CA2311429A1 - Method and device for disintegrating biological cells for the purpose of extracting and analyzing cell contents - Google Patents

Abstract

Disclosed is a method and a device for disintegrating biological cells for the purpose of extracting and analyzing the cell contents, especially nucleic acids. The invention is characterized in that the cells are exposed to an electric field and in that before or after being exposed to the electric field, the cells a are brought into contact with substances supporting cell disintegration.

Description

F97R51 PCT PCTlDE 98/0297 Method of and Apparatus for Disintegrating Biological Cells for Extraction and Analysis of the Contents of the Cells Field of the Invention The present invention relates to a method of as well as an apparatus for disinte-grating biological cells for extraction and analysis of the contents of the cells, par-ticularly the nucleic acids.
For the analysis of biological cells and the analysis of the cell contents in particular, specifically proteins and nucleic acids (DNS and RNS), for instance for the purposes of medical diagnostics, the cell walls and membranes must be disintegrated in order to be able to extract the cell interior in a suitable form from the cells.
Prior Art To this end a number of common methods are appropriate which are described in a general survey in the paper "Cell Fractionation" by Carl A. Schnaitman in:
Manual of Methods for General Bacteriology, American Society for Microbiology, Washington, D.C., 2006, 1981, Chapter 5, pages 52 - 61. The methods described in this paper can be subdivided into different groups which are distinguished by their specific type of cell wall destruction or dissolution, respectively:
Mechanical methods are based on the generation of local pressure variations at the site of the cells to be disintegrated, which cause actually a break-up of the cell walls and membranes. Due to the strong pressure variations, which are required for destroying the cell walls, however, devices with an appropriately stable structure are necessary such as those described in the paper "Modified Press for Disruption of Microorganisms" by J. R. Raper and E. A. Hyatt, in: J. of Bacteriology, vol.
85, pages 712 - 713. Other devices for generating such high pressure or shearing forces are so-called ball or pot mills and the so-called French press, which are illustrated in F97R51 PCT PCTlDE 98/0297 Chapter 9.3.6 on pages 103 - 104 in the "Manual of Industrial Microbiology and Biotechnology", American Society for Microbiology, Washington, D.C., 1986.
The aforementioned devices require a lot of space in order to generate and reliably manage such high pressure forces, in addition to a very high expenditure in engineering and mechanical terms, and are moreover difficult to automate.
Another disadvantage of the known pressurising treatment of biological cells moreover consists in the aspect that the cell contents - by which term the long-chain DNS
molecules are to be understood in particular - are strongly fragmented, which renders a complete DNS analysis more difficult.
Apart therefrom, ultrasonic disintegration methods have become known wherein the cell walls and membranes are really torn up under the action of cavitation effects so that the cell interior may come out. In ultrasonic disintegration, however, a substantial sound energy is dissipated in the sample to be analysed, which involves an unnecessary heating of the sample. Therefore cooling provisions are required.
As a result of the occurrence of temperature variations, however, an process management with a fairly acceptable precision becomes more difficult. To this adds that the known apparatuses for application of the high-power ultrasound are difficult to clean so that the risk of inter-contamination cannot be precluded.
The disintegration of biological cells by exclusively chemical means requires chemicals which are individually matched with the cells, which entails difficulties in the selection of the appropriate chemical in each case for the analysis of unknown cell types, as is the case, for instance, in the diagnosis of unknown pathogenic agents. One example of a cell disintegrating method with the exclusive application of purely chemical disintegrating reactions is described in "Molecular Cloning -a Laboratory Manual", second edition, Cold Spring Harbor Laboratory Press, 1989, pages 1.25 - 1.31. The reaction times necessary to disintegrate cells with the ex-clusive application of chemicals are as long as several hours, despite high con-centrations of the used chemicals which may additionally produce interfering effects in the subsequent analytical steps, e. g. PCR reactions. For some micro-organisms the rate of disintegration is moreover insufficient; for instance it is extremely difficult F97R51 PCT PCTlDE 98/0297 with Micrococcus luteus to achieve a rate higher than 20% with application of chemical processes.
Finally, Methods have become known wherein biological cells such as bacteria or fungi suspended in liquid media are exposed to a strong electrical field. For instance, electrical fields are applied to kill biological cells in a pasteurisation an sterilisation process, as is described,. for instance, in the US Patent 5 235 905. In an attempt to avoid an excessive amount of energy introduced into the sample and overheating of the sample pulsed electrical fields are applied to the sample with respective pulse lengths of a few micro seconds. With a biological cell consisting of electrolytic and therefore electrically conductive contents, from an electrical point of view, which are enclosed by an electrically insulating membrane, the carriers of electrical charges inside are displaced when an electrical field is applied externally. In this manner local potential differences are created between the interior of the cells and the outside of the cell, which result in the cell membrane being subjected to an electrical break-through so that it loses its semi-permeable characteristics whilst its electrical conductivity increases. With low external field strengths and short pulse lengths the modification of the electrical conductivity of the cell membrane is reversible, a phenomenon which plays a central role in electroporation (cf. in this context "Electroporation and Electrofusion in Cell Biology" by E. Neumann, A. E.
Sowers, C.
A. Jordan, Plenum Press, New York, 1989). This effect is utilised, for instance, to introduce genetic material into the cells. The disadvantage of this application of electroporation resides in the aspect that the efficiency typically ranges about 0.01 %, which means that the material is actually introduced into a single cell out of 10,000 cells.
