Title:
Arrangement for treating cells in biology or genetic engineering
Kind Code:
A1


Abstract:
A device for receiving a liquid containing a cell culture includes at least one vessel having a base plate and walls forming an enclosure. A cover is lowerable into the vessel to displace air and, where appropriate, excess liquid. The cover has a liquid admission opening and a liquid discharge opening.



Inventors:
Marotzki, Stefan (Tornesch, DE)
Application Number:
10/252804
Publication Date:
01/30/2003
Filing Date:
09/23/2002
Assignee:
MAROTZKI STEFAN
Primary Class:
Other Classes:
435/305.3
International Classes:
C12M1/22; (IPC1-7): C12M1/22
View Patent Images:



Primary Examiner:
REDDING, DAVID A
Attorney, Agent or Firm:
ALIX, YALE & RISTAS, LLP (HARTFORD, CT, US)
Claims:

What is claimed is:



1. A device for receiving a liquid containing a cell culture comprising: a vessel having a base plate and a wall structure defining an enclosure; and a cover lowerable into the vessel to displace air and, where appropriate, excess liquid, said cover having a liquid admission opening and a liquid discharge opening.

2. The device according to claim 1 further comprising one or more walls for defining a plurality of separate chambers inside the vessel.

3. The device according to claim 2, wherein said vessel has a first width and further comprising a second vessel having a second width which is less than said first width, said second vessel being disposed in said first vessel.

4. The device according to claim 3, wherein said cover closes only the portion of the wider first vessel which is situated outside of the second vessel.

5. The device according to claim 3, wherein the cover defines a cutout which is configured to be complementary with the circumference of the second vessel.

6. The device according to claim 5, wherein the cutout is defined by an opening of the cover.

7. The device according to claim 3, wherein the cover is a holder for the second vessel.

8. The device according to claim 7, wherein the cover connects with the second vessel to permit joint handling.

9. The device according to claim 3 further comprising a separate cover for covering the second vessel.

10. The device according to claim 2 further comprising a ring enclosing a separate chamber disposed between the base plate and a base of the cover and the cover has an opening communicating with the chamber.

11. The device according to claim 1, further comprising at least one cell culture insert.

12. The device according to claim 1, wherein at least one of the openings is provided with a screen.

13. The device according to claim 1, wherein the cover has an upper side defining a trough in fluid communication with at least one of the openings.

14. The device according to claim 13, wherein the trough has a lower base which is not disposed lower than a mouth of the associated opening.

15. The device according to claim 14, wherein the upper side of the cover defines first and second troughs associated with the liquid admission opening and liquid discharge opening, respectively, the first trough being separated from the second trough.

16. The device according to claim 13, wherein a plurality of vessels are enclosed by the cover, the trough being in fluid communication with each of the vessels through a one of the openings disposed proximate to the vessel.

17. The device according to claim 1, wherein the cover has a plurality of openings, at least one of the openings being a liquid admission opening and at least one of the openings being a liquid discharge opening.

18. The device according to claim 1, wherein the device comprises a plurality of contiguous vessels.

19. The device according to claim 4, wherein the cover defines a cutout which is configured to be complementary with the circumference of the second vessel.

20. The device according to claim 4, further comprising a separate cover for covering the second vessel.

21. An arrangement for biological treatment or genetic engineering of cells comprising: at least one vessel each having a base plate and a wall defining an enclosure for receiving cells and a treatment liquid; a machine having means for exchanging the liquid; and a cover having at least first and second openings; wherein the liquid exchange means introduces liquid into the first opening and removes it from the second opening.

22. The arrangement of claim 21, wherein said cover has an upper side defining first and second troughs in fluid communication with the first and second openings, respectively, and the liquid exchange means introduces liquid into the first trough and removes it from the second trough.

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent application Ser. No. 09/787,948, filed on Mar. 23, 2001.

