Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND APPARATUSES HARVESTING, MODIFYING AND
REIMPLANTATION OF DERMAL MICRO -ORGANS
FIELD OF THE INVENTION
[001] The invention relates to the field of tissue based micro-organs,
therapeutic tissue
based micro- organs and methods and apparatuses for harvesting, processing,
implanting
and manipulating dermal tissue.
BACKGROUND OF THE INVENTION
[002] Various methods for delivering therapeutic agents are known. For
example,
therapeutic agents can be delivered orally, transdermally, by inhalation, by
injection and by
to depot with slow release. In each of these cases the method of delivery
is limited by the body
processes that the agent is subjected to, by the requirement for frequent
administration, and
limitations on the size of molecules that can be utilized. For some of the
methods, the
amount of therapeutic agent varies between administrations.
[0004] A dermal micro-organ (DMO), which can be sustained outside the body
("ex vivo"
or "in vitro") in an autonomously functional state for an extended period of
time, and to
which various manipulations can be applied, may then be implanted
subcutaneously or
within the body for the purpose of treating diseases, or disorders, or for
plastic surgical
purposes. The DMO can be modified to express a gene product of interest. These
modified dermal micro-organs are generally referred to as Dermal Therapeutic
Micro-
Organs (DTM05).
[0005] Skin micro-organs (MO), including layers of epidermal and dermal
tissues, for
example; as outlined in PCT/1L02/0880, have been observed to be associated
with a
number of clinical challenges. Harvesting of a skin sample leaves a
superficial wound on
the patient that may last several weeks and may leave scars. The harvested
skin sample
requires significant processing to generate micro-organs from this sample.
Also,
implantation of skin micro-organs subcutaneously or deeper in the body have
been found to
result in the development of keratin cysts or keratin micro-cysts.
Additionally, implantation
of skin micro-organs as a graft onto the skin surface in "slits" requires
significant technical
expertise in order to handle the MO while maintaining its proper orientation.
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[0006] Harvesting of dermis, e.g., to be used as a "filler material" in a
plastic surgical or
cosmetic procedure, is known in the art. Conventional harvesting techniques
include using a
dermatome or scalpel to peel away a layer of epidermis in order to expose a
section of
dermis. The dermatome or scalpel may then be used again to manually harvest
the exposed
section of dermis.
[0007] Another conventional apparatus for harvesting dermis, albeit not
commonly used, is
the Martin Dermal Harvester marketed by Padgett (Part No. P-225) for the
indication of
harvesting dermal cores from the back for subsequent implantation into the
lips during
cosmetic lip augmentation procedures. To operate this device, which is not
commonly used,
to a sharpened cutting tube, which includes a reusable thick walled tube
with an inner
diameter of approximately 4.5 mm, is manually rotated at a very slow speed.
Using this
type of device generally requires applying pressure to the skin surface
directly above the
harvest site and installing sutures with active tugging as the cutting tube is
pushed forward.
Furthermore, the resulting harvested dermis is generally not uniform in
dimensions and
includes "plugs" of epidermis at either end of the dermal core.
SUMMARY OF THE INVENTION
[008] Embodiments of some aspects of the present invention provide a DMO/DTMO
with
the ability to be maintained ex-vivo in a generally viable state, which may
allow various
manipulations to be performed on the DMO, while keeping a high production and
secretion
level of the desired therapeutic agent, as disclosed in United States
Application Publication
No. US-2012/0201793-A1, which is incorporated herein by reference in its
entirety. In
addition, embodiments of some aspects of the present invention provide a
method of
harvesting a DMO and subsequently implanting a DTMO without forming keratin
cysts or
keratin microcysts, e.g., upon implantation of the DTMO subcutaneously or
deeper in the
body. Furthermore, it will be appreciated by persons skilled in the art that
the methods and
devices according to some embodiments of the present invention may be
relatively
uncomplicated and, therefore, the level of skill required from a professional
to carry out the
methods and/or to use the devices of the present invention may not be as
demanding as
those required in conventional procedures.
[009] Some exemplary embodiments of the invention provide a dermal micro-organ
(DMO) having a plurality of dermal components, which may include cells of the
dermal
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tissue and a surrounding matrix. The DMO according to embodiments of the
invention may
generally retain a micro-architecture and three dimensional structure of the
dermal organ
from which it is obtained and the dimensions of the DMO may allow passive
diffusion of
adequate nutrients and gases to the cells and diffusion of cellular waste out
of the cells so as
to minimize cellular toxicity and concomitant death due to insufficient
nutrition and
accumulation of waste.
[0010] In some exemplary embodiments of the invention, the DMO of the
invention does
not produce keratin or produces negligible amounts of keratin.
[0011] In some embodiments of the invention, the DMO does not produce keratin
and/or
to keratin cysts following subcutaneous or deeper implantation in a body.
