EP1244770A2

EP1244770A2 - Device for transferring molecules into cells

Info

Publication number: EP1244770A2
Application number: EP00990583A
Authority: EP
Inventors: Friedrich Ueberle
Original assignee: Dornier Medizintechnik GmbH
Current assignee: Dornier Medizintechnik GmbH
Priority date: 1999-12-23
Filing date: 2000-12-23
Publication date: 2002-10-02
Also published as: CA2397271A1; WO2001048181A3; JP2003533974A; US20030017578A1; WO2001048181A2

Abstract

The invention relates to a medium in which cells and molecules to be transferred in these cells are located. Said medium is moved inside a hollow cylindrical device, which generates acoustic pulses that induce cavitation, in relation to an area in which the acoustic pulses occur in a focussed manner.

Description

Device for the transfer of molecules into cells.

The invention relates to a device according to the preamble of claim 1

From the German application 19834612.3-41 a device and a method is known in which the intracellular transfer of oligonucleotides is described under the influence of shock waves. In the invention described there, a sample container with the molecules to be transferred and the target cells is placed in a type of water bath and exposed to a predetermined number of shock waves. The molecules and the target cells are in a medium that conducts sound waves and in which the vital functions of the target cells remain. These shock waves create a so-called cavitation, which makes the target cells transiently permeable to the molecules to be transferred.

The sample container is then changed. This has the disadvantage that the molecules to be transferred and the target cells have to be brought into the sample container and then into the water bath in a relatively complex manner. This results in a quantitative limitation of the target cells doped with molecules, which makes the known prior art unsuitable for commercial use, for example in the pharmaceutical industry.

It is known from DE 3821354 to generate cavitation in larger volumes. A so-called sonotrode is used there to generate the cavitation. However, it is also known from this document that even high-performance sonotrodes alone cannot efficiently supply large volumes with short-term cavitation, so additional activators are introduced into the suspension, which in turn are brought into resonance and then generate cavitation in their environment in the resonant state. The disadvantage here is that these activators have to be removed from the suspension again.

It is also known from WO 99/58637 to generate cavitation in a volume from piezo transducers. Controllable, constructive and destructive sound interference is generated with phase-shifted control with the aim of achieving a kind of focus at locations of interference maxima. The disadvantage here, however, is that the so generated Focus area includes only small volumes and the technical effort for the control and regulation of the piezo transducer arrays is very high.

In order to effect transfection on the one hand and to destroy as few cells as possible on the other hand, it has surprisingly turned out to be particularly advantageous if the sound pulses only act for a very short time, but at the same time exceed the cavitation threshold of the liquid.

In known devices for generating such cavitation-generating sound pulses that work with the so-called sonotrodes, there is the serious disadvantage that the

Sonotrodes must first be brought into resonance in order to generate vibrations which have such a high amplitude that exceeds the cavitation threshold. In a disadvantageous manner, individual pulses are therefore not possible. This also has the disadvantage that too high a "dose" of cavitation events act on the cells and the cells are thereby destroyed.

The conventional design of the public address system with sonotrodes also promotes foam and aerosol formation, which reduces the reproducibility of the public address results.

In the device known from WO 99/58637, with arrays of piezo transducers driven out of phase, the focus changes as soon as cavitation occurs, as a result of which the focus area can only be predicted stochastically.

It is therefore an object of the invention to provide a device by means of which molecules can be transferred into target cells and thereby overcome the disadvantages of the prior art and, in particular, enable rationally greatly increased production.

It is an object of the invention to provide a device with which cavitation can be generated which, on the one hand, is sufficient to make cells transiently permeable to molecules, but on the other hand only lasts so short that the cells are essentially unsustainable be damaged and, in particular, an efficient, massively increased production is made possible.

This task is accomplished by the characterizing features of the respective main claims.

The respective subclaims relate to further developments and / or particularly advantageous refinements of the invention.

The considerations that led to the creation of the invention adopted the knowledge that a hollow cylindrical device into which a multitude of in-phase sound pulses are introduced radially from the outside produces a reproducible zone of transient cavitation events in the area around the axis of rotation of the hollow cylinder. The considerations that led to the development of the invention assumed that it is irrelevant for the transfer of the molecules into the target cells, how large is the total amount of the medium in which the molecules and target cells to be transferred are located, if any parts from the total amount successively reach the effective range of the acoustic pulses and the target cells contained therein are thus exposed to treatment.

In order to treat a volume of this medium that is large in relation to the effective range of the focused acoustic pulses, it is provided according to the invention that a relative movement takes place between the source of the focused acoustic pulses and the medium to be treated, as a result of which new acoustic pulses that have not previously been focused are always treated Areas of the medium are treated.

