IMPROVEMENTS IN AND RELATING TO SKIN SAMPLE VIABILITY
A method for improving the ex vivo viability of a skin tissue type by mounting samples of the skin tissue type in the skin sample holder, applying separate tensions to at least some of the samples and measuring the ability of at least some of the skin tissue samples to maintain live skin characteristics, such that, for the skin tissue type, the measurements of live skin characteristics determine one or more value of skin tension which improve the ex vivo viability of the skin sample type. The viability may be determined by measuring keratin 17 expression by a wounded skin sample.
The present invention relates to improvements in and relating to skin sample viability and in particular to improving the viability of skin samples which are used to study the biology of skin and to evaluate drug delivery, metabolism, toxicity and efficacy of therapeutics.
BACKGROUND TO THE INVENTIONSkin 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 0.5 mm thick whereas the skin on the palms and the soles of the feet is 4 mm thick and is around 1.4 mm 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, microfibrils, 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 sample types commonly used include human, porcine and murine skin. 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 academic researcher and the cosmetic industry with regards to safety and efficacy assessment for the increasing number of products developed for topical application. Within a global in vitro toxicity market expected to reach $27 billion by 2021, there is a growing demand for ex vivo skin models. 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.
SUMMARY OF THE INVENTIONIn accordance with a first aspect of the invention there is provided a method for improving the ex vivo viability of a skin tissue type, the method comprising the steps of:
mounting samples of the skin tissue type in the skin sample holder;
applying separate tensions to at least some of the samples; and
measuring the ability of at least some of the skin tissue samples to maintain live skin characteristics, such that, for the skin tissue type, the measurements of live skin characteristics determine one or more value of skin tension which improve the ex vivo viability of the skin sample type.
In the present application, ex vivo means any process performed outside the living organism and includes in vitro processes which occur in a test tube, culture dish or the like and explant processes where living cells, tissues, or organs are transferred from animals to a nutrient media.
Preferably, the one or more value of skin tension comprises a continuous range.
Preferably, the step of applying tension comprises stretching the skin sample and holding it in position upon the skin sample holder.
Preferably, the step of measuring the tension comprises applying a measurable force to the surface of a skin sample which is held in the skin sample holder.
Preferably, the force is applied by contacting a probe with the surface of the skin sample displacing the skin a predetermined amount so as to stretch the skin, wherein the amount of force for a given displacement provides a measure of skin tension.
Preferably, where an annular skin sample holder is used, a correction factor is applied to the force measurement to account for the effect of the radius of the skin sample holder.
Preferably, the step of measuring the ability of the first or other sample to maintain live skin characteristics comprises, wounding the skin and measuring keratin 17 expression as an indication of tissue viability.
Preferably, the step of wounding the skin is conducted using a device which ensures that wounds have a consistent depth and coverage across the skin.
Preferably, the step of wounding the skin uses a laser source wound the skin.
Preferably, the step of measuring the ability of the first or other sample to maintain live skin cellular characteristics comprises measuring NRF2 activation in response to small molecules by measuring NQO1 mRNA levels.
Preferably, the step of measuring the ability of the first or other sample to maintain live skin mitochondrial characteristics comprises measuring mitochondrial metabolic activity.
Preferably, the skin sample holder comprises a culture dish with a skin sample holder which holds the skin sample and applies tension to the skin tissue sample.
Preferably, the skin sample holder is adapted to apply different levels of tension to the skin sample.
Preferably, the skin sample holder comprises a base frame, with a skin sample receiving surface upon which at least part skin sample may be placed and which extends across an area defined by the shape of the frame; and
a securing member which is releasably connectable to the base frame and a grip which holds the skin sample under tension.
Preferably, the skin tissue type is porcine, murine or human.
Preferably, for porcine and human skin, the one or more value of skin tension comprises a range of 0.22 to 0.57 N, assuming a specific and constant diameter and deflection distance.
More preferably, the range comprises 0.29 to 0.46 N, assuming a specific and constant diameter and deflection distance.
In accordance with a second aspect of the invention there is provided a skin sample holder for holding an ex vivo skin sample under tension, the skin sample tension being determined by the method of the first aspect of the present invention.
In accordance with a third aspect of the invention there is provided a method for measuring the tension in a skin sample culture apparatus in accordance with the method of the first aspect of the invention, the apparatus comprising:
a spacer which couples the force meter to the skin sample culture apparatus;
a probe which is extendable from the spacer for contact with a skin sample and which applies a force to the skin sample;
a force meter for measuring the force applied
wherein the amount of force for a given displacement, defined by the spacer length, provides a measure of skin tension.
