WO1987002383A1 - Process and medium for field-induced fusion of macromolecules in living cells - Google Patents

Abstract

Process for field-induced fusion of macromolecules into living cells comprising the following sequences, a) enzymatic pre-treatment of the cells; b) suspending the cells in an aqueous, isotonic medium; c) moderating the cell suspension to a temperature of between 0o C and 25o C; d) use of electric field pulses to create passages through the membrane which can be healed; e) heating the cell suspension to heal the membrane passages after application of the field pulses to a temperature above ambient temperature. The process is improved in such a way that significantly higher yields are obtained when fusing these molecules and simultaneously lower concentrations of the substances to be fused are required, this being achieved by the fact that the enzymatic pre-treatment is effected with protolytic enzymes, that the isotonic aqueous medium is used with an electrolyte and non electrolyte portion in order to suspend the cells for the actual incorporation of the macromolecules, that the suspension is cooled to a temperature of between 0o C and 10o C, and that one or several electrical field pulses are applied at a super-critical field strength.

Description

 Description :

Method and medium for field-induced introduction of macromolecules into living cells

The invention relates to a method for field-induced introduction of macromolecules, such as. B. genetic information carriers, in particular DNA and molecules comparable in size, such as. B. proteins, in living cells, comprising the following process steps:

a) enzymatic pretreatment of the cells; b) suspending the cells in an aqueous, isotonic medium; c) tempering the cell suspension to a temperature between 0 ° C and 25 ° C; d) application of electrical field pulses to create curable membrane breakthroughs; e) heating the Zeil suspension to heal the membrane breakthroughs after applying the Eeld pulses to a temperature above room temperature. The invention also relates to a medium for performing this method.

From DE-PS 24 05 119 a method for introducing chemical substances into cells is known, in which the cells are suspended in a physiological electrolyte solution and an electric field with the field strength of 10 ³ to at a temperature between 0 ° C and 25 ° C 10 ⁵ V / cm are exposed, so that membrane breakthroughs occur, through which chemical substances from the liquid surrounding the cell can get into the cell interior. To heal the membrane breakthroughs, the cell suspension is heated in order to accelerate the healing processes of the cell membranes.

It is also known to precede the process of DE-PS 24 05 119 with a pretreatment step in which the cells are subjected to an enzymatic treatment in order to make the cell membranes more stable with respect to electric fields.

In other processes for introducing chemical substances into cells, in addition to the isotonic, pure electrolyte solutions mentioned above, isotonic pure non-electrolyte solutions of inositol or sucrose have also been used.

In all cases, however, the yield of the injection of larger biomolecules, such as. B. DNA, relatively small in living cells, even if larger concentrations in the medium surrounding the cells of the substances to be introduced. Very often the membrane breakthroughs were limited to relatively small areas of the cell membrane, which greatly reduced the ability to heal the membrane.

The object of the invention is to improve a method for introducing macromolecules into living cells in such a way that significantly improved yields are obtained when introducing macromolecules, and that at the same time lower concentrations of the substances to be introduced are necessary.

This object is achieved according to the invention in a method of the type described in the introduction in that the enzymatic pretreatment is carried out using prototype enzymes, in that the isotonic aqueous medium with an electrolyte and a non-electrolyte component is used to suspend the cells for the actual sinus transfer of the macromolecules, that the Suspension is cooled to a temperature between 0 ° C and 10 ° C, and that one or more electric fields are applied with supercritical field strength.

The introduction of macromolecules into cells makes it necessary to create larger breakthroughs in the cell membranes, which must additionally have a relatively long lifespan, so that the macromolecules are noticeably absorbed into the cell interior. The creation of larger ones Breakthroughs in the cell membrane can only be achieved with electrical field pulses whose field strength is above the critical field strength, the critical field strength being the lowest field strength. The first breakthroughs of the membrane occur in places of the cell membrane that are penetrated by the electrical field lines. The above-mentioned process features of the invention now work together in such a way that the larger breakthroughs that arise from the supercritical fields in the cell membrane are distributed almost symmetrically over the cell membrane surface, ie the breakthroughs are not only concentrated on a few locations on the cell membrane.

