GB2466816A

GB2466816A - High throughput DNA damage assay and separable multi-well plate apparatus

Info

Publication number: GB2466816A
Application number: GB0900239A
Authority: GB
Inventors: Simon Laurence John Stubbs; Nicholas Thomas; Rahman Aziz Ismail
Original assignee: GE Healthcare Ltd
Current assignee: GE Healthcare Ltd
Priority date: 2009-01-08
Filing date: 2009-01-08
Publication date: 2010-07-14
Also published as: GB0900239D0

Abstract

The present invention relates to a multi-chamber plate or matrix 2, containing a series of channels 3, which is sealed onto an optically transparent supporting surface 1a to create a channelled block or array of wells 5. The liquid-tight seal is reversible so that the matrix and support can be detached and separated. The apparatus is used in a comet assay (single-cell gel electrophoresis) as follows: the cell samples to be screened are added to the wells as a suspension, solidified, lysed, denatured and incubated with electrophoresis buffer. The matrix is then separated from the support 1 leaving processed samples 6 on the slide, which is electrophoresed, fixed, stained, and then imaged. The apparatus is used in high-throughput drug or genotoxicity screening. The matrix may be a 96-well plate or silicone matrix clamped onto a support, which may be a planar glass slide. The sample may be suspended in gelatinous medium, solidified by cooling to form a gel. The denaturing may be by alkali.

Description

Measurement of Cellular DNA Damage

Technical Field

The present invention relates to high-throughput measurement of DNA damage in eukaryotic cells, specifically to an apparatus and method for quantitative measurement of DNA damage by single cell electrophoresis. The invention is particularly suited for use in Comet assays and for automation.

Background to the Invention

Assessment of genotoxicity, cellular DNA damage resulting in an inheritable mutation, is applied across a wide range of industrial and research sectors. Genotoxicity testing of new chemical entities is a key step in drug development and a variety of methods are currently deployed (Walmsley RM.

2005 Expert Opin Drug Metab Toxicol. 1(2):261-8). Measurement of DNA damage is also widely used in epidemiological and environmental monitoring to assess the impact of chemicals or ionising radiation on humans and other species (M�ller P, et al 2000 Cancer Epidemiol Biomarkers Prey. 9(10):1005-15).

Measurement of DNA damage by single cell electrophoresis, commonly referred to as the Comet assay (Ostling 0 and Johanson KJ 1984 Biochem. Biophys. Res. Commun. 123, 291 -298).), is a widely used technique which offers a number of advantages in sample compatibflity over alternative genotoxicity assays. The Comet assay detects damaged DNA by subjecting cells embedded in an agarose gel to lysis and DNA denaturation, followed by electrophoresis.

Cells are subsequently stained with a fluorescent DNA binding dye and imaged *. 25 by microscopy. In cells without DNA damage high molecular weight DNA remains S..

associated with the cell nucleus, appearing as a bright spot in microscopy images.

In cells where DNA damage has occurred, producing breakage of DNA strands, : lysis and denaturation of the damaged DNA leads to unwinding of super-coiling within the DNA structure allowing DNA to migrate out of the nucleus when subjected to an electrophoretic field. Subsequent staining and microscopy reveals the migrated DNA as a tail emanating from the nuclear head giving an appearance very similar to an astronomical comet, after which the assay is named. Comet assays may be performed on a variety of specimens obtained as a single cell suspension including in vivo samples such as peripheral blood lymphocytes, buccal and nasal epithelia orin vitro samples using cell lines such as CHO, V79, mouse lymphomas or cultured human lymphocytes.

Current methods for performing the Comet assay to measure DNA damage are manually intensive and cumbersome, involving many separate steps and operations, making the assay time consuming to perform and unsuitable to automation. These factors limit the throughput of the assay and can lead to significant variance in the data produced, both within assays and between different laboratories. Some attempts to increase the throughput of the assay by using multiwell plates for sample processing have been reported (Kiskinis E, et al 2002 Mutagenesis. 17(1):37-43), however these have not addressed the rate limiting steps in the assay methodology -the embedding of cells in agarose on an imaging substrate and subsequent processing.

