U.S. patent application number 16/302792 was filed with the patent office on 2019-07-18 for improvements in and relating to skin sample viability.
The applicant listed for this patent is The University of Dundee. Invention is credited to Michael John Conneely, Robyn Patricia Hickerson.
Application Number | 20190219564 16/302792 |
Document ID | / |
Family ID | 56369691 |
Filed Date | 2019-07-18 |
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United States Patent
Application |
20190219564 |
Kind Code |
A1 |
Hickerson; Robyn Patricia ;
et al. |
July 18, 2019 |
IMPROVEMENTS IN AND RELATING TO SKIN SAMPLE VIABILITY
Abstract
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.
Inventors: |
Hickerson; Robyn Patricia;
(Balmullo, GB) ; Conneely; Michael John; (Dundee,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of Dundee |
Dundee |
|
GB |
|
|
Family ID: |
56369691 |
Appl. No.: |
16/302792 |
Filed: |
May 22, 2017 |
PCT Filed: |
May 22, 2017 |
PCT NO: |
PCT/GB2017/000081 |
371 Date: |
November 19, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 23/48 20130101;
G01N 1/36 20130101; G01N 33/5005 20130101; C12M 21/08 20130101;
C12M 35/04 20130101; C12N 5/0629 20130101; A01N 1/0242 20130101;
A01N 1/0263 20130101; C12Q 1/6876 20130101; A01N 1/0278 20130101;
C12M 23/38 20130101; C12M 23/46 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50; C12Q 1/6876 20060101 C12Q001/6876; C12N 5/071 20060101
C12N005/071; G01N 1/36 20060101 G01N001/36 |
Foreign Application Data
Date |
Code |
Application Number |
May 20, 2016 |
GB |
GB1608899.9 |
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.
Description
INTRODUCTION
[0001] 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 INVENTION
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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.
[0011] 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 INVENTION
[0012] In 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:
[0013] mounting samples of the skin tissue type in the skin sample
holder;
[0014] applying separate tensions to at least some of the samples;
and
[0015] 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.
[0016] 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.
[0017] Preferably, the one or more value of skin tension comprises
a continuous range.
[0018] Preferably, the step of applying tension comprises
stretching the skin sample and holding it in position upon the skin
sample holder.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] Preferably, the step of wounding the skin uses a laser
source wound the skin.
[0025] 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.
[0026] Preferably, the step of measuring the ability of the first
or other sample to maintain live skin mitochondrial characteristics
comprises measuring mitochondrial metabolic activity.
[0027] 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.
[0028] Preferably, the skin sample holder is adapted to apply
different levels of tension to the skin sample.
[0029] 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
[0030] a securing member which is releasably connectable to the
base frame and a grip which holds the skin sample under
tension.
[0031] Preferably, the skin tissue type is porcine, murine or
human.
[0032] 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.
[0033] More preferably, the range comprises 0.29 to 0.46 N,
assuming a specific and constant diameter and deflection
distance.
[0034] 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.
[0035] 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:
[0036] a spacer which couples the force meter to the skin sample
culture apparatus;
[0037] a probe which is extendable from the spacer for contact with
a skin sample and which applies a force to the skin sample;
[0038] a force meter for measuring the force applied
[0039] wherein the amount of force for a given displacement,
defined by the spacer length, provides a measure of skin
tension.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] Preferably, the probe further comprises computing means for
calculating the correction factor.
[0044] 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.
[0045] Preferably, the probe is spherical.
[0046] Optionally, the probe is conical.
[0047] Optionally, the probe is cylindrical.
[0048] 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.
[0049] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] The present invention will now be described with reference
to the accompanying drawings in which:
[0051] FIG. 1 is a flow diagram illustrating an example of a method
in accordance with the present invention;
[0052] FIG. 2A is a perspective view of an example of a device used
in the method of the present invention; FIG. 2B is a side view of
the device of FIG. 2A;
[0053] FIG. 3 is a side view of a force meter fitted with a spacer
collar used to measure skin sample tension;
[0054] FIG. 4 is a schematic representation of a skin tension
measurement;
[0055] FIG. 5 is a schematic representation of a skin tension
measurement for a device having a first radius;
[0056] FIG. 6 is a schematic representation of a skin tension
measurement for a device having a second radius;
[0057] FIG. 7 is a schematic representation of a skin tension
measurement for a device having a third radius;
[0058] FIG. 8 is an example of a calibration curve for correcting
measured skin tension measurements with respect to device
radius;
[0059] FIG. 9a shows K17 levels in a large section of wounded skin
without tension and FIG. 9b shows K17 levels in a large section of
wounded skin with tension;
[0060] FIGS. 10a to 10f show keratin 17 levels for a porcine skin
sample under varying levels of tension for unwounded skin and FIGS.