With stronger fields and sufficient pulse lengths, particularly with a repeated treatment of the biological cells, these modifications are permanent and result in the final death of the cell. This approach is used as a standard method for the destruction of micro-organisms in food items.
The German Patent DE 35 37 261 A1 discloses a method of field-induced transfer of macro molecules into living cells, which represents a process reciprocal to the F97R51 PCT PCTlDE 98/0297 selective transfer of cell contents out of cells and is hence subject to other process conditions. The transfer of particles into and out of cells can therefore not be con-sidered to be equivalent operations, specifically as this transfer is of an asymmetrical nature.
The asymmetrical or unidirectional transfer of various substances through the cell wall is rather part of the most important functions of the cell membrane. For instance, the cell membrane has, on the one hand, an asymmetrical structure and, on the other hand, the cell contents are substantially different from the external environment of the cell. The size of the transferred molecules is often comparable to the cell size as such, so that, not least as a result of this fact, the different transfer mechanisms which constitute the basis of transfer into and out of the cell, cannot be described by simple diffusion models - with simple diffusion not being a purely symmetrical process either. The transfer mechanisms are complex, involve several steps and are highly specific of the respective direction of transfer.
The same applies also to the method of transformation of Bacillus stearothermophilus with a plasmid, which is disclosed by the Japanese Patent JP 04173093 A:
All the methods known for the disintegration of biological cells involve the disad-vantage that the processes cannot be realised in a fully automatic manner, apart from the partly very substantial expenditure in terms of apparatus and technology. To this adds that the cell disintegration rate is not always sufficiently high.
Brief description of the invention The present invention is therefore based on the problem of improving a method of disintegrating biological cells for extraction and analysis of the cell contents in such a way that the method can be implemented in a fully automated manner without any high expenditure in terms of apparatus and technology. In particular, the extracted fraction of the cell contents to be analysed should be high independently of the cell type, while the duration of the process time for extraction and isolation of the cell contents should be substantially reduced. Moreover, the invention intends to provide F97R51 PCT PCTlDE 98/0297 an apparatus which permits a low-cost, high-speed and reliable realisation of the method.
In accordance with the invention it has been found that the disadvantages which occur with the known methods of the exclusively chemical disintegration of biological cells can be avoided by a selective sequence or superimposition of electrical and chemical treatments of biological cells.
According to the present invention, the method of disintegrating biological cells for the extraction and analysis of the cell contents excels itself by the provisions that the cells are exposed to an electrical field and that the cells are contacted with a substance promoting the cell integration before or after the application of the electrical field.
The biological cells, which are, as a rule, suspended in a liquid medium, are exposed to an electrical field with typical field strengths between 5 and 100 kV/cm which is operated in pulsed mode with a pulse length of roughly 0.5 to 50 micro seconds. The cells may equally be present in cell unions such as tissue fragments or applied and immobilised on a support such as a filter.
Due to the action of the electrical field and the electrical break-through pores are created in the lipid layers whereof the cell membrane consists. With these pores being very small, however, and with certain cell types being able to obstruct the diffusion of large-size molecules such as those of nucleic acid, the treatment of the cells by means of an electrical field alone is not sufficient in many cases for a release of the cell contents.
In accordance with the invention it is proposed to utilise the creation of the pores in the cell membrane in order to promote and accelerate the action of chemicals for the disintegration of the cells.
According to one variant chemicals are used which produce such an effect on the cell contents and particularly on the molecules to be analysed that they are easier and more quickly to liberate. These chemicals diffuse into the cell interior through the pores which are created by the electrical treatment, and disintegrate, for instance, F97R51 PCT PCTlDE 98/0297 proteins and cut DNA molecules selectively into smaller fragments which, however, are a subsequent DNS analysis. The DNS fragments so created can then freely diffuse through the pores to the outside. These processes are promoted by the addition of a surface-reactive substance (detergent) such as sodium dodecyl sulphate (SDS).
In another variant the fact is utilised that the pores created by the electrical treatment represent weak sites which substantially facilitate and accelerate the attack by the chemicals on the membrane. Chemicals appropriate for use in the complete disintegration of the cell walls are, for instance, chaotropic reagents such as urea, guanidine hydrochloride or thiocyanate and others. Dimethyl sulfoxide =may be employed as well.
With the biological cells suspended in a medium being exposed to an electrical field excessively high concentrations are not required for the admixture of the additional chemical substances, which can preclude negative effects which are due to the presence of the chemical substance in the subsequent analytical steps.
For an optimisation and acceleration of the intended chemical effect the cells contacted with the chemicals, e. g. in the form of a cell suspension, are heated to a temperature level between 35 and 100 °C.
In accordance with the invention an apparatus for realising the method is so structured that a sample volume receiving cells or cell unions suspended in a me-dium is provided with two two-dimensionally configured and mutually opposing electrodes, which can be occluded by a lid element on the upper side of the sample volume, which lid element has a convex configuration on the side facing the sample volume. Due to the convex configuration of the lid element the occlusion of air bubbles can be expediently avoided when the liquid medium is trapped within the sample volume, which bubbles would result in an inhomogeneous distribution of the electrical field and therefore possibly in undesirable electrical break-through in the treating apparatus.

