GB2472384A

GB2472384A - Modified microplate

Info

Publication number: GB2472384A
Application number: GB0913313A
Authority: GB
Inventors: Simon Stafford
Original assignee: Individual
Current assignee: Individual
Priority date: 2009-07-31
Filing date: 2009-07-31
Publication date: 2011-02-09
Also published as: GB0913313D0

Abstract

A microplate comprises a plurality of open wells 2, wherein at least one of the wells comprises an area for communication with the tip of the pipette 1. The area for communication with the pipette tip may comprise one or more raised areas or protrusions 6 located on the base and/or wall of the well interior. The area for communication with a pipette tip can comprise one or more recesses or indentations 5 in the wall and/or base of the well. At least one of the wells may comprise a guide channel 4. Preferably, the guide channel accommodates a pipette tip of a size appropriate for an assay using the microplate. The microplate aids the introduction of fluid 3 into and removal of fluid from the wells of the plate and minimises the damage caused to biological matter growing therein.

Description

A Modified Microplate

Field of Invention

The present invention relates to microplates, and a means for improving fluid exchange when using such plates. More specifically, the invention relates to a microplate which comprises one or more internal modifications which aid the introduction and removal of fluids from the wells of the plate and minimize the damage caused to biological media located within the well, such as cells growing therein, upon introduction or removal of a pipette tip and/or fluids from the plate.

Background of the Invention

Microplates (also known as microtitre, microtiter, microtitration or multi-well plates) are a standard laboratory tool, which are used in screening assays, analytical research and diagnostic techniques. Primary applications for microplates include enzyme linked immunosorbent assays (ELISA), cytotoxic assays, protein assays, and many cell-based assays used in high-throughput screening during the drug discovery process.

Microplates are, in effect, small reaction vessels. They are typically small rectangular trays or cassettes that ate covered with wells or dimples arranged in orderly rows. These wells are used to conduct separate chemical reactions. The number of wells included in microplates is commonly 6, 12, 24, 48, 96, 384 or 1536, depending upon the size of the microplate and the size of the wells.

Microplates typically comprise a base plate fixed to an upper plate which comprises a plurality of wells. Each well comprises a wall and a base. The wells are typically arranged in a matrix arrangement of rows and columns. Microplates may be provided with a lid, which fits over the plate to prevent spillage or contamination of the wells and/or to seal the wells.

While some microplates are designed for reuse, and some reactions can be carried out repeatedly in the same microplate, in general, microplates are laboratory consumables, and are disposed of after use.

Microplates are typically made from polymeric plastic materials such as polypropylene, and the most common form of manufacturing process for microplates is injection moulding. In some cases other materials such as glass may be incorporated into the microplate, typically as the base of the well.

While most microplates are of standard manufacture, specialized microplates are available. Clear bottom microplates are ideal for fluorometric applications as well as cell and tissue culture. UV-treated microplates may be used with protein and nucleic acid concentrations, and in research involving DNA testing or sequencing.

Fluorescence microplates are available with black or white pigments to reduce background signals or to enhance reflectivity. Luminescent vessels provide high reflectivity, medium binding and low cross talk. Additional designs include microplates that are designed to resist corrosives or solvents.

Microplates can be provided with wells of a certain cross-sectional shape (in vertical view), for example, microplates can have wells that have flat or round bottoms; and wells that have vertical, chamfered or conical walls. US 5,017,341, which is concerned with increasing the sedimentation rate of particles in agglutination assays, discloses a plate with a well having a bottom surface at least part of which is inclined.

Wells of a microplate are typically filled using a probe (a single probe, used for repeated filling/aspirating; or multiple aligned probes, which can be used to fill/aspirate several wells at the same time) or a pipette.

Pipettes (also called a pipet, pipettor or chemical dropper) can be individual or multi-channel pipettes. Multi-channel pipettes generally comprise 8 or 12 channels, and are used to aspirate fluid from and/or dispense fluid to multiple wells in a microplate simultaneously. For example, when a microplate comprises 96 wells, the wells are typically arranged in 12 columns of 8 wells. An 8-channel pipette can be used to aspirate fluid from or dispense fluid to an entire column of wells simultaneously, and a 12-channel pipette can be used to aspirate fluid from or dispense fluid to an entire row of wells simultaneously.

Pipettes are available for use in dispensing different volumes of fluid. For example, pipettes ate available for dispensing up to 5i.tl, lOj.tl, 50il, 100d, 200tl, 300 jil, 500fl or imi, or 5ml of fluid.

Disposable pipette tips are available for use with each pipette. For example, pipette tips are available for use in dispensing different volumes of fluid, such as 5tl, lOiil, 50il, 100d, 2OO.tl, 300.d, 500d or lml, or 5m1. These disposable pipette tips are of an appropriate size for use with the corresponding pipette. For example, the outer diameter of the point of a 1000tl pipette tip is approximately 1.5mm, the inner diameter is approximately 0.7 5mm; the outer diameter of the point of a 200 1fl pipette tip is approximately 0.77mm, and the inner diameter is approximately 0.4mm; the outer diameter of the point of a SOul pipette tip (typically used with a robotic' automated systems) is approximately 0.6-0.75 mm, and the inner diameter is approximately 0.3-0.4mm; and the outer diameter of the point of a lOp.l pipette tip is approximately 0.8mm, and the inner diameter is approximately 0.4mm. By point' is meant the open (i.e. dispensing) end of the pipette tip.

Whilst there are many different types of pipette, the most commonly used pipette for filing or aspirating a microplate is a piston-driven air displacement pipette.

Depression of the pipette plunger creates a vacuum. The fluid to be dispensed is then drawn up into the disposable pipette tip. The plunger is depressed to dispense the fluid.

Normal operation consists of depressing the plunger button to the first stop while the pipette is held in the air. The tip is then submerged in the fluid to be transported and the plunger is released in a slow and even manner. This draws the liquid up into the tip. The pipette is then moved to the desired dispensing location.

The plunger is again depressed to the first stop, and then to the second stop, or blowout, position. This action will fully evacuate the tip and dispense the liquid.

Obviously, when dispensing and removing fluid from a microplate, a pipette is chosen which has a tip of an appropriate size for the wells of the microplate.

Biological material may be introduced to, cultured and/or immobilized within individual wells of a microplate. For example, proteins may be immobilised within wells of the microplate, by processes such as adsorption, streptavidin-biotin capture and covalent linking.

Alternatively, living biological cells may be introduced to, and cultured within individual wells of the microplate. The cells are typically introduced as a suspension, wherein a certain volume of the suspension is placed in each well. The loaded microplate is then placed under cell culture conditions (for example in an incubator at 37°C with 5% C02) for a period of time to allow the cells to settle and adhere to the base of the wells. If desited, the culture time can be extended, to allow the cells which have adhered to the base of the wells to grow and/or multiply, with the aim of providing a confluent monolayer of cells on the base of each well.

A typical procedure for using a microplate in a cell based assay is as follows. The culture medium in which the cells have been adhering/growing is removed, either by aspiration using a probe or pipette (either individual or multichannel), or by tipping out the medium into a waste receptacle or onto paper towels. The culture medium is replaced with a washing solution, which is introduced via a pipette and then removed by aspiration or tipping. The washing step may be repeated.

