WO2005005595A1

WO2005005595A1 - Cell treatment chamber

Info

Publication number: WO2005005595A1
Application number: PCT/EP2004/006661
Authority: WO
Inventors: Michael Blumentritt; Vinh Duong; Rüdiger HUHN
Original assignee: Eppendorf Ag
Priority date: 2003-07-08
Filing date: 2004-06-19
Publication date: 2005-01-20
Also published as: DE10330674A1; DE10330674B4

Abstract

The invention relates to a chamber for treating cells contained in a suspension in an electrical field, said chamber comprising a receiving element for the suspension, at least two electrodes which are associated with the receiving element and between which an electrical field for treating cells in a suspension contained in the receiving element can be produced by applying a voltage, and a plastic structure which is connected to at least one electrode by shaping or by a material composition and is used to hold the same.

Description

Cell treatment chamber

The invention relates to a chamber for treating cells contained in a suspension in the electric field.

The chamber according to the invention serves e.g. the electrofusion of cells. In electrofusion, at least two cells are fused in an electrical field that is built up between two electrodes. The electrofusion can e.g. run in three steps. In a first step (pre-alignment), the cells are aligned by applying a high-frequency AC voltage and brought into contact with one another. In a second step (pulse), the cell membranes are broken up using a short, strong current pulse. In a third step (post-alignment), the broken cells are fused by applying a high-frequency AC voltage again.

Cell fusion chambers are known in which two thin platinum wires, which form the electrodes, are wound in parallel in a pair of helical grooves on a conical plastic body. Furthermore, the cell fusion chambers have a conical vessel into which the plastic body can be screwed. The production of the wire electrodes is complex and expensive, so that only a multiple use of the cell fusion chambers is economical. For this purpose, the cell fusion chambers have to be cleaned and sterilized between different applications.

^" EP 1 245 669 A1 discloses a chamber for treating cells contained in a suspension in an electrical field, in which the plate-shaped and closed-surface electrodes are made entirely of electrically conductive Material are formed or have a core with a coating of conductive material forming the electrode surface. With a view to simplifying the production process, for example, electrically conductive plastics (plastics which are filled with carbon) or semiconductor materials can preferably be used. These materials can be processed particularly easily by means of injection molding technology, which is particularly advantageous in the case of chambers which are intended to be disposable. But it is also conceivable that metallized plastic plates are used. The chambers described are still relatively complex and only suitable for use as a disposable item to a limited extent.

This also applies to the electrode chamber disclosed in DE 101 27 247 AI, which has a suspension container in which at least one electrode carrier with at least one pair of electrodes is arranged, the electrode carrier being formed by a carrier body with at least one planar side surface and that at least one pair of electrodes is arranged on the side surface. According to one embodiment, the plate-shaped carrier body of the electrode carrier is releasably attached to positioning devices in the suspension container. The carrier body consists e.g. made of plastic or glass and can be produced, for example, as an injection molded part. The electrodes are each formed from straight electrode strips as interdigitated comb electrodes. The electrodes are preferably made of precious metals, e.g. Gold or platinum, inert alloys, or optionally copper or aluminum.

For the rest, the application background and the prior art for cell fusion chambers are detailed in the introductory descriptions of EP 1 245 669 AI and DE 101 27 247 AI, which are included in the present application. Proceeding from this, the object of the invention is to create a chamber for treating cells contained in a suspension, which is less complex and is better suited for execution as a disposable item.

The object is achieved by a chamber with the features of claim 1. Advantageous embodiments of the chamber are specified in the subclaims.

The chamber according to the invention for treating cells contained in a suspension in the electric field has a receptacle for the suspension,

at least two electrodes assigned to the receptacle, between which an electrical field for treating cells of a suspension contained in the receptacle can be generated by applying a voltage, and

- A plastic structure connected to at least one electrode by means of master forms or material connections for holding the electrode.

In the chamber according to the invention, at least one electrode is connected to a structure for holding the electrodes by means of primary shaping or material bonding. This enables the electrodes to be fixed inexpensively relative to one another or with respect to a receptacle, which is advantageous for inexpensive production in large numbers and the execution of the chamber as a disposable item. At least one pair of electrodes for generating an electrical field is preferably connected to one or more structures.