[0012] In another embodiment of the invention, the DMO of the invention
produces micro
keratin cysts following that will atrophy within a relatively short period of
time, e.g., days
or weeks after subcutaneous implantation.
[0013] In another embodiment of the invention, the DMO contains hair follicles
and
sebaceous glands.
[0014] Further, exemplary embodiments of the invention provide a method and
apparatus
of harvesting a dermal micro-organ. The method may include stabilizing and/or
supporting
a skin-related tissue structure from which a dermal micro-organ is to be
harvested, e.g.,
such that the skin-related tissue structure is maintained at a desired shape
and/or position,
separating at least a DMO from the skin-related tissue structure, and
isolating the separated
DMO from the body. According to some of these exemplary embodiments, a support
structure may include a vacuum chamber able to hold the skin¨related tissue
structure in a
desired shape and position to enable a cutting tool to cut a DMO from the skin-
related
tissue structure. In one embodiment the support structure includes one or more
vacuum
channels to fluidically connect the vacuum chamber with at least one vacuum
source.
[0015] In one embodiment an apparatus for harvesting a dermal micro-organ
comprises (a)
a support structure to support a skin-related tissue structure from which the
DMO is to be
harvested, the support structure comprising a first tubular element, and the
first tubular
element comprising a site of insertion into the apparatus; (b) an introducer;
and (c) a cutting
tool. In some embodiments, the first tubular element is a guide channel that
may guide
additional elements, for instance a cutting tool, for insertion into the
supported skin-related
tissue.
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[0016] In one embodiment, an apparatus of the invention includes a vacuum
chamber
further comprising: (a.) two elevated protrusions, a near, i.e., proximal,
elevated protrusion
and a distal elevated protrusion relative to the site of insertion, wherein
the elevated
protrusions are able to support a plateau of at least epidermal and dermal
skin layers from
the skin-related tissue structure above the trajectory of a cutting tool; and
(b) a central
channel located between the two elevated protrusions, wherein the central
channel supports
epidermal and dermal skin layers from the skin-related tissue structure so
that the dermal
skin layer is within the trajectory of a cutting tool when the cutting tool is
inserted in the
first tubular element of the apparatus.
to [0017] In another embodiment, an apparatus of the present invention
includes an introducer
comprises a second tubular element and a fourth tubular element, wherein the
second
tubular element inserts through the fourth tubular element and extends beyond
the distal
end of the fourth tubular element and the second and fourth tubular elements
together insert
at the site of insertion coaxially within the first tubular element; and
further, the fourth
tubular element remains coaxial and within the first tubular element upon
withdrawal of the
second tubular element.
[0018] In yet another embodiment, an apparatus includes a third tubular
element, for
instance a cutting tool that inserts within and through the fourth tubular
element.
[0019] In one embodiment, the cutting tool comprises a coring tube able to cut
through the
skin-related tissue structure when advance along a cutting axis, wherein the
cutting axis is
substantially coaxial with the first tubular element. In another embodiment,
the coring tube
is a rotatable coring tube attached to a power source.
[0020] In one embodiment, a vacuum chamber includes a vacuum control
mechanism.
Implementation of a vacuum condition may for example, include placing a finger
over a
hole in the vacuum chamber, i.e., a vacuum hole, that when covered creates a
vacuum
condition. Alternative, release of a vacuum condition, may for example include
removal of
the finger from over the vacuum hole. In alternate embodiments, any covering
or
uncovering of the vacuum hole may be used to control vacuum and release
conditions,
respectfully. In one embodiment, a vacuum control mechanism relies on clamping
and
unclamping the vacuum line or opening and closing a valve in the vacuum
control line.
[0021] In one embodiment, a method of harvesting a DMO of the invention
includes the
steps of: positioning an apparatus at a harvest site in contact with an
epidermal surface of a
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subject; supporting a skin-related tissue structure at the harvest site from
which the DMO is
to be harvested; puncturing the skin-related tissue structure; cutting the DMO
from the
supported skin-related tissue structure; and recovering the DMO. In another
embodiment, a
harvesting method includes making only a single puncture point in the skin-
related
structure.
[0022] In one embodiment, a method of harvesting includes the use of a vacuum
to recover
the DMO from the coring tube into a closed container. In one embodiment, the
closed
container is a syringe body. In certain instances, the syringe may have an
attached septum.
In another embodiment, the DMO remains within the coring tube after retraction
from the
to harvest site and recovering the DMO comprises flushing the DMO from the
coring tube.
[0023] Further, exemplary embodiments of the invention provide a method of and
apparatus for implanting a dermal micro-organ. In one embodiment, the dermal
micro-
organ to be implanted is a genetically modified dermal micro-organ, which may
also be
referred to herein as a dermal therapeutic micro-organ (DTMO).
