DE19945441A1 - Introducing nucleic acids, proteins or peptides into eukaryotic cells comprises exposure to ultrasound or gravitational forces in the presence of an adjuvant comprising calcium and phosphate - Google Patents

Abstract

Process (A) for introducing nucleic acids, proteins or peptides into eukaryotic cells comprises mixing the cells in suspension with a transfection agent (I) comprising the desired nucleic acid, protein or peptide and an adjuvant comprising calcium and phosphate and exposing the mixture to ultrasound or gravitational forces. Independent claims are also included for the following: (1) transfected cells obtainable by process (A); (2) a process (B) for preparing an adjuvant for (I), comprising: (a) mixing a solution containing calcium ions with a solution containing phosphate, hydrogen phosphate, dihydrogen phosphate or phosphoric acid and optionally chloride, fluoride or hydroxide ions; (b) incubating the mixture to form a precipitate comprising calcium and phosphate; (c) separating the precipitate and discarding the supernatant; and (d) optionally resuspending the precipitate; (3) a process (C) for preparing (I), comprising performing steps (a)-(c) of process (B) and either: (i) resuspending the precipitate in a solution of the desired nucleic acid, protein or peptide and optionally separating and discarding the precipitate; or (ii) resuspending the precipitate, mixing the suspension with a solution of the desired nucleic acid, protein or peptide and optionally separating and discarding the precipitate; (4) an adjuvant prepared by process (B) or a transfection agent prepared by process (C); (5) apparatus for introducing nucleic acids, proteins or peptides into eukaryotic cells, comprising: (a) a sonicating chamber with an ultrasound source, an inlet and an outlet; and (b) a reservoir that is connected to the inlet and optionally contains a mixture of eukaryotic cells and (I).

Description

The present invention relates, inter alia, to methods for introducing Nucleic acids and proteins in eukaryotic cells, usable for this purpose Additives and transfection agents, as well as devices for carrying out the method according to the invention.

Various methods for the transfection of eukaryotic cells. Some of these procedures affect you certain additives that the nucleic acid to be introduced or the bind or contain protein to be introduced.

A known transfection method is calcium phosphate coprecipitation (see, for example, Graham and von der Eb, Virology 52 (1973), 456-467). It is based on the co-precipitation of nucleic acid and calcium phosphate, e.g. B. by adding an excess of Ca ²⁺ ions to a solution containing DNA and phosphate. The coprecipitated soil body, which contains DNA and calcium phosphate, is then used for the transfection of eukaryotic cells.

Coprecipitation with calcium phosphate has also been used to make proteins in cells introduce. Howcroft et al. the introduction of β-galactosidase in hela-, COS-7 and CHP-126 cells using a calcium phosphate coprecipitation method (Anal. Biochem. 244 (1997), 22-27).

The calcium phosphate coprecipitation method is a relatively inexpensive one The method, however, has the disadvantages that its reproducibility is low and none Possibility of "upscaling", that is, to enlarge the scale.  

In the calcium phosphate coprecipitation method, this must also be done according to this method obtained transfection agent for each new approach, since it is one undergoes rapid "aging" which adversely affects transfection. It is also disadvantageous in that the formation of optimal nucleic acid for transfection Calcium phosphate coprecipitates of many, sometimes difficult to control Factors depends, so that the transfection efficiency sometimes with separate approaches turns out to be significantly different, as each newly manufactured transfection solution is different Contains molecular species or aggregates.

Other known methods for introducing nucleic acids into cells include Use of additives such as DEAE-dextran and poly-L-lysine. This Transfection procedures are e.g. B. Ehrlich et al. (Biochim. Biophys. Acta 454 (1976), 397-409).

Liposome-forming agents have also been used as transfection-promoting additives used (see, e.g., Felgner et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7413-7417; Gao and Huang, Biochem. Biophys. Res. Commun. 179: 280-285 (1991). It is currently assumed that the liposomes bind to the DNA, then these complexes with the cell membrane interact and the DNA eventually through an unknown Mechanism can enter the cell.

Synthetic activated have also been used as a transfection-promoting additive Dendrimers used which are commercially available under the SuperFect ™ brand name are (Qiagen).

Furthermore, Appel et al. (Proc. Natl. Acad. Sci. USA 85 (1988), 7670-7674) exogenous plasmid DNA using serpentine asbestos fibers as carrier material in Introduced monkey COS-7 cells.

On the other hand, transfection methods have been described in the prior art which the temporary perforation of the cells - and thus the absorption of the desired substance - with the help of physical, especially mechanical, electrical or reach acoustic means. So z. B. Bao et al. (Ultrasound in Med. & Biol. 23 (1997), 953-959) a reporter plasmid by exposure to ultrasound in CHO cells  (Chinese hamster ovary cells), and then that of the plasmid encoded protein (luciferase) expressed in the transfected cells.

Ultrasound was also developed by Fechheimer et al. to introduce a macromolecular chemical pH probe, in this case fluorescein-labeled dextran, in cells of Amoeba used (Eur. J. Cell Biol. 40 (1986) 242-247).

Kim et al. describe the use of different ultrasound exposure times and their effect on transfection efficiency in mammalian cells (Human Gene Therapy 7 (1996), 1339-1346). Generally it can be stated that the ultrasound based Transfection methods do not allow high transfection efficiency, d. H. the the The yield of transfected cells in these methods is low.

As another mechanical process to increase the uptake of macromolecules in cells enable the use of laser-generated pressure pulses in US Pat. No. 5,658,892 for the temporary permeabilization of cells. With this technique Laser pulses aimed at an absorbent material so that the resulting Absorption heat an expansion of the surrounding medium and thus a Generates a pressure pulse that acts on the cell and permeabilizes it transiently.

Various mechanical methods for introducing nucleic acids into cells and Organs that are based on brief pressure increases are from US Pat. No. 5,766,901 known.

Another mechanical process is described by Clarke and McNeill, the shear forces use for introducing fluorescently labeled dextran into cells (J. Cell Sci. 102 (1992), 533-541). The shear forces are passed through the cell suspension generates a hollow needle or similar device, resulting in a short-term reversible injury to the cell membrane and subsequent uptake of the transfecting material leads.

In connection with the therapeutic administration of substances, the Use of gas-filled microspheres, e.g. B. Liposomes described (U.S. Patent 5,770,222). Thereby nucleic acids or other contained in the microspheres become therapeutic  effective substances in the patient's target tissue through ultrasound treatment released.

