Introduction The present invention relates to an apparatus for skin sanple culture which is suitable for testing using natural membranes such as skin, synthetic membranes and other materials in sheet form. The present invention relates in particular, to a multiwell skin cell culture device which is suitable for use in high throughput screening. Background to the Invention

Mammal.an skin is composed of two primary layers, the epidermis and the dermis. In order for skin to retain its normal appearance and to function fully in a normal manner, both layers of the skin need to be present.

The epidermis is composed of the outermost la ers of the skin. It forms a protective barrier over the body's surface, is -esponsible for keeping water in the body, protecting from UV light and preventing pathogens from entering. The epidermis contains no blood vessels and cells in the deepest layers are nourished by diffusion from blood capillaries extending to the upper layers of the dermis.

The dermis is the layer of skin beneath the epidarnis; it comprises connective tissue and cushions the body from stress and strain. The dermis provides tensile strength and elasticity to the skin through an extracellular matrix composed of collagen fibrils, microfibrils, and elastic fibers. The dermis is tightly connected to the epidermis through a basement membrane and is structurally ci pded into two areas: a superficial area adjacent to the epidermis, called the papillary region, anc a deep thicker area known as the reticular region. Samples of skin may be removed from an an mal body for the purpose of analysis or in order to grow a sample of skin where a skin graft is required.

Analyse may be undertaken using high throughput screening (HTS) - a technique which is used extensively in drug discovery, biology and chemistry. A typical HTS is performed in a multi-well plate containing target rrolecules and/or cells. Using robotics, data processing and control software, liquid handling devices, and sensitive detectors, HTS allows a researcher to quickly conduct millions of chemical, genetic, or pharmacological tests. Through this process one can rapidly identify active compounds, antibodies, or genes lhat modulate ε particular biomclecular pathway. The results of these

experiments provide starting points for drug design and for understanding the interaction or role of a particular biochemical process in biology.

A multiwell plate is typically a flat plate with multiple wells which function as small test tubes. The multiwell plates used for HTS typically have 96, 384 or 1536 sample wells arranged in a 2:3 rectangular matrix. Each well typically holds somewhere between tens of nanclitres up to 100 microliters of liquid.

The use of multiwell p ates for the analysis of membrane samples is described in WO 2005012549 which discloses an apparatus and method for HTS in which a lamina such as skin is positioned between a donor plate and a receptor plate which has a plurality of wells. The receptor plate and donor plate are both in fluid contact with the lamina and a means for applying an electric current 1o test the response of the lamina in the presence of test formulations is also provided. US 6043027 describes a multiwell single membrane permeation device which has a top member with apertures, a base member which has a plurality of wells, a membrane sheet upon which cell sample is grown and a gasket which provides a seal between the top member and the membrane sheet. Summary o~ the Invention

It is an object of the present invention to provide a test device for use with a sample of natural or synthetic sheets and membranes which is suitable - ^' or HTS.

It is an object of the present invention to provide a multiwell p ate device that allows HTS on tissue, specifically murine or porcine skin tissue and human skin tissue.

It is another object of the present invention to provide a skin culture apparatus which retains a skin sample and maintains the viability of the skin sample in a suitable condition for growth and/or testing.

In accordance with a first aspect of the invention there is provided an apparatus for high throughput screening the apparatus comprising:

a base comprising a plurality of channels for receiving a reagent, the channels being spaced across the su-face of the base and having one or more walls which extend through the base from a first base surface to a second base surface,

a compression member containing a plurality of openings which extend through the corrpression member and which are positioned across the surface of the compression member, one or more of said openings being positioned for alignment with a

corresponding channel in the base;

a grip for removably securing the compression member to the base such that when a compressible sheet is positioned across the channel between the base and the compression member and fixed by the grip, parts of the comp-essible sheet are compressed between the base and the compression member to form a seal between the base and the compression member and the compressible sheet and walls form one or more well for containing the reagent.