A fixative is then introduced via a pipette and left for a period of time. The fixative is generally a toxic substance, such as a paraformaldehyde solution (of a final concentration of 4 or 8% (v/v)). The fixative is used with the aim of fixing the cells in position in the wells of the microplate. The use of such fixative can represent a health and safety hazard, as it is highly undesirable for the fixative to come in contact with the skin of the operator. In addition, it is undesirable for the fixative to be inhaled by the user, and it is therefore desirable to prevent aerosolisation of the fixative.

The fixative is then removed by aspiration or tipping, often with the additional step of banging the plate on a paper towel, in an effort to remove excess solution. The cells are washed free of fixative, a process which may require repeated washing efforts. The cells are then ready for the reagents for use in the assay to be applied.

An example of an assay for which a microplate is used is a screening assay wherein it is desirable to investigate the effect of a reagent or compound on a certain property of the cells. For example, it may be desirable to investigate the effect of a compound on a channel protein, such as a calcium channel protein. An appropriate assay could involve incubation of the cells with a photosensitive dye, such as the fluorescent dye fluo-3AM, which binds to the cells, with excess dye removed by washing. The reagent and appropriate controls are then applied, and removed, and the cells are washed, before the cells are then challenged in order to elicit a fluorescent signal, which is proportionate to the activity of the calcium channel protein as affected by the reagent. The fluorescent signal is detected and quantified by appropriate screening machinery, such as a microplate reader.

As an example of such an assay: it may be desirable to investigate the genotype-correlated sensitivity of selective kinase inhibitors in tumour cell line profiling, as predictive of clinical efficacy. An appropriate assay could involve incubation of diverse epithelial cancer cells with the selective kinase inhibitors; fixing the cells in 4% formaldehyde in phosphate buffered saline(PBS); and staining with the cell-permeant, fluorescent nucleic acid stain Syto6O (Molecular Probes). Fluorescence is quantified using a microplate reader. The sensitivity of each cell line to a given concentration of compound can be calculated as the fraction of viable cells relative to untreated cells. (McDermott etal., PNAS. 2007: 104(50): 19936-19941).

A microplate reader, or spectrophotometer, can be used to quantify the fluorescent signal emitted by a microplate assay, using excitation and emission wavelengths selected in accordance with the fluorescent stain used in the assay. Positive controls (microplate wells containing cells and assay reagents but no test compound) and negative controls (microplate wells containing no test compound and no cells) are included in the microplate.

A measure of the quality of the assay is provided by calculating the Z and Z'-factors.

The Z'-factor or Z' is a dimensionless statistical characteristic. It is calculated from four parameters: the means and standard deviations of both the positive (p) and negative (n) controls (llp,ap, and i,,,a,,): 3 x (ci + a) Zfactor = 1 -

ILLTI -

The Z-factor is as above, but includes the intervention of test compounds.

The closer the value for Z-factor is to 1, the higher the assay quality, as explained further by the table, below, (from Zhang et a!., J Biornol Screen. 1999;4(2):67-73).

Z-factor Interpretation 1.0 Ideal (Z_factors can never actually greater than or equal 1).

An excellent assay. Note that if a = a, 0.5 is equivalent to a between 0.5 and 1.0 separation of 12 standard deviations between lip and li1 between 0 and 0.5 A marginal assay.

less than 0 The signal from the positive and negative controls overlap, making the assay essentially useless for screening purposes.

If the Z-factor approaches or is close to the Z'-factor or Z', the assay is suitably optimized. If the Z-factor approaches 0, the assay requires further optimization.

The steps of applying the challenge solution and commencing screening by the screening machinery are typically very time dependent. Signals from the cells are elicited by the plate reader by exposure to specific wavelengths of light. The dyes used in such assays are, therefore, light sensitive, and subject to bleach' if exposed to light for prolonged periods. It is therefore necessary for the operator to minimize exposure of the dye, and the microplate once it contains the dye, to light (see Friedrich et al. Nature Protocols. 2009: 4(3): 309-324, and page 317 in particular, where avoiding exposure of the substrate solution to light is considered "critical") and quick, efficient removal and addition of substrates facilitates this.

It is critical in such assays that the signal that would be produced by each well under control conditions is consistent, i.e. that the standard conditions in each well are the same, so that any variation in the signal that is produced is directly attributable to the reactant applied, and not to variations between the wells.

To act as a control, and to verify the reliability of the signal and consistency of the signal across the plate, it is usual for each reactant to be applied to more than one well, and typically, each reactant is applied to an entire row or column of wells.

There are various factors which can influence the signal that is produced per well.

For example, the amount of fluid introduced and removed from each well must be consistent. Inconsistencies in the amount of fluid can result in the cells being exposed to variable amounts of reactant and variations in well volume can affect the signal produced and detected as a result of variable light diffraction. Ineffective removal of fluid also results in a residual amount being left in the well, which then dilutes subsequently added solutions. This can inhibit the development of assay substrates.

For example, in ELISA studies, the signal to be read by the microplate reader develops over time, due to enzymatic turnover of a substrate. The developing signal can be measured in a time-course experiment, or the signal can be allowed to develop and then halted at a certain time point by the addition of a stop solution', such as hydrochloric acid. In both cases, residual amounts of assay and/or wash buffer can affect development of the signal.

It is therefore desirable for as much fluid as possible to be removed from each well during each stage of the procedure. To achieve this, the tip of the pipette used to aspirate the fluid needs to touch the base of the well. However, this can cause significant problems, as the pipette tip disrupts biological material located on the base of the well, for example, cells growing on the base of the well.

It is essential that the number of cells in each well is consistent. As the assays for which microplates are typically used rely upon detection of a signal associated with a change in one or more cellular properties of the cells, variations in the number of cells per well will result in variations in the signal produced per well, making it impossible to assess the effect of the reagent.

Variations in cell numbers can be attributable to many factors, such as poor adhesion of the cells to the plate; non-homogeneous cell suspension when seeding the wells and inadequate fixing of the cells, for example due to the use of an inadequate fixing time or inappropriate/weak fixative.

Further to this, the cells are delicate, and can easily be removed from the base and/or sides of the wells of the microplate, particularly before fixing. Removal of cells can result from the whip' or vortex effect of introducing fluid to the well (see, for example, Vichai and Kirtikara. Nature Protocols. 2009: 1(3): 1112-1116, and page 114 in particular, wherein introduction of wash solutions without injecting them directly onto the bottom of the well is considered a critical step), from the suction effect when removing fluid from the well with a pipette, and as a result of tipping or banging out of the fluid from the microplate. A further very important factor is the scraping off of cells by the end of a pipette tip as fluids are introduced or removed.

These factors occur both in plates which are manually prepared by a human operator, and in automated systems which can be used to prepare microplates for screening and/or for carrying out screening.