When developing new and / or improved active pharmaceutical ingredients, e.g. B. on proteins or peptides produced by recombinant methods are known to the person skilled in the art Different approaches are available to make the most of a meaningful one Impact analysis, e.g. B. Experiments in vivo, required amount of the desired substance to provide. One possibility is the development of transgenic organisms or Animals containing the active substance encoded by the nucleic acid introduced in accordingly express large quantities. Another possibility is the production more stable recombinant cell lines (e.g. mammalian cell lines very similar to the human model can produce similar glycoproteins). However, both approaches are high Work and time involved, and there is no certainty whether in the end a suitable transgenic organism or a stable cell line is obtained.

In addition, certain nucleic acid sequences in mammalian cell cultures or in transgenic organisms only with great difficulty or not at all permanently be expressed. Likewise, certain proteins or peptides can only be used for a short time are introduced into cells without the cells being damaged or dying. This stems among other things from the fact that the expressed or introduced proteins or peptides are sometimes toxic to the cells, as z. B. in proteases, nucleases, lipases or Pore formers and also the case with membrane proteins or hydrophobic proteins can.

On the other hand, in the case of transient expression, where there is no stable integration in the genome of the transfected cells is selected, as when introducing Proteins or peptides in eukaryotic cells, the problem of using the known Transfection methods to achieve relatively few possibilities, the method in perform larger scale, for example, in a short period of time larger Obtain amounts of protein. Also known by using transfection-promoting additives, e.g. B. calcium phosphate, caused undesirable toxic side effects, such as the premature death of the cells immediately at or shortly after the transfection, as well as the lack of reproducibility of the respective Transfection approach, have a negative impact in this context. Finally  are the amounts of additional reagents required in many previously known processes, such as B. commercially available transfection reagents or additives, relatively expensive, what the already mentioned "upscaling" or even the large-scale use extremely makes it uneconomical.

The person skilled in the art wants the properties of a particular recombinant produced by him or isolated protein or peptide, it is often desirable to To produce antibodies against this protein or peptide, or its effect on the To determine the organ system of an animal. These steps are made easier when one sufficient amount of protein is available within a very short time, so that after appropriate tests can be immediately decided whether for the protein in question the effort of further development, accompanying z. B. permanent with the establishment expressing cell lines or transgenic organisms appears worthwhile. Desirable is often also the possibility to influence the introduction of the protein or peptide into to research a large number of cells in culture. Also in this context only a small amount of protein or peptide affects that known Transfection procedures can be obtained or introduced into cells, disadvantageous.

Accordingly, it is an object of the present invention to provide a method for Introduction of nucleic acids and proteins or peptides into eukaryotic cells to provide, which offers a high reproducibility. Another task is in the provision of a method for introducing nucleic acids and proteins or Peptides in eukaryotic cells, which is an inexpensive large transfection Cell quantities allowed with good transfection efficiency. It is also a task of the present invention, additives and transfection agents containing them To provide that can be used for a long time after their manufacture.

These and other tasks are the subject of claim 1 and the following claims solved. These concern procedures for the introduction of Nucleic acids and proteins in eukaryotic cells, the invention Transfection agents and additives, and for carrying out the invention Devices that can be used.  

Accordingly, in one aspect, the present invention relates to a method for introducing nucleic acids and proteins or peptides into eukaryotic cells, which comprises the following steps:

• a) Mixing the cells with a transfection agent that is to be introduced Nucleic acid or the protein or peptide to be introduced and a calcium and Contains an additive comprising phosphate,
• b) treatment of the mixture obtained in step i) with ultrasound or Shear forces.

Surprisingly, it was found that the combination of Transfection methods in which nucleic acids or proteins with a calcium and Phosphate-containing additive to be introduced into eukaryotic cells Transfection methods using physical means to improve Transfection efficiency versus transfection methods, the physical means use, which leads specifically to "upscaling", i.e. H. when interpreting procedures in larger scale, is important. The method according to the invention can also be used for transfection of adherent cells also apply to non-adherent cells.

Another aspect of the invention relates to a method in which step ii) and optionally step i) in a continuous process, d. H. in the flow, is carried out.

The invention also relates to cells transfected with proteins or nucleic acids one of the above-mentioned methods are available or can be produced.

In a further aspect, the present invention relates to a method for producing an additive comprising calcium and phosphate for a transfection agent for introducing nucleic acids or proteins and peptides into eukaryotic cells, which comprises the following steps:

• a) Mixing a solution containing Ca ²⁺ ions with a solution containing P _i and optionally X, so that a precipitate is formed in the mixture, where P _{i is} phosphate, hydrogen phosphate, dihydrogen phosphate or phosphoric acid and X is Cl ^- , F ^- or OH ^- and incubating the mixture to form a calcium and phosphate-containing precipitate;
• b) separating the precipitate formed from the mixture from step a) and Discard the supernatant;
• c) optionally resuspending the precipitate obtained in step b).

Surprisingly, it was found that one of the above. Process manufactured Additive obtained or available either after step b) (i.e. that in this step soil body or precipitate obtained) or after step c) (i.e. after resuspension of the precipitate obtained) has a long shelf life (several days or Weeks under appropriate conditions, e.g. B. storage at 4 ° C), d. H. just one low "aging" is subject. In contrast to the way of the conventional Calcium phosphate / DNA coprecipitation obtained can be the so obtained Additive thus also after long storage according to the procedure shown below for the production of a transfection agent, possibly also in portions, each with in cells mixed nucleic acid or proteins or peptides to be introduced and incubated (see step c) of the method for producing a Transfection agent).

In a further aspect, the present invention relates to a method for producing a transfection agent for introducing nucleic acids and proteins or peptides into eukaryotic cells, comprising:

• a) Mixing a solution containing Ca ²⁺ ions, a P _i and optionally X, so that a precipitate is formed in the mixture, where P _{i is} phosphate, hydrogen phosphate, dihydrogen phosphate or phosphoric acid and X is Cl ^- , F ^- or OH ^- and incubating the mixture to form a calcium and phosphate-containing precipitate;
• b) separating the precipitate formed from the mixture from step a) and Discard the supernatant;
• c) resuspending the precipitate obtained in step b) with a nucleic acid or the solution containing the protein or peptide and optionally separating and discarding the precipitate; or
• d) resuspending the precipitate obtained in step b) and subsequent Mixing the resuspended precipitate with the nucleic acid or protein  or peptide-containing solution and optionally separating and discarding the Precipitate.

Surprisingly, it was found that in step c) above or d) transfection agent obtained, in particular the one carried out according to a preferred embodiment of the present invention Separation of the soil body or precipitate obtained supernatant (i.e. that in the Steps c) and d) by fractionation transfection agent), when used for the transfection of eukaryotic cells in the method according to the invention leads to particularly high transfection efficiency and high reproducibility.