BACKGROUND OF THE INVENTION

[0002] For treating or examining cells in molecular biology or genetic engineering, vessels are used (for example, Petri dishes) which receive the cells and a reaction liquid and can be provided with a cover (“Lab-Tek II” brochure from Nalge Nunc International, Naperville;

[0003] “EasiSeal” brochure from HYBAID Limited, Teddigton; “Gene Frame” brochure from Advanced Biotechnologies Ltd., Espom; EP-A 611 598; U.S. Pat. Nos. 4,634,676; 3,745,091; DE-A 19624 917; DE-C 479 304; U.S. Pat. Nos. 2,348,448; 4,294,924; 4,321,330; FR-A 2 698 375). If the treatment (for example, in situ hybridization or in situ PCR) requires exchange of liquids, this can only be done manually in most of the known vessels, because it involves removal and application of a cover. If the treatment requires rapid temperature change, it must be ensured that the substrate layer in the vessel is very thin so as to be able to follow the temperature change quickly and in a precisely controllable manner. In treatments in automatic machines, these requirements have hitherto been satisfied using vessels (Lab-Tek II brochure from Nalge Nunc International, Naperville; U.S. Pat. No. 3,745,091) which consist of a slide with walls detachably arranged thereon. In a first method step which takes place outside the automatic device, the vessels are used with the walls placed on the slides, in which case liquid exchange does not take place or has to be carried out manually. As soon as the automatic treatment is to start, the walls are removed and the slides with the cells adhering to them are introduced into the automatic machine in which the liquid exchange takes place by means of the slides being lowered in groups into optionally alternating baths. If the cells do not adhere, or adhere insufficiently, to the slide, and they are instead completely or partially in suspension, they are centrifuged on the slide so that they adhere thereto after removal of the medium. This is not only complex but also has the disadvantage that the cells are deformed and slight morphological changes can no longer be identified under the microscope. In addition, the required centrifugation steps can not be automated or can be automated only with difficulty.

[0004] The removal of the walls from the slides complicates the procedure, particularly in the case of cells adhering inadequately to the slide. Finally, it may be undesirable to eliminate the vessel division between adjacent cultures by removing the walls.

[0005] It is admittedly known (FR-A-2 698 375) to provide a Petri dish with a cover which has an opening. This is used for removing air and excess liquid when the cover is lowered into the vessel. However, this does not include the concept of automatic liquid exchange using the cover opening as an admission opening and outflow opening.

SUMMARY OF THE INVENTION

[0006] It is an object of the invention to make available a device for receiving a liquid containing a cell culture comprising a vessel having a base plate and walls defining an enclosure, and a cover lowerable into the vessel to displace air and, where appropriate, excess liquid, the cover having a liquid admission opening and a liquid discharge opening.

[0007] The cover can further comprise a plurality of openings, where at least one opening is a liquid admission opening and at least one opening is a liquid discharge opening.

[0008] If displaced liquid is to be collected, or if a reservoir of liquid is to be formed which is intended to replace the liquid present in the treatment chamber, a trough can be formed on the upper side and communicate with a corresponding opening in the cover.

[0009] For liquid which is to pass into the treatment chamber by diffusion or flow, the trough base should not be lower than the mouth of the opening. A plurality of openings can be provided per vessel in order to permit liquid exchange by flow. For this purpose, a plurality of openings should be provided, of which at least one is connected to a trough which is separate from the trough or troughs of the other openings and which is not arranged lower than the mouth of the one opening.

[0010] Particularly in machine treatment of the devices, it may be expedient to combine a plurality of vessels and provide them with a common cover which has the requisite openings in the area of each individual vessel.

[0011] The troughs have the advantage that the mechanical means for introducing liquid can be designed alternately for introduction into the openings or into the associated troughs. Similarly, they can be alternately designed for removing liquid off from a trough or opening.

[0012] In this context, the term trough is intended to signify any vessel-like depression without stating its width or height, unless this is expressly mentioned.

[0013] The cover can, as is known, be designed such that air and a possible excess amount of reaction liquid is displaced through the opening. The cover can be lowered inside the vessel until the desired thickness of the substrate layer is achieved between the base plate of the vessel and the opposite cover base, which desired thickness can range from several hundredths of a millimeter to several millimeters.