A special role for the survival rate of the cells is played by the enzymatic pretreatment with prototypical enzymes, through which the cell membrane becomes more stable against high-energy electric fields. A certain amount of siectrolyte in the isotonic aqueous medium is also essential, due to the symmetry of the distribution of the breakthroughs in the cell membrane can be controlled within certain limits. The cooling of the cell suspension to a temperature between 0 ° C and 10 ° C serves to extend the life of the membrane breakthroughs, so that the absorption rate of macromolecules in the cell interior assumes practical values, even if the macromolecules are only present in comparatively low concentrations in the injection medium. To maintain the viability, ie the ability to divide Zella, at the end of the injection process, it is necessary to raise the temperature in order to accelerate the annealing process of the membrane breakthroughs after the macromolecules have been absorbed into the interior of the cell.

The use of dispase or pronase as protolytic enzymes has proven to be particularly favorable, whereby when using pronase precautions must be taken that this protolytic enzyme is no longer present in the medium surrounding the cells during the infiltration process, since the pronase Activity within the cell causes it to die.

If the cell suspension is kept at around 4 ° C during the introduction, then for a large selection of cell systems there are optimal ratios of the rate constants for the healing of the membrane breakthroughs, the exchange rate itself and other exchange processes which have a negative effect on the rate of wetting of the cell.

The best results are achieved in the method according to the invention if the supercritical field strength is approximately two to three times the critical field strength defined above.

The healing process of the membrane breakthroughs preferably takes place by raising the temperature to approximately 37 ° C. after the injection process has ended. In the case of particularly sensitive cell types, it is sometimes appropriate to transfer the cells into a special healing medium after the introduction process, in which the healing process is then also carried out at elevated temperature.

Another object of the invention is to provide a medium for performing the method.

This object is achieved in a generic medium in that the electrolyte content is 25 mmol to 60 mmol and that the non-electrolyte component has a concentration by which the osmolarity of the medium is increased to such an extent that the medium is isotonic. The medium also contains the macromolecules to be introduced.

As previously mentioned, the electrolyte portion in the aqueous isotonic medium makes it possible to control the distribution of the openings on the membrane surface of the cell.

A particularly symmetrical breakthrough pattern is obtained with an electrolyte content of approximately 30 mmol. The breakthroughs with an assumed spherical shape of the cell are almost symmetrically distributed on both "pole caps" of the cell, so that on the one hand a maximum absorption rate for the macromolecules is achieved and on the other hand not too large a concentration of large membrane breakthroughs on a relatively small surface of the Cell takes place, which significantly improves the ability of the cell to survive. Appropriately, the electrostatic part is composed of aine or divalent cations and mono- or divalent anions. No further demands are made of the cations and anions themselves than that they must be cell-compatible in the usual way.

The non-electrolyte tail of the medium is preferably made from one or more of the non-electrolytes sugar, sugar alcohol, amino sugar and related compounds such as. B. inositol.

Additional buffering of the medium with a phosphate buffer, which is added to the medium, for example in a concentration of 1 mmol, is not absolutely necessary, but is recommended for ampfindiicheran Zeilsystamen. It should be noted that the proportion of phosphate buffer must be taken into account as part of the elecolyte.

These and other advantages of the invention are explained in more detail below using an example:

Introducing E. coli plasmids into mouse lymphocyte cells

Mouse lymphocyte cells were propagated in RPMI 1640 medium (available from Boehringer, Mannheim), which was mixed with 5% FCS (fetal calf serum). For the inclusion experiment, the RPMI medium was decanted and replaced with an isotonic solution with low ionic strength (280 mmol inositol and 1.1 mmol phosphate buffer). The slide was then centrifuged at approx. 140 g. The centrifuged lines were suspended in a solution of low ionic strength to which 0.1 mg / ml Dispasa was added (6 units / mg, quality level 1, Boehringer, Mannheim). After this treatment with protolytic enzyme, the cells were washed with the actual infiltration medium and suspended in it. The composition of the medium was as follows:

30 mmol KCl 20 mmol inositol and 1.1 mmol phosphate buffer (pH 7.2).

The field pulses were applied to the gel suspension cooled to 4 ° C., the density of the cells being 2 × 10 ⁵ to 2.3 × 10 ⁶ cell / ml. The DNA to be introduced was added to the medium in a concentration of 1 μg / ml before the application of the field pulses.

The circular form of a piasmid pSV 2-neo, which comprises a neomvcin-resistant gene, was used as DNA. The partially linearized one

Plasmid DNA was exposed to 3 x 50 units of Eco R 1 by exposure at 37 ° C for 3 hours

50 μg of plasmid DNA were prepared, each time

50 units of Eco R 1 after 0, 1 and 2 hours were added. This is followed by two cycles of a phenol / chloroform extraction, from which the DNA is divided into two parts. Ethanol is precipitated. The plasmid itself was isolated from E. coli.

