WO2011161480A1

WO2011161480A1 - Multi-well assay plate

Info

Publication number: WO2011161480A1
Application number: PCT/GB2011/051216
Authority: WO
Inventors: Marie Lis Kirsten
Original assignee: GOEHRING NATALIA; Imperial Innovations Ltd
Current assignee: GOEHRING NATALIA; Ip2ipo Innovations Ltd
Priority date: 2010-06-25
Filing date: 2011-06-27
Publication date: 2011-12-29
Anticipated expiration: 2012-12-25
Also published as: GB201010736D0

Abstract

A multi-well assay plate I comprises a plurality of wells II defined in a substantially planar substrate. Each well is in fluid communication via a gateway III with at least one neighbouring well to form a well grouping. Each gateway is configured to receive a partition for separating the neighbouring wells.

Description

MULTI-WELL ASSAY PLATE

[0001] This invention relates to a multi-well assay plate.

BACKGROUND

[0002] Multi-well assay plates have a broad application in biological and life sciences, including cell biology, microbiology, biophysics, pharmacology, toxicology and

immunology, as well as other scientific fields. Typically, such assay plates have a standardised design and arrangement of wells so that they are compatible with a multitude of plate readers that determine spectroscopic readouts of each well, for example absorbance, luminescence, fluorescence or microscopy results.

[0003] Multi-well assay plates are known from, for example, WO 2009/097099, US

2003/0215940, US 5,583,037, US 5,962,250, US 5,578,490, US 5,468,638, EP 0 620 274, US 5,183,760, US 5,916,526, US 7,494,623, US 6,558,631 , US 6,410,310, EP 2 306 189, WO 2010/09199, WO 201 1/031386, WO 201 1/047023 and Ubeira et al., Journal of Immunological Methods, 1993, 159, 107-1 13.

[0004] Embodiments of the present invention find application in a wide spectrum of biological tests related to binding and partitioning and also co-culture of cells. Such studies are important for determining interactions between different entities such as cells, proteins, lipids or DNA. These experiments can be performed by mixing the two entities and separating the unbound entities again, followed by detection of the resulting signal.

However, this method has certain drawbacks, such as loss of kinetic information, increase in errors due to a multitude of pipetting and separation steps as well as time consumption.

[0005] In many biological experiments co-culture of two cell types is required to investigate how secretion of one cell type triggers responsive processes in the other cell type. In existing devices, such as the Transwell® plate from Corning, the Multiscreen cell culture system from Millipore and the Unicell® cell culture system from Whatman, the two cell types are spatially separated by a horizontally aligned semi-permeable membrane. However, this design does not allow simultaneous observation of both cell types by spectroscopy and neither compartment can be visualised by microscopy while the experiment is being performed. These systems therefore suffer from a number of drawbacks regarding online analysis and kinetic studies.

[0006] The present invention, at least in the presently preferred embodiments, seeks to provide multi-well assay plates having advantages not known from the prior art.

BRIEF SUMMARY OF THE DISCLOSURE [0007] In accordance with the present invention there is provided a multi-well assay plate comprising a plurality of wells defined in a substantially planar substrate, each well in fluid communication via a gateway with at least one neighbouring well to form a well grouping, wherein each gateway is configured to receive a partition for separating the neighbouring wells.

[0008] Thus, in accordance with the invention, neighbouring wells of the assay plate can be connected via a partition and observed independently.

[0009] The partition may be located permanently in the gateway. For example, the partition may be moulded into the substrate. In embodiments of the invention, the partition is removably received in the gateway. In this way, the partition may be selected and/or positioned to match the requirements of the particular assay.

[0010] The removable partition may be solid, whereby to prevent any fluid

communication between the neighbouring wells when received in the gateway. A solid partition is useful where reagents are to be separated for a period of time and then allowed to come into contact. Thus, a method of using the assay plate of the invention may include the step of removing the solid partition from a gateway in the course of an experimental procedure, whereby to allow fluid communication of a reagent in one well to a

neighbouring well through the gateway. In this way, the solid partition can be used to release reagents or to retain reagents, for example while they solidify.