F97R51 PCT PCTlDE 98/0297 Brief description of the drawing The invention will be described in the following exemplary specification, without any restriction of the general inventive idea, by embodiments with reference to the drawing wherein:
Fig. 1 shows one embodiment of an apparatus for realising the method of disinte-grating biological cells, Fig. 2 illustrates an alternative apparatus with a metal sheet on the lid element, Fig. 3 represents an alternative embodiment with a metal separating sheet within the sample volume, and Fig. 4 shows an alternative embodiment with friction elements for comminution of the tissue.
Brief description of one embodiment Fig. 1 illustrates an expedient apparatus for realising the method of disintegrating biological cells, showing a treatment cell including a synthetic housing 2 into which a lower electrode 3 is incorporated by casting and providing for an upper electrode 4 which is the lid element at the same time. The biological cells to be disintegrated, which are suspended in a solution, are present inside the sample volume 1 between the electrodes. In an alternative the cells may be immobilised on a support such as a filter, with the residual volume of the apparatus being topped up with an electrolyte solution. On account of the convex curvature of the surface of the lid element 4 it is ensured that when the lid element is placed on top air bubbles will not be trapped in the sample volume 1. The excessive suspension 5, which is urged out through the lid element 1 when the lid element is placed on top of the sample volume 1, accumulates in a recess 6 provided in the synthetic housing 2 circularly around the lid element 4. The lower electrode 3 opposite the lid element 4, by contrast, has a concave shape so that the distance between both electrodes is chosen to be constant. Moreover, the configuration of the electrode shape facilitates the filling of a liquid into the sample volume 1 without air bubbles and furthermore a largely complete removal of the sample after completion of the cell disintegration process.