The use of a fractionated transfection agent according to the present In addition, the invention differs from previously known ones based on calcium phosphate Coprecipitation-based transfection procedures, in which always the bottom body is used. An advantage of using the above method (step c) and d)) Fractionated transfection agent obtained or available is in this Context in that no macroscopic amounts of calcium phosphate to the transfecting cells are given and thus annoying, due to large calcium phosphate Precipitates caused side effects such as B. cell death in culture, be reduced or avoided.

The invention further relates to a method for introducing nucleic acids and proteins or peptides in eukaryotic cells as set forth above in which the additive or the transfection agent according to one of the two methods mentioned is manufactured or is available.

The invention also relates to an additive for a transfection agent or a Transfection agent for introducing nucleic acids or proteins and peptides into eukaryotic cells obtained by one of the above methods or is available, and the use of the additive or transfection agent mentioned in a method for introducing nucleic acids and proteins or peptides into eukaryotic cells.  

Furthermore, the invention relates to a device for performing one of the aforementioned methods, which comprises the following components:

• a) an ultrasound chamber with an ultrasound source
• b) an inflow and an outflow, each connected to the ultrasonic chamber are;
• c) a reservoir chamber connected to the inflow, which may be a Mixture of cells with a transfection agent according to the present Invention contains.

The invention also relates to devices as mentioned above, in which the Ultrasound source is a metal tip, or the ultrasound chamber as an ultrasound bath is designed.

The invention also relates to a device as mentioned above, in which the Reservoir chamber has a drain and an inflow, the reservoir chamber can be connected via the drain to a culture vessel or bioreactor, and wherein the drain or inflow connected to the reservoir chamber by pumps, e.g. B. Peristaltic pumps can be regulated.

Finally, the invention relates to the use of a device as above mentioned in a method for introducing nucleic acids and proteins or Peptides in eukaryotic cells.

The figures show examples of preferred devices according to the invention shown, as well as the results of the embodiments 1-8.

Fig. 1 shows part of a device for performing the method according to the invention for introducing nucleic acids and proteins into eukaryotic cells, which allows sonication of a mixture of eukaryotic cells and transfection agent carried in a continuous flow. The arrows indicate the direction of flow of the suspended cells, which may have been previously mixed with the transfection agent in a separate reservoir chamber (not shown). The device shown is also suitable for methods for transfecting large amounts of cells. In the embodiment shown, a decoupler 1 is used which acoustically decouples the ultrasound source (tip) 2 from the surrounding ultrasound chamber 3 . The device also contains an inlet Z and an outlet A,

Fig. 2 shows a device for performing the method according to the invention for introducing nucleic acids or proteins and peptides into eukaryotic cells with subsequent expression of the transfected nucleic acid or further cultivation of the cells. The device shown is also suitable for methods for transfecting large amounts of cells. In the embodiment shown, a decoupler 1 is used which acoustically decouples the ultrasound source (tip) 2 from the surrounding ultrasound chamber 3 . The device also comprises a container with transfection agent 12 , a container with fresh (cell culture) medium 7 and a container for used medium 8 . These containers are connected to the reservoir chamber (or the mixing tank) 6 via pipelines, which comprise the pumps 9 , 9 ′ and 9 ″. The pipeline for the discharge of used medium into the container 8 further comprises a device 10 (e.g. sieve, filter or membrane) which prevents the transfer of the cells into the container for used medium 8 . The device further contains a pump 9 '''which is connected between the reservoir chamber 6 and the ultrasonic chamber 3 . Finally, the device contains a (bio) reactor 11 ,

Fig. 3 shows the results of the experiments described in Example 1, the absolute measured SEAP activity in mU being plotted on the ordinate and the batch number on the abscissa,

Fig. 4 shows the results of the experiments described in Example 2, the absolute measured SEAP activity in mU being plotted on the ordinate and the number of the batch plotted on the abscissa,

Fig. 5 shows the results of the experiments described in Example 3, with the absolute measured SEAP activity in mU on the ordinate and the number of the approach on the abscissa. The three incubation times of the cell culture (24 h, 48 h, 72 h) are shown by differently patterned bars, see legend in the graphic,

Fig. 6 shows the results of the experiments described in Example 5, with the absolute measured SEAP activity in mU on the ordinate and the number of the approach on the abscissa,

Fig. 7 shows the results of the experiments described in Example 6, the absorption at 405 nm (SEAP activity test) being plotted on the ordinate and the number of the batch being plotted on the abscissa,

Fig. 8 shows the results of the experiments described in Example 7, the absorption at 405 nm (SEAP activity test) being plotted on the ordinate and the number of the batch on the abscissa,

Fig. 9 shows the results of the experiments described in Example 8, with the absolute measured SEAP activity in mU on the ordinate and the serial number of the individual fractions on the abscissa.

The method according to the invention for introducing nucleic acids and proteins into eukaryotic cells involve two steps. In the first step (i), eukaryotic Cells mixed with a transfection agent that contains a calcium and phosphate Containing additive that facilitates transfection, as well as one in the cells nucleic acid to be introduced or a protein or peptide to be introduced. The one in The term "transfection" used in connection with the present invention means the introduction of exogenous molecules, in particular nucleic acids or proteins and peptides into eukaryotic cells.

For the method according to the invention, a cell density of 10 ⁴ to 10 ⁹ cells mL ^-1 after mixing the cell solution with the transfection agent is advantageous, 10 ⁵ to 10 ⁷ cells mL ^{-1 are} preferred, and a cell density of about 10 ⁶ cells mL ^-1 is particularly preferred .

The eukaryotic cells to be transfected can be plant cells however, they must be treated beforehand by known methods so that the cell wall is removed is and only the plant cell protoplasts for the inventive method be used. However, animal cells or yeast cells are preferred in animal cells e.g. B. cells from primary cell cultures or preparations, such as lymphocyte preparations or primary fibroblasts. Are preferred also cell lines, which can also be cell lines that are not from mammals originate, such as yeast cell lines or insect cell lines (e.g. that of Spodoptera frugiperda derived Sf21 and Sf9 cells or the BTI-TN-5B1- derived from Trichoplusia ni 4 cells). Mammalian cell lines are also preferred, being adherent both in cell culture as well as non-adherent growing cells are suitable, e.g. B. COS cells, CHO cells, HEK 293 cells, NIH 3T3 cells, BM2 cells, Huh7 cells, Jurkat cells, BAEC Cells, MDCK cells, BHK cells and Hela cells. For use within the In the present invention, CHO cells and HEK 293 cells are particularly preferred. If the cells to be transfected are adherently growing cells, then the cells before mixing with the transfection agent according to the invention according to step i) mentioned above, first enzymatically (trypsinization), chemically (e.g. using EDTA-containing buffers) or mechanically (vigorous shaking, cell Scraper) detached from the substrate. Non-adherent cell lines are particularly preferred.