US patent publication US20030175821 (Hoover & Hoover) describes an automated comet assay system wherein light filtered to the excitation frequency of a fluorescing compound is directed onto the sample. A second filter isolates the emission frequency and the resulting image is directed at a low-light camera.

While this system provides means for automating the acquisition of Comet assay images, it does not address the throughput limitations of the entire process.

Japanese patent publication JP2007024612 (Matsushita Ecology Systems) describes a method for improving the analysis of comet assay images, but does not provide solutions to improving assay throughput. Japanese patent publication *, 25 JP2003210155 (Hitachi Plant Eng and Const. Co.) describes an automated analyzer for processing multiple microscope slides supporting Comet assay samples, but similarly with the preceding prior-art documents does not address the problems inherent in standard Comet assay protocols All current Comet assay protocols use glass microscope slides of varying dimensions as the assay support. Typically cells suspended in molten agarose solution are manually pipetted onto the slide as one or more drops and the drops allowed to solidify. Subsequent assay steps are then performed by dipping the slide in a series of solutions in order to perform the lysis, denaturation and neutralisation stages embodied in a standard Comet assay protocol. Slides are then placed in a horizontal electrophoresis chamber for separation, stained and imaged by fluorescent microscopy. Glass microscope slides, while suited to the final imaging stage of the assay, do not provide an ideal surface for performing the assay, the planar surface offering no effective inter-sample separation, limiting the number of samples that can be applied to a given surface area and providing the potential for cross-contamination of cells between samples. In addition the use of microscope slides or other planar surfaces precludes the automation of Comet assays using laboratory automation and robotic equipment designed to work with standard micro-plate formats, such as 96 well microtitre plates.

In order to overcome the limitations of the current Comet assay and provide a higher throughput and a more reproducible assay we have developed an apparatus and method for integration of all procedural steps which is compatible with industry standard laboratory automation equipment.

Summary of the Invention

According to a first aspect of the present invention, there is provided a method for measuring DNA damage in eukaryotic cells comprising the steps of: a) providing a reversible liquid-tight seal between a first surface of an optically transparent support and a first surface of a matrix wherein the matrix comprises a second surface opposed to the first surface and a *"*** 25 plurality of channels extending therebetween to thereby form an apparatus having a plurality of wells therein; b) adding a suspension of eukaryotic cells to the plurality of wells; c) solidifying the suspension of cells in the plurality of wells; d) lysing the suspension of cells to provide a plurality of Iysed samples; e) denaturing the lysed samples to provide a plurality of denatured samples; f) incubating the plurality of denatured samples with an electrophoresis buffer; g) removing excess liquid from the plurality of denatured samples; h) separating the support from the matrix by breaking the liquid-tight seal therebetween, the first surface of the support comprising the plurality of denatured samples; i) subjecting the denatured samples on the support to electrophoresis to provide a plurality of separated samples; j) fixing the separated samples to provide a plurality of fixed samples; k) staining the fixed samples to provide a plurality of stained samples; I) detecting an optical signal in the plurality of stained samples as a measure of DNA damage.

In one aspect, the second surface of the matrix is diametrically opposed to the first surface of the matrix.

In another aspect, the first surface of the optically transparent support is a planar surface. Preferably, the optically transparent support is a glass slide.

Optionally, the support may be composed of a suitable optically transparent plastic polymer.

In another aspect, the suspension of cells is added in a gelatinous medium.

Preferably, the gelatinous medium is molten at room temperature. More ** 25 preferably, the gelatinous medium is agarose. *..I

lii a further aspect, the support is pre-coated with a gelatinous medium.

In one aspect, step 1(c) is effected by cooling the plurality of wells. It will be understood by the person skilled in the art, that solidification of the suspension of cells may be effected by other means such as the addition of a polymerising agent.

