GB2365126A

GB2365126A - Multichamber device for cell investigation

Info

Publication number: GB2365126A
Application number: GB0117297A
Authority: GB
Inventors: Lothar Poschen; Ralf Wilhelm
Original assignee: Forschungszentrum Juelich GmbH
Current assignee: Forschungszentrum Juelich GmbH
Priority date: 2000-07-22
Filing date: 2001-07-16
Publication date: 2002-02-13
Anticipated expiration: 2021-07-16
Also published as: DE10035750A1; GB2365126B; NL1018571C2; GB0117297D0

Abstract

The device has multiple chambers for cell treatment and analysis by a luminescent process and comprises an upper plate 1 with extension tubes 2 and a lower plate 3 with openings 4 in which removable filters are disposed and e.g. a pump for applying suction. The device is typically in microtitre plate format. Utility of the device can be in a cell hybridisation process using gene probes where a microscopic examination can be subsequently carried out by removing from the device, the filters containing treated cells. Also claimed is a filter combination where filters are joined together by webs.

Description

2-36-5126 AN APPARATUS COMPRISING A MULTIPLICITY OF SAMPLE CHMBERS FOR THE

TREATMENT OF CELLS AND A FILTER COMBINATION.

This invention relates to an apparatus comprising a multiplicity of sample chambers for the treatment of cells, and also relates to a filter combination.

In environmental microbiology, fluorescence staining is a commonly used method of determining the cell count and for the phylogenetic assignment of bacteria from environmental samples. There is an ever-increasing number of fluorescence dyes, with a multiplicity of properties, which are available on the market. For example, some of these dyes specifically bind to free, functional groups of proteins, others intercalate with or bind to nucleic acids, and there are others which are employed analogously to substrates for certain enzymes, and others which indicate the concentration of certain ions. In particular, the binding of probes to cell-specific nucleic acids (hybridisation) constitutes an area of interest. This binding has hitherto been effected on filters or on special microscope slides used for diagnosis.

During the investigation of cells with fluorescence- labelled gene probes, the following process steps have to be performed. First of all, the cells are immobilised or fixed. Immobilisation or fixation is an operation in which the activity of cell proteins is restricted due to degeneration to such an extent that they do not decompose 2 nucleic acids. According to a method described by F. 0.

G16ckner, M. F. Fuchs and R. Amann, 1999, 11Bacterioplankton Compositions of Lakes and Oceans: A First Comparison Based on Fluorescence In Situ Hybridization", Appl. Environ. Microbiol. 65: 3721-3726, the cells are treated with a fixative, and are incubated and subsequently filtered off on a filter.

Standard filters with a diameter of 25 or 47 mm are used for filtration. In an alternative method described by Amman, the cells are treated with a fixative, incubated, centrifuged and re-suspended, and are deposited on special microscope slides comprising cavities and air-dried. This method is published in the article by R. I. Amann, L. Krumholz and D.A. Stahl, 1990, "Fluorescent- oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology,,, J. Bacteriol. 172: 762-770. The method due to Amman has the disadvantage that the solutions do not always evaporate homogeneously on drying, and annular accumulations of objects can be formed. Moreover, a plurality of operations, such as centrifuging, re-suspension and dilution, can damage the cells and can even destroy them. Drying of the microscope slides.is very time-consuming, so that the samples cannot be further processed immediately. For these reasons, the filter method due to G18ckner is generally preferred.

Fixation of the cells is followed by hybridisation as a second process step.

3 In the method of H. M. Christensen, H. M. Hansen and J.

Sorensen, 1999, "Counting and size classification of active soil bacteria by fluorescence in situ hybridisation with RNA oligonucleotide probe", Appl. Environ. Microbiol.

65: 1753-1761, hybridisation is conducted in a reaction vessel. During hybridisation, fluorescence-labelled gene probes are added to the cell suspension. Depending on the type of gene probe used, hybridisation requires a time of to 90 minutes at a temperature of 40 to 500C. The temperature also depends on the gene probe which is selected. If the suspended cells have been hybridised in reaction vessels by the method of Christensen, a centrifugation step is carried out, although this reduces the quality of the cell material, as mentioned above.