Nucleic acids to be introduced (or transfected) into the cells in the sense of The present invention can be naturally occurring molecules, e.g. B. isolates of DNA, mRNA from animal, plant or fungal cells and RNA or DNA from viruses, Viroids, virusoids, bacteria or microorganisms. Furthermore, it can be recombinant constructs produced using genetic engineering, such as plasmids, cosmids, cDNA, phage and viral DNA or RNA, mRNA, tRNA, as well as synthetic oligo- and polynucleotides. The nucleic acid to be transfected can be single-stranded (ssDNA or ssRNA, ss = "single stranded") or double-stranded (dsDNA or dsRNA, ds = "double stranded"). The transfection of is particularly preferred Expression plasmids which contain sequences coding for a desired protein, e.g. Legs Reporter gene sequence or one for a structural protein or enzyme that is of interest coding sequence, combined with suitable expression signals and optionally one or more resistance genes (e.g. antibiotic resistance for amplification in bacteria and / or genes for selection for stable integration in eukaryotic cells).  

Furthermore, the method according to the invention is also suitable for the transfection of Nucleic acids, the non-naturally occurring nucleotides as well as their naturally occurring form containing base changes, insertions and deletions.

The method according to the invention is also particularly suitable for transfection and subsequent expression of nucleic acid sequences necessary for cytotoxic proteins encode (e.g. proteases, nucleases, lipases, channel proteins etc.), as well as for proteins that problems in expression in eukaryotic systems often cause problems such as B. Membrane proteins, since the method according to the invention does not proliferate the cells requires a longer period of time and therefore a high yield of these proteins enables.

The transfection method according to the invention is also suitable for introducing Mixtures of different of the above nucleic acids.

Proteins and peptides to be introduced (or transfected) into the cells in the sense of present invention include viral and bacterial proteins and peptides, proteins and Eukaryotic peptides, prions, as well as synthetic (i.e. non-naturally occurring) Proteins and peptides. The inventive method is also suitable for Transfection of post-translational or otherwise chemically modified proteins and Peptides, including proteins and peptides that are not naturally occurring Contain amino acids, amino acid exchanges, insertions and deletions. The The transfection method according to the invention is also suitable for introducing Mixtures and aggregates of various of the above proteins or Peptides.

The additives according to the invention are calcium and phosphate substances or aggregates containing or derived from calcium phosphate. As Also suitable are additives derived from calcium phosphate or Fabric aggregates that are already alone, d. H. without additional use of physical Aids such as sonication or shear forces, the introduction of nucleic acids or Promote proteins and peptides in eukaryotic cells.  

In a preferred embodiment of the invention, calcium phosphate is used as an additive, various forms of calcium phosphate being suitable here. A preferred form of the calcium phosphate is hydroxyapatite as used e.g. B. can be obtained by bringing together a solution containing Ca ²⁺ with a phosphate-containing buffer and precipitates by exceeding the solubility product of calcium phosphate. Suitable Ca ^{2+ -containing} solutions and suitable buffers as well as concentrations and pH conditions can be found, for example, in the calcium phosphate coprecipitation processes practiced in the prior art, but in the present invention the precipitation is carried out in the absence of nucleic acid or protein or peptide. Suitable are also other ions also containing, preferably F ^-, Cl ^- and OH ^- containing derivatives of calcium phosphate, for example. B. Calcium fluorophosphate.

These additives according to the invention can be in coarse crystalline, fine crystalline or in dissolved form, as fibers or beads, and as a gel. It is always important that the calcium and phosphate containing additive either interact with the molecule to be introduced, i.e. the nucleic acid or the protein or peptide, where interaction means that the additive either serves as a carrier or else by attachment to the molecule to be transferred and, if appropriate, to the cell itself enables accelerated entry of the molecule to be transfected into it, the mechanisms that allow passage can be of various types.

In step ii) of the transfection method according to the invention, an additional physical treatment of the cells made that improved penetration of the substances to be transfected through the cell membrane barrier. In a preferred embodiment, this physical treatment involves the use of Shear forces on the cells to be transfected in the presence of the transfection agent. This can e.g. B. by the passage of the cell suspension through a cannula or a suitable Extrusion device can be achieved with the appropriate membrane. One is advantageous Cannula inner diameter of 0.05 to 1 mm, a diameter of 0.1 to is preferred 0.8 mm, particularly preferred are 0.2 mm. When using membranes (e.g. Extrusion membranes) an average pore size of 0.5 to 50 µm is advantageous, 1 to 20 µm is preferred and 5 µm is particularly preferred. The passage takes place the cell suspension through the cannula or membrane takes place at least once, preferably  several times, in particular two to ten times, particularly preferably three to six times. The occurring Shear forces cause the temporary formation of membrane defects, which can Passage of the nucleic acid contained in the transfection agent or that contained therein Protein or peptide through the membrane barrier. The test conditions, and therefore the appropriate shear forces, vary from cell type to cell type and can vary from Experts can be determined experimentally without further notice. Parameters to consider are the number of passages through the cannula or membrane, the diameter of the Opening or pores, as well as the cell density. As a result, it is important to compromise between occurring membrane defects, which are lethal for the cells, and one sufficient number of membrane defects that prevent the passage to be transfected Allow substance to find. Suitable for the person skilled in the art are readily suitable Modifications of these techniques based on shear forces are conceivable.

In a further embodiment of the present invention, the transfecting cells in step ii) treated with ultrasound. For this are in particular Devices can be used that are normally used for cell disruption using ultrasound and are commercially available. The use of a Metal tip connected to an ultrasonic generator (e.g. devices from the company Bandelin, Berlin), particularly suitable. Ultrasound treatment of those to be transfected Cells preferably take place in a sonication vessel or an ultrasound chamber, wherein a system that a continuous inflow or outflow of the cell suspension and the Transfection agent allowed, is particularly preferred, what z. B. by using Pumps, such as peristaltic pumps, can be achieved. Commercial use Available ultrasonic baths as an ultrasonic chamber is within the scope of the present Invention also possible.

The transfection method according to the invention is suitable for batch methods, with the mixture from step i) in a suitable ultrasonic chamber Ultrasound is treated and the sonicated cells are then treated further be, e.g. B. mixed with culture medium and in a culture vessel or a bioreactor be transferred.