Preferably a -im of the channel on the first base surface or the second base surface is in contact with the rim of the opening. In use, the apparatus s constructed, then arranged such that the compressible sheet is between the compression member and the base with the compressible sheet forming the bottom surface of a well. A reagent is added to the channels and the reagent, under the action of gravity, is in contact with the compressible sheet a: the part which extends across the channel.

Preferably, the rim of the channel which is not in contact with tne compressible sheet is open.

Prefsrably, the compressible sheet is a membrane. Preferably, the compressible sheet is a natural membrane. Preferably, the compressible sheet is skin. Preferably, the skin is murine or porcine skin. Preferably, the skin is human skin.

Preferably, the compressible sheet is a synthetic membrane.

Preferably, the channel is substantially cylindrical in shape. Optionally, the channel is substantially cuboid in shape.

Other channel shapes may be used such as conic, or may have a polygonal cross section. Preferably, the grip comprises one cr moire fixings which connect the compression plate to the base.

Optionally, the grip comprises a snao fit connection which connects the compression plate to the base.

Optionally, the grip comprises a magnetic connection which connects the compression plate to the base. Preferably, the base comprises one o? more base holes positioned for alignment with one or more corresponding compression plate through holes.

Preferably, the grip comprises a fixing w ich is sized to connect the one or more compression plate through hole to an aligned base hole.

Optionally, the channel with a skin sample receiving surface upon which at least part skin sample may be placed and which exiends across an area defined by the shape of the frame; and

a securing member which is releasaoly connectable to the base frame and a grip which holds the skin sample under tension.

Preferably, the grip comprises a releasable connection between the base frame and the securing member. More preferably, the grip comprises one cr more fixings which connect the base frame to the securing member.

Optionally, the grip comprises a snap fit connection between the base frame and the securing member. Optionally, the grip comp ises a magnetic connection between the base frame and the securing member.

Preferably, the base comprises one or more base holes positioned for alignment with one or more correspond! -g compression member through holes.

Preferably, the grip composes a fixing which is sized to connect the one or more compression member through holes to aligned base holes. Preferably, the grip provides a substantially even tensi e force across the skin sample.

Preferably, the apparatus further comprises a tensioner which applies a tensile force across the surface of the compressible sheet. Preferably the tensioner applies a substantially constant tension across the surface of the compressible sheet.

Optionally, spacers inserted between the compress ble sheet and the compression member can be used to optimise the tension across the surface of the compressible sheet.

Optionally, a pattern des gned on the compression sheet can be used to optimise the tension across the surface of the compressible sheet. Preferably, the apparatus further comprises one or more spacer which sets the distance between the base and the compression member. Optionally, the one or more spacer creates a distance between the base and the compression member which is substantially uniform across the surface of the apparatus. Optionally, the one or more spacer cr sates a distance between the base and the compression member which is greater at one part of the apparatus than at another.

Preferably, the one or more spacer is positioned between the base and the

compression member.

Preferably, the spacer is in contact with the compression member and the compressible sheet

Preferably, the apparatus further comprises a fluid cap for introducing a fluid into the well.

Preferably, the fluid cap is positioned on the base at the end of the well remote from the ccmpression member. Preferably, the fluid cap comprises an inlet located at a first position on the fluid cap and an outlet located al a second position on the fluid cap.

Preferably, the fluid cap is adapted to receive a gas. Optionally, the fluid cap is adapted to receive a liquid.

In accordance with a second aspect of the present invention there is provided a method for conducting high throughput screening using the apparatus in accordance with the first aspect of the invention, the methcd comprising the steps of:

Placing a compressible sheet between a base and a comoression member; Securing the compressible sheet in position;

Invert ng the apparatus;

Adding reagent to the well formed by the channel and compressible sheet. In accordance with a third aspect of the invention there is provided a method for concuctirtg high throughput screening using the apparatus in accordance with the first aspect of the invention, the method comprising the steps of:

Placirg a compressible sheet between a base and a compression member;

Securing the compressible sheet in position;

applying reagent to either surface of the compressible sheet before inverting the apparatus and adding culture medium to the well.