This relative movement can now take place by moving the source relative to a fixed liquid container or by moving the liquid container relative to a fixed source.

Accordingly, according to the invention, the medium with the target cells and the molecules to be transferred can also move through a line system while it is under the influence of the focused acoustic pulses. In general, there must be a relative speed between the medium and the source of acoustic pulses that is adapted to the spatial extent of the effective range of the acoustic pulses and the repetition frequency of the acoustic pulses. The parameters must be matched to one another in such a way that the target cells are exposed to at least the predetermined number of pulses as they move through the line system.

The invention is explained in more detail below on the basis of a possible exemplary embodiment.

Figure 1 shows a possible embodiment of the invention. Acoustic pulses are generated by means of an essentially hollow cylindrical source 1, through which a tubular line 2 is guided. The medium is located in this line 2. In the case shown, it is a liquid F that has a viscosity that allows it to flow through line 2.

The source 1 is designed in such a way that it focuses the acoustic pulses in its interior and thus in the area in which the line 2 runs. The focus here is essentially axial. The area of the focus is the so-called effective area of the acoustic pulses, which is why it is called the focus area in the following. In the example shown, the source 1 is designed to be rotationally symmetrical cylindrical. The wall of line 2 is permeable to acoustic pulses at least in the area of the focus. Between the source 1 and the line 2 there is a suitable coupling medium which transmits the acoustic pulses from the source 1 to the line 2.

The principle of excitation for the acoustic pulses is to use a large number of piezo elements, all of which are concentrically directed in the direction of the axis of rotation and are excited simultaneously, in phase. In this way it is possible to create an elongated focus area.

It is also possible to implement the cylindrical source instead of a plurality of piezo elements concentrically aligned with the axis of rotation by means of a plurality of piezo rings lying next to one another, the center of which lie on the axis of rotation. It is also possible to use magnetostrictive actuators to generate the acoustic pulses

Elements to use.

A further embodiment of the invention provides for the source 1 to be designed as a hollow body with a semicircular cross section as a more channel-shaped body. In these embodiments, there is a focus area that extends approximately over the length of the source. The line in which the liquid F is located runs through this elongated focus area. This has the advantage that the source can be coupled to an existing line and the line does not first have to be laid through the hollow cylindrical source 1.

It is also possible to provide a gap in the hollow cylindrical source through which the line can be brought into the interior of the hollow cylindrical body. In this embodiment too, the source can advantageously be coupled to an existing line.

The design of a source 1 according to FIG. 2 with a semicircular cross section offers the advantage that the source can easily be attached to a line from the side without the line having to be laid through the interior of the source.

It is also possible to make the hollow body divisible and then to lay it around the line from both sides.

It is also possible to use an exploding wire as the source of the acoustic pulses, this wire being stretched along the focal line of an elliptically or parabolically shaped hollow tube.

Likewise, a coil can also be used for the excitation, which acts on a membrane located within the coil and thus generates an inwardly directed acoustic pulse. Acoustic pulses are emitted by the source 1 onto the liquid F and briefly create conditions there (pressure or negative pressure with sufficient intensity, cavitation) in order to bring about a transfer of the molecules into the target cells.

It has been shown that the molecules can be transferred into the cells within wide pressure or negative pressure ranges. The pressure ranges are between 10 Mpa and 150 Mpa. The vacuum ranges from -5 Mpa to -50 Mpa. The intensity is between 0.5 mJ / mm2 and 5.0 mJ / mm2.

In addition, it has been shown that a longer duration of the cavitation events does not contribute to an increase in the transfection rate, but rather disadvantageously increases the damage to the cells with increasing duration of the cavitation events.

Therefore, only a short-term cavitation event is generated with the source according to the invention.

The flow rate Vf of the liquid F can be changed by a pump, which is not shown for reasons of clarity.

The source 1 is excited by a unit 3 in pulse form. When flowing through line 2, the target cells and the molecules to be transferred are exposed to a predetermined number of acoustic pulses of a predetermined intensity.

Due to the flow and cavitation-related eddies within line 2, all cells of the suspension come into the effective range of the short-term cavitation sufficiently often when flowing through the source and thus become transiently permeable.

It is also possible to let the liquid F flow through the line 2 in a pulsed or clocked manner. In this case, the liquid F is always conveyed in such a way that the area which was just in front of the source 1 in the line is conveyed so far that it then comes to a standstill at the end of the source 1. While the liquid area within source 1 is in the focus of the acoustic pulses, a predetermined number becomes emitted by acoustic pulses that stimulate the transfer of molecules into the target cells.

The acoustic pulses that act on the liquid or generally formulated on the medium can consist of ultrasound pulses or one or more successive shock waves.