According to one embodiment, master shaping is injection molding, ie when the structure is injection molded, at least one electrode is simultaneously with the structure injection connected. According to one embodiment, the material connection is injection molding, ie a prefabricated structure is injection molded with at least one electrode. According to one embodiment, the material connection is an adhesive, ie the prefabricated structure is bonded to at least one electrode, for example by means of an adhesive adhesive or hot adhesive. According to one embodiment, the material connection is welding, ie the prefabricated structure is welded to at least one electrode. The welding is, for example, a laser beam welding.

To connect to the structure e.g. at least one electrode is introduced into a special injection molding tool and is completely or partially back-molded or overmolded with at least partial release of the electrode. The chamber according to the invention can be made available as a disposable article due to the use of inexpensive materials and processing techniques. The injection molding technology enables contamination-free / low-contamination production. The materials can be chosen so that the chamber is autoclavable. If necessary, the chamber can already be manufactured in the desired purity quality by the manufacturer or brought and packaged by sterilization or autoclaving, so that sterilization or autoclaving is not necessary for the user.

The invention makes it possible to manufacture inexpensive plastic disposables and, if necessary, to provide them in the desired purity quality for the user.

The electrodes are e.g. Wire electrodes made of electrically conductive wire, which are connected to the structure by master forms or material connections. Then the structure is e.g. designed as an isolator.

According to one embodiment, the electrodes are formed by electrically conductive layer structures arranged on at least one insulating film. These electrodes can be manufactured inexpensively. For example, layer structures made of metal are applied to a film in an inexpensive production process (for example plasma process, lamination or galvanic process, which are known from printed circuit board technology).

According to another embodiment, the electrodes are formed from a plurality of electrically conductive foils or foil sections. These are e.g. metallic foils or plastic foils with inclusions of electrically conductive particles, e.g. made of carbon.

The use of foils for the electrodes is favored in that the foils are connected to a structure made of plastic to hold the foil by means of primary shaping or material bonding. The plastic structure is considerably stiffer than the film, so that it stabilizes the film.

The material for the film can be provided or processed inexpensively in the form of a film web or a film strip. After the electrodes have been applied or after the structure has been connected, foils can be cut or punched out in a size suitable for the chamber. A film web or a film strip can e.g. guided by an injection molding tool for overmolding or overmolding with the structure and separated during demolding or later.

According to one embodiment, at least two electrodes, between which the electric field is generated, are formed on a single film. On a single film there can also be several pairs of electrodes, between which electric fields are generated, be formed. As a result, the electrodes are particularly easy to manufacture and can be integrated into the chamber.

According to one embodiment, at least two electrodes are formed on different foils. The electrical field can be generated between the two electrodes on different foils. A plurality of pairs of electrodes can be formed on the different foils, between which an electric field can be generated in each case. The formation of electrodes on different foils favors the treatment of relatively large suspension volumes and cell quantities.

The layer structures forming the electrodes are e.g. Areas and / or webs arranged parallel to one another at a distance from one another or interdigitated comb structures (“interdigital structures”) on one or more foils. The layer structures can be made very thin and approximately flush with the foil.

Distances of the electrodes e.g. in the μm range are possible.

The electrodes are preferably shaped so that they have a favorable influence on the formation of the electrical field. For example, the aim is to generate an inhomogeneous electric field. According to one embodiment, at least one film is profiled for this purpose. According to one embodiment, the film is profiled overall, ie overall has a profile that describes a profile (for example a corrugated or trapezoidal profile corresponding to a corrugated or trapezoidal sheet). According to one embodiment, the film is profiled on at least one surface. Another surface may not be profiled or smooth, or both surfaces may be profiled the same or different. The electrodes generate larger electric field strengths between adjacent protrusions of the profiles than between distant recesses. According to one embodiment, the conductive layer Structures or film sections arranged essentially on the projecting areas of the profiles and the recessed areas essentially free of them.

According to one embodiment, the structure is connected to the side of the film facing away from the receptacle or is injection-molded on this side. This in particular mechanically stabilizes the film. In the case of back injection, the film can be profiled in a suitable injection mold by the injection pressure. According to one embodiment, the back-molded structure is essentially plate-shaped or lattice-shaped. According to one embodiment, the structure has ribs, in particular for stabilization.

According to one embodiment, the film forms the bottom of the receptacle. This configuration is e.g. usable as "microfusion chamber". The microfusion chamber is used to determine the optimal electrical parameters for the fusion. For this purpose, the effects of the electric field are preferably observed under the microscope. The film is profiled, for example. According to one embodiment, the film is transparent. This is for the Illumination of the test volume is advantageous for the examination with the microscope and is a condition for the examination with the aid of an inverse microscope.