Manual preparation of microplates requires human processes such as visual observation and hand dispensing of volumes of fluids into individual wells on the plate. Such meticulous work can be challenging for the operator, particularly when the microplate being used has a large number of wells, and/or when the microplate needs to be located in a sterile hood or a fume hood in order to reduce the risk of contamination of the assay, or to reduce the exposure of the operator to the solutions being used. Hoods can be confined spaces to work in and may be poorly lit, making observation and hand pipetting difficult. Human error can occur as a result of fatigue, eye strain, strain on the operator's pipetting arm and failure to remember which well or group of wells have received fluid. Difficulties in pipetting can lead to errors in dispensing and aspirating solutions from the microplate, such as mis-pipetting or double pipetting; cross contamination of wells; and accidental scraping of cells from the wells.

Automated systems are aimed at reducing the human involvement in preparing and, optionally screening, microplates, in order to reduce errors associated with manual preparation, and the time required to prepare and screen plates. This is particularly the case in high-throughput systems which can be used to prepare and/or screen numerous plates per hour.

Automated systems must be able to manipulate conventional microplates and their contents, for example to allow addition (or removal) of reagents or other materials to (or from) multiple wells of a microplate simultaneously.

Accordingly, automated systems for preparing cells for screening typically comprise a mobile head which locates above the microplate and which has an array of pairs of tubes. One of each pair of tubes is for aspirating fluid from the well, the other for dispensing fluid into the well.

In some automated systems, the aspiration and dispensing tips are separated from each other, so that both enter the same microwell, but are located at a distance from each other.

In typical automated systems, the tips are aligned with the centre of the well, with the operator responsible for setting the height alignment. Poor height alignment can result in the tips touching the bottom of vell and damaging the integrity of biological material located on the bottom of the well, such as a cell monolayer.

-10 -Some systems are designed so that neither the aspiration nor the dispensing tube tip extends to the base of the well. This can mean, as a result of the gap between the base of the well and the tube tip, that the aspiration tube in unable to remove all of the solution from the well, which results in dilution of subsequently added reagents, thereby reducing their effectiveness. What is more, the dispensed fluid is dropped into the well from a height, which can result in damage to cells located on the base of the well.

In some systems, the microplate is moved in a circular and/or cross-wise manner during aspiration. This is to aid the removal of residual liquid, which may collect at, for example, the junction between the wall and the base of the well. This process can increase the time needed to prepare the microplate for screening, which is a disadvantage in many high throughput screening (HTS) systems, and can scrape the base of the well if the tube tips are positioned incorrectly, causing damage to cells adhered to the well base.

Further to this, the suction and dispensing pressures used to aspirate and dispense solutions by such systems can cause loosening and dislodging of cells.

Studies have shown that such automated systems can produce a typical pattern of cell loss (see, for example, the Biotek poster presentation, which can be found at: http: / /wT.biotek. corn/resources /docs /ELx4O5 CW Lab Automation Poster2.p The factors discussed above can result either in the complete lifting off of cells from the well, so that the cells are then lost during subsequent fluid removal, or partial lifting of a layer of cells, which can lead to an erroneous signal during the assay, as a result of variable light diffraction.

Obtaining a consistent signal across the microplate is critical and, in view of this, various ways have been suggested for introducing and/or removing solution from a well of a microplate in order to avoid cell disruption. 11 -

For example, in manual systems, fluids may be removed by the operator by turning the microplate upside down in one motion, which allows the liquid to fall from the well in an uncontrolled manner. However, this can result in inconsistent residual solution volume across the plate, cross contamination between wells caused by splashing, and may also cause mechanical shock to cells leading to cell detachment.

Furthermore, such methods of fluid removal are generally not possible in an automated high throughput screening system, and even if they were incorporated, would considerably increase processing times.

An alternative method is for the operator to thtow or flick the liquid from the microplate into a waste receptacle or onto a paper towel. However, this can result in inconsistent removal of solution, and the whip effect which can dislodge cells. It can also cause cross contamination between wells. Furthermore, this technique carries an increased risk of splash-back and aerosolisation of the solution, which is undesirable given the toxic nature of the fixative.

Laboratory manuals also teach the use of a good pipetting technique', which emphasizes the importance of the operator dispensing the correct amount of fluid, and introducing and removing fluids at the edge of the well, by carefully aiming the pipette tip at a well wall, and allowing solution to slowly trickle down to fill the well, thereby reducing physical scraping of the pipette tip on the adhered cells, and the whip effect caused by fluid introduction or removal. However, this is not a practical solution for most screening assays which, as discussed above, are time dependent.

Furthermore, pipette tips applied to a multi-channel pipette rarely align perfectly, and even a careful pipetting technique cannot avoid variations in pipette tip angle, which can result in inconsistent contact between the pipette tips and wells/cells.

Means of aiding the operator in the introduction and removal of fluids from io microplates are also known. For example, US Publication No. 2007/0009396 discloses a multi-well plate "guide protector", which covers the all of the microplate apart from the column of wells to be filled or aspirated. The operator places the guide protector over the plate, and fills the column of exposed wells, before sliding -12 -the guide to the next column, thereby covering the wells which have been filled.

The guide protector aims to prevent double filling of wells, however, it does not prevent inaccuracies of the operator during pipetting, or damage to the cells in the wells as a result of scraping by the pipette tips or fluid addition/removal.

Furthermore, the guide protector could not be incorporated into an automated systcm.

The Hong Kong CH Gene Limited has devised a series of "WeilMatch pipetting Guide Manuals" which are distributed by Gene Company Limited. These comprise a base, which comprises a platform on which is located a raised rectangle, into which a microplate is placed. The base has two screws located along one edge, which can be adjusted to angle the microplate, and a guide cover, similarly to that disclosed in US Publication No. 2007/0009396, which can be placed over the plate to guide pipetting. However, because the raised rectangle is fixed to the platform, the WeliMatch base can only be used with a specific size of microplate. Further to this, the screws need to be adjusted manually to obtain the desired angle. This can be time consuming, and it can be difficult to ensure that each screw is adjusted to the same height, in order to prevent pipetting inaccuracies as a result of a variation in the angle of incline across the plate, or rocking of the platform -a factor which is particularly important as the operator of the assay may apply a small amount of downward pressure to the pipette tip before/during pipetting, in order to ensure that the tip is in contact with the bottom of the microplate well, thereby preventing the solution from being dropped from a height, which can cause damage to the cells, or residual fluid being left in the well.

US Publication No. 2009/0010811, which is concerned with the provision of an illumination system for enhancing manual handling of a multiwall plate discloses, as part of the system, an elevation apparatus which allows the illuminator and multiwell plate to be inclined to facilitate observation during dispensing of fluid into the plate. The illuminator may have an alignment feature, to provide proper alignment between each element of the light source and each well, however, there is no means of securing the plate to the illuminator or the inclination mechanism, and -13 -the use of an illuminator plate is contraindicated where a photosensitive dye is used in the screening process.

With respect to automated systems, Biotek� provide an automated Microplate Washer system, which has a software controlled flow rate control valve which restricts the flow rate, and an angled dispensing tube to allow the outlet of the tube to be offset from the centre of the well. The angled dispensing of fluid was not considered to significantly affect cell loss. However, Biotek� report, in studies associated with the automated Microplate Washer system, that the critical parameter in preventing cell loss was the fluid dispensing rate. It was found though, that using low flow rates caused premature aspiration of the fluid being dispensed, resulting from formation of a droplet at the end of the dispensing tube, which, as it grew, came in sufficiently close proximity to the end of the aspiration tube for it to be aspirated without having ever entered the well. To eliminate this problem, Biotek� incorporate into their system a "Vacuum on Volume" featute, which allows the user to delay the initiation of aspiration for a brief period, to allow the fluid droplets to flow into the well before aspiration commences.