In one aspect, the lysing step 1(d) is effected by treating the suspension of cells with means selected from the group consisting of electromagnetic radiation, ultra sonic waves, chemical reagents and enzymes. Laser light, for example, can be used to lyse cells. Chemical reagents are commonly used in lysing cells, detergents being particularly effective (e.g. NP-40, Triton X-100, Saponin, Digitonin, CHAPS). A wide range of Iytic enzymes are also available for this purpose (e.g. Lysing Enzymes from Sigma-Aldrich). It will be understood by the skilled person that other means for lysing cells are available, including mechanical disruption, manual grinding, freeze-thawing and liquid homogen isation.

In another aspect, the denaturing step 1(e) is effected by treating the lysed samples with a chemical selected from the group consisting of alkali, acid, EDTA and formamide.

In a further aspect, the fixing stepl(j) is effected by treating the separated samples with a dehydrating agent such as an alcohol or ketone.

In one aspect, the staining step 1(k) is effected by staining the fixed samples with S.' means selected from chemical, enzyme and antibody. Preferably, the chemical means is a DNA binding dye. * S S.'.

In another aspect, the optical signal is a fluorescent signal.

.: In a further aspect, an automated imager is used to detect the optical signal. An example of a suitable automated imager is the IN Cell Analyzer 1000 (GE Healthcare).

In one aspect, a test chemical is added to the suspension of cells in the plurality of wells after step 1(b) or step 1(c). DNA damage can be determined by comparing the optical signal obtained in the presence of the test compound with the optical signal obtained in the absence of the test compound.

According to a second aspect of the present invention, there is provided an apparatus for measuring DNA damage in eukaryotic cells comprising an optically transparent support and a matrix; the optically transparent support comprising a first surface; the matrix comprising a first surface and an opposed second surface having a plurality of channels extending therebetween; characterised in that a reversible liquid-tight seal is provided between the first surface of the optically transparent support and the first surface of the matrix In one aspect, the first surface of the matrix is diametrically opposed to the second surface of the matrix.

In another aspect, the first surface of the optically transparent support is planar.

In a further aspect, the matrix is composed of a plastic or silicone polymer.

In a further aspect, the optically transparent support is composed of glass or a plastic polymer. S.

In one aspect, an adhesive provides the liquid-tight seal. Suitable examples *..e. 25 include, but are not limited to, silicone based adhesives.

In another aspect, fastening means provide the liquid-tight seal. An example of : suitable fastening means is a clamp, such as an adjustable clamp which can be manually adjusted to provide a secure liquid-tight seal.

According to a third aspect of the present invention, there is provided the use of the apparatus as hereinbefore described in high-throughput drug screening and/or high-throughput genotoxicity screening.

In a fourth aspect of the present invention, there is provided a kit comprising a) an optically transparent support; b) a matrix comprising a first surface and an opposed second surface having a plurality of channels extending therebetween; c) sealing or fastening means to provide a reversible liquid-tight seal between a first surface of the optically transparent support and the first surface of the matrix.

In one aspect, the first surface of the matrix is diametrically opposed to the second surface of the matrix.

In another aspect, the kit additionally comprises a lysis reagent and/or a denaturing reagent.

Brief Description of the Invention

The apparatus and method of the invention are described with reference to the *:::: following figures: Figure 1: Schematic drawing of the assay apparatus showing the planar support and matrix array in reversibly affixed or bonded and separated orientations.

*.... 25 Figure 2: Representative images from an assay performed in the apparatus according to the method of the invention.

Figure 3: Representative data from an assay performed in the apparatus **. according to the method of the invention.