[0014] In order to avoid undesired void volume, the circumferential surface of the cover can fit closely, with an essentially identical shape, to the internal surface of the walls. This fit can also constitute the sealing of the cover with respect to the walls, for example, by means of a ground-glass joint or a plastic seal under elastic pressure. However, this does not need to be the case, since, in addition to a close, but not tight fit of the circumferential surface of the cover to the internal surface of the walls, a special seal can be provided which can cooperate, for example, with the edge of the vessel walls or a shoulder thereof. In these cases, it is often expedient to provide a holder for maintaining the sealing position of the cover with respect to the vessel, for example, a screw-on, snap or spring closure; however, the tight fit of the cover on the vessel walls can also be self-supporting, for example, as a result of the frictional forces which exist between two ground-glass surfaces.

[0015] It is expedient for the lower surface of the cover base to extend approximately parallel to the base of the vessel in order to be able to achieve an essentially constant layer thickness. The cover and the vessel can be provided with cooperating contact surfaces, which define the size and constancy of the layer thickness.

[0016] The openings of the cover can be provided with a closure device. However, the possibility also exists of providing sealing by means of a subsequently applied oil layer. This also applies to the circumferential sealing. In many cases a tight seal is only required in respect of the external atmosphere in order, for example, to avoid evaporation or the admission of oxygen. In these cases, it may suffice if the seal is formed not on the vessel and cover, but is instead formed by an apparatus which receives the device, for example, a type of autoclave in which there is an internal atmosphere which is chosen in accordance with the intended purposes. To avoid evaporation, for example, it can have sufficient moisture. To avoid admission of harmful gases, it can consist of nitrogen or noble gas.

[0017] To ensure that it is not only the air in the area of the cover opening that is removed, but also that air quantity which may be contained between the circumferential surface of the cover and the vessel walls, it is possible for the cover opening to be provided with an ascending section whose mouth is at least approximately level with the upper end of the gap situated between the circumferential surface of the cover and the vessel walls. On slow insertion of the cover into the vessel, the static pressure of the liquid column present in the ascending section of the cover opening then ensures that the air in the circumferential gap is also displaced.

[0018] When the cells have deposited sufficiently firmly on the base of the vessel, the excess liquid can be drawn off or displaced through the cover opening without the risk of losing cells. So that it is also possible to work with cells, which are in suspension, the cover opening can be provided with a screen which holds back the cells during displacement of the liquid.

[0019] A plurality of openings can be provided which are designed for attachment of an admission and discharge line for a medium. The device can then also be used as a so-called reactor (Meenen et al.; “Semi Continuous Reactor System . . . ”, Poster 1994 Biomaterials 21:905-908).

[0020] The cells involved can be adherent cells, which stick to the base of the cell culture dish, or suspension cells which swim in the culture medium. Alternatively, tissue sections can also be cultured in the dish. In general, both eukaryotic and prokaryotic cells can be cultured in the vessel. Treatments and examinations which can be carried out using the device according to the invention can, for example, be all types of in situ hybridization and in situ PCR.

[0021] The cover openings are expediently provided inside the cover, at a distance from the edge of the latter. However, this does not exclude the possibility of an opening being formed by means of a spacing provided, at least in places, between the edge of the cover and the vessel walls. A particularly advantageous design in this connection is one in which the edge of the cover comprises a collar which rises upwards from the bottom of the cover plate, which forms in its entirety or in places the said spacing for forming an opening, and whose upper edge cooperates with the vessel walls to form a seal. As the cover is being lowered, quantities of gas and liquid enclosed between cover and vessel can then escape, and it is only at the end of this procedure, when the cover reaches its end position that the seal is obtained.

[0022] If the intention is to subject a plurality of cultures to the same thermal conditions, it is expedient to connect a plurality of vessels to one another. In this case, a cover of the type indicated above, can be provided for each one of the vessels. However, it is also possible to connect a plurality of covers to one another for joint actuation. It is of course possible to make the walls of a plurality of vessels integral with one another, in which case either the bases are also connected in one piece with the walls or they can be separated from these, for example in the form of a slide.

