the duration being of the order of 10 μs to 10 s .
En règle générale, on sélectionne les caractéristi¬ ques de la décharge de courte durée de manière telle que le champ électrique engendré par celle-ci soit compris:As a general rule, the characteristics of the short-term discharge are selected so that the electric field generated by it is understood:
- entre 400 et 2500 V/cm lorsqu'on traite des cellules euca- ryotiques provenant d'organismes pluricellulaires;- between 400 and 2500 V / cm when treating eukaryotic cells originating from multicellular organisms;
- entre 2000 et 5000 V/cm lorsqu'on traite des cellules eucaryotiques unicellulaires telles que des levures, et - entre 10000 et 25000 V/cm lorsqu'on traite des cellules procaryotiques.- between 2000 and 5000 V / cm when treating unicellular eukaryotic cells such as yeasts, and - between 10000 and 25000 V / cm when treating prokaryotic cells.
Par ailleurs, la décharge de longue durée peut avantageusement consister en une tension essentiellement continue, par exemple fournie par un générateur basse tension. Le champ électrique engendré peut être de l'ordre de 50 à 1000 V/cm, en fonction de la durée de l'impulsion et du type de cellules à traiter. La tension est généralement appliquée pendant un temps cumulé de l'ordre de 1 ms à 1 s. Selon une autre variante, l'impulsion de longue durée est éventuellement une impulsion à décroissance expo¬ nentielle.Furthermore, the long-term discharge can advantageously consist of an essentially continuous voltage, for example supplied by a low-voltage generator. The electric field generated can be of the order of 50 to 1000 V / cm, depending on the duration of the pulse and the type of cells to be treated. The voltage is generally applied for a cumulative time of the order of 1 ms to 1 s. According to another variant, the long duration pulse is possibly an expo¬ nential decay pulse.
Selon une forme d'exécution préférée, la première décharge et/ou la seconde décharge peuvent consister en un train d'ondes. Dans ce cas, on peut encore prévoir de permu- ter la polarité de la différence de potentiel aux bornes de la solution au cours de la décharge.According to a preferred embodiment, the first discharge and / or the second discharge may consist of a train of waves. In this case, provision can also be made to swap the polarity of the potential difference across the solution during discharge.
Pour ce qui concerne la décharge de courte durée, un effet de résonance dû à l'inversion de la polarité peut augmenter la création de pores dans la paroi cellulaire.With regard to the short-term discharge, a resonance effect due to the reversal of the polarity can increase the creation of pores in the cell wall.
D'autre part, l'inversion de polarité lors de la décharge de longue durée peut provoquer des mouvements de va- et-vient de la molécule, par exemple l'ADN, à introduire dans les cellules, ce qui augmente les chances de pénétration dans la cellule.On the other hand, the reversal of polarity during the long-term discharge can cause back and forth movements of the molecule, for example DNA, to be introduced into the cells, which increases the chances of penetration. in the cell.
Le procédé de la présente invention permet de transformer des souches cellulaires que l'on n'était pas encore arrivé à transformer jusqu'à présent. Des expériences sur lymphocytes humains et sur certaines souches de levure ont été particulièrement concluantes.The process of the present invention makes it possible to transform cell strains which we had not yet managed to transform until now. Experiments on human lymphocytes and on certain yeast strains have been particularly conclusive.
Dans la mesure où la durée de vie des cellules dans le milieu considéré le permet, on peut encore augmenter le taux de pénétration en effectuant successivement plusieurs démarches conformes à l'invention.Insofar as the lifetime of the cells in the medium in question allows, the penetration rate can be further increased by successively carrying out several steps in accordance with the invention.
Le dispositif qui permet la mise en oeuvre du procédé de l'invention comporte au moins une source de haute tension et une source de basse tension, la tension dégagée par l'une et l'autre étant alternativement mise aux bornes d'électrodes adaptées à une cuvette connue en soi destinée à contenir la suspension.The device which allows the implementation of the method of the invention comprises at least one high voltage source and one low voltage source, the voltage released by one and the other being alternately placed at the terminals of electrodes adapted to a well known per se intended to contain the suspension.
La source de haute tension peut avantageusement consister en un condensateur associé à un circuit de charge; il peut également s'agir d'un générateur haute tension. La source de basse tension quant à elle peut être par exemple un générateur basse tension, ou encore un conden¬ sateur associé à un circuit de charge.The high voltage source can advantageously consist of a capacitor associated with a charging circuit; it can also be a high voltage generator. The low voltage source can for example be a low voltage generator, or a capacitor associated with a charging circuit.