[0024] In one embodiment, an apparatus for implanting a DMO or a DTMO includes
(a) a
loading syringe comprising a first tubular element; (b) an implanting tool
comprising a
second tubular element; (c) a support structure to hold a skin-related tissue
structure in
place, wherein the DMO is to be implanted within the skin related tissue
structure; (d) an
introducer for puncturing the skin at a penetration site; (e) a stopper tool
able to be
connected to the support structure, the stopper tool comprising a tubular
element, and the
stopper tool assisting in maintaining the position of a DMO during retraction
of the
implanting tool.
[0025] In an exemplarily embodiment of the invention, a method for implanting
a DTMO
includes the steps of: (a) loading a DTMO into a loading syringe, the loading
syringe
comprising a first tubular element; (b) transferring the DTMO from the loading
syringe into
an implanting tool, the implanting tool comprising a second tubular element;
(c) placing an
implanting apparatus at an implantation site, wherein the apparatus is in
contact with an
epidermal layer of the subject and the implanting axis is generally parallel
with the
epidermal layer; (d) supporting a skin-related tissue structure at the
implantation site
wherein the DTMO is to be implanted into the skin-related structure; (e)
puncturing the skin
within the skin-related tissue structure at the penetration site, wherein the
skin is punctured
using an introducer including an inner needle and an outer sleeve element; (f)
removing the
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inner needle of the introducer and advancing the implanting tool into the skin-
related tissue
structure along the implanting axis; and (g) withdrawing the second tubular
element
wherein the DTMO remains within the skin-related tissue structure. In one
embodiment, a
stopper tool is used to assist in maintaining the position of a DTMO during
retraction of the
implanting tool. In certain instances, the first and second steps may be
optional, as a DTMO
may be loaded directly into the distal end of the implantation needle by
suctioning from the
back end of the needle with a syringe.
[0026] In another embodiment, a DTMO may be implanted by directly injecting
the
DTMO from a syringe through a needle whose distal end is positioned under the
skin or at
to another anatomical location, if linear implantation is not important.
[0027] Further exemplary embodiments of the invention provide a genetically
modified
dermal micro-organ expressing at least one recombinant gene product the DMO
having a
plurality of dermal components, including cells and matrix of the dermal
tissue, which
retain the micro-architecture and three dimensional structure of the dermal
tissue from
which they are obtained, and having dimensions selected so as to allow passive
diffusion of
adequate nutrients and gases to the cells and diffusion of cellular waste out
of the cells so as
to minimize cellular toxicity and concomitant death due to insufficient
nutrition and
accumulation of waste, wherein at least some of the cells of the DMO express
at least one
recombinant gene product or at least a portion of the at least one recombinant
gene product,
as described in United States Publication No. US-2012-0201793-A1, and
incorporated
herein in full. In still other exemplary embodiments, the at least one
recombinant gene
product is an at least one recombinant protein.
[0028] In some embodiments of the invention, the genetically modified DMO of
the
invention produces substantially no keratin.
[0029] In some embodiments, the invention provides a method of delivering to a
recipient
a recombinant gene product produced by the DMO.
[0030] In some embodiments, the invention provides a method of inducing a
local or
systemic physiological effect by implanting a DMO in a recipient.
[0031] In another embodiment the invention provides a method of delivering a
protein of
interest to a subject. The method includes implanting the genetically modified
DMO into
the skin, under the skin or at other locations in the body.
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[0032] In another embodiment, the invention provides a method of implanting a
DTMO so
as to avoid or to reduce keratin cyst formation.
[0033] In one embodiment, the invention provides a method for removal of an
implanted
DTMO.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Non-limiting embodiments of the invention are described in the
following
description, to be read with reference to the figures attached hereto. In the
figures, identical
and similar structures, elements or parts thereof that appear in more than one
figure are
to generally labeled with the same or similar references in the figures in
which they appear.
Dimensions of components and features shown in the figures are chosen
primarily for
convenience and clarity of presentation and are not necessarily to scale.
[0035] FIG. 1 is a schematic block diagram of an exemplary method of producing
and
utilizing dermal therapeutic micro-organs (DTM05), in accordance with an
exemplary
embodiment of the invention;
[0036] FIG. 2 is a schematic flowchart illustrating a method of harvesting a
DMO
according to some exemplary embodiments of the invention;
[0037] FIGS. 3A-3G are schematic illustrations of exemplary stages of
harvesting a DMO
in accordance with a method of FIG. 2;
[0038] FIGS. 4A-4E show embodiments of some elements of a harvesting
apparatus, a
medical drill for use with a harvesting apparatus and a syringe, septum and
collet for use
harvesting the DTMO. 4A shows an embodiment of a syringe (4002) for harvesting
and a
septum (4004). 4B shows an embodiment of a collet. 4C shows an embodiment of a
support
structure with a vacuum hole being covered by a finger. 4D shows an embodiment
of an
introducer: inner needle (4006¨needle) and outer guide (4008-white sleeve). 4E
shows an
embodiment of a cutting tube (4010), a drill (4012) and a drill hand piece
(4014).