The mixing of the cells to be transfected with the transfection agent according to step i), as shown in FIG. 2, preferably takes place in a reservoir chamber 6 , in which the transfection agent can act on the cells. However, the mixing can also be carried out in a suitable manner in an external vessel.

In particular for large batches, ie for the transfection of large volumes of cell suspensions, step ii) and preferably also step i) of the method according to the invention are carried out in a continuous flow. For example, the procedure can be as follows: As shown in Fig. 2, the cells to be transfected are placed in the appropriate cell culture medium in the reservoir chamber (or the mixing tank) 6 , the transfection agent from a container 12 through an inlet using the pump 9 '' passed into the reservoir chamber 6 and mixed with the cells to be transfected. In addition, if necessary, fresh cell culture medium is passed from a container with medium 7 via pump 9 'into the reservoir chamber. The reservoir chamber 6 is also provided with a drain through which it is connected to the container for used medium 8 , to which intermediate pumps 9 are connected, so that a discharge of used medium is possible. In a preferred embodiment, filters or membranes 10 , which prevent a transfer of the cells into the collecting vessel, are located between the collecting vessel for used medium and the reservoir chamber (or the mixing tank) 6 . The mixture of medium, transfection agent and cells located in the reservoir chamber 6 is then pumped by a fourth pump 9 '''into the ultrasound chamber 3 , where it is guided past the ultrasound source 2 attached to a decoupler 1 and sonicated. In the example shown, the mixture flows after passing through the ultrasound source into a further chamber (bioreactor) 11 , in which the expression of a protein under cultivation conditions (e.g. after transfection with exogenous DNA) or the further cultivation of the cells (e.g. after the introduction of an exogenous protein or peptide).

In the context of the present invention, it is advantageous if the cell suspension is guided past the ultrasound source at a constant flow rate. It is readily possible for the person skilled in the art to set suitable flow rates or theoretical exposure times for each volume element containing cells, both in the case of sonication carried out continuously and in the form of pulses, in such a way that a high transfection efficiency with at the same time as gentle treatment of the cells and, in the case of transfection nucleic acids to be expressed, high yield of expressed product is achieved. For a given volume of the mixing chamber 6 of 1 ml and the sonication times and amplitudes mentioned below, a flow rate of 15-60 ml / min is advantageous, with a range of 20-40 ml / min. especially 30 mL / min. is preferred.

The ultrasound treatment in step ii) preferably takes place in cycles, i. H. in the form of Pulse instead, both in the batch process and in the process with continuous Flow. Suitable values for sound frequency, pulse duration and pulse intensity can be from Cell type to cell type vary and should be adapted from case to case, which the Expert in preliminary tests is easily possible. For a stationary approach, i.e. H. at the flow rate 0 (batch process), result at a constant frequency of 20 kHz and an amplitude of 40% of the maximum amplitude advantageous pulse lengths between 0.01 s and 1 s, a pulse length between 0.05 s and 0.5 s being preferred and a pulse length between 0.1 s and 0.2 s is particularly preferred. The number of In an advantageous embodiment, (similar) pulses are between 1 and 20, between 1 and 10 pulses are preferred, and between 2 and 4 pulses are particularly preferred same energy and duration. Here is the use of ultrasound frequencies between 18 kHz and 3 MHz is advantageous, frequencies between 19 kHz and 2 MHz preferred, particularly preferred are 20 kHz. For conversion to flow rates, the are not equal to 0, the average residence time of a volume element results in the ultrasound chamber from the action of a preferred number of pulses on the respective volume element results. If, for example, 2 pulses are preferred, then the Flow rate should be set so that after the 2nd Ultrasound pulses the respective volume element, preferably the capacity corresponds to the ultrasound chamber that should have left the ultrasound chamber.

In an advantageous embodiment, the present ultrasound sources Invention of commercially available sources (e.g. the device UW 2070 from Bandelin, Berlin) with the associated tips, the use of tips with a diameter between 1-10 mm is advantageous, a diameter of 2-8 mm preferred and a diameter of 2-6 mm is particularly preferred. It can for example the tips MS 73 (3 mm diameter) and KE 76 (6 mm diameter) from the company Bandelin can be used. There are generally peaks for the flow method preferred with a larger diameter than for the stationary approach. The exact Operating conditions of the tips and the ultrasound source can be understood by those skilled in the art without Further information can be found in the operating instructions of the corresponding manufacturers. The peaks  should always be cleaned and sterilized before use. B. by Exposure to chemicals such as ethanol or by autoclaving can happen.

In a further preferred embodiment of the present invention, in sonicating mixture of cells and transfection agent from step i) additionally contain acoustically unstable additives that disintegrate when exposed to ultrasound Promote cavitation and thus permeabilization of the cells during the Advantageously influence the sonication step. Many of these additives are known as Ultrasound contrast media commercially available.

In the context of the present invention can also during Sonication process in addition low molecular weight organic substances in the transfecting cells are introduced, this being in a preferred Embodiment effectors are, that is, activators or inhibitors of enzymes. However, other cellular components (such as e.g. Cell membrane or ER) or substances (e.g. DNA or lipids) interacting molecules be introduced, such as. B. coenzymes, hormones and hormone precursors.

In a further preferred embodiment, after passing the ultrasound source, the cell suspension is transferred directly into a reactor (e.g. a bioreactor) or into a vessel (e.g. a cell culture vessel 11 ) in which it is then preferably mixed with cell culture medium and e.g. B. can be cultivated for expression of the transfected DNA.

The vessels used in the present invention can both be specially manufactured for the purpose of the method according to the invention and also comprise combinations of commercially available vessels (for a selection of suitable sonication vessels, see, for example, the operating instructions from the Bandelin company). The geometry of the ultrasound chamber is of great importance for the method according to the invention, since the type and strength of the acoustic effects which act on the cells depend on the shape of the chamber. Parameters such as heat dissipation and mixing also depend on the vessel geometry and should be taken into account when designing the device. The following statements refer to the device shown in Fig. 1. In the context of the present invention, it is advantageous to use a slightly conical (glass) vessel as the ultrasonic chamber ( 3 ). The preferred dimensions of the ultrasound chamber and the ultrasound source (tip) located therein are as follows: diameter of the tip 6 mm, average inside diameter of the ultrasound chamber 1 cm, distance of the tip from the bottom of the chamber 1 cm, average wall distance of the tip 2 mm, minimum height of the drain (A) 2.5 cm above the chamber bottom, position of the inflow (Z) directly above the bottom of the ultrasonic chamber, inner diameter of Z and A 1-6 mm, depending on the flow rate.