After centrifugation, the cells are washed. The cells are then filtered off and the filter is transferred to a microscope slide for microscopic examination. In the method of G16ckner, the filter, which is situated on the microscope slide, is coated with a hybridisation solution and is placed in a humid hybridisation chamber. In order to wash the product, the filter is transferred by a washing buffer into a small glass bottle, and is subsequently dried and drawn on to a microscope slide for microscopic examination. In this method, at least 10 per cent of the cells are lost, even under the optimum conditions. This loss of cells cannot be controlled in environmental samples, however. This process is described in the publication by F. 0. G16ckner, R. Amann, A. Alfreider, J. Pernthaler, R.

4 Psenner, K. Trebesius and K. H. Schleifer, 1996,---Anin Situ Hybridization Protocol for Detection and Identification of Planktonic Bacteria System'', Appl. Microbiol. 19: 403-406. 5 The methods of fluorescence staining which are known from the prior art have the disadvantage that a plurality of operations, using different equipment, are necessary, in which cell material can be lost, or in which cells can be damaged due to mechanical stresses, e.g. due to centrifugation or due to re-suspension. An apparatus for the hybridisation of cells is known from DE 199 54 713.041. In this apparatus, a suction filtration apparatus in the form of a frit is provided in a hybridisation volume which can be heated, so as to be able to remove the reaction solution by suction when required. In this apparatus, the cells to be investigated, together with a hybridisation solution, are introduced into the hybridisation volume and are incubated. After the desired period of incubation, the liquid phase can be removed by means of a vacuum pump. The cells are retained by a filter which is situated at the base of the hybridisation volume. The filter can subsequently be removed from the apparatus to permit further microscopic evaluation. This method has the disadvantage that a high consumption of material is required for the necessary apparatuses if there is a plurality of samples which are treated and investigated simultaneously. Moreover, a relatively large volume of sample and reagent solution is required (about 2 ml).

What are termed microtitration plates have been increasingly used in applied environmental microbiology in recent years. These plates have dimensions of about 125 x 81 mm, and are provided with 32, 96 or 396 cavities which can accommodate a volume from 60 to 300 gl, for example.

These cavities are filled individually by means of auxiliary equipment. Pipettes comprising one channel or comprising 8, 48 or 96 channels are used for this purpose.

The advantage of microtitration plates is that a multiplicity of identical or different samples, including reference solutions, can be treated and evaluated in parallel. There is a multiplicity of specific microtitration plates which are commercially available, e.g. for DNA purification and for immunoassays, as well as correspondingly adapted accessories such as photometers and heater blocks. The prior art also comprises filtration systems which are used in combination with microtitration plates. Filters are used here which are fixedly welded into the cavities in the microtitration plates, or whole filter mats are used which are placed on the microtitration plate as a whole. One disadvantage of using fixedly welded filters in the microtitration plate is that it is not possible to make a direct microscopic evaluation of the filter, since accurate focusing on the samples by a microscope is impossible due to the thickness of the microtitration plates. A further disadvantage of these microtitration plates is that they can only be used once, and sample treatment is therefore relatively expensive 6 (about 30 DM/plate). Moreover, the usual designs of microtitration plates, which are made of rigid plastics, constitute an obstacle to controlled heating, due to the reduced thermal conductivity of these plates compared with metals. A disadvantage of using whole filter mats is the occurrence of cross-contamination by reagents contained in adjacent samples, and the occurrence of problems related to optical interference effects. This is because it is quite possible for diffusion to occur over the entire filter mat without any delimitation of the individual sample areas from each other. Moreover, microscopic evaluation of whole filter mats is not very practicable, since they cannot be completely placed on a commercially available microscope stage, due to their size.

It would be desirable to create an apparatus which enables a large number of samples to be treated under identical conditions using small amounts of reagent solution, and which makes it possible to effect direct optical evaluation, and to create an apparatus which comprises a large proportion of reusable elements, in order to make the use of said apparatus less costly.

According to a first aspect of the present invention there is provided an apparatus including a multiplicity of sample chambers for the treatment of cells and for analysis by means of luminescent processes, comprising an upper plate with extension tubes and a lower plate with 7 openings in which mobile filters are inserted, and means for filtering liquid under suction.

According to a further aspect of the invention there is provided a filter combination in which the individual filters are joined to each other via webs.