[0039] FIG. 5A-B are schematic illustration of some components of a dermal
harvesting
apparatus according to another exemplary embodiment of the invention. FIG. 5A
is a
schematic illustration of a lateral view of embodiments of a harvesting
apparatus. FIG. 5B
is a schematic illustration of a cross-sectional view of the apparatus of FIG.
5A externally
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supporting a skin-related tissue structure from which a dermal micro-organ may
be
harvested at a desired position;
[0040] FIG. 6 is a schematic illustration of some components of a dermal
harvesting
apparatus according to yet another exemplary embodiment of the invention;
[0041] FIG. 7 is a flow chart illustrating a DTMO implanting method, according
to some
exemplary embodiments of the present invention;
[0042] FIGS. 8A-E show an embodiment of an implanting apparatus. 8A is an
embodiment
of a loading syringe, 8B is an embodiment of an implanting tool, 8C is an
embodiment of
an introducer, 8D is an embodiment of a support structure, and 8E is an
embodiment of a
stopper.
[0043] FIG. 9A-E are schematic illustrations of exemplary stages of implanting
a DTMO in
accordance with a method of FIG. 7;
[0044] FIGS. 10A-B show embodiments of a syringe with a septum and collet.
FIG. 10A
shows a syringe with a septum and collet inserted through a support structure
guide channel
and an outer sleeve, wherein the support structure is connected to a vacuum
source. FIG.
10B shows an embodiment of a syringe with a collet and needless valve attached
to the
back end of a coring needle. The T-end of an introducer (1008) is identified
for orientation
purposes;
[0045] FIG. 11 shows a schematic illustration of an embodiment of a support
structure.
[0046] FIG. 12 is a flow chart illustrating a method of removing a previously
implanted
DTMO;
[0047] FIG. 13 shows an embodiment of a syringe (1306) being attached to a
glued-on
male luercap (1302) near the back end of a coring needle (1304)
[0048] FIGS. 14A and 14B show embodiments of a harvested dermal micro-organ
(FIG.
14A) and harvesting (1402) and implanting (1404) sites on a human subject.
[0049] It will be appreciated that for simplicity and clarity of illustration,
elements shown
in the figures have not necessarily been drawn to scale. For example, the
dimensions of
some of the elements may be exaggerated relative to other elements for
clarity. Further,
where considered appropriate, reference numerals may be repeated among the
figures to
indicate corresponding or analogous elements.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
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[0050] In the following detailed description, numerous specific details are
set forth in order
to provide a thorough understanding of the invention. However, it will be
understood by
those skilled in the art that the present invention may be practiced without
these specific
details. In other instances, well-known methods, procedures, and components
have not been
[0051] The following description is presented to enable one of ordinary skill
in the art to
make and use the invention as provided in the context of a particular
application and its
requirements. Various modifications to the described embodiments will be
apparent to
those with skill in the art, and the general principles defined herein may be
applied to other
[0052] I. Exemplary Definitions of Terms Used Herein
[0053] The term "explant" as used herein, refers in some embodiments of the
invention, to
a removed section of living tissue or organ from one or more tissues or organs
of a subject,
[0054] The term "dermal micro-organ" or "DMO" as used herein, refers in some
embodiments of the invention, to an isolated tissue or organ structure derived
from or
identical to an explant that has been prepared in a manner conducive to cell
viability and
function, while maintaining at least some in vivo interactions similar to the
tissues or organ
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epithelial cells, other cell types, bases of hair follicles, nerve endings,
sweat and sebaceous
glands, and blood and lymph vessels. Wherever used herein below, the
description of the
embodiments related to DMO relates also to a MO. Further, whenever the term
"dermal
tissue" is used, it also relates to "dermal organ" and a DMO.
[0055] As used herein, the term "microarchitecture" refers, in some
embodiments of the
invention, to the characteristic of the explant in which, in one embodiment at
least about
50%, in another embodiment, at least about 60%, in another embodiment at least
about
70%, in another embodiment, at least about 80%, and in another embodiment, at
least about
90% or more of the cells of the population, maintain, in vitro, their physical
and/or
to functional contact with at least one cell or non-cellular substance with
which they were in
physical and/or functional contact in vivo. Preferably, the cells of the
explant maintain at
least one biological activity of the organ or tissue from which they are
isolated.