Slowing the rate of flow obviously increases the time needed to prepare the microplate for screening, and this is disadvantageous in high throughput screening systems. Further to this, the inclusion of software to control flow rate, additional flow rate valves and the accompanying vacuum system have an impact on the cost of the screening process.

Modification of microplates to avoid some of the problems associated with cell ioss and plate inconsistencies has also been considered.

WO 99/20394 discloses a microplate assembly comprising a plurality of vent tubes and caps. The vent tubes, which are for the purpose of permitting the pressure within the interior volume of the well to be equalized with the ambient pressure, terminate in a vent that communicates with the interior of the well. Material may be added to, or removed from the wells via the vent passage. However, the vents disclosed in WO 9 9/20394 are located in the centre of each well, and introduction of fluid via a standard pipette tip would result in fluid being dropped from a height directly onto, and removed at a height from the centre of the well, which could result in physical damage to cells growing in this area. WO 99/20394 also discusses the use of a probe, which is narrow enough to allow insertion into the base of the well via the vent, in order to add or remove fluid. Ejection of fluid through such a narrow probe though, particularly under the conditions required of an automated system where plates need to be prepared and screened rapidly, could cause damage to cells growing in the wells.

US 7,326,385 discloses a multi-well plate wherein each well is coupled to an adjacent aspiration hole, so that the well and the hole are in fluid communication. Media can thus be aspirated and replaced from the wells without disturbing the tissue samples in the wells. However, the plate described in US 7,326,385 is, from a manufacturing perspective, quite different from standard microplates, as it requires an additional hole to be placed next to each well with a channel between the hole and the well, and this has cost implications. Furthermore, the entry point for fluid into the welis of the plate disclosed in US 7,326,385 is at the base of the well, i.e. underneath any cells growing on the base of the well. Introduction of fluid could, therefore, disrupt cells growing in the well.

It is therefore desirable to provide a means for improving the introduction and removal of fluids ("fluid exchange") to and from the wells of a microplate which minimizes the damage caused to cells within the wells, and minimizes the health and safety risks associated with the use of substances during the assaying procedure.

Accordingly, it is an object of the present invention to provide a simple and inexpensive means for introducing and removing solutions to and from a well of a microplate, which minimizes damage to biological material located within the wells.

The term "biological material" or "biological matter" encompasses any material harvested, expressed or purified from an organism or biological source, such as proteins, including recombinant proteins, antibodies and cells.

-

In particular, it is an object of the present invention to provide a means for fluid exchange which provides a specific area for communication of a pipette tip with a microplate well.

It is a further object of the invention to provide a means for facilitating and standardizing the introduction of a pipette tip into a well of a microplate.

It is a further object of the invention to provide a means for introducing and removing solutions from a microplate that minimizes the exposure of the user to the solutions being used.

A further object of the present invention is to provide a means for improving the ease of operation and comfort of the pipette operator when solutions are introduced and removed from a microplate manually.

It is a further object of the present invention to provide a means for introducing and aspirating solutions to and from a well of a microplate which decreases the amount of time required to introduce or aspirate the fluid in comparison to known means which incur the same level of damage to biological material located in the

wells upon introduction/aspiration of fluid.

It is a further object of the invention to provide a means of improving and aiding the removal of fluid from microplate wells, and in particular, a means for ensuring that a consistent amount of fluid is removed per well, so that the amount of residual fluid per well is minimized, and is consistent across the wells in the microplate.

It is a further object of the invention to provide an improved microplate for the introduction and removal of fluid. In particular, it is an object of the invention to provide an improved microplate for the introduction and removal of fluid wherein the improvements result from modifications to the plate which are easily incorporated into the manufacturing process. -16

Accordingly, in a first aspect of the invention, there is provided a microplate comprising a plurality of open wells, wherein one or more of the wells comprise an area for communication with the tip of a pipette.

In particular, the area of the well for communication with the tip of a pipette is shaped to receive the tip of the pipette. This shaping helps to provide the tip of the pipette with access to any fluid in the well, facilitates removal or aspiration of said fluid, and in particular, facilitates removal or aspiration of residual fluid which remains following aspiration of the majority of the fluid from the well. The area of communication is preferably provided so that the addition or removal of fluid to or from the well using a pipette will cause minimal and/or predictable and controlled disruption to any biological matter, such as cells, located in the well.

Providing a specific area for communication of the pipette tip with the well has numerous advantages. It controls the contact between pipette tip and the microplate well, thereby ensuring that the specific contact area between microplate well and the pipette tip is consistent across the wells in the plate. This means that in each well, only the biological matter at the specific pipetting point of contact, or in the immediate vicinity thereof will be physically disrupted by the pipette tip. This not only reduces the amount of biological matter, such as the number of cells scraped off per well, but standardizes the amount or number of cells that are damaged or removed per well.

A specific contact point also provides the operator with a specific point to aspirate fluid from, and/or dispense fluid to, or can act as a reference point to allow the operator to consistently dispense fluid at an alternative location within the well if desired. These factors reduce the effect upon biological matter located in the wells caused by variations in the height from which fluid is dropped, and facilitate introduction and removal of fluids from the wells, thereby reducing the time taken to carry out the assay. Providing a specific point of contact within each microplate well provides the operator with feedback' regarding the location of the pipette tip.

This can help to improve the accuracy of pipetting, and allows the operator to apply a small amount of downward pressure to the pipette tip before/during pipetting, -17 -safe in the knowledge that the pipette tip is located within the correct place within the well. Further to this, the microplate may be rotated by the operator or automated system through, for example 180°, following aspiration of a solution from the wells of the plate, in order to perform the related dispensing step at the opposite side of the well. This means that the location points for aspiration and dispensing of fluid are separated from each other, which can help to minimize thc disruption caused to biological matter located within the microplate wells, as any damage caused by aspiration is not exacerbated by dispensing of fluid at the same location, and vice versa. Providing a microplate with a specific area for communication with a pipette tip at one or both of the aspiration and dispensing locations allows the operator to aspirate and dispense fluids knowing that the pipette tip is positioned consistently.

The introduction of a modification to a microplate well which serves as a contact point for the pipette tip has the potential to create a natural weak point' in the biological matter located within the well. Contact of a pipette tip with biological matter in a microwell plate inevitably results in damage to the matter, due to its delicate nature. The creation of a weak point could have the advantage that the damage caused by the pipette is more likely to be limited to this area, as the biological matter at the weak point is sacrificed' by contact with the pipette tip whilst the integrity of the surrounding cells, which do not form part of the weak point, is maintained.

The presence of a contact point also acts to reduce turbulence in the fluid being aspirated or dispensed, particularly when a channel is formed between the pipette tips and specific areas of communication within the well provided for that purpose.

As a result of the microplate modifications according to the invention, the amount of biological matter (for example, the number of cells) per well will be consistent across the microplate. This leads to an improvement in assay quality, as it provides more reliable results, reduces the number of experimental repeats that are required, and reduces the number of false positives. All of these factors contribute to a reduction in the cost associated with the assay.