Optionally, the apparatus is constructed to ANSI/SBS dimension standards offers advantages for compatibility with currently available automated handli ng apparatus.

Brief Description of the Drawings

The present invention will now be described with reference to the accompanying drawings in which:

Figure 1 shows an exploded perspective view of a first embodiment of a skin sample culture and membrane test device in accordance with the present invention;

Figure 2 a plan view of the embodiment of figure ;

Figures 3A, 3B and 3C are cress sectional view of the embodiment of figure 1 and illustrate the process of using the apparatus as a multi-well plate; and

Figures 4A and 4B are examples of suitable compressible materials. Figure 5A is a perspective view of a second embodiment of the present invention and figure 5B is a side view of the embodiment in figure 5A;

Figure 6 shows a perspective view of another embodiment of a skin sample culture and membrane test device with a fluid cap in accordance with the present invention

Figure 7 is a plan view of an apparatus in accordance with the present invention, in which leakage from well to well has been measured; Figure 8A is a perspective view of another embodiment of the present invention, figure 8B illustrates experimental data in which skin cultured in the oresent invention responds to a small molecule drug and figure 8C is a graph which plots NQ01 mRNA levels versus time of treatment; and Figure 9 shows an embodiment of the present invention whicn is designed to standard ANSI dimensions.

Detailed Description of the Drawings

High throughput screening (HTS) is the core of drug discove . A typical HTS is performed in 384-well plates containing target molecules and/or cells. Here we describe a plate device that allows HTS on tissue, specifically murine or porcine skin tissue and hurnsn skin tissue obtained from abdominoplasty surgery.

The present invention comprises, a base and matching compressian plate between which the compressible sheet is placed.

Figure 1 is an exploded perspective view of a first embodiment of he present invention. Figure 2 is a plan view of the same and figure 3 is a cross section of part of the device. An embodiment of the present invention will be described with reference to figures 1 to 3.

Figures 1 to 3 show an apparatus 1 which comprises a base 3, a compression plate 5 and a compressible sheet 7 which is positioned between the base 3 and the

compression plate 5. The base comprises an array of channels 15 which are substantially cylindrical in cross section and extend from a first surface 4 which, in this example is positioned towards the compression ptate 5, to a second surface 6 which is remote from the compression plate 5. The channel is open ended at the first surface 4 and at the second surface 6 which defines a circular rim or lip.

The compression plate 5 comprises a substantially planar member which has a plurality of openings 17 arranged in an array. The size and position of the openings 17 matches the size and position of the channels 15 in the base 3 such that when the compression plate 5 is aligned with and placed upon the base 3, the openings 17 of the compression plate 5 and the channels 15 of the base 3 are aligned to have a common centre point.

A grip mechanism is included in order to secure the compression plate 5 to the base 3. In this example, the grip comprises a series of screws 9 which are connectable to the compression plate via through holes 11 and is connectable to the base via base holes 13.

In this example, screws 9 fasten together the compression plate 5, compressible sheet 7 and base 3 around the perimeter of the compression plate 5 and base 3. Additional screws 18 are used towards the centre of the compression plate as shown in. figure 2.

When the compression plate 5 is secured through the compressible sheet 7 to the base 3, the parts of the compressible sheet 7 between the base and the compression member are compressed to form a seal between the base and the compress on member and the compressible sheet and walls forms a well for containing the reagent. As is shown in f gure 3A. In this example of the present invention, the compressible material is porcine or human skin, hat single sample acting as a gasket between the upper and lower plates. In use, a single skin sample 7 is placed across all wells 15 of base 3 either unstretched or under a user defined tension. Orce the skin 7 is in place and the compression plate 5 is secured with he screws 9, the skin 7 acts as a gasket. As shown in figures 3B and 3C, the entire device can then be turned over and the wells 15 filled with a reagent medium 19 (with or without test compound). The entire device is then incubated in the "upside down" crientation in a standard incubator with appropriate secondary

containment. Breathable plate seals can be used with this plate.