An advantage of the device according to the invention is the ability to Flow method to transient cell transfection on a large scale to reach. This leads to the fact that transient transfection on an industrial scale Scale can be done, which is the production of larger amounts of protein made possible without the skilled person having to create a stably transfected cell line. Thereby there is the possibility to quickly and efficiently to protein quantities in milligrams or even gram range, the test series in the animal model (or also in the patient) and allow the efficient generation of antibodies directed against the protein. Consequently, the expert only needs one after these experiments have been carried out to perform permanent expression of promising "candidate proteins" and saves unnecessary effort for the not always unproblematic Generation of permanently transfected cell lines. The method according to the invention provides a valuable addition to many screening processes with high sample throughput which usually only contain small amounts of substances and their interactions and Can investigate properties in vitro.

Also when expressing or introducing proteins that are toxic, such as Nucleases, proteases, lipases, prions or pore-forming proteins make this possible The inventive method an increased transfection yield and thus opens the Possibility of larger amounts of these substances in transiently transfected cells to manufacture or incorporate into this. Also hydrophobic proteins, which are usually used in the permanent transfection can cause major problems in large quantities in one short timeframes can be obtained.

Furthermore, the present invention comprises a method for producing an efficient, phosphate-based additive which facilitates transfection and which offers numerous advantages over the previously known additives. While in the calcium phosphate coprecipitation method from the prior art nucleic acid or protein and calcium phosphate are precipitated together, which, for. B. can be done by presenting a solution containing DNA and phosphate with subsequent addition of CaCl ₂ , in the process according to the invention the precipitation is first carried out (step a)), the precipitate (step b)) or after resuspending the precipitate from step b) the suspension obtained (step c)) can then be used for several days or even weeks for the production of the transfection agent according to the invention.

In order to achieve the precipitation in step a), two solutions are mixed, one solution being Ca ²⁺ ions and the other PO ₄ ^3- , HPO ₄ ^2- , H ₂ PO ₄ ^- , or H ₃ PO ₄ , and optionally F ^- , Cl ^- or OH ^- contains. The person skilled in the art can readily see that the precipitation process is a pH and temperature-dependent reaction. In a particularly preferred embodiment, the two solutions are immediately mixed completely in step a), so that the precipitation process takes place suddenly.

In step b), after an incubation period of several minutes to hours, the precipitate formed separated. The separation can be done by various means Processes known to those skilled in the art, such as centrifugation (for example 4 krpm, 10 min. Heraeus Minifuge) or filters. The supernatant is discarded and the precipitate before Incubation with the nucleic acid or protein or peptide or before further processing if necessary kept.

In an advantageous embodiment, the precipitate from step b) is resuspended. The Resuspension takes place in a solution that is suitable for further processing of the calcium and Phosphate-containing additive is suitable for the transfection agent, wherein isotonic biocompatible buffers such as B. Hepes-buffered NaCl solution are preferred. It should be emphasized here that the present method is not only the introduction of Nucleic acids in cells, but also an efficient introduction of proteins and Peptides enables.

Accordingly, in a preferred embodiment, the resuspending of the in Step b) obtained precipitate in a solution that is already the one to be transfected Contains nucleic acid or the protein or peptide to be transfected. Here the Calcium phosphate soil from step b), for example by multiple suction  and ejection suspended in said solution by means of a pipette. After that it will Incubated mixture, which is preferably a few minutes to hours, and then used for transfection (step i) of the invention Transfection procedure). Before further use in step i), the The transfection method separated the precipitate from the mixture mentioned, e.g. B. by Centrifugation (4 krpm, 10 min. Heraeus Minifuge) or filtration, and only the one obtained Supernatant used for transfection.

It is also preferred to first precipitate the precipitate from step b) in a suitable one Solution containing no nucleic acids, proteins or peptides, e.g. B. a biocompatible isotonic buffer (see above), and then resuspend the suspension with a Mix solution containing the nucleic acid to be transfected or the one to be transfected Contains protein or peptides, and the mixture thus obtained, if necessary after incubation for several minutes to hours for transfection according to step i) of the invention Use transfection procedures. In a preferred embodiment, the Precipitate is first separated from the mixture by centrifugation or filtration, and only the supernatant thus obtained is used for transfection.

In contrast to the calcium phosphate coprecipitation method, the preferred embodiments of the method according to the invention the supernatant Transfection is used while discarding the soil body. This has compared to the known method the advantage that no macroscopic amounts of calcium phosphate what is brought into the transfection batch and thus into the cell culture medium be particularly problematic with regard to the adsorption of proteins present there (Hydroxyapatite, a calcium phosphate containing hydroxyl ions, becomes Protein purification used) and also negatively affects the cell homeostasis, because in the co-precipitation method, the slurried calcium phosphate / DNA soil settles on the cells.

The inventive method and the use of the inventive Transfection agent with the calcium phosphate based additive also perform as u. a. shown in Example 3, for an improved expression of the introduced Nucleic acid sequence, low cell damage and long cell life after  Transfection and thus high (overall) product yields per culture approach, which one represents an important economic factor.

The following are exemplary embodiments which are intended to explain the present invention in more detail, but are not to be understood as restricting the invention.

example 1 Transfection of CHO cells with DNA using Calcium phosphate and ultrasound treatment, optimization of pulse duration and number

First, a DNA-containing transfection agent is made using the additive calcium phosphate. For this purpose, a calcium phosphate precipitate is first produced. This is done by mixing equal volumes of sterile-filtered 0.25 M CaCl ₂ solution and 2 × HBS (0.28 M NaCl, 0.05 M HEPES, 1.5 mM Na ₂ HPO ₄ , adjusted to pH 7.05 with NaOH) . The reaction mixture is incubated for 10 min at room temperature and then centrifuged for 10 min at 4 krpm (Heraeus minifuge). The resulting pellet is finally taken up in 1/10 of the starting volume (sterile 20 mM HEPES buffer, 150 mM NaCl (pH 7.4)) and stored at 4 ° C.

Then 30 μL of a resuspended calcium phosphate suspension prepared as described above are mixed with 30 μg of the plasmid pCSEAP in 20 mM HEPES, 150 mM NaCl, pH 7.4 in order to obtain the transfection agent in a final volume of 100 μL. The plasmid pCSEAP codes for the secretory alkaline phosphatase (SEAP). The plasmid also contains a CMV promoter, the activity of the SEAP in the supernatant of transiently transfected cells using the method of Berger et al. (Berger et al., Gene 66, pp. 1-10, (1988)) can be easily determined. The 100 µL transfection agent thus obtained is then carefully mixed with 1 mL serum-free cultured CHO cells (cell density = 2 × 10 ⁶ cells / mL) in a sterile vessel. The CHO cells used are harvested in the exponential growth phase and the desired cell density (2 × 10 ⁶ cells / mL) is set by adding appropriate amounts of preheated cell culture medium.