-18 -Providing a specific area for communication of the pipette tip with the well of a microplate also has the advantage that it allows solutions to be introduced and removed to and from the well without significantly extending the amount of time required to introduce or aspirate fluid in comparison to known means (i.e. introducing or removing fluid by simply inserting a pipette tip into the well without any guidance mechanism or contact point), a factor which is very important in time-sensitive assays. In fact, providing a specific area for communication of the pipette tip with the well of a microplate can allow more rapid introduction or removal of fluid to the well in comparison to known means, as the fluid can be introduced or aspirated more rapidly as the damage caused to the biological matter within the well is localised and/or reduced.

A further advantage achieved by the present invention is the improvement in fluid removal from the well, which facilitates the job of the user, or the automated system, and in particular, ensures that the amount of residual fluid per well is minimized, or substantially removed completely. Residual amounts of assay buffer or reagents can dilute subsequently added solutions, and inhibit the development of assay substrates. For example, residual wash solution or buffer affects ELISA substrate development. Residual levels of wash buffer also reduce the signal-to-noise ratio, and hence affect the Z' and Z-factors.

Providing an effective means for removing fluids from a microplate also obviates the need for the solutions to be tipped, flicked or banged out of the plate. This reduces the likely damage to biological matter, and in particular, cells within the plate and minimizes the exposure of the user to the solutions being used in the assay. This can have significant health and safety benefits, particularly where the solutions being used are toxic.

Providing a means for facilitating the introduction of a pipette tip into a well of a microplate by, for example, providing a guide channel, also has numerous advantages. Firstly, it guides the pipette tips into a standard position, which ensures that each well is subjected to the same contact with the pipette tip, thereby -19 -preventing unnecessary cell disruption. Providing a guidance mechanism allows the operator to insert the pipette tips accurately and quickly, thereby reducing human error and increasing the speed of the assay.

Providing an angled guidance channel into each well also has the advantage that it improves the comfort of pipetting by decreasing the stress placed on the user's arm.

In certain embodiments, the area for communication with the pipette tip comprises one or more areas located on the base and/or wall of the well interior, which are raised from the surface of the well base or wall respectively. This has the advantage that the pipette tip does not come into contact with the biological matter (such as a cell monolayer) located on the microwell base. Accordingly, any cell disruption resulting from introduction of the pipette to the microplate well is restricted to the raised areas, thereby minimizing damage to the cell monolayer on the well base.

In certain embodiments, at least two raised areas are located on the well base. In preferred embodiments, at least one such raised area or protrusion is located on the well base. In alternative preferred embodiments, one such raised area or protrusion is located on the well base with another such raised area located at a substantially adjacent point on the well wall.

In preferred embodiments of the invention, one area for communication with the pipette tip is provided per well. This allows the operator to identify the location for the aspiration step by the presence of the area for communication with the tip, and then, having performed the aspiration step at this location, rotate the plate through 180° and perform the dispensing step at the opposite side of the microplate wells by carefully releasing the fluid against the wall of the wells. This embodiment has the advantage that the aspiration step can be performed relatively quickly, and at a consistent location within the microplate wells, due to presence of the area for communication with the pipette tips on which the tips can be located; whilst the dispensing step can be carried out at a separate location within the niicroplate wells.

This arrangement aims to minimize disruption to biological matter located within the wells, whilst increasing the speed at which fluid can be aspirated from the wells.

-20 -Alternatively, at least two areas for communication with the pipette tip, each comprising one or more raised areas, may be provided per well, wherein the areas are located at substantially opposite sides of the well. The provision of areas for communication with the pipette tip at both the intended aspiration and dispensing points ensures that contact between the pipette tip and the biological matter on the base of the well is minimized. Furthermore, the microplate can be rotated by the operator or automated system through substantially 180° following aspiration, in order to perform the related dispensing step. The location points for aspiration and dispensing of fluid are thus separated from each other, so that any damage to biological matter in the well caused by aspiration is not exacerbated by dispensing of fluid at the same location, and vice versa.

The specific area for communication between the pipette tip and the microplate well may be located in the area of the microplate well where, following aspiration of the majority of fluid from the well, residual fluid typically collects, to facilitate removal of residual fluid from the microplate, for example the junction between the bottom of the well and the wall of the well. Accordingly, in certain embodiments, the raised area extends between the wall of the well and the base of the well, and the edges of the pipette tip can be entirely located on the raised area.

In preferred embodiments the raised area(s) may be spaced so that when the pipette tip is introduced, one or more edges of the pipette tip rest on raised area(s), or on the raised area and on the well wall/base, thereby forming a channel between the pipette tip and the raised area(s)/well wall/base for the release and aspiration of fluid.

Accordingly, in embodiments of the invention comprising two or more raised areas, the raised areas are located at a distance from each other which is approximately comparable to the diameter of an appropriately sized pipette tip for the assay, so that the pipette tip can be located on the two raised areas, rather than touching the base of the well. For example, when a 20tl or a 200d pipette is used, the raised areas may be located between 0.1 and 0.75 mm apart, more preferably between 0.25 -21 -and 0.5mm apart, and even more preferably between 0.35 and 0.45 mm apart; and when a 10 1 pipette tip is used, the raised area(s) may be between 0.1 to 0.775 mm apart, and more preferably between 0.25 and 0.5mm apart, and even more preferably between 0.35 and 0.45 mm apart.

In certain embodiments, the raised areas may be located so that one or both of the aspiration and dispensing tubes provided in an automated system will locate thereon.

The raised areas may have any two-dimensional or three-dimensional shape, including cuboidal, pyramidal, hemispherical, conical, cylindrical, or any form of prism, and located in any orientation. In preferred embodiments, the raised areas are approximately the shape of prisms.

The area(s) for communication with the pipette tip may comprise, or communicate with means for preventing lateral movement of the pipette tip.

For example, the raised areas may be shaped to prevent lateral movement of the pipette. For example, a raised area may comprise one or more substantially cuboidal areas on which an aspect of the pipette tip can be located, and additional raised areas, against or between which the pipette tip can be located. Such additional raised areas may be located on either side of one or both of the raised area(s) on which the pipette tip is intended to be located, and may be greater in height than the raised area(s) which is intended to communicate with the pipette tip. This helps to prevent lateral movement of the pipette tip. For example, a raised area intended for communication with the pipette tip located in the junction between the bottom of the well and the wall of the well, may have an additional raised area on either side.

The raised areas may be any size suitable for location within a well of a microplate, and/or for location of an appropriately sized pipette tip or pipette tip edge on or substantially on the raised area. For example, for a microplate wherein the appropriate pipette tip is a 3001, 2001d, 501.d or 10fl tip, the maximum diameter of -22 -the surface of the raised area intended for contact with the pipette tip, or an edge of the pipette tip may be between 0.1mm and 2mm, more preferably between 0.5mm and 1.5mm and even more preferably between 0.7mm and 1 mm. It should be noted that the pipette tip may be blocked or compromised with regard to fluid aspiration or release by the raised area(s) if the surface of the raised area intended for contact with the pipette tip is either too small or too largc.