[0011] In embodiments of the invention, the partition comprises a semi-permeable membrane which, in use, controls passage of reagents between the wells separated by the partition. The semi-permeable membrane allows the passage of molecules between wells dependent on the pore size of the membrane.

[0012] In embodiments of the invention, the partition may comprise a test material located within a frame. Thus, the plate may be used to test the effect of two reagents on opposite sides of the material or to test the barrier properties of the material.

[0013] The test material may be a human or animal tissue sample, for example a skin sample or a slice taken from an organ. Alternatively, the test material may comprise a cell culture on a support structure. The support structure may be a semi-permeable membrane.

[0014] Typically, the partition is arranged in the gateway substantially perpendicularly to the plane of the substrate. Thus, for a substantially horizontal substrate, the partition is substantially vertical. This has the advantage that the partition does not obscure the wells which can be viewed from above (or below). [0015] Desirably, the partition sealingly engages the gateway. Thus, the partition may form a water tight (liquid-tight) seal at the interface with the gateway. In this way, the wells are separated effectively and, where the barrier comprises a semi-permeable membrane, any transfer between the wells is through the membrane. In this regard, the perimeter of the partition may be formed of elastomeric material to provide sealing engagement with the gateway. Where the partition is solid, the entire partition may be formed of elastomeric material. The elastomeric material may be reinforced to provide stability to the partition.

[0016] The partition may be rectangular, circular, elliptical or any other suitable shape.

[0017] The wells may be positioned on the planar substrate in positions corresponding to those of a standard micro-plate, whereby interactions in the wells can be monitored by a standard micro-plate reader. In this way, a standard micro-plate reader can be used with the plate of the invention. It is not necessary for the plate of the invention to comprise a well corresponding to every well of a standard micro-plate. Thus, the plate of the invention may comprise fewer wells than a standard micro-plate, with each well in a position corresponding to the position of a well of a standard plate.

[0018] The plate may comprise at least two well groupings, each comprising a plurality of wells in fluid communication via at least one mutual gateway, wherein the wells of each grouping are separated from the wells of the other grouping(s) by the substrate material. In this way, multiple assays may be carried out on the plate in discrete well groupings. Each well grouping may comprise more than two wells. In embodiments of the invention, each well grouping may comprise three, four, five or more wells.

[0019] At least one well of the grouping may be in fluid communication with a plurality of neighbouring wells via respective gateways. Thus, one well may be surrounded by a plurality of wells. The central well may be defined substantially by the partition(s) between the central well and the neighbouring wells. In embodiments of the invention, a central well is adjoined by two, three, four or more neighbouring wells. Such an arrangement has the advantage that a single reagent in the central well can be applied to a plurality of other (different) reagents in the surrounding wells.

[0020] In this case, a method of using the assay plate of the invention may include depositing a first reagent in the well of the grouping that is in fluid communication with a plurality of neighbouring wells via respective gateways, depositing different second reagents in each of the neighbouring wells and monitoring simultaneously the interaction of the first reagent with each of the second reagents. The first and second reagents may be deposited in any order, as required. Further reagents may be deposited before or after the first and/or second reagents. The further reagent(s) may be the same reagent(s) for each of the neighbouring wells. This method has the advantage that the effect of the first reagent on a plurality of second reagent may be monitored under the same conditions.

[0021] In a particular method, the second reagents are human or animal cells. The first reagent may be a biologically-active, for example pharmacologically-active substance. Other reagents used in accordance with the invention may be microorganisms, such as E- coli, yeast, fungi and the like, in particular for the study of biotechnological production of substances, for example the selection and/or comparison of clones that convert a substance enzymatically into a desired product. Thus, for method using the invention, the reagent may be bacterial, human or animals cells. In general terms, the reagents may be prokaryotic or eukaryotic organisms.

