Patent Description:
Skin is the strong outer covering of vertebrate animals. Other animal coverings such as the arthropod exoskeleton have a different developmental origin, structure and chemical composition.

In mammals, the skin is an organ made up of multiple layers of ectodermal tissue covering the underlying tissue, ligaments, bones and internal organs. It acts as a sensory organ, protects the body against pathogens, ultraviolet damage, excessive water loss, provides insulation, temperature regulation and produces vitamin D.

The thickness of skin varies from location to location on an organism. In humans for example the skin located under the eyes and around the eyelids is the thinnest skin in the body at <NUM> thick whereas the skin on the palms and the soles of the feet is <NUM> thick and is around <NUM> thick on the back.

Mammalian skin is composed of two primary layers, the epidermis and the dermis. The epidermis is a stratified, cornified epithelium with specific barrier functions which is supported by the complex extracellular matrix environment of 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 forms a protective barrier over the body's surface, is responsible 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 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, microfibr Is, and elastic fibers. The dermis is tightly connected to the epidermis through a basement membrane and is structurally divided into two areas: a superficial area adjacent to the epidermis, called the papillary region, and a deep thicker area known as the reticular region.

Samples of skin may be removed from an animal body for the purpose of analysis or in order to grow a sample of skin where a skin graft is required.

Skin grafting is an essential component of reconstructive surgery after burns, trauma, tumor excision, and correction of congenital anomalies. The best possible skin available for grafting is skin from the same patient taken from a donor site elsewhere on the body which is referred to as an autograft. Suitable skin graft donor sites are limited by body surface area and may also be affected by previous graft harvest or trauma. In patients suffering from large burns with limited donor skin sites, cadaver allografts are commonly used for temporary skin coverage.

In all cases, there is a need to maintain the skin sample in a healthy state and to slow or completely arrest deterioration of the quality of the sample whilst it is being stored.

Reliable skin models which recapitulate the features of live skin are essential for the investigation of cutaneous biology and drug discovery. Full-thickness ex vivo skin culture systems have been used extensively. The global market for ex vivo skin models includes the academic researcher, the pharmaceutical industry and the cosmetic industry with regards to safety and efficacy assessment for the increasing number of products developed for topical application. Furthermore, the development of such a model system is of an increasing priority due to the European Community regulation that bans the use of animal testing for cosmetic ingredients.

<CIT> discloses a method of growing vertebrate skin in vitro. Part of the process involves creating a method for stretching the skin so as to enhance skin growth by positioning the skin segment in an artificial cell-growth medium and subjecting the skin segment to stretching forces while the skin segment is in the medium.

<CIT> describes an apparatus for measuring transdermal diffusion. It comprises a cylinder body with a cover featuring a groove arranged on the upper surface of the cover body. The cross section of the cover body is concave and a clamping groove is arranged on a side wall of the groove and the cover body and the cylinder body are connected through screw threads or are clamped. The other end of the cylinder body <NUM> is used for fixing excised skin.

The two prior art documents above do not address the same technical problem as the present invention.

<CIT> discloses a tissue stretching device configured for orbicular expansion and culturing of a tissue. A bottom solid support of the device is configured to receive an expandable membrane, upon which the tissue may be placed and under which a fluid may be delivered. In some embodiments the bottom solid support comprises a fluid inlet and a fluid outlet, which allow for an increase in the volume/pressure of the fluid in the bottom solid support under the expandable membrane in order to stretch the tissue.

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

The embodiments illustrated in <FIG> and <FIG> are outside the scope of the appended claims.

The present invention provides a novel means by which cultured skin samples may be kept under tension. Maintaining the skin samples under tension is beneficial because it extends the viable lifetime and the quality of the skin. In addition, the present invention allows the skin to be quickly mounted with minimal waste, cultured in a standard CO<NUM> incubator, without the need to suture the skin sample in place. Suturing is very time consuming both when the sample is being stored in a sample dish and when the sample is subsequently to be removed from the dish.

<FIG> shows a first embodiment of a skin sample storage apparatus. <FIG> shows the apparatus <NUM> which has a base <NUM> upon which a skin sample is placed in use, and a cap <NUM> which is placed on top of a skin sample, in use. Through holes <NUM> in the cap <NUM>, are aligned with holes <NUM> in the base <NUM> and screws <NUM> or other suitable fixings are used to attach the cap <NUM> to the base <NUM>.