D'une manière préférée, la tension et la constante de temps du ou des condensateur(s) sont programmables. Le procédé ainsi que le dispositif de l'invention conviennent particulièrement bien pour la perméabilisation de parois cellulaires afin d'y introduire des molécules telles que l'ADN, des protéines, des anticorps, des drogues ou autres produits chimiques. Une autre application réside dans l'extraction de molécules, notamment de l'ADN, de cellules rendues perméa¬ bles. Cette technique permet notamment d'obtenir de l'ADN "propre", simplifiant grandement les étapes de purification ultérieure.Preferably, the voltage and the time constant of the capacitor (s) are programmable. The method and the device of the invention are particularly suitable for permeabilization of cell walls in order to introduce molecules such as DNA, proteins, antibodies, drugs or other chemicals. Another application lies in the extraction of molecules, in particular DNA, from cells made permeable. This technique makes it possible in particular to obtain "clean" DNA, greatly simplifying the purification steps later.
On peut encore trouver une application du procédé de l'invention dans la modification de cellules et éventuel¬ lement dans la fusion de celles-ci. La figure 1 est une représentation schématique d'une forme d'exécution d'un dispositif permettant la perméa¬ bilisation de cellules vivantes selon l'invention. Celui-ci comporte un condensateur 1 associé à un circuit de charge 3. Une fois que ce condensateur 1 est chargé à une valeur pré- déterminée par le circuit de contrôle 11, un dispositif de commutation 2 (de type relais, thyristor, GTO ou assimilé) le met en contact avec une cuvette d'électroporation 7 conte¬ nant, en suspension, des cellules vivantes et des macromolé¬ cules biologiques. La première décharge est alors appliquée. A la fin de cette décharge, le dispositif 2 passe en position ouverte et un autre dispositif de commutation 4, de même type, met ensuite en contact le générateur basse- tension 5 avec la cuvette d'électroporation. La deuxième décharge peut alors avoir lieu. Ses caractéristiques (forme, amplitude, durée) sont aussi déterminés par le circuit de contrôle 11.One can also find an application of the method of the invention in the modification of cells and possibly in the fusion of these. Figure 1 is a schematic representation of an embodiment of a device for permeability of living cells according to the invention. This comprises a capacitor 1 associated with a charging circuit 3. Once this capacitor 1 is charged to a value predetermined by the control circuit 11, a switching device 2 (of the relay, thyristor, GTO or assimilated) puts it in contact with an electroporation dish 7 containing, in suspension, living cells and biological macromolecules. The first discharge is then applied. At the end of this discharge, the device 2 switches to the open position and another switching device 4, of the same type, then brings the low-voltage generator 5 into contact with the electroporation cuvette. The second discharge can then take place. Its characteristics (shape, amplitude, duration) are also determined by the control circuit 11.
A la fin de la seconde décharge, le dispositif de commutation 4 retourne à l'état ouvert et le dispositif de l'invention est prêt pour une nouvelle électroporation. Un dispositif de shuntage 9 commandé par le circuit de contrôle 11 est placé en parallèle sur la cuvette d'élec¬ troporation 7 pour permettre la détermination de la constante de temps de décharge du condensateur 1.At the end of the second discharge, the switching device 4 returns to the open state and the device of the invention is ready for a new electroporation. A shunting device 9 controlled by the control circuit 11 is placed in parallel on the electrolytic basin 7 to allow the determination of the discharge time constant of the capacitor 1.
Suivant une autre forme d'exécution non représen- tée, le dispositif de l'invention ne comporte pas de conden¬ sateur, ni de circuit de charge. Dans ce cas, un générateur haute tension génère la première décharge, de courte durée, qui est alors de type rectangulaire. Dans cette forme d'exé¬ cution, le dispositif de commutation 2 reste fermé en perma- nence pendant chaque décharge de type rectangulaire.According to another embodiment not shown, the device of the invention does not include a condenser, nor a charging circuit. In this case, a high voltage generator generates the first discharge, of short duration, which is then of the rectangular type. In this form of execution, the switching device 2 remains permanently closed during each discharge of the rectangular type.
Eventuellement, la deuxième décharge peut aussi être de type exponentiel décroissant, auquel cas le généra¬ teur basse tension sera remplacé par un ensemble circuit de charge et condensateur, la valeur de charge de ce dernier étant également déterminée par le circuit de contrôle.Optionally, the second discharge may also be of decreasing exponential type, in which case the low voltage generator will be replaced by a circuit assembly. load and capacitor, the latter's charge value also being determined by the control circuit.