According to one embodiment, the chamber has an opening opposite the film. The chamber can be filled or emptied through the opening and, if necessary, an objective of a microscope can be inserted. This is advantageous for focusability. In the case of inverse microscopes, in which the objective is brought up to the film from below, the small wall thickness of the film is advantageous for the focusability. According to one embodiment, the ability to insert the lens is further promoted by a cross section of the receptacle that widens toward the opening.

According to one embodiment, the receptacle of the microfusion chamber is surrounded by a shield for the electrical field. In microscopic examinations, the shielding prevents harmful effects of the high-frequency alternating voltage of high power on the laboratory personnel.

According to one embodiment, parallel foils are kept at a distance from one another by means of a frame connected to them. This “application fusion chamber” favors the treatment of larger cell quantities or suspension volumes and, in contrast to the microfusion chamber, is particularly advantageous for the technical implementation of cell fusions if the electrical parameters are fixed connected or back-injected and / or laterally connected or extrusion-coated with the structure which forms the frame The foils are arranged or can be used, for example, with the frame in a container which has the receptacle for the suspension The frame has on at least one side at least one opening to fill or withdraw the suspension.

According to one embodiment, the parallel foils are profiled and the projecting areas and the recessed areas of different foils are each aligned with one another. This favors a field which is advantageously designed for cell fusion.

According to one embodiment, the structure connected to the film is at least part of a wall delimiting the receptacle. For example, the bottom of the microfusion chamber is through the film and the entire wall of the microfusion chamber is through the structure sprayed on the edge with the film. The structure is thus, for example, sleeve-shaped or plate-shaped or block-shaped with a passage for forming the receptacle, which is closed at the bottom by the film and has an opening at the top. In an application fusion chamber, for example, the structure simultaneously forms the wall and the frame, which keeps the two parallel foils at a distance from one another.

According to another embodiment, the electrodes are electrically conductive layer structures or support structures which are applied to support structures and which are overall electrically conductive. The electrodes are relatively rigid due to the support structures. They are e.g. very thin-walled, with a rigid construction e.g. contributes to the material and / or dimensional stability, e.g. through profiling. For the formation, arrangement and manufacture of the layer structures on the support structures and the electrically conductive formation of the support structures and the arrangement of the support structures in the chamber, what has been said above regarding the corresponding features of the foils or chambers equipped with foils applies.

According to one embodiment, the electrodes are connected to contacts outside the receptacle. The layer structures or conductive foils or substrate structures are e.g. led outward from the area sprayed with the structure and form contacts on an outside area. The contacts serve e.g. the connection of an electrical system for generating the alternating voltage or the pulse for cell fusion.

According to one embodiment, the film and / or support structure is made from a plastic material. According to one embodiment, the film and / or support structure is made of PP, PE, PC, PS or PMMA. According to one embodiment, the layer structure is made of a metal. According to one embodiment, this is Metal aluminum, stainless steel, platinum or titanium. The layer structure or the conductive film or support structure is preferably made of a corrosion-resistant and / or a non-cytotoxic material. Non-corrosion-resistant and / or cytotoxic materials are, for example, provided with a corrosion-resistant and / or non-cytotoxic coating.

According to one embodiment, the structure is made of a plastic material. The plastic material is e.g. PP, PE, PC, PS or POM or an essentially non-conductive plastic material.

According to one embodiment, the material of the film or support structure has an approximately the same or lower melting temperature than the material of the structure. This favors a melt connection between the film or support structure and structure during injection molding. According to one embodiment, the material of the film or stucco structure has a higher melting temperature than the material of the structure. This creates a less firm connection between the film or support structure and structure, e.g. by gluing or clamping (e.g. after a shrinking process), which can promote peeling off of the film, which is advantageous for certain applications.

The invention is explained in more detail below with reference to the attached drawings of exemplary embodiments. The drawings show:

1 shows a microfusion chamber in a perspective view obliquely from above.

2 shows a section of an application chamber with back-injected, parallel foils in a perspective view obliquely from below; 3 shows an enlarged section of the same application chamber;

Fig. 4 shows a section of another application chamber with profiled, parallel foils in a perspective view obliquely from above.