In this example, the screws provide for the removable attachment of the cap <NUM> to the base <NUM>. The base further comprises lower channels <NUM> and two upper channels <NUM> which extend radially through the circumference of the base <NUM>. The lower channels <NUM> are positioned towards the bottom surface <NUM> of the base <NUM>, and the upper channels are placed at the top surface <NUM> of the base <NUM>. In this example, the lower channel <NUM> is a closed channel at the bottom of the base <NUM>.

The upper channel <NUM> is open at its top.

<FIG> shows a second embodiment of skin sample culture apparatus base.

<FIG> shows the apparatus <NUM> which has a base <NUM> upon which a skin sample is placed in use, and a cap <NUM> which is placed on top of a skin sample, in use. Through holes <NUM> in the cap <NUM>, are aligned with holes <NUM> in the base <NUM> and screws <NUM> or other suitable fixings are used to attach the cap <NUM> to the base <NUM>.

In this example, the screws provide for the removable attachment of the cap <NUM> to the base <NUM>. The base further comprises lower channels <NUM> and upper channel <NUM> which extend radially through the circumference of the base <NUM>. The open lower channels <NUM> are positioned towards the bottom surface <NUM> of the base <NUM>, and the enclosed upper channel is placed towards the top surface <NUM> of the base <NUM>.

In this example, the upper channel <NUM> is closed at the top surface of the base <NUM>. The lower channels <NUM> are open at the bottom surface.

<FIG> show the base <NUM> of a third embodiment of a skin sample culture apparatus base.

The base <NUM> comprises base holes <NUM>, lower channels <NUM> and upper channel <NUM> which extend radially through the circumference of the base <NUM>. In this embodiment, the lower channels are open at the bottom surface <NUM> of the base <NUM>.

The upper channel <NUM> is enclosed. As shown in <FIG>, the top surface <NUM> of the upper channel <NUM> is at an angle with respect to the bottom of the base <NUM> such that the end of the channel <NUM> at the outermost part of the base <NUM> is higher than the end of the channel at the innermost part of the base <NUM>. The angle <NUM> illustrated in this embodiment of the invention is approximately <NUM>°.

The angle of inclination of the top surface assists with the passage of trapped air from under a skin sample when the apparatus is in use.

In this and other examples, a single upper channel and multiple lower channels are arranged at an equal spacing around the base <NUM>. In other examples, multiple upper or lower channels may be present or multiple upper and lower channels may be arranged alternately or in groups; for example, one side of the base may have upper channels and another may have lower channels. This may further assist the removal of air bubbles from the underside of the skin sample.

<FIG> shows an example of a method by which the skin sample <NUM> is attached to a skin sample culture apparatus. <FIG> shows a mounting mat <NUM> upon which is placed a skin sample <NUM>.

<FIG> shows the mounting mat <NUM> and the skin sample <NUM> which has been stretched across the mounting mat <NUM> and secured in position by pins <NUM> which are pushed through the skin sample <NUM> into the mounting mat <NUM>.

<FIG> shows a base <NUM> positioned upon the mounting mat <NUM>.

<FIG> shows that the base <NUM> has been moved from its position in <FIG> and has been moved under the stretched skin sample to position <NUM>. <FIG> is a side perspective view at the base <NUM> in position <NUM> between the skin sample <NUM> and the mat <NUM>.

<FIG> shows a cap <NUM> placed on top of the skin <NUM> aligned with the base <NUM> at position <NUM>.

<FIG> shows another example of a skin sample culture apparatus.

<FIG> shows the apparatus <NUM> which has a base <NUM> upon which a skin sample is placed in use, and a cap <NUM> which is placed on top of a skin sample, in use. Through holes <NUM> in the cap <NUM>, are aligned with holes <NUM> in the base <NUM> and screws <NUM> or other suitable fixings are used to attach the cap <NUM> to the base <NUM>. It is mounted on mat <NUM>.

In this example, the screws <NUM> provide for the removable attachment of the cap <NUM> to the base <NUM>. The base is similar to that described with reference to <FIG>. The base <NUM> comprises base holes <NUM>, lower channels <NUM> and upper channels <NUM> which extend radially through the circumference of the base <NUM>. The upper channels <NUM> are enclosed and as with the example of <FIG>, the top surface of the upper channel is at an angle with respect to the bottom of the base such that the end of the channel <NUM> at the outermost part of the base <NUM> is higher than the end of the channel at the innermost part of the base <NUM>. The angle of inclination of the top surface assists with the passage of trapped air from under a skin sample when the apparatus is in use.