Avantageusement, les valeurs des différents paramè¬ tres (résistance du dispositif de shuntage, capacité du condensateur, voltage et durée de la décharge émise) sont réglables et programmables.Advantageously, the values of the various parameters (resistance of the shunting device, capacitance of the capacitor, voltage and duration of the discharge emitted) are adjustable and programmable.
Le cas échéant, un condensateur peut être remplacé par plusieurs condensateurs de capacités différentes sélec¬ tionnées par le circuit de contrôle 11 montés en parallèle. L'invention est décrite plus en détail ci-dessous à l'aide d'exemples d'exécution de perméabilisation de cellu¬ les réalisées grâce à un dispositif selon l'invention.. Exemple 1 Perméabilisation de cellules hypophysaires de rat Clignée GH. GC . GH3b6. GH4 ,If necessary, a capacitor can be replaced by several capacitors of different capacities selected by the control circuit 11 mounted in parallel. The invention is described in more detail below using examples of cell permeabilization carried out using a device according to the invention. Example 1 Permeabilization of rat pituitary cells Cligne GH. GC. GH3b6. GH4,
On prépare les cellules comme pour une électropora¬ tion classique. On les concentre à 4 millions de cellules/800 μl, dans le milieu de culture habituel de ces cellules. On ajoute l'ADN, de 3 μg à 100 μg suivant le plasmide ou le fragment d'ADN utilisé. On dépose 800 μl du mélange dans une cuvette d'électroporation classique de 4 mm. On applique une première décharge de 300 V (750 V/cm), avec une résistance de shuntage de 74 Ω et un condensateur de 40 μF. On applique ensuite, une seconde décharge de 100 V (250 V/cm) avec une résistance de shuntage de 132 Ω et un condensateur de 1800 μF. Les cellules sont immédiatement transvasées dans du milieu de culture frais. Par la méthode de mesure enzymatique "CAT", on obtient une réponse 10 à 100 fois supérieure par rapport à l'état de la technique. Exemple 2The cells are prepared as for a conventional electropora¬ tion. They are concentrated at 4 million cells / 800 μl, in the usual culture medium for these cells. DNA is added, from 3 μg to 100 μg depending on the plasmid or the DNA fragment used. 800 μl of the mixture are placed in a conventional 4 mm electroporation cuvette. A first discharge of 300 V (750 V / cm) is applied, with a shunt resistance of 74 Ω and a capacitor of 40 μF. Then apply a second discharge of 100 V (250 V / cm) with a shunt resistance of 132 Ω and a capacitor of 1800 μF. The cells are immediately transferred to fresh culture medium. By the enzymatic measurement method "CAT", a response 10 to 100 times greater is obtained compared to the state of the art. Example 2
Perméabilisation de levuresYeast permeabilization
On procède de façon analogue à l'exemple 1 en utilisant les paramètres suivants :The procedure is analogous to Example 1 using the following parameters:
- première décharge: 750 V à 1500 V suivant la souche shunt 74 Ω condensateur 40 μF cuvettes de 2 mm- first discharge: 750 V to 1500 V depending on the 74 Ω shunt strain 40 μF capacitor 2 mm cuvettes
- deuxième décharge: 100 V shunt 282 Ω condensateur 1800 μF On obtient 103 à 107 transformants/μg ADN, c'est-à- dire jusqu'à 10 à 100 fois plus que selon l'état de la tech- nique. Exemple 3 Perméabilisation de bactéries- second discharge: 100 V shunt 282 Ω capacitor 1800 μF We obtain 10 3 to 10 7 transformants / μg DNA, that is to say up to 10 to 100 times more than according to the state of the art. EXAMPLE 3 Permeabilization of Bacteria
On procède de manière analogue avec les paramètres suivants: - première décharge: voltage de 2000 à 2500 V shunt de 74 Ω condensateur de 40 μF cuvettes de 2 mmWe proceed in an analogous manner with the following parameters: - first discharge: voltage from 2000 to 2500 V shunt of 74 Ω capacitor of 40 μF 2 mm cuvettes
- deuxième décharge: voltage de 100 V shunt de 132 Ω condensateur de 1800 μF Dans le cas de bactéries gram-, on obtient 106 à- second discharge: voltage of 100 V shunt of 132 Ω capacitor of 1800 μF In the case of gram- bacteria, we obtain 10 6 at
ÎO11 transformants/μg ADN, c'est-à-dire jusqu'à 10 fois plus que selon l'état de la technique. Dans le cas des bactéries gram +, on obtient 10* à 10^ transformants/μg ADN, c'est-à-dire jusqu'àÎO 11 transformants / μg DNA, that is to say up to 10 times more than according to the state of the art. In the case of gram + bacteria, 10 * to 10 ^ transformants / μg DNA are obtained, i.e. up to
100 fois plus.100 times more.