[0023] Of particular importance in the context of the invention is the possibility of being able to create a plurality of chambers by means of forming walls inside one and the same vessel, which chambers, during the course of treatment of a culture, are at times separated from one another, or are not separated, depending on the requirements. For this purpose, the cover is provided with an arrangement for dividing off such chambers from one another. A particularly advantageous design is one in which a narrower vessel is so designed that it can be accommodated in a wider vessel. The cover of the wider vessel is in this case generally designed in such a way that it serves solely to close the area of the wider vessel situated outside the narrower vessel. For the closure of the narrower vessel, a special closure is then optionally provided, for which the general comments made above in respect of the closure of the vessels also apply. This means that in addition to a simple air-permeable or airtight closure, a cover can also be provided which is equipped with an opening for displacement of the atmosphere, and possibly excess liquid, arising in the narrower vessel, and whose air opening can be closed. However, there is also the possibility of structurally connecting the covers of the wider vessel and of the narrower vessel to one another for joint actuation.

[0024] Where, in the present connection, mention is made off “a” narrower vessel, this is intended to mean that it is at least one; a plurality of narrower vessels can, however, also be provided in a wider vessel. The arrangement of a narrower vessel in a wider vessel affords the possibility of subjecting the cultures within the narrower vessel and within the wider vessel to exactly the same thermal conditions. They can have separate bases. Of particular importance, however, is a design in which the narrower vessel shares the base with the wider vessel. This affords the possibility of subjecting one and the same culture inside the wider vessel to different reaction conditions (for example, another reaction liquid) during all or some of the reaction steps. Accordingly, the wall of the narrower vessel is used only in those reaction steps, inside the otherwise uniform culture, in which the reaction conditions are intended to be different, e.g., for positive control or negative control in PCR.

[0025] There are cases in which the narrower vessel can remain in the wider vessel during the entire reaction or sequence of reactions. In these cases, it can be permanently connected to the base and to the wider vessel from the outset at the factory stage. In other cases, the compartmentalization is desired at the start of the reaction or chain of reactions; the narrower vessel can then be connected, likewise at the factory stage, to the base of the wider vessel, but it can be detached therefrom so that the compartmentalization can be annulled at the desired time. The factory-stage arrangement has the advantage that the narrower vessel can be accurately positioned in relation to the walls of the wider one, and this therefore guarantees that the cover fits. In other cases, one will want to have the freedom to be able to carry out the compartmentalization at any desired time. In these cases, the wall of the narrower vessel can be fitted by the user.

[0026] If the cover is intended to close off the wider vessel completely, it must not only have its circumferential surface adapted to the wall of the wider vessel, but must also be able to be connected sufficiently tightly to the narrower vessel. This can be achieved by its having a cutout which is adapted to the shape of the narrower vessel and which can join to the latter with the desired tightness. This cutout can be formed by an outflow opening. In another embodiment, the cover is connected in one piece to the wall of the narrower vessel, so that the compartmentalization made possible by the narrower vessel comes about with the insertion of the cover. In both cases, the cover serves as a holder or guide during insertion and, if appropriate, also during use of the narrower vessel.

[0027] In some cases an absolutely tight seal between the wall of the narrower vessel and the base of the wider vessel is not necessary; the narrower vessel can then simply be formed by a small tube which is held by the cover and reaches down to the base of the wider vessel. If a tight connection of the narrower vessel wall against the base is required, the lower edge of the wall of the narrower vessel can be provided with a seal, the wall being pressed against the base by the cover or an additional clamp in order to generate the sealing pressure. The wall of the narrower vessel can also be bonded to the base. In the simplest case, the vessel wall of the narrower vessel comprises a sealing ring which is fitted between the base plate and the cover base, fits tightly against these and divides the chamber enclosed by it from the space surrounding it. To ensure that the enclosed chamber can be subjected to a different treatment, it must be accessible by way of a cover opening which is located in its area and which can be the outflow opening.