Preferably, the step of measuring the tension comprises applying a measurable force to the surface of a skin sample which is held in the skin sample holder.
Preferably, the force is applied by contacting a probe with the surface of the skin sample displacing the skin a predetermined amount so as to stretch the skin, wherein the amount of force for a given displacement provides a measure of skin tension.
Preferably, where an annular skin sample holder is used, a correction factor is applied to the force measurement to account for the effect of the radius of the skin sample holder.
Preferably, the probe further comprises computing means for calculating the correction factor.
For example, the computing mean may be contained in the probe as hardware, firmware and/or software or may be a separate module or device.
Preferably, the probe is spherical.
Optionally, the probe is conical.
Optionally, the probe is cylindrical.
In accordance with a fourth aspect of the invention thee is provided a method of culturing a skin tissue sample at a tension determined by the method of the first aspect of the invention.
Preferably, the skin tissue type is porcine, murine or human and is cultured at a tension in the range 0.22 to 0.57 N, assuming a specific and constant diameter and deflection distance. More preferably, the range comprises 0.29 to 0.46 N.
The present invention will now be described with reference to the accompanying drawings in which:
As shown in
The ability of the first sample to maintain live skin characteristics 7 is determined. In this example of the present invention, the live characteristics of the skin sample are measured by wounding the skin using a laser and measuring the excretion of keratin 17 (K17) in response to the wound as described in
One or more separate samples of the skin tissue type are then separately mounted in the skin sample holder 9 and separate tensions are applied to each of the subsequent samples 11. In each case the ability of the skin tissue sample to maintain live skin characteristics is measured 13, such that, for the skin tissue type, a value or range of values of surface tension is determined which improve the in-vitro viability of the skin sample type.
The optimal tension window can then be determined based on the ability to respond to wounding (15). It will be appreciated that the method described above shows sample measurements being made one after another, such measurements may be made at the same time using separate skin sample holders for each sample.
When a known displacement h 65, is exerted upon the skin sample 61, a force reading is recorded. This provides a measure of skin tension.
In cases where the device is annular, devices having different radii may be used.
Where the radii are different as shown in
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.
In essence 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,
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,
Acone=πr√{square root over (r2+h2)}
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 E over the stretched membrane is given by,
Where ΔA, the change in area of the membrane, is given by,
ΔA=Acone−A0
ΔA=πr√{square root over (r2+h2)}−πr2
Therefore ε can be written as,
The elastic modulus E can now be fully defined as,
In examples of the present invention described herein the skin sample holder our has a 15 mm diameter culture device with a 3 mm indentation/push depth (15-3) to characterise the optimum tension F0 required in our membrane.
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.
Using the values for our membrane characterisation (15-3) 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,
F15-3=λFx-y
Here, we see that, as expected, the correction factor for a 15 mm diameter device with an indentation depth of 3 mm is unity or 1.
Maintaining the device diameter at 15 mm 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 10% during tension measurements, i.e. the membrane is not stretched by an amount greater than 10% by the probe.
Defatted skin was cultured at a user defined tension in the device of
In this example of the present invention, the process for ensuring skin was mounted at a specific tension was an iterative process which required the skin sample to be fully mounted in the culture device before measuring the tension of the sample. Following mounting, the force meter 41 of
In order to determine the manner in which ex vivo viability of a skin tissue type might be improved, measurements of the ability of the skin sample to maintain live skin characteristics such as metabolic activity, response to small molecule drugs, and the ability to respond to injury were made.
1. Tissue Viability (Keratin 17 Expression Following Wounding)Human and porcine skin was cultured under a range of tensions (i.e., no tension up to fully stretched) for 24-48 hours before being exposed to wounding using an Er-YAG laser set to a constant ablation depth and coverage. After 48 h, the skin was harvested, formalin fixed and stained for keratin 17 expression, which is a marker of the ability of skin to respond to wounding. A healthy skin sample which exhibits live skin characteristics would be expected to express keratin 17 in response to wounding. In the following pictures, K17 expression is shown by the white and/or lighter shaded areas in the slide.