F97R51 PCT PCTlDE 98/0297 For contacting the electrodes 4 and 3 for conveying and fixing the parts of the treatment cell in an automatic device in particular, the lower electrode 3 is provided with a plug 7 whilst the upper electrode 4 has a recess 8.
The electrode spacing, which is preferably set in this embodiment, amounts to 2 to 5 mm while the electrode diameter is roughly 1 cm. With these dimensions and with a voltage of approximately 20 kV, which is still comparatively easy to manage, the strength of the electric field ranges between 40 and 100 kV/cm, which is a field intensity which is well suitable for cell disintegration.
The electrode surface may preferably be made of aluminium because this rriaterial is biologically insert and has a sufficient chemical stability. Moreover, it is easy to process and inexpensive and therefore suitable for disposable articles in particular.
After the treatment with the electrical field the chemical treatment may be continued in the same volume by adding the chemicals, or alternatively the cell suspension may be transferred by means of a pipette into another vessel.
Even though the embodiment illustrated in Fig. 1 may be troublesome for removal of the sample due to the structural design of the lid element because the lid element must be removed before the sample volume 1 is emptied and a substantial part of the solution may adhere thereto as a drop. This, in turn, could result in the contamination of the environment and hence in the inter-contamination of further samples, which should, however, be avoided particularly in case of an automated handling of such samples.
To facilitate the removal of the samples particularly in an automatic apparatus and in order to ensure avoidance of inter-contamination the apparatus according to the embodiment of Figure 2 provides for a two-piece configuration of the lid element 4.
The lid element 4 is provided with a central bore 9 and is enclosed by an electrically conductive sheet 10 which wraps the lid element 4 and is preferably made of aluminium. After completion of the inventive method a pipette is introduced through the passage 9 to pierce the sheet 10 so that the suspension contained in the sample F97R51 PCT PCTlDE 98/0297 volume 1 can be exhausted by suction. Even though in this embodiment the sheet must be replaced each time this approach appears to be yet expedient specifically since the treatment cell as a whole may be configured as disposable article in order to avoid inter-contamination.
Figure 3 shows a further embodiment which comprises a lid element 4 comparable to that of the embodiment according to Fig. 1. However, this design provides for a metal sheet 11 separating the sample volume 1, which sheet constitutes at the same time the lower electrode. A cavity 12, which is connected to a passage 7, is provided underneath the electrode 11. Like in the embodiment according to Fig. 1 the treatment cell is filled with a suspension of biological cells while a high-voltage treatment can be performed with chemical additives for cell disintegration.
Upon completion of the application of an electrical field the cavity 12 is evacuated through the passage 7. Due to the difference in pressure the sheet 11 is torn open so that the processed suspension can be exhausted by suction through the passage for further analysis.
This variant is particularly suitable for a fully automatic realisation of the method, wherein the risk of inter-contamination can be completely precluded.
In all cases described in the foregoing a subsequent chemical treatment is necessary in order to transfer the cell contents efficiently to the outside. The chemicals may be added to the solution prior to the application of the electrical field; as they are electrolytes in many cases, however, or produce optimum effects in electrolytic buffers they should be added to the cell suspension only after completion of application of the electrical field.
In a variant of the chemical treatment a mixture of 15 different restriction endo-nu-cleases (3 U/ml of each) are added to the cell suspension and the suspension is then incubated at 37 °C for a period of 10 to 30 minutes approximately. The regents diffuse through the membrane pores, which are created by the application of the electrical field, into the cell interior and selectively cut the DNS strands to fragments F97R51 PCT PCTlDE 98/0297 including some thousands of base pairs which may be used later on for a PCR
reaction. After the treatment proteinase K in a concentration of 1 Ng/ml is added to the suspension, and the temperature level is set to 60 °C. Proteinase K
equally diffuses through the pores and causes the lysis of both cell proteins and the enzymes added in the previous step so that any interfering influence on the subsequent PCR
reactions is precluded. After incubation at 60 °C for 10 to 30 minutes the temperature is raised to 95 °C. In this step the proteinase K is denatured and the DNS diffuses through the pores in the membrane to the outside. With such a treatment the PCR
inhibitors - both the added ones and those inherent to the cell - are largely disintegrated.
In another variant of the chemical treatment, upon completion of the application of the electrical field, guanidine hydrochloride or thiocyanate (GdnHCI or GdnSCN) are added to the cell suspension so as to adjust a concentration of 0.3 to 3 M.
The suspension is heated to a temperature of 60 to 100 °C and is incubated for 10 to 30 minutes. With such a treatment the cell walls and membranes are destroyed and the DNS is consequently liberated. This method is simpler than the approach described before but it entails the disadvantage that guanidine compounds, which are here used in a concentration lower than that common for a purely chemical disintegration of cells, may inhibit the subsequent PCR reaction and must therefore be eliminated from the solution.
In the embodiment according to Fig. 4 the surface on the electrodes 13 and 3 is roughened so that the tissue fragments 16 introduced into the sample volume 1 may be comminuted.
In correspondence with the embodiment according to Fig. 4 the upper electrode and the housing 2 are so configured that the electrode is axially guided for rotational movement. The residual volume of the sample volume 1 is topped up with a liquid 17 and the tissue sample 16 is compressed between the electrodes by pressure exerted onto the upper electrode 13. In this case the liquid is only required for precluding electrical discharges between the electrodes because the sample as such contacts F97R51 PCT PCTlDE 98/0297 the electrodes directly. The liquid introduced between the electrodes is therefore not definitely electrically conductive so that that pure water or even oil may be used.
Following the high-voltage treatment and the addition of appropriate chemical ad-ditives the tissue sample 16 is crushed by the application of a high pressure on the upper electrode 13, possibly in combination with rotational movements. The upper electrode 13 and the housing 2 with the lower electrode 3 hence play the role of a die and a female mould. The electrode surfaces should preferably have a rough profile 17 so as to render the tissue disintegration more efficient.
The liquid liberated in this step or the paste so formed, respectively, which -contains the nucleic acids, rises in the gap 14 between the upper electrode 13 and the housing 2 and can be exhausted by suction through the passage 15 for further analysis.