By means of the apparatus and filter combination according to the invention, it is now possible to investigate a large number of samples (e.g. 96) under identical conditions, particularly under identical thermal conditions. The apparatus enables smaller amounts of sample to be used, and thus also results in a lower consumption of probes and of cell material, as well as permitting the direct microscopic evaluation of the samples by removing the mobile filters. In addition, it is possible to re-use the apparatus after removing the filters.

The drawings illustrate an example of an embodiment of the apparatus according to the invention, and also illustrate the filter combination.

The drawings are as follows:

Figure 1 is a plan view of the upper plate; Figure 2 is a plan view of the lower plate; Figure 3 is a side view of the upper and lower plates; Figure 4 shows the filter combination; Figure 5 is a plan view of the baseplate; and 8 Figure 6 is a side view of the baseplate.

The upper plate 1 which is illustrated in Figure 1 comprises a plate with 96 cavities. These cavities form extension tubes 2, the openings of which can be seen from above in Figure 1. The cavities are arranged in a regular pattern.

In the plan view of the lower plate 3 which is shown in Figure 2, the filters 5 can be seen which rest on the openings 4 in the lower plate 3. The filters are each joined to each other via a web 6 and are each delimited by a ring 7.

In the side view shown in Figure 3, both the upper plate 1 with the extension tubes 2 and the lower plate 3 with the openings 4 can be seen. When the apparatus is used,' the extension tubes 2 in the upper plate 1 are inserted in the openings of the lower plate 3.

Figure 4 shows a filter combination comprising 8 filters 5, which are joined to each other via webs 6 and which are each surrounded by a ring 7.

Figure 5 is a plan view of the baseplate 8 which comprises an internal volume 11. It also comprises a sealing ring 9, which it seals the internal volume 11, and a suction tube 10.

9 In the view of the baseplate which is shown in Figure 6, the suction tube 10 can be seen which leads into the internal volume 11 in the baseplate 8.

The apparatus according to the invention and according to claim 1, which comprises an upper plate 1 with extension tubes 2 and a lower plate 3 with openings 4 in which mobile filters 5 are inserted, and which also comprises means for removing a liquid by suction, enables a large number of samples to be treated and investigated under identical conditions. The openings 4 in the lower plate 3 can take the form of round bores, which amongst their other features taper conically, or can also be angular punched-out shapes. Filters 5 are inserted in the openings 4. These filters retain the material which is to be investigated and are only permeable to the liquid with which the samples are treated or in which the samples are dissolved. The liquid can be removed from the apparatus through the filters 5 with the aid of the means for removing liquid or by suction.

The advantageous embodiment of the apparatus according to claim 2, which comprises an associated chamber which can be heated, enables cell material to be treated under constant, temperature-controlled conditions. Thus, for example, hybridisation of cells can be carried out without the cells having to be removed from the apparatus for filtration or for the further addition of reagents. The apparatus can be placed in a heater block, for example. It is it particularly advantageous here that a temperature gradient can be covered in order to determine the optimum temperature in each case.

Ey means of the embodiment of the apparatus according to claim 3, which comprises a pump for the removal of liquid by suction, it is possible to perform separation processes directly with the apparatus. The pump can be a vacuum pump, a water pump or a diaphragm pump, for example. The liquids can be removed completely by suction, either via the entire lower plate 3 or by connecting the pump to a baseplate 8 which seals the entirety of the openings 4, or if need be the pump can also be connected to the individual openings 4, so that each individual sample can be removed by suction (claim 4). This has the advantage of enabling different incubation times to be used under conditions which are otherwise constant.

The advantageous embodiment of the apparatus according to claim 5, in which the filters 5 are the joined to each other via webs 6, enables a plurality of filters 5 to be removed simultaneously from the apparatus. The number of filters which are to be removed is fixed by the joining of the individual filters 5 to each other by means of the webs 6, and can be varied as required.

The embodiment of the apparatus according to claim 6, in which the filter diameter corresponds to the diameter of the openings 4, serves to seal the openings 4 in the lower 11 plate 3 and thus serves to form the base for the reaction volume of the apparatus.

The advantageous embodiment of the apparatus according to claim 7, in which the spacing between the filters 5 corresponds to the spacing between the openings 4 in the lower plate 3, enables the filters 5 to be arranged by means of the webs is 6 so that a number of 8 or 16 filters, for example, can be inserted in the openings 4 in one operation, without the filters 5 having to be inserted individually.