Detailed Description of the Invention

With reference to Figure 1, the apparatus [4] or device of the current invention comprises an optically transparent support [1j having a first surface [la], which is preferably planar in nature, reversibly bonded to a first surface [2a} of a matrix [2]. The support [1] comprises a glass or plastic sheet with optical characteristics which render it suitable for fluorescence microscopy. The external dimensions of the support are chosen to make the device compatible with handling by laboratory automation equipment. Dimensional standards for microplate devices suitable for handling by such automated equipment are published by the American National Standards Institute (ANSI) in conjunction with the Society for Biomolecular Sciences (SBS) Microplate Standards Working Group (http://www.sbsonline.com/msdc/approved.php). The external dimensions of the matrix [2] are similarly selected to render the apparatus or device compatible with microplate device standards. The matrix [2] is furnished with an array of channels [3] opening onto the lower or first surface [2a] and upper or second surface [2b] of the matrix [2]. The internal dimensions and spacing of the channels [3] are chosen to render the apparatus or device compatible with automated liquid dispensing in accordance with the ANSI/SBS standards.

Suitably the number of channels in the array is 96 or 384.

A first surface [Ia] of the support [1] is reversibly bonded to the lower or first surface [2a] of the matrix [2] to form a liquid-tight seal and thus provide an array of wells [5] open for access at the top surface [2b] and sealed at the bottom surface [2a] to the support surface [la], thereby providing a plurality of separated test sites or wells [5] for performing the assay. The matrix [21 may be *.. 25 fabricated from any suitable plastic or polymer material by injection moulding or casting. For injection moulding polystyrene or other plastics routinely used in the fabrication of microplates according to the ANSI/SBS standards may be used.

For fabrication by casting silicone or other flexible polymers may be used.

Reversible bonding of the matrix [2] to the support [1] may be achieved by gluing with a silicone or other suitable flexible adhesive or welding in the case of rigid materials such a polystyrene. Where silicone or similar polymers are used to cast the matrix the inherent adhesion of the polymer surface to smooth glass or plastic is sufficient to form a liquid-tight seal between the containers formed. Where necessary for additional bonding and rigidity for the purposes of handling the device, the bonding between the matrix and support plate may be augmented by clamping the components together by use of clips or a rigid frame, providing the overall dimension of the device conform to the ANSI/SBS standards. Alternatively an additional intermediate non-bonded thin sealing gasket of compressible material, such as a silicone polymer, may be used to form a seal between the first surface [2a] of the matrix [2] and the first surface [la] of the support [1], the resulting assembly being held together by clamping with clips or a rigid frame.

All cellular treatment steps in the assay protocol are performed in the wells of the apparatus or device. The standard dimensions of the wells and their spacing allows the assay to be performed manually using multi-channel pipettes or in an automated workflow using robotic liquid handling equipment to add and remove reagents from the wells. Once all processing steps have been completed the matrix [2] is separated from the support [1] and discarded, leaving an array of processed samples [6] adhering to the first surface [la] of the support [1] for electrophoresis, staining and imaging. Electrophoresis is readily performed by immersion of the support in a horizontal submarine electrophoresis tank of suitable dimensions such as a Sub-Cell GT 170-4404 (Bio-Rad). Following staining the support is imaged by automated high-throughput fluorescence ::::. microscopy using suitable instrumentation such as IN Cell Analyzer 1000 (GE Healthcare). The resulting images are analysed for DNA damage by automated image analysis using suitable software such as IN Cell Investigator (GE i... 25 Healthcare). S...

Example: Integration of Assay Protocol in a High-Throughput Apparatus *, *.: The apparatus or device of the invention was assembled using a microscope slide (BDH), pre-coated with I % agarose, adhered and clamped to a FIexiPERM 12 well silicone block (Sigma). One surface of the Flexiperm block comprises a silicone surface which forms a liquid-tight seal on glass. The block further comprises a silicone matrix vertically pierced by a 2x6 array of holes at ANSI/SBS standard 96 well micro-plate size and spacing. Jurkat (ATCC) cells were grown under standard tissue culture conditions, washed twice in phosphate buffered saline (PBS) by centrifugation at 1000rpm for 3 minutes, the supernatant removed and the cells re-suspended in PBS at 2 x i05 cells/mi. Cells were diluted 1:10 with pre-wamied (37°C) LMAgarose (Trevigen) and mixed gently by pipette. An aliquot (2Oj.tl) was dispensed into each well of the device and incubated at 4°C for 30 minutes to solidify the agarose suspension.