[0028] According to the invention, the cover can also be provided with further arrangements, which are desired, for the treatment of the cultures. For electroporation, the cover and the base can support electrodes, for example, planar electrodes, and the cells to be treated are arranged on a porous membrane between these.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The invention is explained in greater detail below with reference to the drawing, which diagrammatically depicts advantageous illustrative embodiments, and in which:

[0030] FIG. 1 shows a schematic representation;

[0031] FIG. 2 shows a device with ground-in cover;

[0032] FIG. 3 shows a device with elastically sealing cover;

[0033] FIG. 4 shows a device with a screw-on cover;

[0034] FIG. 5 shows a device with a snap-locking cover;

[0035] FIGS. 6, 7, and 8 show different embodiments of a compartmentalized device;

[0036] FIG. 9 shows a partial section illustrating one sealing possibility;

[0037] FIG. 10 shows a device with electrode;

[0038] FIG. 11 shows a further compartmentalized device;

[0039] FIG. 12 shows a device with a plurality of vessels and common cover;

[0040] FIG. 13 shows the device according to FIG. 12 with special recesses at the cover openings;

[0041] FIG. 14 shows the device according to FIG. 12 with a plurality of openings per vessel; and

[0042] FIGS. 15 and 16 show a vessel with a plurality of openings in various stages of operating.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The device basically comprises the vessel 1, for example, a Petri dish, and a cover 2. The vessel 1 has a base plate 3 and walls 4. These can be designed in one piece or as multiple parts. FIG. 3 indicates that the base plate is formed by a slide 3 feet on which the walls 4 are secured in a known manner by means of a releasable adhesive 5. Instead of this, a sealing ring can be used in conjunction with an arrangement for holding the walls, with inclusion of the sealing ring on the slide. Vessel and cover are made of a material, which does not negatively affect the substrate, and the process that may be carried out, for example, glass or suitable plastic. The vessels may be individual vessels; alternatively, a plurality of vessels are provided contiguously as one group.

[0044] If the intention is to cultivate the cells in the dish or to subject them to molecular biological or genetic engineering treatments or examinations, the cover 2 is used, which cover 2 closes the dish off tightly against the walls 4. It has at least one opening 6. The atmosphere which is displaced upon insertion of the cover into the vessel and, where appropriate, a part of the liquid, can flow off through this opening. The lower mouth of the opening is arranged in the part of the cover base 7 submerging in the liquid, and in fact it is preferably arranged in the highest region thereof. If so required, the opening can be closed after the cover has been applied. In this respect, the vessel is known (FR-A 2698375). The distance between the bottom surface of the cover base 7 and the top 8 of the base generally amounts to 20 μm for observation purposes, but can also be substantially larger or smaller, depending on requirements.

[0045] The type of sealing of the cover 2 with respect to the walls 4 which is chosen in each particular case depends on the material that has been chosen. If glass is used, this can be achieved by means of grinding. This is illustrated in FIG. 2. The cover 2 and the vessel 1 are provided with collars, 9, 10 which have been ground to match and complement each other. The cover opening 6 is situated at a suitable location within the cover area surrounded by the collar. Such a shape of the vessel and of the cover can also be used with materials other than glass.

[0046] If elastically yielding plastic material is used for the vessel walls and/or the cover, the sealing can be achieved by resilient pressing of a sealing edge, as is indicated in FIG. 3. The walls 4 of the vessel, made of glass or plastic for example, extend parallel to one another and perpendicular to the base 8, for example, cylindrically. The cover 2 is made of an elastic plastic. Jutting upwards from its preferably flat cover base 7, there is a collar 11 which, in the lower area 12, extends approximately parallel to the walls 4 and, with respect to the latter, has no clearance space or only a slight clearance space. The upper edge 13 of the collar 11 has a slightly greater diameter so that it is elastically compressed upon insertion into the wall 4 and thus bears tightly and with prestressing against the inner surface of the wall 4 about its entire circumference. The position of the cover in the vessel is secured by means of its friction on the vessel walls.

[0047] In the illustrative embodiment according to FIG. 4, the collar 11 of the cover likewise adjoins the walls 4 of the vessel with only a slight gap width. At the free edge, the collar 11 and the walls 4 are provided with a cooperating thread 25. A sealing ring 14 is placed between the end faces of these parts. The ring 39 placed between the base plate 3 and the cover base 7 is discussed in detail below.