In this and other examples, a single upper channel and multiple lower channels are arranged at an equal spacing around the base <NUM>. In other examples, multiple upper and lower channels may be arranged alternately or in groups; for example, one side of the base may have upper channels and another may have lower channels. This may further assist the removal of air bubbles from the underside of the skin sample.

<FIG> and <FIG> show a method by which an example of a skin sample culture apparatus is used.

<FIG> shows a mounting mat <NUM>, a skin sample <NUM> and a base <NUM>. The base <NUM> is placed in a mounting mat inset <NUM>. The mat <NUM> has sufficient thickness such that the depth of the inset ensures that the top surface of the base <NUM> is flush with the top surface of the mounting mat <NUM>.

<FIG> show the process by which a skin sample is placed upon a mounting mat which has a base <NUM> positioned in the inset <NUM>. The skin <NUM> is fixed to the mounting mat on one side with pins <NUM>. <FIG> shows the skin <NUM> being stretched across the top of the base <NUM> and secured to the mounting mat <NUM> with a third pin <NUM>.

<FIG> shows a fourth corner of the skin being stretched across the top of the base <NUM> and secured to the mounting mat <NUM> with a fourth pin <NUM>.

<FIG> shows additional pins <NUM> which attach the skin sample <NUM> to allow even tension throughout the skin.

<FIG> shows a biopsy punch <NUM> which is used to cut holes in the skin to allow the attachment of screws to the base <NUM> through the cap <NUM> which has been positioned on top of the skin sample <NUM> in alignment with the base <NUM>. The screws are placed in the cap holes <NUM> and are driven through the cut holes in the skin <NUM> and held in the base holes which are aligned with the cap holes <NUM>. As shown in <FIG>, this process allows for the removal of the pins <NUM> and retains the skin sample <NUM> under tension clamped between the cap <NUM> and the base <NUM>. <FIG> shows the skin sample culture apparatus being removed from the mat <NUM>. <FIG> shows excess skin from the edges of the sample trimmed off.

<FIG> show the culture dish <NUM> containing a culture medium <NUM> and a skin sample culture apparatus <NUM> being lowered towards the culture dish <NUM>.

<FIG> show the skin sample culture apparatus <NUM> being lowered at an angle towards the culture dish. Advantageously it has been found that, lowering the skin sample culture apparatus <NUM> at an angle with the air channel angled upwards encourages any air which might be between the skin sample and the culture medium towards the higher side of the skin sample culture apparatus and therefore reduces the risk that the air will be trapped under the skin sample. Trapped air prevents the culture medium from having full contact with the skin sample and therefore, may cause the skin sample to degrade.

During the process of lowering the sample and once it is in position as shown in <FIG>, a base which has angled upper channels as shown in the example of <FIG> will further assist with the removal of trapped air by providing an easy path for the trapped air's exit from beneath the skin sample. Once the skin sample culture apparatus is in position, a cover <NUM> is placed upon the culture dish <NUM>.

The above embodiments show the grip element comprising a number of screws which connect the base to the cap. Other grips may be used, such as a snap fit connection or a magnet, for example.

<FIG> is a perspective cut-away view of another embodiment of the present invention.

<FIG> shows the apparatus <NUM> with a base <NUM>, a securing member <NUM> and a tensioner cap <NUM>. The base <NUM> has base holes <NUM> which receive fixings <NUM>. The base is annular in shape and further comprises channels <NUM> which extend through the perimeter of the base allowing culture medium, air and other fluids to move through the space at or below the underside <NUM> of the skin sample <NUM>.

The securing member <NUM> is annular in shape and congruent with the base <NUM>. Securing member holes <NUM> extend through the securing member <NUM> and, in use, are aligned with the base holes <NUM>. The skin sample <NUM> is secured in position upon the apparatus by placing the skin sample <NUM> between the base <NUM> and securing member <NUM> to or slightly beyond the outer circumference of the securing member <NUM> and base <NUM>. A screw is inserted through the securing member hole <NUM> and the base hole <NUM> and the screw is tightened to secure the skin sample in position between the base <NUM> and securing member <NUM>.