Cells were treated with 200p.l of PBS (controls) or 100j.iM hydrogen peroxide/H202 to cause DNA damage and incubated at 4°C for 30 minutes.

Solutions were removed by pipette and 200tl pre-.chilled Comet assay lysis solution (Trevigen) was applied to each well and the apparatus incubated in the dark at 4°C for 60 minutes. Lysis solution was removed and 200pl alkaline denaturation solution (0.6g NaOH + 250p.l of 200mM EDTA in 50m1 water) was added to each well and the apparatus incubated for 60 minutes at room temperature.

All liquid was removed from the wells of the apparatus and 20Oil of lx TBE electrophoresis buffer (Sigma) was added to each well and the device incubated at room temperature for 10 minutes. Washing and equilibration was repeated twice more followed by removal of all liquid from the device.

The clamps were then removed and FIexiPERM block separated from the I..

glass slide. The slide was placed in a horizontal submarine electrophoresis tank (Bio-Rad) in lx TBE buffer and electrophoresis performed at 25 volts for 10 minutes. Following electrophoresis the slide was fixed by immersion in 70% (vlv) ethanol for 5 minutes and then dried overnight at room temperature in the dark. S...

The slide was stained with SYBR Green (Trevigen), placed in a slide holder and imaged on IN Cell Analyzer 1000 (GE Heaithcare) using acquisition settings corresponding to standard 96 well micro-plate well spacing to acquire images from each separate sample.

Representative images from assays performed in the device are shown in Figure 2. Treatment of cells with increasing concentrations of hydrogen peroxide, a well known DNA damage causing agent, produced a dose-dependent increase in DNA damage as indicated by an increase in Comet frequency, intensity and length with exposure to hydrogen peroxide.

Images were analysed using an algorithm generated using IN Cell Investigator Developer Toolbox software (GE Healthcare) to segment images, identify nucleoids and comets and to generate quantitative measures of DNA damage. Typical data from a Comet assay performed in the device are shown in Figure 3.

The Developer Toolbox algorithm used generated a number of quantitative measures of DNA damage which are commonly used in the analysis of comet assays; i) Tail Intensity; comet head and tail regions are segmented and the total intensity of DNA fluorescence in the tail region is reported.

ii) Tail length; comet head and tail regions are segmented and the length of the tail region is reported.

iii) Percentage DNA in Tail; comet head and tail regions are segmented and the intensity of DNA fluorescence in the tail region is reported as a percentage of the total DNA in the head and tail.

iv) Tail Moment; comet head and tail regions are segmented and the percentage of DNA fluorescence in the tail region is multiplied by the length of the tail.

: The data from the assay performed in the apparatus show a clear dose-dependent increase in all DNA damage parameters derived by image analysis. * S S... * S S... *5 S * S S * S. ** S * * S.

Claims

Claims 1. A method for measuring DNA damage in eukaryotic cells comprising the steps of: a) providing a reversible liquid-tight seal between a first surface [la] of an optically transparent support [1] and a first surface [2a] of a matrix [2] wherein said matrix [2] comprises a second surface [2b} opposed to said first surface [2a] and a plurality of channels [3] extending therebetween to thereby form an apparatus [4] having a plurality of wells [5] therein; b) adding a suspension of eukaryotic cells to said plurality of wells [5]; C) solidifying said suspension of cells in said plurality of wells [5]; d) lysing the suspension of cells to provide a plurality of lysed samples; e) denaturing said lysed samples to provide a plurality of denatured samples; f) incubating said plurality of denatured samples with an electrophoresis buffer; g) removing excess liquid from said plurality of denatured samples; h) separating the support [1] from the matrix [2] by breaking said liquid-tight seal therebetween, the first surface [la] of the support [1] comprising the plurality of denatured samples [6]; i) subjecting the denatured samples [6] on the support [1] to electrophoresis to provide a plurality of separated samples; j) fixing said separated samples to provide a plurality of fixed samples; : k) staining said fixed samples to provide a plurality of stained samples; I) detecting an optical signal in said plurality of stained samples as a measure of DNA damage.*S,... * 25
2. The method according to claim 1, wherein the first surface of the optically transparent support is a planar surface. ** I I S *