The advantageous embodiment of the apparatus according to claim 8, in which the filters 5 are each surrounded by a ring 7, a ensures a leak-tight connection of the filters 5 to the openings 4 in the lower plate 3. At the same time, it enables the filters 5 to be fixed to the openings 4, and stabilises the filters 5, which are otherwise susceptible to breakage.

The embodiment of the apparatus according to claim 9, in which the diameter of the ring 7 is greater than the diameter of the openings 4 in the lower plate 3, ensures that the ring 7 around the filters 5 is placed around the openings 4 in the manner of a collar, and thus seals the openings.

The advantageous embodiment of the apparatus according to claim 10, in which the ring 7 has a thickness which does 12 not exceed 0.2 mm, enables the filters 5 to be removed from the apparatus directly after staining or after another chemical and or biological treatment, and placed under a microscope. Due to the low thickness of the filters 5 together with the rings 7, it is possible for the objective lens of the microscope to be moved sufficiently close to the filter in order to achieve good focusing.

The embodiment of the apparatus according to claim 11, in which the filters 5 have a pore size of 0.20 to 0.45 tm, enables a large range of different microorganisms to be retained by the filters 5, and thus enables treatments, such as hybridisation operations for example, to be performed on microorganisms of different sizes.

The embodiment of the apparatus according to claim 12, in which the filters 5 consist of polycarbonate, alumina or cellulose nitrate, enables different requirements to be taken into consideration which may result from different usages of solvents and acids, or which may also result from a different protein binding behaviour, or from the intrinsic and background fluorescence which form part of the properties of the filters concerned.

The advantageous embodiment of the apparatus according to claim 13, in which the diameter of the extension tubes 2 in the upper plate 1 corresponds to the diameter of the openings 4 in the lower plate 3, results in the 13 extension tubes 2 in the upper plate 1 being accurately sealed to the openings 4 in the lower plate 3 without liquid being able to emerge, and without the formation of dead volumes. 5 The embodiment of the apparatus according to claim 14, in which the extension tubes form a volume of up to 300 Ll after they have been placed on the lower plate 3, enables a small sample volume and a small volume of reagent solution to be employed, which results in cost savings. Compared with the prior art, the requisite amount of hybridisation reagent is reduced by a factor of 10.

The effect of the embodiment of the apparatus according to claim 15, in which the apparatus consists of a thermally conductive material, is that when the apparatus is heated, e.g. by inserting it in a heater block, the desired temperature is reached rapidly. If a temperature gradient is set, the change in temperature is rapidly transmitted from the heater block to the apparatus. An apparatus is suitable and is particularly preferred for this purpose which comprises VA steel (a nickel-chromium steel) (claim 17).

The advantageous embodiment of the apparatus according to claim 16, which consists of a material which is opaque to light, prevents damage to lightsensitive reagents or microorganisms.

14 The use of a chemically inert material as in the embodiment according to claim 18, in which the VA steel is coated with Teflon, glass, plastics, gold or ceramics, enables chemically more reactive reagents to be used, such 5 as solvents and acids for example.

The advantageous embodiment of the apparatus according to claim 19, in which the apparatus can be sterilised, permits multiple use of the apparatus. The apparatus can be used under sterile conditions by treating the apparatus as a whole in an autoclave, for example. The only disposable material which is used is that of the filters 5.

The filter combination according to claim 20, in which the individual filters 5 are joined to each other via webs 6, enables an arbitrarily variable number of filters 5 to be joined to each other and thus enables each to be used according to need. For example, 8 chains of filters 5 can be joined to each other, and can be inserted in a commercially available microtitration plate having 8 x 12 cavities, with the filters being accurately arranged for in a row in said plate. In principle, itis possible to employ a grid of connected filters 5, which depending on the requirements can be arbitrarily separated again at the webs 6 between the filters 5, in that order to produce a 4th chain or even a square arrangement, and for example. Because the filters 5 are only connected via the webs 6, the area for diffusion between the individual filters 5 is very small, so that cross-contamination of adjacent filters 5 is prevented. Due to the interconnection of the filters 5, the relatively small filters 5 can be removed ily from a filter holder, since the entire filter more easi combination can be pulled out of the holder.