According to FIG. 1, a microfusion chamber 1 has a cuboid structure 2 made of plastic, which delimits a conical passage 3 which runs perpendicular to the upper side 4 and the lower side 5 of the structure 2 parallel to it. The passage. 3 widens towards the top 4 of the structure and has an opening 6 there.

The passage 3 is closed at the bottom by a film 7 which is sprayed onto the lower side 5 of the structure 2. The film 7 has a protrusion 8 on the side of the structure 2.

The film 7 consists of a non-conductive, transparent plastic material.

The passage 3 and the film 8 delimit a recording 9 within the structure 2.

The film 7 carries on the top two linear, parallel layer structures 10, 11 made of an electrically conductive metal. The layer structures 10, 11 are only a small distance apart from one another in a region running diametrically over the bottom of the receptacle. There they essentially form floor-flush electrodes. According to an embodiment not shown, the layer structures 10, 11 are arranged on two parallel Wellenpro filen on the top of the film 7. The layer structures 10, 11 are below the structure 2 for covering stood 8 unfinished. There, the layer structures 10, 11 are pulled apart and expanded to form electrical contact surfaces 12, 13 which are accessible from the outside.

The plastic materials of the structure 2 and the film 7 are selected such that a firm connection with the film 7 is achieved by spraying the plastic material of the structure 2 onto the film 7, e.g. by merging.

The Milαofusionkammer 1 is inexpensive to manufacture as a one-off part.

A cell-containing suspension can be applied to the area of the electrodes 10, 11 in the receptacle 9. A microscope can be inserted with the objective into the opening 6 of the receptacle 9. The suspension can be illuminated by means of a light source arranged on the underside of the film. When a voltage for the cell fusion is applied to the contacts 12, 13, the fusion process can be observed under the microscope and the most favorable parameters for the cell fusion can be determined.

2 and 3, an application fusion chamber 20 has two parallel, largely corrugated foils 21, 22 made of a non-conductive plastic material. The foils 21, 22 have layer structures 23, 24 made of an electrically conductive material on the inner sides facing one another, which cover the inner sides over their entire surface.

The outside of the corrugated foils 21, 22 are back-molded with plate-shaped structures 25, 26 made of relatively rigid plastic. This stabilizes the foils in their corrugated form.

The plastic films 21, 22 and the plate-shaped structures 25, 26 are made of a plastic frame by a frame 27 which is completely or partially encircling the outside. material fixed in their arrangement. The frame 27 can consist of the same plastic material as the structures 25, 26 and can be injection molded together with them in a single operation or in an additional operation. ■

Edge-side sections 28, 29 of the foils are guided through the frame 27 to the outside in order to allow contacting of the layer structures 23, 24.

A receptacle 30 is formed between the inner sides of the foils 21, 22 and the frame 27. The frame 27 thus forms a wall delimiting the receptacle 30. The frame 27 has at least one opening, which is not shown. It is e.g. a fully or partially open side of the frame 27 or at least one inlet and / or outlet formed therein.

The microfusion chamber 20 is also filled with a cell-containing suspension. The contacts 28, 29 are also connected to an electrical system in order to carry out the cell fusion.

The cell fusion chamber 20 can also be implemented inexpensively as a one-off part.

FIG. 4 shows a cell fusion chamber 40, in which the foils 41, 42 have a wavy profiling ring 43, 44 only on the mutually facing inner sides and are flat on the outer sides 45, 46.

The foils 41, 42 are extrusion-coated by a frame-like structure 47 on the outer sides. As a result, a receptacle 48 is formed between the foils 41, 42 and the frame-like structure 47. The receptacle 48 in turn has at least one opening, not shown, in the area of the frame-like structure 47. The wave-shaped inner sides of the foils 41, 42 are provided with an electrically conductive layer structure 49, 50 over the entire surface. The bad structures 49, 50 are led outside through the frame 47 in areas not shown and connected to contacts for connecting an electrical system.

In chamber 40, a cell fusion can also be carried out after filling a cell-containing suspension into the receptacle 48 and applying a voltage to the contacts. The chamber 40 is also inexpensive to carry out as a one-off part. The profiling rods of the chambers 20, 40 are e.g. can be produced by embossing.

Claims

Expectations

1. chamber for treating cells contained in a suspension in the electric field with - a receptacle (9, 30, 48) for the suspension, - at least two electrodes (10, 11; 23) assigned to the receptacle (9, 30, 38), 24; 49, 50) between which an electrical field for treating cells in a suspension contained in the receptacle (9, 30, 40) can be generated by applying a voltage, and - one with at least one electrode (7; -21, 22; 41, 42) plastic structure (4; 25, 26; 47) connected by reshaping or composite material to hold the electrode.