[0056] The term "donor" as used herein, refers in some embodiments of the
invention to a
subject, from which the explant is removed and used to form, or which is
already in the
form of, one or more micro-organs. In one embodiment, the donor is a human
subject. In
another embodiment, the donor is a non-human mammalian subject.
[0057] The term "therapeutic micro-organ (TMO)" as used herein, refers in some
embodiments of the invention to a dermal micro-organ (DMO) that can be used to
facilitate
a therapeutic objective, such as, for example, an DMO that has been
genetically altered or
modified to produce a therapeutic agent, such as a protein or an RNA molecule.
The
therapeutic agent may or may not be a naturally occurring body substance.
Wherever used
hereinbelow, the description of the embodiments related to TMO relates also to
DTMO
which is a therapeutic Dermal MO which may be in some embodiments of the
invention
genetically modified.
[0058] The term "implantation" as used herein, refers in some embodiments of
the
invention, to introduction of one or more TMOs or DTMOs into a recipient,
wherein said
TMOs or DTMOs may be derived from tissues of the recipient or from tissues of
another
individual or animal. The TMOs or DTMOs can be implanted in a slit within the
skin, can
be subcutaneously implanted, or may be implanted by placement at other desired
sites
within the recipient body. In one embodiment, a DTMO is derived from tissue of
the
recipient. In one embodiment, a DTMO is implanted substantially into the
dermal layer of
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skin tissue. In one embodiment, a DTMO is implanted between the dermal and fat
layer of
skin tissue.
[0059] The term "recipient" as used herein refers, in some embodiments of the
invention, to
a subject into which one or more TMOs or DTMOs are implanted. In one
embodiment, the
recipient is a human subject. In another embodiment, the recipient is a non-
human
mammalian subject. In some embodiments, a recipient receives one or more
autologous
TMOs or DTMO.
[0060] The term "in vitro" as used herein should be understood to include "ex-
vivo".
[0061] The term "coring tube" as used herein may relate, individually or
collectively, to the
terms "cutting tool", "cutting tube" and "coring needle", "coring tool", as
well as to any
other elements with similar functionalities. In some embodiments, a coring
needle of this
invention is for single use.
[0062] The term "implanting tool" as used herein may relate, individually or
collectively, to
the terms "implantation needle", "implanting needle" and "implanting tube", as
well as to
any other elements with similar functionalities. In some embodiments, an
implanting tool of
this invention is for single use.
[0063] The term "tubular element" as used herein refers to an element having
the form of
or consisting of a tube, wherein a tube refers to any of various usually
cylindrical structures
or devices. In one embodiment, a tubular element is an element having the form
of a hollow
elongated cylinder. In another embodiment, a tubular element is an element
having the form
of a cylindrical channel, e.g., a tunnel or channel cut through a solid mass.
In yet another
embodiment, a tubular element is open at both ends. In still another
embodiment, a tubular
element is open at one end. In a further embodiment, a tubular element
comprises a beveled
needle tip at one end. In another embodiment, a tubular element comprises at
least one
blunt end that is sharpened. In one embodiment a tubular element is an element
having the
form of a solid, non-hollow elongated cylinder, for instance a rod. In one
embodiment, a
tubular element may include a guide channel.
[0064] The term "rod" as used herein refers to a straight three-dimensional
element, which
has a solid geometry. In one embodiment, the rod has a circular cross section.
In one
embodiment, the rod has a non-circular cross-section.
[0065] The term "skin-related tissue structure", as used herein, refers to a
structure of tissue
components that may be stabilized and/or supported by apparatuses defined
herein to enable
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the harvesting of a dermal micro-organ therefrom or for the implantation of a
DMO therein.
A skin-related tissue structure may include components of the epidermal
tissue, and
components of the dermal tissue. Optionally, the skin-related tissue structure
may include
fat tissue and/or muscle tissue in the vicinity of the dermal tissue.
[0066] In one embodiment, a skin-related tissue structure of the present
invention includes
the skin tissue components drawn into the central channel under vacuum
conditions. In one
embodiment, a skin-related structure includes epidermal, dermal and fat
tissue. In another
embodiment, a skin-related structure includes epidermal, dermal, fat and
muscle tissue.
[0067] The term "central channel" as used herein may in some embodiments of
the
to invention be used interchangeable with the term "vacuum chamber".
[0068] The term "coaxial", as used herein, refers to a radial symmetry of
concentrically or
approximately concentrically positioned components. In this way, tubular
elements may be
positions approximately equidistant from a common axis. In one embodiment, a
cutting
tool is aligned approximately equidistant from a common axis presented by a
guide
channel. In one embodiment, a cutting tool is aligned approximately
equidistant from a
common axis presented by a central channel.