Exemple 4Example 4
Perméabilisation de protoplastes On procède de façon analogue aux exemples précé¬ dents avec les paramètres suivants:Permeabilization of protoplasts The procedure is analogous to the previous examples with the following parameters:
- première décharge: voltage de 150 à 250 V shunt de 74 Ω condensateur de 40 μF cuvettes de 4 mm- first discharge: voltage from 150 to 250 V shunt of 74 Ω capacitor of 40 μF 4 mm cuvettes
- deuxième décharge: voltage de 50 à 100 V shunt de 132 Ω condensateur de 1800 μF On obtient 10 à 103 transformants/μg ADN. - second discharge: voltage from 50 to 100 V shunt from 132 Ω capacitor from 1800 μF 10 to 10 3 transformants / μg DNA are obtained.

Claims

REVENDICATIONS
1. Procédé de perméabilisation de membranes cellu¬ laires par décharge électrique (électroporation) caractérisée en ce qu'on applique à une suspension contenant des cellules à rendre perméables ainsi que des molécules destinées à être logées à l'intérieur des cellules, successivement au moins une décharge électrique de courte durée engendrant dans la suspension un champ électrique de valeur élevée et ensuite au moins une décharge électrique de plus longue durée et engendrant dans la suspension un champ électrique de valeur moins élevée.1. Method for permeabilization of cell membranes by electrical discharge (electroporation), characterized in that a suspension containing cells to be made permeable as well as molecules intended to be housed inside the cells is applied successively at least a short duration electric discharge generating in the suspension an electric field of high value and then at least one longer duration electric discharge and generating in the suspension an electric field of lower value.
2. Procédé selon la revendication 1 caractérisé en ce que la décharge de courte durée consiste en une impulsion à décroissance exponentielle, notamment obtenue par la dé- charge d'un condensateur.2. Method according to claim 1 characterized in that the short-duration discharge consists of an exponential decay pulse, in particular obtained by the discharge of a capacitor.
3. Procédé selon la revendication 1 caractérisé en ce que la décharge de courte durée consiste en une impulsion de type rectangulaire.3. Method according to claim 1 characterized in that the short-duration discharge consists of a pulse of rectangular type.
4. Procédé selon l'une quelconque des revendica- tions précédentes, caractérisé en ce que la valeur du champ électrique engendré par la décharge de courte durée varie de 400 à 25000 V/cm, en fonction de la durée de l'impulsion et du type de cellules à traiter, la durée de l'impulsion étant de 10 μs à 10 ms. 4. Method according to any one of the preceding claims, characterized in that the value of the electric field generated by the short-term discharge varies from 400 to 25,000 V / cm, depending on the duration of the pulse and the type of cells to be treated, the duration of the pulse being from 10 μs to 10 ms.
5. Procédé selon la revendication 4 caractérisé en ce que la valeur du champ électrique engendré par la décharge de courte durée varie de 400 à 2500 V/cm lorsqu'on traite des cellules eucaryotiques provenant d'organismes pluricellulai- res, de 2000 à 5000 V/cm lorsqu'on traite des cellules euca- ryotiques unicellulaires telles que des levures et de 10.000 à 25.000 V/cm lorsqu'on traite des cellules procaryotiques. 5. Method according to claim 4 characterized in that the value of the electric field generated by the short-term discharge varies from 400 to 2500 V / cm when treating eukaryotic cells originating from multicellular organisms, from 2000 to 5000 V / cm when treating unicellular eukaryotic cells such as yeasts and from 10,000 to 25,000 V / cm when treating prokaryotic cells.
6. Procédé selon l'une quelconque des revendica¬ tions précédentes caractérisé en ce que la seconde décharge, de longue durée, consiste en une tension essentiellement continue, par exemple fournie par un générateur basse ten¬ sion, engendrant un champ électrique de l'ordre de 50 à 1000 V/cm, en fonction de la durée de l'impulsion et du type de cellules à traiter. 6. Method according to any one of the preceding claims, characterized in that the second discharge, of long duration, consists of an essentially continuous voltage, for example supplied by a low voltage generator, generating an electric field of the around 50 to 1000 V / cm, depending on the duration of the pulse and the type of cells to be treated.