In preferred embodiments, the size and/or location of the raised areas should correspond to the size of the pipette tip or the diameter of the pipette tip used in the assay, so that the pipette tip can be contacted with two or more raised areas, or one raised area and the wall or base of the well, and thus be prevented from sub stantially touching the base of the well.

The raised areas are preferably of minimal height, in order to allow effective aspiration of residual fluid from the wells of the microplate. For example, the maximum height of a raised area is preferably no more than 1.5mm; more preferably no more than 1mm; even more preferably no more than 0.75mm; and most preferably no more than 0.5mm. In certain embodiments wherein the invention comprises more than one raised area, one of the raised areas intended for communication with the pipette may be greater in height than the other raised area(s).

In alternative embodiments, the area for communication with the pipette tip comprises one or more recesses or indentations in the wall and/or base of the well.

In preferred embodiments, the indentation accommodates the point of a pipette tip of a size appropriate for the assay. By this is meant that the indentation is of a size such that the dispensing end of a pipette tip which is chosen as appropriate for the assay being used inserts, at least partially, into the indentation.

Accordingly, in preferred embodiments, the indentation has approximately or substantially the same diameter as the open (i.e. dispensing end) of the pipette tip.

For example, when a 20d or 200d pipette is used, the maximum diameter of the -23 -indentation(s) may be 2 mm-0.lmm. More preferably, the maximum diameter of the indentation(s) is 0.5mm and 1.5mm, and even more preferably between 0.7 and 1mm.

Tailoring the size of the indentation to the size of the pipette tip being used can help to prevent movement of the pipette tip once it has been introduced into the indentation. It should be noted that the pipette tip may be blocked or compromised with regard to fluid aspiration or dispensing if the indentation is too deep.

Accordingly, the indentation(s) should be no more than 1 mm deep. More preferably, the maximum depth of the indentation(s) is 0.1 to 0.5 mm.

In certain embodiments, the indentations or recesses may be located so that one or both of the aspiration and dispensing tubes provided in an automated system will locate therein.

The indentation may be any shape, for example, cuboidal, pyramidal, hemispherical, conical, cylindrical, or any form of prism, and located in any orientation. Pipette tips are typically circular in cross-section, and tailoring the shape of the indentation to the shape of the pipette tip end can help to prevent movement of the pipette tip once it has been introduced into the indentation.

In preferred embodiments, the indentation located on the walls of the well is substantially pyramidal or cuboidal in shape; and the indentation located on the base of the well is substantially pyramidal or cuboidal in shape.

In some embodiments according to the invention, the area for communication with the pipette tip may comprise one or mote raised area located on the wall or base of the well, together with one or more indentation(s) on the wall or base of the well.

In preferred embodiments, all or substantially all of the wells of a microplate comprise an area for communication with the tip of a pipette located substantially at the base of the well.

In certain embodiments, the microplate of the present invention further comprises one or more wells which comprise a guide channel.

In preferred embodiments, the guide channel accommodates a pipette tip of a size appropriate for the assay. By this is meant that the guide channel is of a size such that a pipette tip which is chosen as appropriate for the assay being used inserts, at least partially, into the channel.

In preferred embodiments, the guide channel comprises a tapered indentation in the interior wall of the well, so that the indentation is deeper at the lip of the well, and is less deep at the base of the well (i.e. the indentation extends further into the wall of the well at the top than at the bottom), so that from the perspective of a vertical cross-section through the well, the channel appears angled from the vertical.

In certain embodiments, the end of the guide channel which is located near the base of the well ends (i.e. so that that the indentation ceases) in close proximity to a raised area which is located on the wall or base of the well, so that the pipette tip is guided by the guide channel onto the raised area.

In alternative embodiments, the end of the guide channel which is located near the base of the well terminates in an indentation in the well wall, or the well base. In some embodiments wherein the end of the guide channel terminates in an indentation in the well wall, the indentation communicates with a raised area which is located at the junction between the wall of the microplate well and the base of the microplate well.

In certain embodiments, the guide channel commences substantially at the lip of the well, and ends substantially at, above or below, the base of the well.

In a preferred embodiment, the guide channel commences substantially at the lip of the well.

-25 -In preferred embodiments, the present invention decreases the amount of time required to introduce or aspirate fluid in comparison to an unmodified microplate/known systems which incur the same level of cellular damage to biological material located within the wells upon introduction/aspiration of fluid.

Microplates according to the present invention may be made by injection moulding.

A metal mould used to create standard microwell plates would requite little modification in order to make a microplate in accordance with the present invention. This is an advantage of microplates according to the present invention, which do not require the creation of complex moulds or the use of specialist manufacturing steps, which could be costly and time-consuming.

In particular, embodiments of the present invention wherein the area(s) for communication with the tip of the pipette comprise one or more raised areas on the base of the microplate well, and/or one or more indentations or recessed areas on wall of the microplate well, could be manufactured using moulds based upon those used to create standard microplates, without the need for significant modification.

Furthermore, creation of such microplates would not cause significant technical difficulties; there is potential, during the removal of a metal mould template from a newly created mictoplate, for the microplate to be damaged. Raised areas or protrusions located on the microplate well base, and indentations or recesses located on the microplate well wall would not adversely affect the ease with which the mould could be removed. Accordingly, the potential for damage to the plate is not increased by any significant degree when using a mould suitable for creating a microplate comprising one or more raised areas, indentations, or recesses, in accordance with the present invention, than when creating standard microplates.

Brief Description of the Figures

Figure 1 shows a single well from a standard microplate, with a pipette tip introduced to aspirate or dispense solution. Figure 1A is cross-sectional view; Figure lB shows plan view from above.

-26 -Figure 2 shows a single well from a microplate with areas for communication with a pipette tip, and a guide channel, in accordance with an embodiment of the present invention, with a pipette tip introduced to aspirate or dispense solution. Figure 2A is cross-sectional view; Figure 2B shows plan view from above.

Figure 3 demonstrates the effect of a pipette tip upon a monolayer of adherent cells which are growing on the bottom of a standard microplate well. Figures 3A, B and C show a cross-sectional view; Figures 3D, E, F show a plan view from above.

Figure 4 shows a cross-section through various microplates with areas for communication with a pipette tip in accordance with the present invention. Figure 4A demonstrates a microplate well wherein the area for communication with the pipette tip comprises two raised areas located on the base and wall of the well interior. Figure 4B demonstrates a microplate well wherein the area for communication with the pipette tip comprises an indentation located on the base of the well interior. Figure 4C demonstrates a microplate well wherein the area for communication with the pipette tip comprises a raised area located on the base of the well, and an indentation located on the wall of the well. Figure 4D demonstrates a microplate well wherein the area for communication with the pipette tip comprises indentations located on the base and the wall of the well interior.