10g to 10p show keratin 17 levels for a porcine skin sample under
varying levels of tension for wounded skin;
[0061] FIG. 11a to FIG. 11c show keratin 17 levels for a human skin
sample under varying levels of tension for wounded skin;
[0062] FIG. 12a shows a collection of slides from which K17 levels
have been determined for unwounded and wounded porcine skin, FIG.
12b shows a collection of slides from which K17 levels have been
determined for wounded human skin and FIG. 12c is a graph which
plots estimated keratin 17 levels against tension;
[0063] FIGS. 13a to 13c show unwounded skin samples which are in
vivo, from an industry standard source and held under tension in
accordance with an example of the method of the present invention
and FIGS. 13d to 13f show laser wounded skin samples which are in
vivo, from an industry standard source and held under tension in
accordance with the method of the present invention;
[0064] FIG. 14 is a graph which plots NQO1 mRNA levels as a measure
of NRF2 activity indicating cellular viability in the skin; and
[0065] FIG. 15 is a graph which plots mitochondrial metabolic
activity for fresh skin, skin under tension in accordance with an
example of the method of the present invention, skin under no
tension and devitalised skin.
DETAILED DESCRIPTION OF THE DRAWINGS
[0066] FIG. 1 is a flow diagram 1 which illustrates an example of a
method in accordance with the present invention. The method of the
present invention allows ex vivo samples of a skin type, such as
porcine, human or murine skin to be tested in order to determine
preferred and/or optimum skin tension levels.
[0067] As shown in FIG. 1 a first sample of a skin tissue type is
mounted 3 in a skin sample holder such as, for example, the device
shown in FIGS. 2A and 2B. Using the device of FIGS. 2A and 2B, a
tension is applied across the skin sample and measured 5 using a
force meter or by other means. In this example of the present
invention, the tension is measured using the force meter of FIG.
3.
[0068] 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 FIGS. 3
to 12.
[0069] 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.
[0070] 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.
[0071] FIG. 2A is a perspective view of an example of a device used
in the method of the present invention and FIG. 2B is a side view
of the device of FIG. 2A. A skin culture dish 21 is shown
containing a culture medium 25. The device 23 has a base 33 upon
which a skin sample 27 is placed in use, and a cap 29 which is
placed on top of a skin sample, the skin extending to the outer
circumference 26 of the cap 29 and base 33. Fixings 31 extend
through holes in the cap 29 and corresponding holes in the base 33
and are used to attach the cap 29 to the base 33. The fixings
provide for the removable attachment of the cap 29 to the base 33.
In use, the skin sample 27 is stretched across the base and secured
to the base 33 and cap 29 using fixings 31.
[0072] FIG. 3 shows an apparatus for measuring the tension in a
skin sample which has been placed in the device of FIGS. 2a and 2b.
The apparatus comprises a force meter 41, a spacing collar 43 which
slides over the probe shaft which extends downwards from the body
of the force meter 41. The collar 43 is a cylindrical tube with a
diameter and circumferences which matches that of the cap such that
the end of the collar rests upon the cap 57 of the device of FIG.
4. The spherical indenter 45 exerts a force upon the skin
sample.
[0073] FIG. 4 is a schematic representation 51 of a skin tension
measurement. It shows a spherical indenter 53, a skin sample
culture device 55 similar to that of FIGS. 2a and 2b which has a
cap 57, countersunk fixings 59 which extend through the cap 57 into
the base 63 to hold a skin sample in place and under tension. Both
the cap and the base comprise annuli of substantially identical
inner and outer radius. The radius of the cap 67 as measured at the
inner diameter and the vertical displacement 65 are also shown.
[0074] 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.
[0075] In cases where the device is annular, devices having
different radii may be used.
[0076] FIG. 5 is a schematic representation of a skin tension
measurement for a device having size 71 with a first radius 79, a
cap 73, base 75. The vertical displacement 81 of the skin sample 77
as caused by the indenter 83 is shown.