[0022] A further method of using the assay plate of the invention may include separating the central well of the grouping that is in fluid communication with a plurality of

neighbouring wells via respective gateways by different partitions in each of the gateways, and monitoring simultaneously the interaction of a reagent in the central well with reagents in the neighbouring wells and/or with the different partitions. This method has the advantage that the effect of different partitions on the interaction of reagents may be monitored under the same conditions. The reagents in the neighbouring wells may be the same or different. The different partitions may be membranes of different permeability or may be membranes coated or cultured with different materials or may be tissue samples, for example.

[0023] In particular arrangements, the plate may comprise at least two, preferably at least four, more preferably at least eight, most preferably at least 16, wells in at least four, preferably at least eight, well groupings.

[0024] The wells may be substantially cylindrical in form. The bottom of each well may be substantially flat. Other shapes are possible. For example, the wells or the well bottoms may be hemispherical or conical.

[0025] The substrate may be optically opaque or translucent. In one embodiment, the substrate is substantially optically transparent. This has the advantage that the interactions in the wells can be observed from below through the material of the substrate. Thus, a method of using the assay plate of the invention may include the step of locating the planar substrate substantially horizontally and monitoring the progress of a reaction between neighbouring wells of a well grouping from below. Alternatively, a method of using the assay plate of the invention may include the step of locating the planar substrate substantially horizontally and monitoring the progress of a reaction between neighbouring wells of a well grouping from above. The monitoring of the wells may be by microscopy, spectroscopy, in particular fluorescence spectroscopy or absorption spectroscopy, or any other suitable method.

[0026] The reagents used in assays conducted with the plate of the invention may include, for example, proteins, vesicles, DNA, cells, cell cultures, drug molecules, biomolecules, lipids, sugars, human or animal tissues. In general terms, the reagents may be bioreactive reagents. The invention may be applied to assays in the fields of, for example, cell culture, biophysical studies, lipid transport studies, dialysis for protein crystallisation, immunology, cell migration, chemotaxis, cell proliferation, cell differentiation, endocrinology, pharmacology, toxicology, efficacy, microbiology, parasitology, drug/tissue interaction, drug partitioning, drug non-specific binding, cell proliferation, gene expression, cell differentiation, oncology and cell invasion.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 is a top plan view of an exemplary embodiment of a micro-well plate in accordance with the invention with two-well connectivity;

Figure 2 is a cross sectional side view along line IV of Figure 1 ;

Figure 3 is a top plan view of an exemplary embodiment of a micro-well plate in accordance with the invention with four-well connectivity;

Figure 4 is a cross-sectional side view along line VI of Figure 3;

Figure 5 is a top plan view of an exemplary embodiment of a micro-well plate in accordance with the invention with five-well connectivity;

Figure 6 is a cross-sectional side view along line VIII of Figure 5;

Figure 7 is a top plan view of an exemplary embodiment of a micro-well plate in accordance with the invention with total connectivity of all wells and some exemplary inserts;

Figure 8 is a perspective side view of a membrane insert surrounded by a stabilising and insulating support;

Figures 9a and 9b are top plan views of two possible embodiments of the invention (two-well and four-well connectivity) with inserted membrane barriers;

Figure 10 is a top plan view of a five-well connectivity design in accordance with the invention with inserted membrane barriers; Figure 1 1 is a schematic depiction of an exemplary experiment to investigate nonspecific binding and efficacy of drugs to different tissues;

Figure 12 is a schematic depiction of an exemplary experiment to investigate cell interaction through secretion of soluble compounds;

Figure 13 is a schematic depiction of an exemplary experiment to investigate competitive binding to vesicles of different lipid compositions; and

Figure 14 is a schematic depiction of an exemplary experiment to investigate transporting proteins.

DETAILED DESCRIPTION

[0028] Referring to Figure 1 , an embodiment of the invention consists of a multi-well

(micro-well) plate I into which a multitude or at least two wells II are embedded. The assay plate I of this embodiment matches the standard design and dimensions for micro plate reader analysis. In other words, the centres of the wells II correspond to the locations of the centres of wells in a standard micro-well plate. For example, in the plate I of Figure 1 , the location of the wells II corresponds to the locations of wells of a standard 96 well plate, i.e. eight rows (A - H) by twelve columns (1 - 12). However, in the embodiment of Figure 1 , the wells II are arranged in four rows of six pairs of interconnected wells II, so that the plate I includes only 48 wells. Each of the 48 wells II of the plate I of Figure 1 is located in a position corresponding to the position of one of the wells in a standard 96 well plate, but less than all of the standard well locations are occupied by a well II in the embodiment of Figure 1.