The introduction by means of electrically induced breakthroughs in the cell membranes was carried out analogously to the instructions of Zimmermann et al (U. Zimmermann, G. Pilwat and F. Riemann, Biophys. J. 14, 881 (1974). The pulse duration was set to 5 μs. After application of the pulses, the suspension was kept at 4 ° C. for one to two minutes, after which the suspension was removed from the chamber using a micropipette and placed in a volume of 15 ml of a healing medium which was preheated to 37 ° C. The composition of the healing medium was as follows:

120 mmol sodium chloride 3.5 mmol KCl 3.5 mmol K ₂ HPO ₄ 3 mmol KH ₂ PO ₄ 0.5 mmol magnesium acetate 0.1 mmol calcium acetate and 10 mmol glucose

The cells were held in the healing medium at 37 ° C for about 20 minutes.

After the cell membranes had completely healed, the cells were centrifuged off and transferred to a nutrient medium (RPMI 1640) with 5% FCS and incubated for 48 hours. After treatment with In a selection medium, the yield of cloned lines was determined by counting the colonies formed (within 12 to 18 days after the addition of the selection medium). The results of the experiments are almost kept in Tables 1 and 2.

In the experiments, the fall strength of the field pulses was varied. A field strength of 8 kv / cm was sufficient for good clone yields, although 10 kv / cm gave better and more reproducible results in most experiments. A further increase in the field strength up to 20 kv / cm or multiple use of pulses (e.g. 3 pulses in an interval of 20 sec at 10 kv / cm) do not result in a correspondingly increased yield of stable clones.

Table 1

Yield of stable, i.e. H. viable cloning under various field conditions and using circular plasmid DNA. Unless otherwise noted, the DNA concentration is 1 μg / ml. Numbers in brackets refer to dia

Number of clones at 1 μg DNA / ml. The number of cells fluctuated between 6 x 10 ⁵ and 7 x 10 ⁶ in the

Experiments. Contral experiments in which the Zallan were exposed to the same experimental conditions, but without the use of electric Faid pulses, produced no or only individual clones.

Table 2

Comparison of the yields of stable clones obtained with circular and linear plasmid DNA. The results were obtained in parallel experiments, using 1 μg / ml DNA and 1.3 or 2.4 × 10 ⁶ cells / ml for the circular and the linear form of DNA.

Claims

Patent claims: 1. Method for field-induced introduction of macromolecules, such as. B. from genetic information carriers, in particular DNA, and in size comparable biomolecules, such as. B. Protainen, in living cells, which comprises the following procedural steps: a) enzymatic pretreatment of the cells; b) suspending the cells in an aqueous, isotonic medium; c) tempering the cell suspension to a temperature between 0 ° C and 25 ° C; d) application of electrical field pulses to create curable membrane breakthroughs; a) heating the cell suspension to heal the membrane breakthroughs after applying the Feidpulse to a temperature above room temperature, the method being characterized in that f) the enzymatic pretreatment with protolytic enzymes is carried out in that g) the isotonic aqueous medium with an electrolyte and a non-electrolyte portion is used to suspend the cells for the actual introduction of the macromolecules, that h) the suspension is at a temperature between 0 ° C and 10 ° C is cooled, and that i) one or more electric field pulses with supercritical field strength are applied. 2. The method according to claim 1, characterized in that dispase or pronase are used as protolytic enzymes. 3. The method according to claim 1 or 2, characterized in that the dial suspension to approximately 4 ° C is cooled. 4. The method according to any one of claims 1 to 3, characterized in that the supercritical field strength is two to three times the critical field strength. 5. The method according to any one of claims 1 to 4, characterized in that after the application of the fall pulse or the field pulses to approximately 37 ° C is heated. 6. Medium for performing the method according to any one of claims 1 to 5, characterized in that the electrolyte portion to 25 mMoi Is 60 mMoi and that the non-electrolyte antail has a concentration by which the osmolarity of the medium is increased to such an extent that the medium is isotonic. 7. Medium according to claim 6, characterized in that the electrolyte content is approximately 30 mMoi. 8. Medium according to claim 6 or 7, characterized in that the electrolyte portion comprises mono- or divalent cations and mono- or divalent anions. 9. Medium according to one of claims 6 to 8, characterized in that the non-alkali metal portion one or more of the series of non-electrolytes of sugar, sugar alcohol, amino sugar and related compounds such as. B. Inositol. 10. Medium according to one of claims 5 to 9, characterized in that the electrolyte portion comprises a phosphate buffer.