[0069] As used herein, the term "approximately" refers to a range of values
within plus or
minus 10% of an ideal. For example, approximately coaxial tubular elements may
share
the identical central axis or may be have central axes that are within 10% of
a shared
identical central axis.
[0070] In one embodiment, one tubular element is contained within another
tubular
element, but the central axis of both tubes need not be aligned.
[0071] While, for clarity and completeness of presentation, all aspects of the
production
and utilization of DTMOs are described in this document, and embodiments of
the
invention are described from the start of the processes to their ends, it
should be understood
that each of the aspects described herein can be used with other methodologies
and/or
equipment for the carrying out of other aspects and can be used for other
purposes, some of
which are described herein. The present invention includes portions devoted to
the
preparation and maintenance of dermal micro-organs for transformation into
DTMOs. It
should be understood that the dermal micro-organs produced according to these
aspects of
the invention can be used for purposes other than for transformation into
DTMOs
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[0072] In some embodiments of the invention, the micro-organ is a DMO
including a
plurality of dermis components, for example, fibroblasts and/or epithelial
components
containing nerve endings and/or sweat glands and/or sebaceous glands and/or
blood and
lymph vessels and/or elastin fibers and/or collagen fibers and/or endothelial
components
and/or immune system derived cells and/or extra-cellular matrix. Conventional
subcutaneous implantation of a micro-organ including epidermal layers ("split
thickness
skin MO") in mice and pigs, may result in formation of keratin cysts or macro-
keratin cysts.
In contrast, when skin tissue is sampled to obtain a DMO or when a DMO is
directly
harvested, according to exemplary embodiments of the invention, after
subcutaneous
to implantation or implantation in other anatomical locations, no cysts or
macro cysts are
observed in mice, pigs or in humans. It should be noted that the biological
activity (for
example, secretion of a therapeutic protein, e.g., erythropoietin and
elevation of hematocrit
as a result) of a DTMO according to embodiments of the invention may be
comparable to
or even higher than split thickness skin derived TMO.
[0073] In general, production of DTMOs may include DMO harvesting, maintaining
the
DMO and/or modifying the DMO and/or genetically altering them and, in some
embodiments, verifying the production of a desired agent (for example
proteins) by the
DMO. Utilization of the DTMO may include production, within a patient's or
animal's own
body, of therapeutic substance, such as proteins, for treatment of a subject.
For example, the
DTMO can be implanted into or under the skin or within the body of the subject
to produce
the agent/protein in vivo.
[0074] In one embodiment, a DTMO is not encapsulated in an immunoprotective
capsule
or sheath.
[0075] In some embodiments of the invention, the DMO may contain tissue of a
basal
epidermal layer and, optionally, other epidermal layers of the skin. In other
embodiments,
the dermal micro-organ does not include basal epidermal layer tissue.
[0076] In some embodiments of the invention, the DMO does not include
epidermal layers.
In other embodiments, the DMO may contain a few layers of epidermal tissue. In
some
embodiments, the dermal micro-organ may lack a complete epidermal layer. In
certain
instances, a DMO may include invaginations of the epidermis within the dermal
tissue
layers, while still lacking a complete epidermal layer.
[0077] In one embodiment of the invention, the DMO includes the entire cross-
section of
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the dermis. In another embodiment of the invention, the dermal micro-organ
includes part
of the cross-section of the dermis. In a further embodiment, the DMO includes
most of the
cross section of the dermis, namely, most of the layers and components of the
dermis
including the papillary and reticular dermis. In a further embodiment, the DMO
includes
primarily dermal tissue, but may also include fat tissue. In some embodiments
of the
invention, the DMO does not produce keratin or produces a negligible amount of
keratin,
thereby preventing the formation of keratin cysts following implantation in a
recipient.
H. Methods and Apparatuses for Harvesting a DMO
[0078] The DMO to be harvested can be removed from the body by any means of
to removing tissue known in the art, such as biopsy procedures. The
harvesting procedure
keeps intact the micro-architecture of the tissue from which it is removed. In
one
embodiment the DMO may be obtained by direct biopsy and then be cut to the
required
size. In another embodiment, a tissue sample may be obtained by direct biopsy,
in which the
desired size of the dermal micro-organ is obtained. In another embodiment, non-
desired
tissue may be cut from the harvested biopsy or directly harvested micro-organ.
In one
embodiment, a DMO may be obtained by direct biopsy and then processed to
become a
DTMO by genetic modification of the DMO in vitro. In one embodiment a DMO or a
DTMO may be labeled in vitro for identification purposes, e.g., a DMO or a
DTMO may be
colored prior to implantation by an inert, biocompatible ink or stain
containing, for
example, a chromophore, which may be visible to the naked eye or may require
special
illumination conditions to visualize it.