[0029] In a potential alternative to the embodiment of Figure 1 , additional rows (B, D, F, H) of five pairs of interconnected wells II may be located in the space between the rows (A, C, E, G) shown in Figure 1 , offset by one well position from the six-pair rows. In this case, the first well II of each of the rows is located at column 2.

[0030] In the example of Figure 1 , the positions of the centres of the wells II correspond to positions in a standard 96-well plate. However, other designs are possible in which the centres of the wells II correspond to positions in a standard 6-well, 12-well, 24-well, 48- well, 384-well or 1 ,536-well plate, for example.

[0031] The embodiment of Figure 1 consists of a parallelepipedal base (plate) with dimensions compatible with standard micro-plate readers. The plate comprises a multitude of cylindrical open-topped reservoirs (wells) II of which at least pairs of wells are interconnected via a short connecting slot III that allows insertion of a rectangular membrane IX surrounded by a sealing material (e.g. rubber or other elastomer) making the separation between the wells watertight to stops leakage of fluid between wells so that the only connection is via the membrane pores.

[0032] The plate I may be moulded from a plastics material that does not interfere with the mode of measurement, such as polystyrene, polymethylmetacrylate, low protein binding cyclo olefin polymer, or others. Other non-plastics materials are possible. For cell culture or protein studies the surface of the plate I may also be treated appropriately. If very low non-specific protein binding is required, the surface may furthermore be coated with suitable materials. Typically, the material of the plate I should be biocompatible. It is desirable for the plate material to be transparent in order that interactions in the wells II can be observed, for example by microscopy, from below the plate I through the transparent bottom of the wells II. Alternatively, the interactions can be observed from above the wells II.

[0033] The plate (base) I consists of two or more ideally cylindrical flat-bottomed open- topped wells II of which at least two wells are interconnected. The plate I is designed so that all wells II can be connected to their neighbouring wells II by a short slot III. The interconnection is a slot III designed to allow vertical insertion of at least one separate barrier IX. Into these slots III the user may insert separating barriers IX that either fully separate the wells II and seal the connection watertight, or according to the user's needs, the barrier discs IX may contain a semi-permeable membrane XI of a suitable material and pore size to allow lateral diffusion of small molecules. Depending on the choice of pore size the membrane barrier IX can separate a wide range of biological motifs including vesicles, proteins, DNA, cells etc. while permitting smaller particles to pass through. In this way each plate I can be custom-designed to each experiment by varying the pattern of connected wells II and choosing suitable membrane materials and pore sizes. Thus, the insertable barrier IX may either be impermeable to completely block the interconnection between the wells II or it may contain a semi-permeable membrane XI that allows passage of particles below a defined size.

[0034] An advantage over existing similar devices provided by the plate I of

embodiments of the invention is the possibility to monitor all separated wells II in parallel, for example by fluorescence spectroscopy, ultra violet to visible absorption spectroscopy and microscopy, thereby allowing on-line detection and kinetic studies without the need to remove the sample into a separate container for analysis. Due to the modular insertion of the membrane barriers IX, wells II can be connected and blocked in a customised manner. Furthermore, a choice of different membrane materials and pore sizes and different plate materials allows the final set-up to be adjusted to the user's needs. [0035] It is desirable to study the interaction of biomolecules including, but not restricted to, drug molecules with different types of human (or animal) tissues in order to compare properties as for example non-specific binding or efficacy in different tissues, such as tumour cells and/or healthy cells. As all wells II are accessible for microscopy and spectroscopy the interaction of the drug with the different tissues can be compared immediately.

[0036] Furthermore, the plate I according to the invention permits co-culture of two or more cell types in the same assay volume while keeping the cells spatially separated and allowing only molecules below a defined size to pass between the separated wells.