2. Chamber according spoke 1, in which the structure (4; 25, 26; 47) is injection-bonded to at least one electrode during injection molding of the structure.

3. Chamber according spoke 1 or 2, in which the structure (4; 25, 26; 47) with at least one electrode (10, 11, 23, 24; 49, 50) is injection molded and / or glued and / or welded. ^"

4. Chamber according to one of claims 1 to 3, in which the electrodes are formed by at least one insulating film (7; 21, 22; 41, 42) or support structure arranged, electrically conductive layer fractures.

5. Chamber according to one of claims 1 to 4, wherein the electrodes of a plurality of electrically conductive films (7; 21, 22; 41, 42) or film sections or support structures are formed.

6. Chamber according to one of claims 1 to 5, in which at least two electrodes (10, 11) are arranged on a single film (7) or support structure.

7. Chamber according to one of claims 1 to 6, wherein the at least two electrodes are arranged, or supporting structures (23, 24;; 49, ^'50) on different foils (41, 42 21, 22).

8. Chamber according to one of claims 1 to 7, in which the layer fractures (10, 11; 23, 24; 43, 50) at least one film (7; 21, 22; 41, 42) or support structure over the entire surface or along electrically conductive Cover webs.

9. Chamber according to one of claims 1 to 8, in which at least one film (21, 22; 41, 42) or Sffitz structure is profiled.

10. Chamber according spoke 9, in which the film (21, 22; 41, 42) or support structure is profiled as a whole or on at least one surface.

11. Chamber according to one of claims 1 to 10, wherein the structure (25, 26) is connected to the side of the film (21, 22) or support structure facing away from the receptacle.

12. Chamber according to one of claims 1 to 11, wherein the structure (25, 26) is plate-shaped or lattice-shaped.

13. Chamber according to one of claims 1 to 12, wherein the structure (25, 26) has ribs.

14. Chamber according to one of claims 1 to 13, wherein the film (7) or support structure is the bottom of the receptacle (9).

15. Chamber according to one of claims 1 to 14, wherein the film (7) or support structure is transparent.

16. Chamber according to one of claims 11 to 15, wherein the structure connected to the opposite side of the film (7) or support structure is transparent.

17. Chamber according to one of claims 14 to 16, which has an opening (6) opposite the film (7) or support structure.

18. Chamber according spoke 17, in which the receptacle (9) has a cross section widening towards the opening (6).

19. Chamber according to one of claims 14 to 18, wherein the receptacle (9) is surrounded by a shield for an electric field.

20. Chamber according to one of claims 1 to 19, in which parallel foils (23, 24; 49, 50) or support structure are kept at a distance from one another by means of a frame (27, 47) connected to them.

21. Chamber according to claim 20, in which the projecting and recessed areas of parallel profiled foils (23, 24; 49, 50) or support structures are aligned with one another.

^'22. The chamber of claim 20 or 21, wherein the frame (27, 47) formed from a connected with a portion of the foils or supporting structures structure.

23. Chamber according to one of claims 1 to 22, in which the structure (4; 25, 26; 47) connected to the film (7; 21, 22; 41, 42) or support structures has at least a part of the receptacle (9, 30, 48) bounding wall forms.

24. Chamber according to one of claims 1 to 23, wherein the electrodes (10, 11; 23, 24) are connected to contacts (12, 13; 28, 29) outside the receptacle (9, 30).

25. Chamber according to one of claims 1 to 24, wherein the film (7; 21, 22; 41, 42) and / or support structure is a PP, PE, PC, PS or PMMA film and / or support structure and / or the layer structure (10, 11; 23, 24; 49, 50) is an aluminum or stainless steel or platinum or titanium layer and / or the structure (4; 25, 26; 47) made of PP, PE, PC, PS or POM is made.

26. Chamber according to one of claims 1 to 25, wherein the plastic material of the film or support structure (7; 21, 22; 41, 42) has an approximately equal or lower melting temperature than the plastic material of the structure (4; 25, 26; 47) having.

27. Chamber according to one of claims 1 to 26, wherein the plastic material of the film or support structure (7; 21, 22; 41, 42) has a higher melting temperature than the plastic material of the structure (4; 25, 26; 47).