Optionally, prior to turning over the device and filling wells 15 with reagent medium a topical treatmert to the membrane 7 can be applied.

The clamping fcrce compresses the skin 7 in between each channel 15, effectively sealing each channel 15 using the skin itself to form the well. In this manner each well can be considered a discrete sample where an individual experiment can be performed. In some embod ments of the present invention the skin is tensioned before clamping the top plate in place. It has been noted that the act of compressing the skin around each channel without additional tensioning causes the free skin over the well opening to bulge into the opening. This seems to stretch the piece of free skin to a degree sufficient enough to maintain it in culture. Tensioning is preferred where the channel cross sectional area is large.

Figures 4A and 4B show examples of a compressible sheet. Figure 4A shows a membrane such as porcine or murine skin. Figure 4B shows a compressible substrate 31 upon which a sample 33 may be mounted. Figire 5A shows a perspective view of another embodiment of the present invention with the addition of optional spacers 121. In use the spacers 121 are inserted between the compression member 5 and the compressible sheet or sample of skin 7. Figires 5A and 5B show an apparatus 101 wnich comprises a base 103, a compression plate 105 and a compressible sheet 107 which is positioned between the base 3 and the compression plate 5. The base comprises an array of channels 115 which are substantially cyl ndrical in cross section ar d extend from a first surface 114 which, in this example is positioned towards the compr ^e ssion plate 105, to a second surface 116 which is remote from the compression plate 105. The channel is open ended at the first surface 114 anc at the second surface 116 wnich defines a circular rim or lip.

The compression plate 105 comprises a substantially planar member which has a plurality of openings 1 17 arranged in an array. The size and position of the openings 117 matches the size and position of the channels 115 in the base 103 such that when the compression plate 5 is aligned with and placed upon the base 103, the openings 117 of the compression plate 105 and the channels 115 of the base 103 are aligned to have a common centre point. Figure 5B shows spacers 121 of varying thickness which have been compressed between the compressible member 105 and the compressible sheet 07. The spacers 121 bear some of the compression load experienced by the compressible sheet 107 and the base 103 when the compression plats 105 is tightened into position. Therefore, the spacers 121 function as a means of controlling the compression on the

compressible sheet 107 and the tension across the surface of the sheet where it forms part of a well.

In this example, a gradient of tension 122 and compression has been applied to the skin sample through the use of the spacers which imparts a slight angle 123 to the compressible member 105. In another embodiment, a varied number of spacers of differing thicknesses can be used to optimize or change the compression and tension.

Figure 6 is a perspect ve view of another embodiment of the present invention. Figure 6 shows the apparatus 201 with a base 203, a compression plate 205 and a fluid cap 225. The base has holes (not shown) which receive fixings 209 The base is rectangular in shape and further comprises channels (not shown) which extend through the perimeter of the base, allowing the culture medium, air and other fluids o move through the space at or below the underside of the skin sample 207.

The fluid cap 225 is substantially rectangular in shape having an enclosed too surface 227, an enclosed side surface 229 with a seal 231 on its lower perimeter. The seal is designed to retain the fluid in the space at or around the tcp surface of the skin sample 207. The irlet 233 is connectable to a fluid source and the outlet 235 is connected to a fluid collector. In use, the fluid cap 225 is placed over the compression plate 205 and pushed downwards into place and the seal 231 holds the fluid cap in position. A fluid source is connected to the fluid cap inlet 233. The fluid may be introduced as a batch into the fluid cap 225, in which the outlet 235 is closed anc orce the required amount of fluid has been added the inlet 233 is closed. Alternatively, the fluid may be introduced continuously so a con:inuous flow of fluid passes through Ihe fluid cap 225, in this case the inlet 233 and the outlet 235 remain open, the outlet 235 being connected to a fluid collection vessel (not shown).