[0037] The plate according to this embodiment of the invention is different and improved over designs such as described in US 5,962,250 in that:

1 ) more than two wells II can be separated via a semi-permeable membrane XI due to the modular design of the wells II; and

2) each independent cavity can be measured in a standard micro-plate reader with the membranes XI inserted.

This broadly extends the range of experiments that can be carried out with the plate of the invention and also reduces errors and time consumption resulting from transferring the assay volumes into separate measuring containers.

[0038] Additional optional features of the plate I may include a temperature control device attached to the bottom of the plate or within a cavity within the plate I for more efficient heat transfer. Examples may be heating mats, resistive heaters and Peltier heaters etc. to observe biological processes under physiological conditions or to cool down the samples. If required, the plate I may also be equipped with a stirring device in each well.

[0039] As shown in Figure 8, the membrane inserts IX may exemplarily consist of porous materials XI that have appropriate properties for the planned experiments such as low interaction with the involved biomolecules. Furthermore, the pore sizes should be controllable in the manufacturing process of the membranes with a low variance and be definable in a broad range of diameters from a few nanometres to the micrometer range. Such materials include polytetrafluoroethylene, polyethylene, PET, polycarbonate, cellulose, polypropylene and inorganic materials such as aluminium oxide.

[0040] It is desirable that the vertical inserts IX seal the connection between the respective wells II water tight. For ease of manually handling the insertion of the membranes into the slots the inserts IX may comprise an elongated top for example of plastics for gripping the insert IX. The insert IX comprises a rubber or elastomer rim X, which provides sealing engagement with the slot III and may be reinforced for stability. The insert IX and/or rim X may be configured to clip into the slot III.

[0041] It is also possible for the insert IX to support tissue, such as human or animal tissue, rather than a porous membrane. Indeed, cells may be grown on the membrane for study.

[0042] Figure 9a shows a portion of the plate I of Figure 1 with the barriers IX inserted in some of the slots.

[0043] The embodiment of Figure 3 differs from the embodiment of Figure 1 in that in this embodiment the wells are arranged in groupings of four interconnected wells V. Barriers IX can be inserted in the slots provided in the plate to partition the four wells, as shown in Figure 9b.

[0044] The embodiment of Figure 5 differs from the preceding embodiments in that in this embodiment the wells are arranged in groupings of five interconnected wells V. In this case, four substantially cylindrical wells surround a central square well defined by the inserted barriers, as shown in Figure 10.

[0045] In the embodiment of Figure 7, all wells on the plate are interconnected and can be separated by barriers IX.

[0046] Examples of applications of embodiments of the invention include, but are not limited to: biophysical studies, such as protein vesicle binding and partitioning studies, lipid transport studies, dialysis for protein crystallisation; immunology, for example cell interactions through secretion of soluble compounds; cell migration (chemotaxis); cell proliferation and differentiation; endocrinology, e.g. secretion of and response to hormones; pharmacology, e.g. cell toxicity studies; microbiology and parasitology, including interaction between organisms via secreted compounds; drug/tissue interaction; drug partitioning; drug non-specific binding; cell proliferation under drug presence.

[0047] There will now be described some specific examples of the types of experiments that can be carried out with the plate of the invention.

Non-specific binding and efficacy of drugs to different tissues

[0048] One application of an embodiment of the invention provides a method to test competitive binding and efficacy of a drug in different tissue types, as shown in Figure 1 1 . This is a useful way of investigating if an anti-tumour drug preferentially targets or affects tumour cells compared to normal cells.

[0049] As shown in Figure 1 1 , on the plate I a central well is connected to up to four other cells in equal distance to the central well and the wells are separated by semi- permeable membranes that maintain separation of the different cell types but allow passage and interchange of media components and other small molecules (see Figures 5, 6 and 10). The different cell or tissue types in the outer wells are cultured to optimal confluency. The drug molecules are added to the central well at the start of the

experiment. Two possible types of assays are proposed here:

1 ) To investigate and compare non-specific binding to different tissue types, such as tumour tissue and normal tissue, the drug molecules should either exhibit intrinsic fluorescence or be fluorescently labelled (or trigger a fluorescent signal in transfected cell lines) and be quantified by fluorescence (or luminescence) spectroscopy in each tissue after binding;

2) To investigate cell viability and the efficacy of a drug, a plate with transparent bottom wells should be used and the cells observed by light microscopy.