The effect of the advantageous embodiment of the filter combination according to claim 21, in which the web 6 consist of a plastics material, is firstly to produce a mechanically stable joint between the filters 5, and is secondly to produce a chemically inert joint. At the same time, any exchange of material by diffusion between adjacent filters 5 is made more difficult. The effect of the embodiment of the filter combination according to claim 22, in which the individual filters 5 are surrounded by a ring 7, is, to create a leaktight seal between the filters 5 and the filter holder in which the filter combination is inserted. This embodiment also enables the filters 5 to be fixed in the holder and stabilises the filters 5, which may be breakable.

The effect of the filter combination according to claim 23, in which the ring 7 consists of a plastics material, is the f irstly produce a mechanically stable joint between the filters 5, and secondly results in a chemically inert stabilisation of the filters. At the same time, any exchange of material by diffusion between adjacent filters 5 is made more difficult.

16 The embodiment of the filter combination according to claim 24, in which the ring has a thickness which does not exceed 0.2 mm, enables the filters 5 to be removed from the filter holder directly after staining or after another chemical or biological treatment and to be placed under a microscope. The slight thickness of the filter 5 and ring 7 enables the objective of a microscope to be moved sufficiently close to the filters 5 in order to achieve good focusing.

The embodiment of the filter combination according to claim 25, in which the diameter of the ring 7 around the filters 5 is greater than the diameter of the openings 4 in the lower plate 3 of an apparatus according to any one of claims 1 to 19, enables the ring 7 around the filters 5 to be placed round the openings 4 like a collar, so that it seals them.

The advantageous embodiment of the filter combination according to claim 26, in which the filters are linearly joined to each other, enables a filter chain which comprises the requisite filters to be inserted as required and enables this filter chain to be matched to the existing number of filter holders. The filter chains can thus be well matched to the bores in a microtitration plate which are arranged in the form of a matrix.

The embodiment of the filter combination according to claim 27, in which 8 filters 5 are joined linearly to each 17 other, enables these filter chains to be inserted as a whole in a corresponding holder, e.g. as a row in a microtitration plate, and enables them to be removed again on the completion of treatment. 5 The effect of the embodiment of the filter combination according to claim 28, in which the diameter of the filters 5 corresponds to the diameter of the openings 4 in the lower plate 3 corresponding to an apparatus according to any one of claims 1 to 19, is that the openings 4 in the lower plate 3 are sealed and thus form the base for the reaction volume of the apparatus.

The embodiment of the filter combination according to claim 29, in which the filters consist of polycarbonate, alumina or cellulose nitrate, enables different requirements to be taken into consideration which may result from different usage of solvents and acids and which may also result from different protein binding properties which form part of the properties of the respective filter.

The effect of the filter combination according to claim 30, in which the filters have a pore size of 0.2 to 0.45 pim, is that a large range of different microorganisms can be retained by the filters 5, and thus treatments such as hybridisation procedures, for example, can be performed on microorganisms of different sizes.

18 Example: The upper plate 1, comprising 96 extension tubes 2 in a 12 x 8 arrangement, is fitted, forming a liquid-tight seal, to the lower plate 3 which also comprises 96 openings in a 12 x 8 arrangement and which is equipped with 12 filter combinations, each comprising 8 filters 5 per row. The upper plate 1 is completely closed by a cover. The lower plate 3 is sealed so that it is liquid-tight by a baseplate 8 fitted with a sealing ring 9. A vacuum can be applied to the entire apparatus via the baseplate 8, which is connected via a suction tube 10 to a vacuum pump. A clamp can be fitted round the entire apparatus to ensure reliable sealing of the assembled plates. The entire apparatus is inserted in a heater block for temperature- controlled operation.

gl of pre-heated hybridisation buffer with a corresponding content of gene probes is introduced into the reaction volume which is formed by the extension tubes 2 in the upper plate 1 together with the filters in the openings 4 in the lower plate 3. 100 gl of fixed cells are subsequently added thereto. After a hybridisation period of one hour, the liquid is removed by suction, the temperature of the apparatus is controlled at the desired washing temperature, and 200 gl of washing buffer is introduced. After one hour, the washing buffer is removed by means of the vacuum pump, which is connected to.the baseplate 8 via the suction tube 10. A further 200 gl of washing buffer is added and is removed by suction again.