Figure 5 shows a cross-section through a microplate with areas for communication with a pipette tip, and a guide channel comprising a tapered indentation in the interior wall of the well. In Figure 5A, the areas for communication with the pipette tip comprise one indentation located on the base of the well interior, and an indentation in the wall of the well interior; and a guide channel, wherein the end of the guide channel that is located near the base of the microplate well terminates in the indentation in the well wall. Figure 5B shows a microplate well wherein the areas for communication with the pipette tip comprise one raised area located on the base of the well interior, and an indentation in the wall of the well interior; and a guide channel, wherein the end of the guide channel which is located near the base of the microplate well terminates in the indentation in the well wall, Figure 5C shows a microplate well wherein the areas for communication with the pipette tip -27 -comprise raised areas located on the base of the well interior, wherein one of the raised areas is located at the junction between the bottom of the well and the wall of the well, and a guide channel, wherein the end of the guide channel which is located near the base of the microwell terminates in a raised area. Figure 5D shows, in 3-dimensional form, preferred shapes for the raised areas for location on the base of the well interior. Figure 5E and F show, in 3-dimensional form, preferred shapes for the indentation located on the base of the well interior. Figure 5G provides, in 3-dimensional form, an example of two raised areas for location within a microplate well, wherein one of the raised areas is located at the junction between the bottom of the well and the wall of the well. Figure 5H provides an alternative example of two raised areas for location within a microplate well in 3-dimensional form, wherein one of the raised areas is located at the junction between the bottom of the well and the wall of the well. Figure 51 shows, in three-dimensional form, two raised areas for location within a microplate, wherein one of the raised areas is located at the junction between the bottom of the well and the wall of the well, and the other is located on the base of the well, and a guide channel, wherein the guide channel terminates in an indentation in the wall of the well, which communicates with the raised area located at the junction between the bottom of the well and the wall of the well. Figure 5J shows, in three-dimensional form, two raised areas for location within a microplate, wherein one of the raised areas is located at the junction between the bottom of the well and the wall of the well, and the end of a guide channel, wherein the end of the guide channel terminates in the raised area located at the junction between the bottom of the vell and the wall of the well.

Figure 5K shows, in three-dimensional form, two raised areas located within a microplate on which the pipette tip can be located, a guide channel, and two additional raised areas located either side of the raised area which is located at the junction between the bottom of the well and the wall of the well. The additional raised areas prevent lateral movement of the pipette tip.

Figure 6A shows a cross-sectional view and a plan view (from above) of an embodiment of a microplate in accordance with the present invention, wherein the area for communication with a pipette tip comprises two raised areas located on the wall and the base of the microplate well. Figure 6B shows a cross-sectional view -28 -and a plan view (from above) of an embodiment of a microplate in accordance with the present invention wherein the area for communication with a pipette tip comprises two indentations located on the wall and the base of the microplate well.

Figure 7A shows a cross-sectional view and a plan view (from above) of an embodiment of a microplate in accordance with the present invention, wherein the areas for communication with the pipette tip comprise two indentations located on the base and wall of the well interior; and a guide channel, wherein the end of the guide channel which is located near the base of the microplate well terminates in the indentation in the well wall. Figure 7B shows a cross-sectional view and a plan view (from above) of an embodiment of a microplate in accordance with the present invention, wherein the areas for communication with the pipette tip comprise one raised area located on the base of the well interior, and an indentation in the wall of the well interior; and a guide channel, wherein the end of the guide channel which is located near the base of the microplate well terminates in the indentation in the well wall Figure 8 demonstrates the effects of a pipette tip upon a monolayer of adherent cells which are growing on the bottom of a microplate well according to an embodiment of the present invention. Figures 8A, B and C show a cross-sectional view; Figures 8D, E, F show a plan view from above.

Detailed Description of the Figures

In Figure 1, a pipette tip I is introduced into a standard microplate well 2 to remove residual liquid 3.

In Figure 2, a pipette tip 1 is introduced into a microplate well in accordance with an embodiment of the present invention 2' to remove residual liquid 3. The well has a tapered guide channel 4, wherein the end of the guide channel which is located near the base of the microplate well terminates in the indentation in the well wall 5.

The areas for communication with the pipette tip comprise the indentation located on the wail of the well interior 5, and the raised area located on the base of the well, 6.

-29 -In Figure 3A, a pipette tip 1 is introduced to a standard microplate well 2 containing a monolayer of adherent cells 7, to remove the residual fluid 3. In Figure 3B and 3C, the pipette tip contacts the base of well, scraping and dislodging the cells, resulting in removal of some of the cells from an area of the base of the well 8. In addition, some of the cells are dislodged from the base of the well, but remain attached to cells which are affixed to the base of the well 9. These cells could detach from the well upon aspiration.

An illustration of the cell distribution before introduction of the pipette tip is provided by Figure 3D. An illustration of the damage that can be caused to the cells by the introduction of the pipette tip is provided by Figures 3 E and F. In Figure 4A, a microplate well in accordance with an embodiment of the present invention 2' has two raised areas 6. One raised area is located on the well wall, and the other is located on the well base. The raised areas are spaced so that when the pipette tip 1 is introduced, the edges of the pipette tip test on the raised areas, so that a channel is formed between the pipette tip and the raised areas for the release and aspiration of fluid.

Figure 4B shows a microplate well in accordance with an embodiment of the present invention 2' with an indentation 5 located on the well base. An edge of the pipette tip 1 can be located within the indentation 5, whilst the other edge of the pipette tip rests against the well wall.

Figure 4C shows a microplate well in accordance with an embodiment of the present invention 2' with one raised area 6 located on the well base and one indentation 5 located on the well wall. The raised area and indentation are spaced so that when the pipette tip 1 is introduced, the edges of the pipette tip Lest Ofl the raised area 6 and in the indentation 5, so that a channel is formed between the pipette tip 1 and the areas for communication with the tip of a pipette 5 and 6, for the release and aspiration of fluid.

-30 -Figure 4D shows a microplate well in accordance with an embodiment of the present invention 2' with two indentations 5 located on the well base and wall. The indentations are spaced so that the pipette tip 1 can be located within the indentations, thereby forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid.

Figure 5A shows a microplate well in accordance with an embodiment of the present invention 2' with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in an indentation 5 which acts as an area for communication with the pipette tip 1; and a further indentation 5 located on the base of the well, so that the pipette tip 1 can be introduced to the well via the guide channel 4, and located in the two indentations 5 on the wall and base of the well, thereby forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid.

Figure 5B shows a microplate well 2' in accordance with an embodiment of the present invention, designed, in particular, to prevent lateral movement of the pipette tip once located for aspiration or dispensing of fluid. The well comprises a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in an indentation 5 which acts as an area for communication with the pipette tip 1; and a raised area 6 located on the base of the well, so that the pipette tip 1 can be introduced to the well via the guide channel 4, and located in the indentation 5 on the wall of the well, and the raised area 6 on the base of the well, thereby forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid.

Figure SC shows a microplate well in accordance with an embodiment of the present invention 2' with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in a raised area 6' which acts as an area for communication with the pipette tip 1, and a further raised area 6 located on the -31 -base of the well, so that the pipette tip 1 can be introduced to the well via the guide channel 4, and located on the raised areas 6' and 6, thereby forming a channel between the pipette tip I and the areas for communication with the tip of a pipette for the release and aspiration of fluid.

Figure 5D shows, in three dimensional form, two preferred approximately pyramidal shapes for the raised areas 6 for location on the base of the well interior which can act as an area for communication with a pipette tip 1. Figure 5E and F show, in three dimensional form, preferred, approximately pyramidal shapes for the indentation 5 for location on the base of the well interior, which can act as an area for communication with a pipette tip 1.