Cell interaction through secretion of soluble compounds

[0050] The plate according to an embodiment of the invention may be used for the investigation of immunological interactions such as response assays or migration

(chemotaxis) assays, as shown in Figure 12.

[0051] For this experiment two wells are connected and separated by a semi-permeable membrane that only permits passage of small soluble molecules (see Figures 1 and 2). Two cell types of interest are co-cultured in the two wells and at the start of the experiment one cell type (the donor cell) is stimulated and secretes molecules that may trigger a response in the other cell type (the acceptor cell). This response could be in the form of movement, where the acceptor cells are attracted or repelled by the secreted molecules.

[0052] These types of experiments are very common, but generally require purification of the secreted substance and addition to the acceptor cells. This renders the experiment very elaborate and also presumes the knowledge of the identity of the secreted molecules. Furthermore, in all existing similar plates which allow co-culture of the two cell types, the two compartments are horizontally connected which inhibits the observation of both compartments by microscopy during the assay, leading to loss of kinetic information.

[0053] The response of the acceptor cell may furthermore be on the level of protein expression.

[0054] Further still, the secreted molecules may lead to a change in DNA or RNA presence in the acceptor cell type. In this case, fluorescence in situ hybridisation (FISH) may be applied to detect the presence of genes and localise mRNA in order to investigate the spatio-temporal patterns of gene expression. [0055] Gene expression may also be observed by reporter genes. Furthermore, a change of absorption or fluorescence of the substrate of the protein of interest upon reaction may be exploited to quantify gene expression.

[0056] Cell differentiation may also be achieved using the plate according to

embodiments of the invention. Cell differentiation can be attained by supplementing the cell media with certain factors that are secreted by other types of cells. For example, bone marrow cells differentiate into macrophages upon treatment with L cell conditioned medium, which is commercially available. The plate according to embodiments of the invention allows co-culture of conditioning cells, thereby bypassing the need to buy expensive conditioning medium. The conditioning cells can be grown in up to four "feeder" wells surrounding the well with the differentiating cells, thereby increasing the

concentration of differentiation inducing factors.

Protein-lipid interaction studies

1. Competitive binding to vesicles of different lipid compositions, size or geometry

[0057] The investigation of lipid binding molecules such as, for example, proteins is of very high interest. To date no devices have been developed to facilitate assays aimed at the study of molecule or protein interaction with vesicles. As exemplified in Figure 13, observation of competitive binding to different types of lipids (differing in lipid composition) may be achieved by connection of at least two wells and separation with membranes that allow passage of the proteins and other small soluble molecules but not of vesicles. In the example shown, the vesicle suspension is deposited into the outer wells while the protein is added to the middle well. If the protein is intrinsically fluorescent, fluorescently labelled or induces fluorescence at the membrane of the vesicles (e.g. FRET), competitive binding to the differing lipid compositions can be quantified by fluorescence spectroscopy.

2. Investigation of transporting proteins

[0058] Certain types of proteins are known to be involved in delocalisation of molecules such as, for example, lipids, transporting them from one cellular membrane to another. Such processes may be investigated using the plate according to embodiments of the invention.

[0059] Using fluorescently labelled molecules or as in the example in Figure 14, lipids, the kinetics of transport can easily be observed in both wells using fluorescence spectroscopy. Two wells are separated by a semi-permeable membrane that allows passage of the protein-lipid complex, but retains the vesicles in their separate

compartments (wells). Vesicles of different properties such as lipid composition or vesicle size are deposited into the separate wells. The transport of a specific lipid from one vesicle type to the other can be observed by incorporation of a fluorescently labelled analogue of the respective lipid into the "donor" vesicles. Addition of the lipid transporting protein to the respective well enables the time resolved observation of the transport of the labelled lipid from "donor" vesicles to "acceptor" vesicles.