[0048] Instead of the screw-on closure, it is possible to provide any type of snap closure, or a clamp acting from outside on the cover and holding it in its position. A design with a snap closure is illustrated in FIG. 5. The edge 15 of the cover base 7 sits sealingly on a shoulder 16 of the wall 4. It is held in this position by virtue of the fact that the free edge 17 of the collar 11 of the cover 2 cooperates in a locking manner with a circumferential nose 18 on the free edge of the vessel walls. The shoulder 16 also has the advantage that it forms a limit stop, which ensures the desired spacing between the cover base 7 and the vessel base 3.

[0049] As is shown in FIG. 1, the cover base 7 can be shaped in such a way that the opening 6 begins at an elevated part of the base. This makes it easier to eliminate air bubbles without leaving any behind. In general, however, this is not necessary. Remaining air inclusions can be flushed out with an excess of the culture medium or the reaction liquid.

[0050] In the examples according to FIGS. 1, 3, and 4, the opening 6 is connected to an ascending tube 24 which is at least as high as the outflow section for removing air from the gap between the cover collar 11 and the vessel walls 4. By means of the static pressure in the ascending tube, it is possible, if so desired, to ensure that air inclusions are driven out from the annular gap between the circumferential surface of the cover and the internal surface 21 of the vessel walls. This objective can also be achieved by giving the cover opening such a high flow resistance with respect to liquid, and by inserting the cover so quickly, that the overpressure produced in the vessel guarantees the removal of any air from the annular gap. FIG. 5 shows that the opening 6 narrows towards the top to form a gap 23 of small width, which as a throttle has a correspondingly high flow resistance. This gap can be designed in such a way that, upon insertion of the cover into the vessel, it opens in the manner of a nonreturn valve, with elastic deformation of the material, to allow the escape of the air and excess liquid and, thereafter, under the elastic restoring force of the material and/or under the action of a clamp (not shown in the drawing), once again closes in an airtight manner. By contrast, FIG. 4 shows a stopper 19 for closing the opening 6. It is also possible, however, to close the opening by means of a droplet of oil.

[0051] If there is a danger of cells being swept out with the displaced liquid, the cover opening 6 is closed by a screen 22, as is indicated diagrammatically in FIG. 3.

[0052] A plurality of cover openings 6 can be provided if the intention is to use the device as a bioreactor through which a reaction medium is to pass. In such a case, at least one opening is connected to a liquid admission line and at least one other opening is connected to a liquid discharge line.

[0053] In some cases, it is possible to dispense completely with an airtight closure between the cover edge and the vessel walls, namely, if an innocuous atmosphere is guaranteed inside an apparatus receiving the device and if this atmosphere is protected from the external atmosphere by means of an appropriate closure of the apparatus, which in this case replaces the closure provided on the device itself.

[0054] The illustrative embodiments according to FIGS. 6 and 8 are based on the illustrative embodiment according to FIG. 1, although they could readily start from any other embodiment of the invention described above. They illustrate the formation of a narrower chamber 30 inside a wider vessel 1. The walls 31 forming the chamber 30 can involve a simple tube, for example, made of glass or plastic, and in any case, sit with their lower ends on the top 8 of the base plate 3 in order to separate the chamber 30 from its surroundings. If the wall 31 is not designed in one piece with the base plate 3, it can be attached thereto by means of a suitable seal. The seal can be formed by an adhesive or, in accordance with the example shown in FIG. 9, by an elastomer strip 32 placed firmly and sealingly on the lower edge. In some cases it suffices if the lower edge of the wall 31 is placed on the top 8 of the base 3 without an additional sealing means. In this case, and also when using an elastomer seal which is not adhesively bonded, it is expedient to promote the tight contact on the top 8 of the base by means of the wall 31 being prestressed against the base 3. When it is made in one piece with the cover 2, as in FIG. 7, this prestressing can be produced by utilizing the elastic properties of the vessel or cover, by means of the fact that the length of the wall 31 has a certain over-dimension with respect to the base spacing of the cover and the cover is placed firmly on the vessel 1, for example, with the aid of a screw-on, friction-type or snap closure. However, such prestressing can also be produced if, as in the illustrative embodiment according to FIG. 6, the wall 31 is held releasably in a recess or holder 33 of the cover 2, with a certain frictional force being exerted on the wall 31 by the holder 33. The holder 33 expediently encloses the wall 31 tightly. It is formed, for example, by a bore in which the tubular wall 31 is guided with a close fit. As FIG. 11 shows, this bore can be formed by a cover opening 6.