Figure 5G provides a three-dimensional depiction of the interior surface of a microplate well in accordance with the present invention 2" with two raised areas 6' and 6 located in the well interior, wherein one raised area 6' is located at the junction between the bottom of the well and the wall of the well, and the other 6 is located on the base of the well interior, wherein the raised areas act as areas for communication with a pipette tip 1 and form a channel between the pipette tip 1 and the raised areas 6' and 6 for the release and aspiration of fluid.

Figure 5H provides an alternative three-dimensional depiction of the interior surface of a microplate well in accordance with the present invention 2" with two raised areas 6' and 6 located in the well interior, wherein one raised area 6' is located at the junction between the bottom of the well and the wall of the well, and the other 6 is located on the base of the well interior, wherein the raised areas act as areas for communication with a pipette tip 1 and form a channel between the pipette tip 1 and the raised areas 6' and 6 for the release and aspiration of fluid.

Figure 51 provides a three-dimensional depiction of the interior surface of a microplate well in accordance with the present invention 2" with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in an indentation 5 which communicates with a raised area 6', which is located at the -32 junction between the bottom of the well and the wall of the well; a second raised area 6 is located on the base of the well interior. The pipette tip 1 can be introduced to the well via the guide channel 4, and located on the indentation 5 on the wall of the well and the raised area 6, or on the two raised areas 6' and 6, in either case forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid. At least partial location of the pipette rip in the indentation 5 in the wall of the well prevents lateral movement of the pipette tip Figure 5J provides a three-dimensional depiction of the interior surface of a microplate well in accordance with the present invention 2" with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in a raised area 6', which is located at the junction between the bottom of the well and the wall of the well with a second raised area 6 located on the base of the well interior. The pipette tip 1 can be introduced to the well via the guide channel 4, and located on the two raised areas 6' and 6, forming a channel between the pipette tip I and the areas for communication with the tip of a pipette, for the release and aspiration of fluid.

Figure 5K provides a three-dimensional depiction of the interior surface of a microplate well in accordance with the present invention 2" with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in a raised area 6', which is located at the junction between the bottom of the well and the wall of the well with a second raised area 6 located on the base of the well interior, and two additional raised areas 6", located either side of the raised area which is located at the junction between the bottom of the well and the wall of the well 6'. The pipette tip 1 can be introduced to the well via the guide channel 4, and located on the two raised areas 6' and 6, forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid. The additional raised areas 6" prevent lateral movement of the pipette tip once it has been located on raised areas 6 and 6'.

-33 -Figure 6A shows a cross-sectional view and a plan view (from above) of a microplate well in accordance with an embodiment of the present invention 2', wherein the area for communication with a pipette tip comprises two raised areas 6 located on the wall and the base of the microplate well. The raised areas are spaced so that when the pipette tip 1 is introduced, the edges of the pipette tip rest on the raised areas, so that a channel is formed between the pipette tip and the raised areas for the release and aspiration of fluid.

Figure 6B shows a cross-sectional view and a plan view (from above) of a microplate well in accordance with an embodiment of the present invention 2', wherein the area for communication with a pipette tip comprises two indentations located on the wall and the base of the microplate well. The indentations are spaced so that when the pipette tip 1 is introduced, the edges of the pipette tip rest in the indentations, so that a channel is formed between the pipette tip and the indentations for the release and aspiration of fluid.

Figure 7A shows a cross-sectional view and a plan view (from above) of a microplate well 2' in accordance with an embodiment of the present invention with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in an indentation 5 which acts as an area for communication with the pipette tip 1; and a further indentation 5 located on the base of the well, so that the pipette tip 1 can be introduced to the well via the guide channel 4, and located in the two indentations 5 on the wall and base of the well, thereby forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid.

Figure 7B shows a cross-sectional view and a plan view (from above) of a microplate well 2' in accordance with an embodiment of the present invention with a guide channel 4 comprising a tapered indentation in the interior wall of the well, wherein the end of the guide channel that is located near the base of the microplate well terminates in an indentation 5 which acts as an area for communication with -34-the pipette tip 1; and a raised area 6 located on the base of the well, so that the pipette tip 1 can be introduced to the well via the guide channel 4, and located in the indentation 5 on the wall of the well, and the raised area 6 on the base of the well, thereby forming a channel between the pipette tip 1 and the areas for communication with the tip of a pipette, for the release and aspiration of fluid.

In Figures 8A-C, a pipette tip 1 is introduced to a microplate well 2', in accordance with the present invention which contains a monolayer of adherent cells 7, via a guide channel 4, which terminates in an indentation 5. The well has a raised area 6 located on the well base. The indentation 5 and the raised area 6 are spaced so that the edges of the pipette tip I can be located in the indentation 5 and on the raised area 6. Contact with the well base is, therefore, avoided.

As shown in Figures 8D-F, the numbet of cells are disrupted by the introduction of the pipette 1, either resulting in complete removal from the well 8', or partial dislodging of the cells which then remain attached to cells which remain affixed to the base of the well 9' is minimized. -35 -

Claims

Claims 1. A microplate comprising a plurality of open wells, wherein one or more of the wells comprises an area for communication with the tip of a pipette.
2. A microplate as claimed in claim 1, wherein the area for communication with the pipette tip comprises one or more raised areas or protrusions located on the base and/or wall of the well interior.
3. A microplate as claimed in claim i or claim 2, wherein the area for communication with the pipette tip comprises one or more recesses or indentations in the wall and/or base of the well.
4. A microplate as claimed in claim 1, wherein the area for communication with the pipette tip comprises one or more indentations in the wall and/or base of the well and one or more raised areas located on the base and/or wall of the well interior.
5. A microplate as claimed in any of claims 1-4, wherein when the area for communication with the pipette tip is a raised area or protrusion, it is located at the junction between the bottom of the well and the wall of the well.
6. A microplate as claimed in any of the preceding claims, wherein the area(s) for communication with the pipette tip comprise or communicate with means for preventing lateral movement of the pipette tip.
7. A microplate as claimed in claim 6, wherein the area for communication with the pipette tip comprises one or more raised areas upon which the pipette tip can be substantially located, and one or more additional raised areas against or between which the pipette tip can be located in order to prevent lateral movement of the pipette tip.

-36 -
8. A microplate as claimed in claim 6, wherein the means for preventing lateral movement of the pipette tip comprises one or more indentations.
9. A microplate as claimed in any of claims 1-8, wherein the areas for communication with the pipette tip are located at a distance from each other which is comparable to the diameter of an appropriately sized pipette tip for an assay using the microplate.
10. A microplate as claimed in any of claims 1-9, wherein the areas for communication with the pipette tip are located at opposite sides of the microplate well.
11. A microplate as claimed in any of claims 1-10, wherein the one or more wells further comprises a guide channel.
12. A microplate as claimed in claim 11, wherein the guide channel accommodates a pipette tip of a size appropriate for an assay using the mictoplate.
13. A microplate as claimed in claim 11 or 12, wherein the guide channel comprises of a tapered indentation in the interior wall of the well.
14. A microplate as claimed in any of claims 11-13, wherein the guide channel terminates in an indentation in the wall of the microplate well.