[0079] In some embodiments of the invention, the dermal micro-organ is
directly harvested
from the body. In other embodiments, a dermal micro-organ is harvested with
the aid of a
harvesting apparatus. The inner diameter dimension of a cutting tool used to
harvest a
dermal micro-organ may be, for example, about 0.5-4 mm. In another embodiment,
the
dimension may be, for example, 1.71 mm. In yet another embodiment, the
dimension may
be 1.21 mm. In still another embodiment, the dimension may be, for example, 1-
3 mm. In a
further embodiment, the dimension may be, for example, 2-4 mm. In one
embodiment the
dimension may be, for example, 1-2 mm. In another embodiment, the dimension
may be
0.5-1.5 mm. In yet another embodiment, the dimension may be, for example,
about 1.5 mm.
In still another embodiment, the dimension may be, for example, about 2 mm.
[0080] In some embodiments, the cutting tool has dimensions corresponding to
needle size
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dimensions. In one embodiment, the cutting tool is, for example, a 14GA
needle. In another
embodiment, the cutting tool is a 15GA needle. In yet another embodiment, the
cutting tool
is a 16GA needle. In yet another embodiment, the cutting tool is a 17GA
needle. In still
another embodiment, the cutting tool is an 18GA needle. In a further
embodiment, the
cutting tool is a 19GA needle. In one embodiment, the cutting tool is a 12GA
needle. In
another embodiment, the cutting tool is a 13GA needle. The wall thickness of a
cutting tool
corresponding to a needle size dimension, may be for example a regular wall
thickness
(RW), a thin wall thickness (TW), a extra thin wall thickness (XTW), or any
thickness
known in the art.
to [0081] The shape of the tip of a cutting tool may also play a role in
harvesting a DMO. A
sharp tip may be used, as is, e.g., commercially available needles.
Alternatively, a cutting
tool may have a tip, which has been sharpened, e.g., by polishing or through
the use of
chemical treatments or using electro-chemical erosion. In one embodiment, the
sharp tip of
a cutting tool is symmetrical sharpened. The sharpening of the tip may be
either on the OD
surface or the ID surface. For example, the tip may be sharpened by removing
material
from the outer or inner surface of the tip.
[0082] In some embodiments, the harvested DMO may not retain its cylindrical
shape after
harvesting, i.e., at least one dimension of its cross section may expand while
at least another
dimension of its cross section may contract. In one embodiment, for example,
at least one
dimension may be 0.5-3.5 mm and at least one dimension may be 1.5-10 mm.
[0083] In another embodiment, the dimensions of the tissue being harvested may
be, for
example, about 5-100 mm in length. In another embodiment, the dimensions of
the tissue
being harvested may be, for example, about 10-60 mm in length. In another
embodiment,
the dimensions of the tissue being harvested may be, for example, about 20-60
mm in
length. In another embodiment, the dimensions of the tissue being harvested
may be, for
example, about 20-50 mm in length. In another embodiment, the dimensions of
the tissue
being harvested may be, for example, about 20-40 mm in length. In another
embodiment,
the dimensions of the tissue being harvested may be, for example, about 20-100
mm in
length. In another embodiment, the dimensions of the tissue being harvested
may be, for
example, about 30-100 mm in length. In another embodiment, the dimensions of
the tissue
being harvested may be, for example, about 40-100 mm in length. In another
embodiment,
the dimensions of the tissue being harvested may be, for example, about 50-100
mm in
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length. In another embodiment, the dimensions of the tissue being harvested
may be, for
example, about 60-100 mm in length. In another embodiment, the dimensions of
the tissue
being harvested may be, for example, about 70-100 mm in length. In another
embodiment,
the dimensions of the tissue being harvested may be, for example, about 80-100
mm in
length. In another embodiment, the dimensions of the tissue being harvested
may be, for
example, about 90-100 mm in length. In another embodiment the length may be
around 20
mm. In another embodiment, the length may be about 30 mm. In another
embodiment, the
length may be about 40 mm.
[0084] When a DMO has the above listed dimensions, it maybe maintained in
vitro, e.g., in
to a growth medium under proper tissue culture conditions for extended
periods of time, for
example, several days, several weeks or several months. The DMO may be
maintained, for
example, in-vitro in defined growth media. In one exemplary embodiment the
growth
media may include growth factors, fetal calf serum (FCS), human serum, or
Synthetic
Serum Substitute (SSS). In another exemplary embodiment the growth media may
include
serum either from the donor or the recipient subject. In yet another
embodiment the growth
media may include autologous serum. In another embodiment, no serum is added
to the
media.
[0085] In accordance with an aspect of some embodiments of the invention, only
a portion
of a DTMO generated may be used in a given treatment session. The remaining
DTMO
tissue may be returned for maintenance and/or may be stored (e.g.,
cryogenically or
otherwise) for later use. In one embodiment, a DMO is stored, for example
cryogenically or
otherwise, prior to genetic modification processing. In another embodiment, a
DMO is
stored, for example cryogenically or otherwise, following genetic modification
processing.