*
3. The method of claim 1 or 2, wherein the optically transparent support is a glass slide.
4. The method according to any of claims 1 to 3, wherein the suspension of cells is added in a gelatinous medium.
5. The method of claim 4, wherein said gelatinous medium is molten at room temperature.
6. The method according to claim 4 or 5, wherein the gelatinous medium is agarose.
7. The method according to any preceding claim wherein the support is pre-coated with a gelatinous medium.
8. The method according to claim 5 or 6, wherein step 1(c) is effected by cooling the plurality of wells.
9. The method according to any preceding claim, wherein lysing step 1(d) is effected by treating the suspension of cells with means selected from the group consisting of electromagnetic radiation, ultra sonic waves, chemical reagents and enzymes.
10. The method according to any preceding claim, wherein the denaturing I... : step 1(e) is effected by treating the lysed samples with a chemical selected from the group consisting of alkali, acid, EDTA and formamide. e..
11. The method according to any preceding claim, wherein the fixing stepl(j) is effected by treating the separated samples with a dehydrating agent such as an alcohol or ketone.S

*
12. The method according to any preceding claim, wherein the staining step 1(k) is effected by staining the fixed samples with means selected from chemical, enzyme and antibody.
13. The method according to claim 12, wherein said chemical means is a DNA binding dye.
14. The method according to any preceding claim, wherein said optical signal is a fluorescent signal.
15. The method according to any preceding claim, wherein an automated Imager is used to detect the optical signal.
16. The method according to any preceding claim, wherein a test chemical is added to the suspension of cells in the plurality of wells after step 1(b) or step 1(c).
17. The method according to claim 16, wherein DNA damage is determined by comparing the optical signal obtained in the presence of said test compound with the optical signal obtained in the absence of the test compound.
18. An apparatus [4] for measunng DNA damage in eukaryotic cells comprising an optically transparent support [1] and a matrix [2]; said optically transparent support comprising a first surface [la]; said matrix [2] comprising a first surface [2aJ and an opposed second surface [2b] having a plurality of channels [3] extending therebetween; * * characterised in that a reversible liquid-tight seal is provided between the first surface [Ia] of the optically transparent support [1] and the first surface [2a] of the **S**.* 25 matrix [2]. **.* * * ***.
19. The apparatus according to claim 18, wherein the first surface of the optically transparent support is planar.
20. The apparatus according to claim 18 or 19, wherein the matrix is composed of a plastic or silicone polymer.
21. The apparatus of according to any of cairns 18 to 20, wherein the optically transparent support is composed of glass or a plastic polymer.
22. The apparatus according to any of claims 18 to 21, wherein an adhesive provides said liquid-tight seal.
23. The apparatus according to any of claims 18 to 22, wherein fastening means provides said liquid-tight seal.
24. Use of the apparatus according to any of claims 18 to 23 in high-throughput drug screening and/or high-throughput genotoxicity screening.
25. A kit comprising a) an optically transparent support [1]; b) a matrix [2]comprising a first surface [2a] and an opposed second surface [2b] having a plurality of channels [3] extending therebetween; c) sealing or fastening means to provide a reversible liquid-tight seal between a first surface [Ia] of said optically transparent support [1] and said first surface [2a] of the matrix [2].
26. The kit of claim 25, additionally comprising a lysis reagent and/or a S.... : denaturing reagent. S... * S **.*S..... * S S... * . *. S. * * S * * .. * S* *.