It has been shown that the transfer of the molecules into the cells depends on the number of ultrasound impulses or shock waves. The transfection rate increases with a higher number of ultrasound pulses or shock waves. Likewise, the number of cells damaged by the treatment increases with the number of ultrasound impulses or shock waves.

The intensity (unit of measurement mJ / mm2) of the ultrasound impulses or shock waves also influences both the number of intracellularly transferred molecules and the damage to the cells. With increasing intensity, there is an increase in both the number of intracellularly transferred molecules and the damaged cell.

Analogous to the described movement of the medium through the line, it is also possible to realize the relative movement according to the invention between the source of acoustic pulses and the medium in which the molecules and the target cells are located by moving a container in which the medium is located , The source of the acoustic pulses is fixed and the container is moved.

The container can be of any shape here, but it is crucial that the movement of the container takes place in such a way that the entire volume of the container has successively reached the effective range of the acoustic pulses.

It is also possible to fill and seal the container at a sterile location and then treat this container in a "non-sterile area" with the acoustic pulses zα. It is also possible to make the container stationary and one or more

Moving sources of acoustic pulses so that the entire volume of the container successively reaches the effective range of the acoustic pulses.

All movements of the medium relative to the source of acoustic pulses can be continuous or clocked.

In a further development of the invention, it is provided that the molecules to be transferred are fed to the medium in which the target cells are located only directly before the focus area.

A particularly advantageous construction of the source is shown in FIGS. 3 and 4.

The source shown in FIG. 3 consists of an inner ring 3, which can accommodate the line (not shown here) or a sample tube in its inner free space 7.

Piezo elements 4 are arranged distributed around the outer circumference of the inner ring 3. For reasons of clarity, only some of these piezo elements 4 are shown in this illustration.

The piezo elements 4 are held on their side facing away from the inner ring 3 by a further ring 6. The outer ring 6 is designed as a clamping ring. Its inside diameter can be changed using a clamping screw 2. This also changes the gap 1.

In this way, the piezo elements 4 can be firmly clamped between the inner ring 3 and the outer clamping ring 6. This ensures good sound transmission between the piezo elements 4 and the rings 4 and 6.

FIG. 4 shows a further version of the device according to the invention. There, the piezo elements 4 are arranged around the inner ring 3 in the manner known from FIG. 3. An intermediate ring 8 rests on its side facing away from the inner ring 3. Piezo elements 7 are in turn arranged on this intermediate ring 8. These piezo elements 7 are followed by the clamping ring 6 already known from FIG. 3.

Claims

Device for the transfer of molecules into cells

1 .. Device for the transfer of molecules into cells, wherein a medium (F) in which the molecules to be transferred and the target cells are in the focus area of a source of acoustic pulses can be exposed to these acoustic pulses, characterized in that the source at least approximately has a linear focus area in which the acoustic pulses exceed a predetermined pressure or negative pressure and / or a predetermined intensity and a device is provided by which a relative movement between the medium (F) and the focus area is carried out.

2. Device according to claim 1, characterized in that a line (2) is provided, through which the medium (F) can be transported and the line is guided through the focus area of the source (1).

3. Device according to claim 1 or 2, characterized in that the source (1) is designed as an essentially hollow cylindrical body and the focus region runs along the central axis of the hollow cylindrical body.

4. The device according to claim 1 or 2, characterized in that the source (1) of acoustic pulses is designed as a body with a semicircular cross section and the focus region runs along the center line of the semicircle.

5. Device according to one of claims 1 to 4, characterized in that the source (1) comprises a plurality of piezo elements which can be excited in time and in phase.

6. Device according to one of claims 1 to 5, characterized in that the source (1) consists of a plurality of piezo rings lying side by side.

7. Device according to one of claims 1 to 6, characterized in that access to the line (2) is provided, through which the molecules to be transferred can be fed to the medium (F) in which the target cells are located.

8. Device according to one of claims 1 to 7, characterized in that a plurality of sound transducers is applied to a common carrier and a mechanical element (6) presses the sound transducer (4) on the carrier (3).

9. The device according to claim 8, characterized in that the mechanical element (6) touches the sound transducer (4) on the side that touches the side facing away from the carrier (3).

9. The device according to claim 8, characterized in that the mechanical element (6) is designed in the form of a clamping ring.

10. The device according to claim 8, characterized in that the carrier (3) and the mechanical element (6) are annular.

11. Device according to one of claims 8 to 10, characterized in that the sound transducers are arranged in several layers, with different distances from the carrier (3).

12. The device according to claim 11, characterized in that the different layers of!: Chall transducers are separated by separating layers.