7. Procédé selon la revendication 6 caractérisé en ce que la tension essentiellement continue est appliquée pendant un temps cumulé de l'ordre de 1 ms à 1 s.7. Method according to claim 6 characterized in that the essentially continuous voltage is applied for a cumulative time of the order of 1 ms to 1 s.
8. Procédé selon l'une quelconque des revendica- tions 1 à 5, caractérisé en ce que la seconde décharge, de longue durée, consiste en une impulsion à décroissance expo¬ nentielle.8. Method according to any one of claims 1 to 5, characterized in that the second discharge, of long duration, consists of an exponentially decreasing pulse.
9. Procédé selon l'une quelconque des revendica¬ tions 1 à 5 caractérisé en ce que la décharge de courte durée et/ou la décharge de longue durée consistent en un train d'ondes.9. Method according to any one of claims 1 to 5, characterized in that the short-term discharge and / or the long-term discharge consist of a train of waves.
10. Procédé selon l'une quelconque des revendica¬ tions 1 à 5 et 9 caractérisé en ce qu'on permute la polarité de la différence de potentiel aux bornes de la solution pendant la seconde décharge, de courte durée, et le cas échéant pendant la première décharge.10. Process according to any one of claims 1 to 5 and 9, characterized in that the polarity of the potential difference at the terminals of the solution is swapped during the second, short-duration discharge, and if necessary during the first discharge.
11. Dispositif pour la mise en oeuvre du procédé de l'invention caractérisé en ce qu'il comporte au moins une source de haute tension, et une source de basse tension, la tension dégagée par l'une et l'autre étant alternativement mise aux bornes d'électrodes adaptées à une cuvette connue en soi destinée à contenir une suspension contenant des cellules à rendre perméables, ainsi que des molécules desti¬ nées à être logées à l'intérieur des cellules. 11. Device for implementing the method of the invention characterized in that it comprises at least one high voltage source, and one low voltage source, the voltage released by one and the other being alternately applied at the electrode terminals adapted to a well known per se intended to contain a suspension containing cells to be made permeable, as well as molecules intended to be housed inside the cells.
12. Dispositif selon la revendication 11 caracté¬ risé en ce que la source de haute tension consiste en un condensateur (1) associé à un circuit de charge (3).12. Device according to claim 11 caracté¬ ized in that the high voltage source consists of a capacitor (1) associated with a charging circuit (3).
13. Dispositif selon la revendication précédente caractérisé en ce que la source de haute tension consiste en un générateur haute tension.13. Device according to the preceding claim characterized in that the high voltage source consists of a high voltage generator.
14. Dispositif selon l'une quelconque des revendi¬ cations 11 à 13 caractérisé en ce que la source de basse tension consiste en un générateur basse tension (5).14. Device according to any one of claims 11 to 13, characterized in that the low voltage source consists of a low voltage generator (5).
15. Dispositif selon l'une quelconque des revendi- cations 11 à 13 caractérisé en ce que la source de basse tension consiste en un condensateur associé à un circuit de charge.15. Device according to any one of claims 11 to 13 characterized in that the low voltage source consists of a capacitor associated with a charging circuit.
16. Dispositif selon l'une quelconque des revendications 11 à 15 caractérisé en ce que la tension maximum et la constante du temps du ou des condensateur(s) sont programmables.16. Device according to any one of Claims 11 to 15 characterized in that the maximum voltage and the time constant of the capacitor (s) are programmable.
17. Utilisation du dispositif selon l'une quelcon- que des revendications 11 à 16 pour l'introduction dans les cellules de molécules telles que l'ADN, des protéines, des anticorps, des drogues ou autres produits chimiques.17. Use of the device according to any one of claims 11 to 16 for the introduction into the cells of molecules such as DNA, proteins, antibodies, drugs or other chemicals.
18. Utilisation du dispositif selon l'une quelcon¬ que des revendications 11 à 16 pour l'extraction de molécu- les, notamment de l'ADN, de cellules.18. Use of the device according to any one of claims 11 to 16 for the extraction of molecules, in particular DNA, from cells.
19. Utilisation du dispositif selon l'une quelcon¬ que des revendications 11 à 16 pour la modification ou la fusion de cellules. 19. Use of the device according to any one of claims 11 to 16 for the modification or fusion of cells.
PCT/BE1991/000030 1990-05-16 1991-05-16 Method and device for making living cells permeable WO1991018103A1 (en)

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Application Number Priority Date Filing Date Title
BE9000518A BE1004328A3 (en) 1990-05-16 1990-05-16 METHOD AND DEVICE FOR LIVING CELLS permeabilization.
BE9000518 1990-05-16

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