The tensioning adjustment mechanism is a cap <NUM> is annular in shape and is positioned on top of the securing member <NUM> and is congruent with the securing member <NUM> except that a tensioner <NUM> extends downwards from the inner circumference of the tensioning cap, slightly beyond the inner surface of the securing member <NUM>. The tensioner is sized such that it extends beyond the 'bottom surface' of the tensioner cap <NUM> to be longer than the securing cap depth <NUM>.

A securing member/tensioner cap hole <NUM> is threaded. The tensioner cap <NUM> has a tension adjustment mechanism <NUM> comprising screw <NUM> which extends through the tensioner cap <NUM> into the securing member/tensioner cap hole <NUM>.

In use, a skin sample <NUM> is firstly secured in position between the base <NUM> and the securing member <NUM>. The tensioner cap <NUM> is fitted on the top of securing member <NUM>. In order to increase the tension across the surface of the skin the tension cap screws <NUM> are turned. This moves the tensioner cap <NUM> closer to the securing member <NUM>. In this embodiment of the present invention, the tensioner <NUM> is rigidly fixed to the tension adjustment mechanism <NUM> of the tensioner cap <NUM>. Tightening the tensioner cap screw <NUM> moves the tensioner <NUM> vertically towards the upper surface <NUM> of the skin sample <NUM>. Upon contact with the skin sample surface <NUM>, further tightening of the tension cap screw <NUM> will push the tensioner <NUM> and the edges of the skin <NUM> downwards, pulling the skin tight across its surface and increasing the tension across the sample.

<FIG> is a perspective cut-away view of a modified version <NUM> of the embodiment of the present invention shown in <FIG> on which the channels <NUM> of <FIG> have been removed. The embodiments of <FIG> and <FIG> provide a quick and easy way of adjusting the tension in the skin sample.

<FIG> is a perspective view of another embodiment. <FIG> shows the apparatus <NUM> with a base <NUM>, a securing member <NUM> and a fluid cap <NUM>. The base has holes (not shown) which receive fixings <NUM>. The base is annular in shape and further comprises channels <NUM> which extend through the perimeter of the base, allowing the culture medium, air and other fluids to move through the space at or below the underside of the skin sample <NUM>.

The fluid cap <NUM> is substantially cylindrical in shape having an enclosed top surface <NUM>, an enclosed side surface <NUM> with a seal <NUM> on its lower circumference, at the open bottom surface of the cylindrical sloped cap. The seal is designed to retain the fluid in the space at or around the top surface of the skin sample <NUM>. The inlet <NUM> is connectable to a fluid source and the outlet <NUM> is connected to a fluid collector. In use, the fluid cap <NUM> is placed over the securing member and pushed downwards into place and the seal <NUM> holds the fluid cap in position. A fluid source is connected to the fluid cap inlet. The fluid may be introduced as a batch into the fluid cap, in which the outlet <NUM> is closed and once the required amount of fluid has been added, the inlet is closed. Alternatively, the fluid may be introduced continuously so a continuous flow of fluid passes through the fluid cap <NUM>, in this case the inlet <NUM> and the outlet <NUM> remain open, the outlet being connected to a fluid collection vessel.

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

In another embodiment of the present invention, the apparatus incorporates a tensioning cap and a fluid cap thereby allowing the user to alter the skin sample tension and perform experiments which change the medium to which the top layer of the skin sample is exposed. In another embodiment of the present invention, the apparatus incorporates a fluid cap on both the top and bottom sides of the device allowing experiments which change the medium to which both the upper and lower sides of the skin sample are exposed to.

<FIG> is a perspective view of a modified version of the embodiment <NUM> shown in <FIG> with the base channels absent.

<FIG> is a perspective view of an embodiment of the present invention which incorporates features of <FIG> and <FIG>. It shows the tensioning cap <NUM> and base of <FIG> and the fluid cap <NUM> of <FIG> with a base similar to that shown in <FIG> and <FIG>.

In another embodiment of the present invention the tensioner is resiliently mounted and attached to a force meter such that the force applied by the tension adjustment mechanism is measurable.

<FIG> is a perspective view of an embodiment of the present invention shown in <FIG> with the addition of a fluid cap to the bottom of the apparatus. It shows the tensioning cap <NUM> and a base <NUM> with a top fluid cap <NUM> and bottom fluid cap <NUM>. The bottom fluid cap <NUM> features a fluid inlet <NUM> and fluid outlet <NUM> which allow control of the atmosphere at the bottom surface of the skin sample.