For
These findings are further supported by
Staining shows uniform induction of K17 in the wounded in vivo skin. The human skin cultured at a level of tension at which the ex vivo skin sample's viability was improved or optimised in accordance with the method of the present invention showed a wound response similar to what is observed in vivo. This is clearly shown when
One of the genes that is upregulated upon NRF2 activation is NAD(P)H dehydrogenase [quinone] 1 (NQO1).
Improvements and modifications may be incorporated herein without deviating from the scope of the invention.
Claims
1. A method for improving the ex vivo viability of a skin tissue type, the method comprising the steps of:
- mounting samples of the skin tissue type in a skin sample holder;
- applying separate tensions to at least some of the samples; and
- measuring the ability of at least some of the skin tissue samples to maintain live skin characteristics, such that, for the skin tissue type, the measurements of live skin characteristics determine one or more value of skin tension which improve the ex vivo viability of the skin sample type.
2. A method as claimed in claim 1 wherein, the one or more value of skin tension comprises a continuous range of tension values.
3. A method as claimed in claim 2 wherein tension measurements are made in multiple samples of the skin tissue type to populate the range of tension values between unstretched and a maximum stretch.
4. A method as claimed in claim 3 wherein the range of tension values is used used in subsequent experiments which further ex vivo experiments in which the skin tissue type is used.
5. A method as claimed in claim 1 wherein, the step of applying tension comprises stretching the skin sample and holding it in position upon the skin sample holder.
6. A method as claimed in claim 1 wherein, the step of measuring the tension comprises applying a measurable force to the surface of a skin sample which is held in the skin sample holder.
7. A method as claimed in claim 6 wherein, the force is applied by contacting a probe with the surface of the skin sample displacing the skin a predetermined amount so as to stretch the skin, wherein the amount of force for a given displacement provides a measure of skin tension.
8. method as claimed in claim 6 wherein, where an annular skin sample holder is used, a correction factor is applied to the force measurement to account for the effect of the radius of the skin sample holder.
9. A method as claimed in claim 1 wherein, the step of measuring the ability of the first or other sample to maintain live skin characteristics comprises, wounding the skin and measuring keratin 17 expression as an indication of tissue viability.
10. A method as claimed in claim 9 wherein, the step of wounding the skin is conducted using a device which ensures that wounds have a consistent depth and coverage across the skin.
11. A method as claimed in claim 9 wherein, the step of wounding the skin uses a laser source to wound the skin.
12. A method as claimed in claim 1 wherein, the step of measuring the ability of the first or other sample to maintain live skin cellular characteristics comprises measuring NRF2 activation in response to small molecules by measuring NQO1 mRNA levels.
13. A method as claimed in claim 1 wherein, the step of measuring the ability of the first or other sample to maintain live skin metabolic characteristics comprises measuring metabolic activity.
14. A method as claimed in claim 1 wherein, the skin sample holder applies tension to the skin tissue sample.
15. A method as claimed in claim 14 wherein the skin sample holder is mounted in a culture dish.
16. A method as claimed in claim 1 wherein, the skin sample holder is adapted to apply different levels of tension to the skin sample.
17. A method as claimed in claim 1 wherein, the skin sample holder comprises a base frame, with a skin sample receiving surface upon which at least part skin sample may be placed and which extends across an area defined by the shape of the frame; and
- a securing member which is releasably connectable to the base frame and a grip which holds the skin sample under tension.
18. A method as claimed in claim 1 wherein, the skin tissue type is porcine, murine or human.
19. A method as claimed in claim 15 wherein, for porcine and human skin, the one or more value of skin tension comprises a range of 0.22 to 0.57 N, assuming a specific and constant diameter and deflection distance.
20. A method as claimed in any preceding claim 16 wherein, the range comprises 0.29 to 0.46 N, for a given skin sample holder diameter and deflection distance.
21. A method of culturing a skin tissue sample at a tension determined by the method of claim 1.
22. A method of culturing a skin sample as claimed in claim 21 wherein, the skin tissue type is porcine or human and is cultured at a tension in a range of 0.22 to 0.57 N, assuming a specific and constant diameter and deflection distance.
23. A method of culturing a skin sample as claimed in claim 21 wherein, the skin tissue type is porcine or human and is cultured at a tension in a range of 0.29 to 0.46 N, for a given skin sample holder diameter and deflection distance.
Type: Application
Filed: May 22, 2017
Publication Date: Jul 18, 2019
Inventors: Robyn Patricia Hickerson (Balmullo), Michael John Conneely (Dundee)
Application Number: 16/302,792