[0055] If the cover 2 and the wall 31 are separate parts, they must be positioned exactly in relation to one another in the vessel 1. This is most easily done by first inserting the wall 31 into the cover 2 and then inserting the latter, together with the wall, into the vessel 1. Alternatively, the cover is first put into place and the wall 31 is then inserted through the cover. If it is necessary to install the chamber 30 inside the vessel 1 before the cover 2 is put into place, it is best to use a positioning gauge for the wall 31, the dimensions of which positioning gauge in relation to the vessel 1 are the same as those of the cover 2. This positioning gauge can be removed before the cover 2 is inserted, for example, after an adhesive connecting the wall 31 to the top 8 of the base has hardened, or the positioning gauge remains in place underneath the cover 2 when the latter is inserted. In this case, the positioning gauge does not need to be a separate part; rather, it can be structurally connected to the wall 31, for example, by means of the latter having a series of arms protruding all around from it, parallel to the base 3 of the vessel 1 or to the base 7 of the cover, which arms terminate in positioning contact against the internal surface 21 of the vessel walls 4.

[0056] In the same way as the vessel 1 is provided with a cover 2 equipped with an opening 6, the chamber 30 can be provided with a closure member 34 which is suitable for limiting the height of the substrate above the vessel base 8 and which contains a channel 35 which in turn can be closed by means of a stopper (not shown).

[0057] A further example of compartmentalization is illustrated in FIG. 4. A sealing ring 39 of elastomer material, for example, a commercially available O-ring, is placed between the base plate 3 and the cover base 7. The ring material is so thick that, in the end position, it bears with elastic deformation tightly against the base plate 3, and the cover base 7. In this way a chamber is created within the sealing ring, which chamber is separated off from the area located outside the sealing ring. It is accessible via the outflow opening 6 in the cover, or via any other opening created especially for this purpose, in order to permit a treatment of its contents, which differs from the treatment in the surrounding area. The ring 39 can be placed loosely in the vessel 1 before the cover 2 is put into place, in which case care must be taken to ensure that the area enclosed by it lies at the point where the opening 6 is expected. In order to ensure communication with the opening 6 and to simplify the handling, the ring can also be firmly connected to the cover 2 permanently or temporarily, for example, by means of an adhesive or a form-fitting holder (not shown in the drawing).

[0058] The compartmentalization according to the invention makes it possible, in individual reaction steps or in a plurality of reaction steps, to do something inside the chamber 30 that is different from what is being done outside it, but with the thermal conditions remaining the same. The cultures can also be the same if the chamber 30 is inserted only after the culture has been placed in the vessel 1. For example, in one stage of a multi-stage procedure, twice as much enzymes can be introduced inside the chamber 30 as is introduced outside the chamber. Thereafter, the chamber wall 31 is removed, and all the cells are once again treated identically. If a plurality of such insert chambers 30 are used in a vessel, the possible variations multiply accordingly.

[0059] If the cells in the vessel are to be subjected to electroporation or electrofusion, metal electrodes (not shown in the drawing) are provided in the cover and base. As is shown in FIG. 10, the cells are then placed on a porous base 36 or a membrane of a cell culture insert 37 between base 3 and cover 2 and are exposed to an electric field.

[0060] In the embodiments shown in FIGS. 12 to 14, a plurality of vessels with base 3 and walls 4 are combined in one piece. It is possible for this to include more than the two vessels shown. A common cover 40 is provided for a plurality of these vessels and it cooperates sealingly with the walls 4 of the vessels, in one of the ways explained above. In the embodiments according to FIGS. 12 and 13, an outflow opening 6 in the cover 40 is provided for each vessel.