<FIG> shows an apparatus for measuring the tension in a skin sample which has been placed in a skin sample culture apparatus in accordance with the present invention.

The apparatus comprises a force meter <NUM>, a spacing collar <NUM> which slides over the probe shaft <NUM> and extends downwards from the body of the force meter <NUM>. The collar <NUM> is a cylindrical tube with a diameter and circumferences which matches that of the cap such that the end of the collar <NUM> rests upon the cap of a skin sample culture apparatus <NUM>. The spherical indenter exerts a force upon the skin sample.

<FIG> are schematic diagrams which illustrate the operation of the apparatus <NUM>.

<FIG> shows the spherical indenter <NUM> resting upon the skin sample <NUM> at the centre of the sample. The cap <NUM>, fixings <NUM> and base <NUM> are also shown in the diagram. When a known displacement Δh <NUM>, defined by the collar <NUM> length, is exerted upon the skin sample <NUM>, a force reading is recorded. This provides a measure of skin tension.

Where the radii are different, it is necessary to account for the effect of the radius on the measured value of tension.

The elastic modulus E of the membrane mounted in the culture device, defined as the relationship between stress (force per unit area) and strain (proportional deformation), will be used to relate tension measurements in devices of different diameters using variable indentation distances.

When the membrane is mounted in the culture device at the correct tension it will possess a certain elastic modulus.

It is assumed the probe is in frictionless contact and for simplicity that the deformed membrane conforms to a conical geometry with a uniform strain. It is also assumed the membrane is a linearly elastic material.

The probe applies an average stress over the membrane given by, <MAT>.

Where F is the normal force applied by the probe and Acs is the cross sectional area of the membrane.

Due to this stress, the membrane deforms to a ~conical geometry with a surface area defined as, <MAT>.

Where r is the membrane diameter and h is the indentation distance, or equally the height of the cone formed by the stretched membrane. The area strain ε over the stretched membrane is given by, <MAT>.

Where ΔA, the change in area of the membrane, is given by, <MAT> <MAT>.

The elastic modulus E can now be fully defined as, <MAT>.

In examples of the present invention described herein the skin sample holder our has a <NUM> diameter culture device with a <NUM> indentation/push depth (<NUM>-<NUM>) to characterise the optimum tension F<NUM> required in our membrane.

Therefore this will be our reference from which the conversion factors will be calculated, however, this approach can be applied generally to any diameter/depth values used for membrane characterisation.

To find the required probe force reading Fx on a different diameter device we equate the elastic moduli. <MAT> <MAT> <MAT> <MAT>.

Using the values for our membrane characterisation (<NUM>-<NUM>) we can generate a matrix of conversion/correction factors to allow testing of devices of different diameters and also using different indentation depths/distances.

The correction factor (λ) relates the measured force as follows, <MAT>.

Here, we see that, as expected, the correction factor for a <NUM> diameter device with an indentation depth of <NUM> is unity or <NUM>.

Maintaining the device diameter at <NUM> but increasing indentation depth we see the factor decrease which is logical considering the membrane is stretched to a greater extent with an increase indentation. Similarly, with a smaller diameter device/membrane the correction factor decreases for a given indentation depth.

Due to the assumption of a linearly elastic material it is advised that strain is below <NUM>% during tension measurements, i.e. the membrane is not stretched by an amount greater than <NUM>% by the probe.

It will be appreciated that it is convenient for the apparatus and its component parts to have a generally circular form. Other shapes may be used, such as square, oval or rectangular, and as such fall within the scope of the present invention.

Claim 1:
A skin sample culture apparatus (<NUM>, <NUM>) which comprises:
a base frame (<NUM>), with a skin sample receiving surface upon which at least part of a skin sample
may be placed and which extends across an area defined by the shape of the frame;
and a securing member (<NUM>) which is releasably connectable to the base frame and a grip which holds the skin sample under tension, characterised in that the base frame comprises a plurality of channels (<NUM>) which extend through the side of the base frame and
wherein at least one of said channels is an upper channel positioned towards the skin sample receiving surface of the base frame, and at least one of said channels is a lower channel positioned towards the bottom surface of the base frame upon which it rests in use in a culture medium receptacle containing culture medium,
wherein the upper channel is positioned for allowing air or other ambient gas to exit from the position below a skin sample when it is situated on the base frame within a culture medium receptacle containing culture medium.