19 The filters are then removed from the apparatus by withdrawing the entire filter combination, which comprises 8 filters per row, from the lower plate. The filter combination can be subjected directly to microscopic examination. If need be, a complementary staining operation can also be performed in the apparatus. The filters remain in the openings 4 in the lower plate 3 during this entire procedure. This has the advantage that any cells which become detached from the filter can be 10 recaptured by suction.

Claims

1. An apparatus including a multiplicity of sample chambers for the treatment of cells and for analysis by means of luminescent processes, comprising an upper plate with extension tubes and a lower plate with openings in which mobile filters are inserted, and means for filtering liquid under suction.

2. An apparatus according to claim 1 in which a chamber which can be heated at a controlled temperature forms part of the apparatus.

3. An apparatus according to either one of claims 1 or 2 in which the means for filtering liquid under suction comprise a pump.

4. An apparatus according to claim 3 in which the pump is directly connected to the individual openings in the lower plate or to a baseplate which seals the entirety of the openings.

5. An apparatus according to any one of claims 1 to 4 in which the filters are joined to each other via webs.

6. An apparatus according to any one of claims 1 to 5 in which the filter diameter corresponds to the diameter of the openings.

21

7. An apparatus according to any one of claims 1 to 6 in which the spacing between the filters corresponds to the spacing between the openings in the lower plate.

8. An apparatus according to any one of claims 1 to 7 in which the filters are each surrounded by a ring.

9. An apparatus according to claim 8 in which the diameter of the ring is greater than the diameter of the openings in the lower plate.

10. An apparatus according to either one of claims 8 or 9 in which the ring has a thickness which does not exceed 0.2 mm.

11. An apparatus according to any one of claims 1 to 10 in which the filters have a pore size of 0.2 to 0.45 gm.

12. An apparatus according to any one of claims 1 to 11 in which the filters are made of polycarbonate, alumina or cellulose nitrate.

13. An apparatus according to any one of claims 1 to 12 in which the diameter of the extension tubes in the upper plate corresponds to the diameter of the openings in the lower plate.

22

14. An apparatus according to any one of claims 1 to 13 in which after they are fitted to the lower plate, the extension tubes form a volume of up to 300 tl.

15. An apparatus according to any one of claims 1 to 14 and consisting of a thermally conductive material.

16. An apparatus according to any one of claims 1 to and consisting of a material which is opaque to light.

17. An apparatus according to any one of claims 1 to 16 and comprising VA steel.

18. An apparatus according to claim 17 in which the VA steel is coated with Teflon, glass, plastics, gold or ceramic.

19. An apparatus according to any one of claims 1 to 18 which can be sterilised.

20. A filter combination, in which individual filters are joined to each other via webs.

21. A filter combination according to claim 20 in which the webs consist of a plastics material.

22. A filter combination according to either one of claims 20 or 21 in which the individual filters are surrounded by a ring.

23 23. A filter combination according to claim 22 in which the ring consists of a plastics material.

24. A filter combination according to either one of claims 22 or 23 in which the ring has a thickness which does not exceed 0.2 mm.

25. A filter combination according to any one of claims 22 to 24 in which the diameter of the ring around the filter is greater than the diameter of the openings in the lower plate of an apparatus according to any one of claims 1 to 19.

26. A filter combination according to any one of claims 20 to 25 in which the filters are joined linearly to each other.

27. A filter combination according to any one of claims 20 to 26 in which 81 filters are joined linearly to each other.

28. A filter combination according to any one of claims 20 to 27 in which the diameter of the filters corresponds to the diameter of the openings in the lower plate corresponding to an apparatus according to any one of claims 1 to 19.

24

29. A filter combination according to any one of claims 20 to 28 in which the filters consist of polycarbonate, alumina or cellulose nitrate.

30. A filter combination according to any one of claims 20 to 29 in which the filters have a pore size of 0.2 to 0.45 im.

31. An apparatus including a multiplicity of sample chambers for the treatment of cells and for analysis by means of luminescent processes substantially as described with reference to and as illustrated by the accompanying drawings.

32. A filter combination substantially as described with reference to and as illustrated by the accompanying drawings.