F97R51 PCT PCTlDE 98/0297 LIST OF REFERENCE NUMERALS
1 processing space (is filled with the biological material to be treated in the appropriate medium (electrolyte solution)) 2 housing / capsule 3 lower electrode 4 lid excess medium 6 recess / groove therefor 7 plug 8 bore for electrode connection 9 passage in the lid metal sheet on the lid 11 lower electrode made of metal sheet 12 cavity 13 upper electrode 14 gap passage in the housing 16 tissue sample 17 liquid

Claims (22)

1. A method of disintegrating biological cells for extraction and analysis of the cell contents, wherein said cells are exposed to an electrical field, and that before or after the application of said electrical field said cells are contacted with substances promoting the cell disintegration.

2. The method according to Claim 1, wherein said electrical field is a pulsed field having a field intensity higher than 5 kV/cm, preferably higher than 20 kV/cm, and has a pulse length of approximately 0.5 to 50 microseconds.

3. The method according to Claim 2, wherein approximately 1 to 100 field pulses are applied to the cells.

4. The method according to any of the Claims 1 to 3, wherein said cells are present in a cell union or on a support, preferably a filter.

5. The method according to any of the Claims 1 to 3, wherein said cells are suspended in an electrically conductive medium.

6. The method according to Claim 5, wherein said medium in which the cells are suspended has a specific electrical resistance of 20 to 200 .OMEGA..

7. The method according to any of the Claims 1 to 6, wherein said substance is a detergent, a nuclease, or a protease.

8. The method according to Claim 7, wherein said detergent is SDS (sodium dodecyl sulphate).

9. The method according to Claim 7, wherein said protease is proteinase K.

10. The method according to any of the Claims 1 to 6, wherein said substance is dimethyl sulfoxide (DMSO).

11. The method according to any of the Claims 1 to 6, wherein said substance is guanidine thiocyanate or hydrochloride (GdnSCN or GdnHCl).

12. The method according to Claim 11, wherein said guanidine compound has a concentration higher than 0.2 M.

13. The method according to any of the Claims 5 to 12, wherein said medium is exposed to a temperature between 35 and 100 °C.

14. The method according to any of the Claims 1 to 13, wherein said cell contents to be disintegrated are nucleic acids.

15. An apparatus for disintegrating biological cells for extraction and analysis of the cell contents, for realising the method according to any of the Claims 1 to 14, wherein a sample volume with two two-dimensionally structure and mutually opposing electrodes is provided which receives the cell unions suspended in a medium in the form of a tissue union or cells applied on a support, and which is adapted to be closed on the upper side of the sample volume by a lid element having a convex configuration on its side facing said sample volume.

16. The apparatus according to Claim 15, wherein said lid element is configured as electrode.

17. The apparatus according to Claim 16, wherein the electrode opposite said lid element has a side of concave configuration which faces said sample volume.

18. The apparatus according to Claim 16 or 17, wherein said lid element has a passage opening to said sample volume and is coated with an electrically conductive sheet on the side facing said sample volume.

19. The apparatus according to any of the Claims 16 to 18, wherein the lower electrode opposite said lid element is configured as sheet separating said sample volume from a draining passage located underneath said sheet.

20. The apparatus according to any of the Claims 15 to 19, wherein said lid element is configured as die and adapted to be axially guidable for rotational movement inside said sample volume.

21. The apparatus according to Claim 20, wherein the surfaces of said electrodes, which are facing said sample volume, are roughened.

22. The apparatus according to any of the Claims 15 to 21, wherein said electrodes are spaced from each other by a distance of 1 to 5 mm approximately.

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SUMMARY

What is described here is a method of and an apparatus for disintegrating biological cells for extraction and analysis of the cell contents, specifically nucleic acids.
The invention excels itself by the provisions that the cells are exposed to an electrical field and that prior to or after the application of the electrical field the cells are contacted with substances promoting the disintegration of cells.