The fluid cap will allow the ability to culture skin such that Ihe atmosphere (e.g., humidity, gas composition, etc.) at the surface of the skin Gan be controlled separately from the atmosphere of the incubator.

Figure 7 is a plan view of an apparatus in accordance with the present invention, in which leakage from well to well has been measured. In wells labelled 1 and 6, medium containing 1 mM simvastatin was added at time 0 h. All other wells contained medium only After 24 h, the skin in wells 1-1G was removed from the device using a 3 mm biopsy punch. The skin was homogenised in organic solvent to extract simvastatin from the treated and peripheral skin. Simvastatin levels in the solvent were then quantitated by LC-MS. High levels of simvastatin were measured in wells 1 and 6 which v/ere trealed with simvastatin directly. Little (< 0.6%) or no leakage was detected in the neighbouring wells. <LOQ = below the limit of quantitation. ND = not detectec.

Figure 8A is a perspective view of another embodiment of the present invention 341 . Figure 8A shows the apparatus 341 with a base 345, and compression plate 343. The base has holes (not shown) which receive fix ngs 349. Figure 8A shows a par.ial 384- well device 341 that has the overall well dimensions and spacing of a stardarc 384-well plate used for HTS. Figure 8B is a schematic diagram which summarises an experiment n which skin cultured in the apparatus 341 was treated with a small molecule NRF2 activator (TBE-31) in aider tc evaluate the intracellular viabili-y of the tissue. NRF2 transcriptionally regulates multiple genes that play both direct and indirect roles in activating intracellular ant -ox da:ive pathways.

One of the genes that is upregulaied upon NRF2 activation is NAD(P)H dehydrogenase [quinonel 1 JNQ01 ). Figure 8C shows NGOI mRNA levels 353 obtained from total RNA isolated from the skin treated wlh TBE-31 compound (a potent NRF2 aclivator) at the indicated times 355. It is shewn here that the intracellular viability of the skin cultured in tiis device remains viability for at least 4 days as the NRF2 response is statistically ihe same whether the flss je is treated on Day 0 or Day 4. Figure 9 is a perspective view of anolner embodiment of the present invention Figure 9 shows the apparatus 451 which is designed in accordance with AN3I/SBS/SLAS dimensions. Well 459 spacing and apparatus footprint including, width 453, length 455 and height 457 all match standard micrqplate dimensional standards to ensure compatibility with automated handling systems. The device of the present invention can be machined or 3D printed in a variety of materials, including but not limited tc plastics such as ABS, Polypropylene, Polystyrene, PTFE, PEEK or PET and metals such as stainless steel or titanium. Additionally, the devise can be mass produced through methods such as injection moulding, insert moulding and vacuum forming.

The compression plate can be secured using a variety of methods (depending on application and design), including, but not limited to, screw, spr ng clio and magnetic fixation. The device holds the membrane under a user-defined tension at the air-liquid interface and allows it to be maintaired in culture in a format featuring a footprint and well spacing matching ANSI/SBS standard dimensions, affording the device

conpatibi ity with various automated methods of handling. The desig ned number of weBs can range from 12 to 384, preferably arranged in a 2:3 rectangular matrix and the thickness of the plates can range from 2 mm to 15 mm depend ng on application.

Each well is separate, containing its own volume of culture medium. The medium is added through standard automated oipettes which are a standard part of high throughput screening apparatus. Because the plate is handled upside down and each ^■ well is separately filled, this and other embodiments of the invention have no single reservoir of culture medium and thus no requirement to allow air to escape.

In another embodiment of the invenlion, the base may be submerged in a reagent medium prior to attachment of the compression plate in order to fill each well with the same solution. In most cases, different reagents may be used in each well or triplicate of wells as they will contain different compounds dissolved at different concentrations in medium.

Improvements and modifications may be incorporated herein without deviating from the scope of the invention.