[0077] FIG. 6 is a schematic representation of a skin tension
measurement for a device having size 91 with a first radius 99, a
cap 93, base 95. The vertical displacement 101 of the skin sample
97 as caused by the indenter 103 is shown.
[0078] FIG. 7 is a schematic representation of a skin tension
measurement for a device having size 111 with a first radius 119, a
cap 113, base 115. The vertical displacement 121 of the skin sample
117 as caused by the indenter 123 is shown.
[0079] Where the radii are different as shown in FIGS. 5 to 7, it
is necessary to account for the effect of the radius on the
measured value of tension.
[0080] 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.
[0081] In essence when the membrane is mounted in the culture
device at the correct tension it will possess a certain elastic
modulus.
[0082] 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.
[0083] The probe applies an average stress over the membrane given
by,
.sigma. = F A CS ##EQU00001##
[0084] Where F is the normal force applied by the probe and
A.sub.cs is the cross sectional area of the membrane.
[0085] Due to this stress, the membrane deforms to a .about.conical
geometry with a surface area defined as,
A.sub.cone=.pi.r {square root over (r.sup.2+h.sup.2)}
[0086] 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,
= .DELTA. A A 0 ##EQU00002##
[0087] Where .DELTA.A, the change in area of the membrane, is given
by,
.DELTA.A=A.sub.cone-A.sub.0
.DELTA.A=.pi.r {square root over (r.sup.2+h.sup.2)}-.pi.r.sup.2
[0088] Therefore .epsilon. can be written as,
= r 2 + h 2 - r r ##EQU00003##
[0089] The elastic modulus E can now be fully defined as,
E = .sigma. ##EQU00004##
[0090] 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 F.sub.0 required in our membrane.
[0091] 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.
[0092] To find the required probe force reading F.sub.x on a
different diameter device we equate the elastic moduli.
E 0 = E x ##EQU00005## .sigma. 0 0 = .sigma. x x ##EQU00005.2## F 0
/ A CS 0 0 = F x / A CS x x ##EQU00005.3## F 0 = F x A CS 0 0 A CS
x x ##EQU00005.4##
[0093] 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.
[0094] The correction factor (.lamda.) relates the measured force
as follows,
F.sub.15-3=.lamda.F.sub.x-y
[0095] 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.
[0096] 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.
[0097] 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.
[0098] FIG. 8 shows a 3D surface plot 131 of a section of the
calculated conversion-correction factor matrix. Correction factors
137 are related to indentation depths 133 and device diameter 135
with the use of a colour scale 139.
Experimental Results
[0099] Defatted skin was cultured at a user defined tension in the
device of FIG. 2. The tension experienced across the surface of the
skin sample was measured through an indentation measurement using
the force meter of FIG. 3. Force was applied perpendicular to the
surface of the skin and the skin tissue was stretched by a
user-defined fixed distance (in this instance 3 mm).
[0100] 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 FIG. 3, fitted with a
spherical probe 45 and a spacer collar 43 to fix the indentation
distance, was placed centrally over the skin sample holder. The
spacing collar is put into firm contact with the top of the culture
device, pushing the probe into contact with the skin, thus
displacing it a defined distance. A record of the peak force
imparted on the probe is then taken as a measure of tension. In
order to fine tune the tension, the culture device was partially
disassembled by loosening the screw fixings allowing small
adjustments to the skin to be made. Tension was regulated to a
relatively high degree of accuracy using this method.
[0101] 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)
[0102] 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.
[0103] FIG. 9 shows keratin 17 levels in porcine skin with and
without tension. FIG. 9a shows that keratin 17 expression is
observed near the site of wounding when the skin is cultured
without tension. FIG. 9b shows that keratin 17 expression at
consistent levels throughout the skin sample that is cultured at
tension. The results show that the unwounded skin expresses little
or no Keratin 17 until the skin tension reaches a level at which
the skin is damaged by the stretch itself in FIG. 9f. In the case
of healthy wounded skin, the expectation would be that keratin 17
expression would occur.
[0104] For FIGS. 10 and 11, images were taken of skin tissue
between the laser wounds.
[0105] FIGS. 10a to 10f show keratin 17 levels for an unwounded
skin sample under varying levels of tension and FIGS. 10g to 10p
show keratin 17 levels for a wounded skin sample under varying
levels of tension. In each image the skin tension (in newtons; N)
is shown in the top right hand corner of the image.