[0061] In the embodiment according to FIG. 12, the cover 40 has, on its upper side, an upwardly projecting edge 41 which delimits a trough 42 into which the cover openings 6 open. On the one hand, this trough collects the liquid which flows off when the cover is inserted into the vessel. On the other hand, and this is the more important point, the trough can be used for adding liquid which is to be transferred into the treatment chambers 43 in order to modify the properties of the liquid which is present there and in which the cells are situated. It is possible to fill and empty the trough using a suitable known pipetting robot.

[0062] The liquid present in the trough 42 can move through the openings 6 and into the chambers 43 by diffusion. A common trough 42 for a plurality of openings 6 of different treatment chambers 43 is chosen if the liquid in the different treatment chambers 43 is to be modified in the same way.

[0063] If the treatment liquid in adjoining treatment chambers 43 is to be modified in a different way, separate troughs are used. For example, in the embodiment according to FIG. 12, a further wall 41 (not shown) could also be provided on the upper side of the cover in the middle between the mouths of the opening 6. The embodiment according to FIG. 13 achieves this aim in a slightly different way. A depression 44 in the base of the trough 42 is provided in the mouth area of each of the openings 6. Different liquids can be introduced into the depressions 44 and then in each case diffuse into the associated treatment chambers 43. In the claims, the depressions 44 are also referred to as a trough.

[0064] The troughs make it possible to automate the process steps for exchanging the treatment liquid. For this purpose, a suitable machine (for example a known pipetting robot) is provided with nozzles, indicated diagrammatically at 45, which are controlled by a program in such a way that at a given time they introduce a liquid of a desired type and amount into corresponding troughs.

[0065] If exchange of the treatment liquid by diffusion takes too long, the treatment liquid can also be exchanged by forced flow. Two illustrative embodiments of devices, which are suitable for this, are shown in FIGS. 14 to 16.

[0066] In FIG. 14, it has been assumed (as in FIGS. 12 and 13) that a plurality of vessels with treatment chambers 43 are combined in one piece and are provided with a common cover 46. Each part of the cover 46 allocated to a vessel has two openings 6a, 6b which open into a trough 42 on the upper side of the cover. At least the opening 6a is provided at the upper end with a depression 44. If so desired, the cover can also be used for exchanging the treatment liquid by diffusion, but it is mainly suited for forced exchange of the treatment liquid. First, the new treatment liquid to be added is introduced into the depression 44 of the opening 6a by means of a nozzle 45 (which may belong to a pipette robot). The treatment liquid present in the treatment chamber 43 is then suctioned off by means of a pipetting robot or by means of a suction nozzle 48 lowered onto the other opening 6b, by which means the new liquid to be added is at the same time drawn into the treatment chamber free from bubbles. The amount suctioned off is precisely dosed to guarantee exchange. The new liquid to be introduced can be present in excess if the aim is to achieve as complete as possible a replacement of the liquid previously present in the treatment chamber 43, and if this liquid is to be flushed out by the replacement liquid.

[0067] The illustrative embodiment according to FIGS. 15 and 16 permits flow exchange of the liquid without mechanical means. The opening 6a, through which new liquid is to be delivered, is connected at the top to a trough 47 of small cross section and comparatively great height, while the opening 6b, through which the liquid to be replaced is expelled, communicates with the trough 42 of large cross section and comparatively low height. If, as is shown in FIG. 15, the high trough 47 is filled with the new liquid to be added, which is shown by dots, the static pressure difference in the troughs 47 and 42 results in the expulsion of the liquid present in the treatment chamber 43, and symbolized by small circles. According to FIG. 16, the exchange is completed when the levels in troughs 42 and 47 are equal.

[0068] It will be appreciated that more than two openings can be provided. For liquid exchange which is as complete as possible, it may be expedient to provide the opening 6a for the new liquid to be added in a central position and to distribute a greater number of outlet openings uniformly near the circumference.

[0069] The exchange of liquid can also be carried out in the reverse order by means of the fact that the new liquid to be introduced is introduced at excess pressure into one of the openings and the liquid to be replaced is thereby displaced from the other opening. In this case, it may be expedient to design the outlet openings, corresponding to the example in FIG. 5, so as to open under excess pressure and to automatically reclose.