Size dependent diffusion of protein sample

[0060] The time of equilibration of Alexa488 labeled BSA (bovine serum albumin) across two wells separated by a 1 μηη porous membrane and a 12 μηη porous membrane was examined, using the plate I of Figures 1 and 9a. A fluorophore labelled (Alexa488) BSA (approx 66 kDa) protein is administered into a first well of a pair of wells II separated by a porous membrane XI at a concentration of 2 μΜ in a buffer (50 mM

tris(hydroxymethyl)aminomethane (Tris), 1 mM Dithioerythritol (DTE) containing protease inhibitor). The second well of the pair is administered with the buffer only. Both wells are then monitored over time and the fluorescence intensity is measured at 524 nm with 488 nm excitation. In a first experiment the two wells are separated with a 1 μηη Nucleopore™ membrane. In a second experiment the two wells are separated with a 12 μηη

Nucleopore™ membrane. Prior to the experiment, the Whatman Nucleopore membranes were sonicated for five mins in the buffer to remove any trapped air in the pores.

[0061] The ratio of the fluorescence intensity of the two wells was measured over time. For the 1 μηη membrane the ratio reached a maximum value of 0.6 after 320 minutes. However, for the 12 μηη membrane the ratio reached a value of 1 .0 after 320 minutes, indicating equilibrium and demonstrating the mobility of the protein through the larger pore size of the membrane compared to the smaller pore size.

Other examples

[0062] Using the plate I of Figures 1 and 9a, it is possible to co-culture cancer cells in a first well of the pair and stromal fibroblasts in a second well of the pair for oncology study. In this case, it is possible to observe migration through the membrane or observe and/or measure the juxtacrine or paracrine effect of one cell line on the other (e.g. sonic hedgehog stimulation and signalling).

[0063] Using plates according to the invention, it is possible to co-culture stem cells to observe and/or measure the effect of differentiation factors, possibly secreted from differentiated cells in co-culture.

[0064] Using the plate VII of Figures 5 and 10, it is possible to co-culture for different cell lines in four different, separated wells and measure and/or observe drug efficiency or toxicology, for example for drug profiling or clinical trial studies [0065] Using the plate VII of Figures 5 and 10, it is possible to co-culture the same cell line in different wells, and use different markers to observe and/or measure different endpoints in drug efficiency/toxicology studies.

[0066] Cell invasion (e.g. tumour invasion in 2D and 3D) studies can also be carried out. Using the plate VII of Figures 5 and 10, four different collagen concentration matrices can be poured into respective wells of the grouping, with the wells separated by impermeable, removable barriers. The collagen is allowed to set and the barriers are removed. The central space between the wells is filled with a cell line and cell invasion and invasion mechanisms into the different matrices is observed and/or measured. As an alternative, four different matrices (e.g. laminen, matrigel, collagen, fibronectin gel) may be provided in the four wells. It is also possible to assess the effect of a drug on cell invasion/migration.

[0067] Chemotaxis can also be studied using the plate I of Figures 1 and 9a, for example, by co-culturing neutrophils and a cell line producing chemokines in respective wells II of the plate separated by a porous membrane. Rather than using a cell line that produces chemokines, purified chemokines may be provided in the surrounding or neighbouring wells. A concentration gradient can be established and cell migration can be observed and/or measured.

[0068] In general terms, embodiments of the invention provide a means for performing biological or biophysical membrane binding and partitioning studies with the possibility for measurement of resulting signals by spectroscopic methods and microscopy. As an alternative to microscopy, EPI-imaging is also possible.

[0069] Thus, embodiments of the invention provide a plate with interconnected wells separated by exchangeable inserts with porous membranes for lateral diffusion of molecules allowing simultaneous observation of the independent wells.

[0070] Embodiments of the invention provide a micro-well plate with two or more interconnected compartments (wells) separated vertically by a semi-permeable membrane that allows lateral diffusion of molecules below a certain size, defined by the pore size of the membrane barrier.