[0106] FIGS. 10g and 10h show skin samples with no tension and
tension of 0.13 N respectively. At these tension levels, there is
little or no keratin 17 expression. Keratin 17 expression is
observed in FIGS. 10i, 10j and 10k which are for skin samples with
tensions of 0.29 N, 0.34 N and 0.45 N respectively. The largest
apparent keratin 17 expression is seen at a tension of 0.29 N. FIG.
10l shows little or no keratin 17 expression at a tension of 0.57
N. Based on these results, it can be concluded that there is an
optimum range of tension values at which ex vivo skin samples
should be held in order to maximise their viability. The range of
certain viability is between 0.29 and 0.45 N for porcine skin. Skin
cultured at a tension less than or equal to 0.13 or more than or
greater than 0.57 N shows considerably reduced viability.
[0107] FIGS. 11a to 11i show keratin 17 levels for a wounded human
skin sample prepared as described above under varying levels of
tension. In each image the skin tension (in newtons; N) is shown in
the bottom right hand corner of the image.
[0108] FIGS. 11a and 11b, show skin samples at no tension and
tension of 0.22 N respectively. At these tension levels, there is
little or no keratin 17 expression. Keratin 17 expression is
observed in FIGS. 11c and 11d which are for skin samples with
tensions of 0.31 N and 0.46 N respectively. FIG. 11e shows little
or no keratin 17 expression at a tension of 0.63 N. Based on these
results, it can be concluded that there is an optimum range of
tension values at which ex vivo skin samples should be held in
order to maximise their viability. The range of certain viability
is between 0.31 and 0.46 N for human skin. Skin cultured at a
tension less than or equal to 0.22 or greater than 0.63 N shows
considerably reduced viability.
[0109] These findings are further supported by FIG. 12. FIG. 12a
shows a collection of slides from which K17 levels have been
determined for unwounded and wounded porcine skin. FIG. 12b shows a
collection of slides from which K17 levels have been determined for
wounded human skin. FIG. 12c is a graph which plots estimated
keratin 17 levels 155 as measured by the normalised average pixel
value from the images against skin tension 153 for a porcine skin
control sample, a wounded porcine skin sample and a wounded human
skin sample. The key 156 provides information to distinguish
between the results for the different samples used. The peak value
is within the optimum range 157 shown in FIGS. 10 and 11. The
second peak is caused by keratin 17 which has been expressed due to
damage caused by the skin being overstretched 159.
[0110] FIGS. 13a to 13c are pictures of unwounded skin samples
which are, respectively, in vivo, from an industry standard source
and held under tension in accordance with an example of the method
of the present invention. FIGS. 13d to 13f are pictures of laser
wounded skin samples which are, respectively, in vivo, from an
industry standard source and held under tension in accordance with
the method of the present invention. The in vivo skin was prepared
from a biopsy taken from a human volunteer 24 h after wounding with
the Er-YAG laser.
[0111] 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 FIGS. 13d and 13f are compared. The industry standard model
did not show any response to wounding as shown in FIG. 13e.
2. Cellular Viability (NQO1 Expression)
[0112] FIG. 14 is a graph 163 which plots NQO1 mRNA levels upon
treatment with an NRF2 activator. FIG. 14 summarises an experiment
in which skin cultured using the method of the present invention
was treated with a small molecule NRF2 activator (TBE-31) in order
to evaluate the intracellular viability of the tissue. NRF2
transcriptionally regulates multiple genes that play both direct
and indirect roles in activating intracellular anti-oxidative
pathways.
[0113] One of the genes that is upregulated upon NRF2 activation is
NAD(P)H dehydrogenase [quinone] 1 (NQO1). FIG. 14 is a graph which
shows NQO1 mRNA levels 167 obtained from total RNA isolated from
the skin treated with TBE-31 compound (a potent NRF2 activator) at
the indicated concentrations 169. It is shown here that the
cellular viability of the skin cultured in this device responds to
NRF2 activators to a greater extent than does the industry
standard.
3. Mitochondrial Viability (MTT Conversion Assay)
[0114] FIG. 15 is a graph 171 which plots metabolic activity 175
for fresh skin 177, skin under tension in accordance with an
example of the method of the present invention 179, skin under no
tension 181 and devitalised skin 183. The graph shows similar
responses for fresh skin 177 and skin under tension in accordance
with an example of the method of the present invention 179.
[0115] Improvements and modifications may be incorporated herein
without deviating from the scope of the invention.
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