The transfected cells are then immediately subjected to an ultrasound treatment a Bandelin sound system (UW 2070 with step horn SH 70 G and HF generator GM 2070), using a (micro) tip with a 3 mm diameter. The tip is disinfected with 70% ethanol before use. After 48 hours under Standard expression conditions (37 ° C) are taken from 100 µL of the mixture and 10 min heat inactivated at 65 ° C. Then the SEAP activity after the above-mentioned. Standard procedure determined.

In order to achieve optimal transfection efficiency, optimization experiments are carried out with a varying number of ultrasound pulses as well as different ultrasound Pulse lengths performed. The sound energy in each of these experiments is 40% of the Maximum amplitude. The following approaches, listed in Table 1, are used examined:

Table 1

The results of the experiments shown in Table 1 are shown in Fig. 3. The experiments prove that under the given test conditions, longer sonication times (0.2 s) result in a significantly lower transfection efficiency, with an increasing number of pulses also having a negative effect with a longer pulse length. The experiments shown enable the person skilled in the art to optimize and adapt the method according to the invention to different transfection systems.

Example 2 Transfection of CHO cells with DNA using Calcium phosphate and ultrasound treatment, optimization of pulse duration and energy

In this exemplary embodiment, an optimization of the pulse energy was carried out in connection with a different number of ultrasonic pulses for the given system CHO cells / pCSEAP plasmid. The CaP _i precipitate and the transfection agent were prepared as described in Example 1. Table 2 shows the various experimental approaches schematically. A constant pulse duration of 0.1 s was used in each case.

Table 2

The results of the experiments shown in Table 2 are shown in Fig. 4. It shows that a particularly high transfection efficiency is achieved for the system used with a pulse duration of 0.1 s, a pulse energy of 40% power (device setting for the Bandelin Sonifier) and two pulses.

Example 3 Transfection of CHO cells with DNA using Calcium phosphate and ultrasound treatment, optimization of the Expression duration and the amount of DNA and calcium phosphate

The test conditions are identical to those given in Example 1, with the Difference that the amount of plasmid (pCSEAP) and an Calcium phosphate, as well as the expression duration is varied. Table 3 shows the different experimental approaches schematically. The plasmid concentration of Stock solution is 1 mg / mL, the calcium phosphate solution was prepared as below Example 1 described.

Table 3

The results of the experiments schematically listed in Table 3 are shown in Fig. 5. The absolute SEAP activity [mU] is plotted on the ordinate, the numbers of the corresponding approaches are noted on the abscissa. Except in batches 1 and 17, three expression times, 24 h, 48 h and 72 h, were measured in each case. It turns out that the cells were still able to produce proteins even after three days and that the use of 40 .mu.L plasmid (1 mg / mL) and 20 .mu.L calcium phosphate suspension for optimal expression of the enzyme and little cell damage and thus leading to the highest product yields.

Example 4 Transfection of CHO cells with DNA using Calcium phosphate and ultrasound treatment, use of HEK 293 cells

In this experiment, HEK 293 cells are used instead of CHO cells in order to document the applicability of the transfection method according to the invention to different cell lines. For this purpose, HEK 293 cells are cultivated in DMEM medium, which contains 10% fetal calf serum. The cells are then harvested using an enzyme-free dissociation solution (Sigma) and centrifuged. Then the cells are resuspended in serum-free medium, so that a cell density of 2 × 10 ⁶ cells / mL results. 30 μg of pCSEAP in 20 mM HEPES, 150 mM NaCl, pH 7.4 are then mixed with 30 μL of the calcium phosphate suspension described in Example 1, so that a final volume of 100 μL results. 1 mL cell suspension is added to this mixture, then the mixture is transferred to an incubation vessel. Finally, 2 × 0.1 s with 40% power is sonicated with the Bandelin ultrasound device using a 3 mm tip. After 48 h of incubation (expression), 100 μL of the mixture are removed and examined for SEAP activity. The result is an activity of 1.275 mU total SEAP activity. This experiment proves that cell lines other than CHO cells can also be used in the context of the present invention.

Example 5 Transfection of CHO cells with DNA using various Calcium phosphate fractions and ultrasound treatment

30 µg pCSEAP in 20 mM HEPES, 150 mM NaCl, pH 7.4 are mixed with 20 µL CaP _i suspension (prepared as in Example 1), so that a total volume of 100 µL results. The suspension is then centrifuged for 5 min at 13 krpm (Heraeus minifuge). The supernatant is then used directly for transfection; the pellet is resuspended in 100 µL 20 mM HEPES, 150 mM NaCl, pH 7.4 and then also used for transfection. The 2 × 100 µL transfection agent thus obtained are then carefully mixed with 1 mL serum-free cultured CHO cells (cell density 2 × 10 ⁶ cells / mL) in a sterile vessel. The CHO cells used are harvested in the exponential growth phase and the desired cell density is set by adding appropriate amounts of preheated cell culture medium. The cells to be transfected are then immediately subjected to an ultrasound treatment using a Bandelin sonication device (Bandelin, Berlin), sonicating 2 × 0.1 s at 40% power and using a (micro) tip with a diameter of 3 mm. The tip is disinfected with 70% ethanol before use. After 40 hours under standard expression conditions (37 ° C.), 100 μL of the mixture are removed and heat inactivated at 65 ° C. for 10 minutes. The SEAP activity is then determined using a standard procedure. Table 4 shows the various experimental approaches schematically.

Table 4

The results of the test series shown schematically in Table 4 are shown in Fig. 6. It is shown that the use of the supernatant after mixing a calcium phosphate suspension with DNA and centrifuging for transfection is far more suitable than the resuspended soil body (pellet) containing high concentrations of calcium phosphate (see approach No. 6).

Example 6 Transfection of CHO cells with DNA using Calcium phosphate and shear forces

CHO cells cultured under serum-free conditions are harvested in the exponential growth phase and brought to a cell density of 2 × 10 ⁶ cells / mL. The additive is prepared as indicated in Example 1. Then 30 µg pCSEAP are mixed with 20 µL or 200 µL calcium phosphate suspension and added to 1 mL cell suspension. The mixture is then drawn into a 1 mL syringe equipped with a 0.4 × 20 mm hollow needle. The suspension is then expelled several times and sucked up again (see table). After 20 h of incubation, 50 μL of the culture medium heat-inactivated at 65 ° C. for 10 min are removed and a SEAP activity test is carried out. In this case, the absorption at 405 nm was determined after 2 h of incubation at 37 ° C. Table 5 shows a summary of the experiments carried out.