[0071] In summary, a multi-well assay plate I comprises a plurality of wells II defined in a substantially planar substrate. Each well is in fluid communication via a gateway III with at least one neighbouring well to form a well grouping. Each gateway is configured to receive a partition for separating the neighbouring wells.

[0072] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0073] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1 . A multi-well assay plate comprising a plurality of wells defined in a substantially planar substrate, each well in fluid communication via a gateway with at least one neighbouring well to form a well grouping, wherein each gateway is configured to receive a partition for separating the neighbouring wells.

2. An assay plate as claimed in claim 1 , wherein the partition is removably received in the gateway.

3. An assay plate as claimed in claim 2, wherein the partition is solid, whereby to prevent any fluid communication between the neighbouring wells when received in the gateway.

4. An assay plate as claimed in claim 1 or 2, wherein the partition comprises a semipermeable membrane which, in use, controls passage of reagents between the wells separated by the partition.

5. An assay plate as claimed in claim 2, wherein the partition comprises a test material located within a frame.

6. An assay plate as claimed in claim 5, wherein the test material is a human or animal tissue sample.

7. An assay plate as claimed in claim 5, wherein the test material comprises a cell culture on a support structure.

8. An assay plate as claimed in any preceding claim, wherein the partition is arranged in the gateway substantially perpendicularly to the plane of the substrate.

9. An assay plate as claimed in any preceding claim, wherein the partition sealingly engages the gateway.

10. An assay plate as claimed in claim 9, wherein the perimeter of the partition is formed of elastomeric material to provide sealing engagement with the gateway.

1 1 . An assay plate as claimed in any preceding claim, wherein the wells are positioned on the planar substrate in positions corresponding to those of a standard micro-plate, whereby interactions in the wells can be monitored by a standard micro-plate reader.

12. An assay plate as claimed in any preceding claim comprising at least two well groupings, each comprising a plurality of wells in fluid communication via at least one mutual gateway, wherein the wells of each grouping are separated from the wells of the other grouping(s) by the substrate material.

13. An assay plate as claimed in any preceding claim, wherein each well grouping comprises more than two wells.

14. An assay plate as claimed in claim 13, wherein at least one well of the grouping is in fluid communication with a plurality of neighbouring wells via respective gateways.

15. An assay plate as claimed in any preceding claim comprising at least eight (preferably at least 16) wells in at least four (preferably at least eight) well groupings.

16. An assay plate as claimed in any preceding claim wherein the bottom of each well is substantially flat.

17. An assay plate as claimed in any preceding claim wherein the substrate is substantially optically transparent.

18. A method of using an assay plate as claimed in any preceding claim, the method including the step of locating the planar substrate substantially horizontally and monitoring the progress of a reaction between neighbouring wells of a well grouping from above.

19. A method of using an assay plate as claimed in claim 17, the method including the step of locating the planar substrate substantially horizontally and monitoring the progress of a reaction between neighbouring wells of a well grouping from below.

20. A method as claimed in claim 18 or 19, wherein the monitoring is by microscopy, spectroscopy, in particular fluorescence spectroscopy or absorption spectroscopy.

21 . A method of using an assay plate as claimed in claim 3 or any of claims 6 to 17 when dependent directly or indirectly on claim 3, the method including the step of removing the solid partition from a gateway in the course of an experimental procedure, whereby to allow fluid communication of a reagent in one well to a neighbouring well through the gateway.

22. A method of using an assay plate as claimed in claim 14 or any of claims 15 to 17 when dependent directly or indirectly on claim 14, the method including depositing a first reagent in the well of the grouping that is in fluid communication with a plurality of neighbouring wells via respective gateways, depositing different second reagents in each of the neighbouring wells and monitoring simultaneously the interaction of the first reagent with each of the second reagents.

23. A method as claimed in claim 22, wherein the second reagents are human or animal cells.

24. A method as claimed in claim 22 or 23, wherein the first reagent is a biologically- active substance.