Table 5

The results of the tests listed in Table 5 are shown in Fig. 7. A combination of 200 µL calcium phosphate suspension with 8 aspirations and expulsions proved to be optimal for maximizing the transfection yield of the system used.

Example 7 Transfection of CHO cells with DNA using Calcium phosphate and ultrasound, optimization of sonication times

CHO cells cultured under serum-free conditions are harvested in the exponential growth phase and brought to a cell density of 2 × 10 ⁶ cells / mL. The additive is prepared as indicated in Example 1. Then 10 µg pCSEAP are mixed with 10 µL calcium phosphate suspension and added to 1 mL cell suspension. The cell suspension is sonicated at 70% output with different sonication times (see Table 6). After 20 h of incubation, 50 μL of the culture medium heat-inactivated at 65 ° C. for 10 min are removed and a SEAP activity test is carried out. In this case, the absorption at 405 nm was determined after 2 h of incubation at 37 ° C. Table 6 shows a summary of the experiments carried out.

Table 6

The results of the experiments listed in Table 6 are shown in Fig. 8. As can be seen in Fig. 8, the sonication time for the system used at 70% (sound) power is 0.5 s maximum transfection yields.

Example 8 Flow transfection

A transfection batch analogous to example 5 is set up, a volume of 35 ml of cell suspension being used. The CHO suspension cells used came from a spinner culture. The cells are adjusted to a cell density of 2 × 10 ⁶ cells / mL with fresh medium and mixed with the transfection agent. The mixture is passed through the reservoir chamber, which contains a 6 mm ultrasound tip, using a peristaltic pump. The flow rate is set to 30 mL / min, the sound power is set to 40% of the maximum amplitude. The pulse duration is 0.1 s, which are interrupted by pauses of 0.9 s. On average, each mL of cell suspension receives two pulses lasting 0.1 s. Six medium fractions of 1 mL each are collected separately and 100 µL of them are removed after 40 h of incubation or expression. The SEAP activity is then determined using the standard method mentioned.

The results of the above experiments are shown in Fig. 9, which shows the activity of 6 removed fractions of 1 mL each. The transfection rates achieved in the flow-through transfection method according to the invention are consequently similar to those achieved in the stationary batches and show that a constant high level of expression is achieved in cells transfected in the flow-through method.

Claims (20)

1. A method for introducing nucleic acids and proteins or peptides into eukaryotic cells, comprising the following steps:

• a) mixing the cells in suspension with a transfection agent which contains the nucleic acid or the protein or peptide to be introduced and an additive comprising calcium and phosphate,
• b) treatment of the mixture obtained in step i) with ultrasound or shear forces.

2. The method according to claim 1, wherein step ii) and optionally also the Step i) is carried out in a continuous process. 3. The method according to any one of claims 1 or 2, wherein it is in the nucleic acid to be introduced around an oligonucleotide or polynucleotide, ssRNA, dsRNA, ssDNA or dsDNA. 4. The method according to any one of claims 1-3, wherein the protein to be introduced Is prion or a fragment of a protein. 5. The method according to any one of claims 1-4, wherein the cells used are adherent Are cells. 6. The method according to any one of claims 1-5, wherein the cells used are non- are adherent cells. 7. The method according to any one of claims 1-6, wherein the ultrasonic exposure in step ii) is carried out in cycles. 8. Cells transfected with proteins or nucleic acids, obtainable or obtained from a method according to any one of claims 1-7.   9. A process for producing a calcium and phosphate-containing additive for a transfection agent for introducing nucleic acids or proteins and peptides into eukaryotic cells, which comprises the following steps:

• a) Mixing a solution containing Ca ²⁺ ions with a solution containing P _i and optionally X, so that a precipitate is formed in the mixture, where P _{i is} phosphate, hydrogen phosphate, dihydrogen phosphate or phosphoric acid and X is Cl ^- , F ^- or OH ^- and incubating the mixture to form a calcium and phosphate-containing precipitate;
• b) separating the resulting precipitate from the mixture from step a) and discarding the supernatant;
• c) optionally resuspending the precipitate obtained in step b).

10. A method for producing a transfection agent for introducing nucleic acids and proteins or peptides into eukaryotic cells, comprising:

• a) Mixing a solution containing Ca ²⁺ ions with a solution containing P _i and optionally X, so that a precipitate is formed in the mixture, where P _{i is} phosphate, hydrogen phosphate, dihydrogen phosphate or phosphoric acid and X is Cl ^- , F ^- or OH ^- and incubating the mixture to form a calcium and phosphate-containing precipitate;
• b) separating the resulting precipitate from the mixture from step a) and discarding the supernatant;
• c) resuspending the precipitate obtained in step b) with a solution containing the nucleic acid or the protein or peptide and optionally separating and discarding the precipitate; or
• d) resuspending the precipitate obtained in step b), and then mixing the resuspended precipitate with a solution containing the nucleic acid or the protein or peptide and optionally separating and discarding the precipitate.

11. The method according to any one of claims 1-7, wherein the additive or Transfection agent can be produced by a method according to claim 10 or 11 is.   12. Additive that prevents the uptake of nucleic acids or proteins and peptides promotes eukaryotic cells, or transfection agent for the introduction of Nucleic acids and proteins or peptides in eukaryotic cells available by a method according to claim 9 or 10. 13. Use of the additive or the transfection agent according to claim 13 in a method for introducing nucleic acids and proteins or peptides into eukaryotic cells. 14. Device for introducing nucleic acids and proteins or peptides into eukaryotic cells, comprising the following components:

• a) an ultrasound chamber with an ultrasound source
• b) a drain and an inflow, each connected to the ultrasonic chamber
• c) a reservoir chamber connected to the inflow and optionally containing a mixture of eukaryotic cells with a transfection agent as defined in claim 1.

15. The apparatus of claim 15, wherein the ultrasound source is a metal tip. 16. The device according to one of claims 14 or 15, wherein the reservoir chamber is connected via the drain to a culture vessel or bioreactor. 17. The device according to any one of claims 14-16, wherein the reservoir chamber one Outflow and has an inflow. 18. The apparatus according to any one of claims 14-17, wherein the drain and the inflow be regulated by pumps. 19. The device according to any one of claims 14-18, wherein the ultrasonic chamber Is ultrasonic bath.   20. Use of a device according to one of claims 14-19 in one Process for the introduction of nucleic acids and proteins or peptides into eukaryotic cells.