[0086] It is a feature of some embodiments of the invention that a large
number of dermal
micro-organs may be processed together in a batch process into DTMOs, as
described
below. This may allow for more convenient processing, but will not allow for
determination
of the secretion level of each DTMO separately. In other embodiments, a DMO
may be
processed independently into a DTMO, as described herein.
[0087] In some exemplary embodiments of the invention a potency assay may be
performed for the therapeutic agent, which may be produced and/or secreted by
either a
single DTMO or a batch of DTMOs. The potency assay may include, for example, a
cell
proliferation assay in which the proliferation response of the cells is mainly
dependent on
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the presence of the therapeutic agent in the growth media of the cells. In one
embodiment,
analysis of a DTMO may use for example an ELISA assay in order to quantify
secretion
levels of an at least one secreted therapeutic agent.
[0088] According to some embodiments of the invention, a method of harvesting
the DMO
may include stabilizing and supporting a skin-related tissue structure from
which a DMO is
to be harvested, e.g., such that at least the DMO and/or one or more other
tissue segments in
its vicinity are maintained at a desired shape and/or position, separating at
least a portion of
the DMO from surrounding tissue, and extracting the separated DMO, as
described in detail
below.
to [0089] FIG. 1 shows an overview of a methodology for producing and
utilizing DMOs and
DTMOs, in block diagram form, in accordance with an exemplary embodiment of
the
invention. Similarly, a DTMO may be produced by following the steps described
independent of a bioreactor. At block 202 a DMO is harvested from a subject.
In some
embodiments of the invention, the DMO is harvested from the same subject to
which
therapy will later be applied. In an embodiment of the invention, the DMO is
from dermal
tissue. Optionally, other tissues are harvested and used in a manner similar
to that described
below with reference to dermal tissue. While the method described below is
exemplary,
other methods of harvesting tissue samples can be used in some embodiments of
the
invention. If desired, the DMO or DTMO can be cryogenically stored for later
use (i.e.,
introduction at the same stage of the process). Alternatively, for certain
embodiments, the
DMO can be implanted directly back into the patient from which it was
harvested to
produce a therapeutic, cosmetic, or other physiological affect.
[0090] In order for a DMO to be a viable micro-organ, it must have at least
one dimension
that is small enough that nutrients can diffuse to all the cells of the DMO
from a nutrient
medium which contacts the DMO and that waste products can diffuse out of the
DMO and
into the medium. This enables the DMO to be viable in vitro long enough for
the further
processing described below and for the optional further utilization of the DMO
as a source
for a therapeutic agent, such as a protein. The method of harvesting a DMO
generally
results in a DMO having an in vitro life of several months.
[0091] A suitable genetic modification agent is prepared (block 208).
Alternative
exemplary methods of preparing the agent include creation of aliquots with a
desired
amount of a modifying agent using a predefined dilution buffer containing
modifying agent,
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such as for example a viral vector, and validating the activity of the
modifying agent. All of
these processes are well known in the art. At this point the DMO can be stored
cryogenically, for later introduction at the same place in the process. This
can be performed
using known protocols for gradual freezing of tissues and cells, using for
example, DMEM
medium containing 10% DMSO
[0092] At block 210 the DMO is genetically altered. As described above, many
methods of
genetic alteration are known and may be used in conjunction with the present
invention. As
an example, the following description is based on using a viral vector to
insert a gene into
the cells of the DMO. This process is well known and will not be further
described, except
to as to the particular methodology and apparatus for introducing the virus
to the DMO.
[0093] At block 212 the genetically altered DTMO is optionally tested for
production and
secretion rates of the therapeutic agent. There are various methods of
determining the
quantity of secretion, for example, ELISA, other immunoassays, spectral
analysis, etc. In
addition the quality of the secretion is optionally tested, for example for
sterility and/or
activity of the secreted protein. This may be performed periodically or
continuously on-line.
At this point the DTMO can be cryogenically stored for later use.
[0094] At blocks 214 and 216, the amount of DTMO required for producing a
desired
therapeutic effect is determined. As indicated below, the therapeutic dose
requirements can
be estimated from measured secretion rates, patient parameters and population
statistics on
the estimated or known relationship between in vitro secretion and in vivo
serum levels.
[0095] At block 218 the desired number of the DTMOs are selected. A DTMO is
loaded
into an implantation tool. Exemplary implementation tools are described below.
[0096] If the DTMOs must be transported prior to being transferred to the
implantation
tools, it is optionally held (220) in a maintenance station or under
maintenance conditions,
in which the temperature, humidity, etc. are held at levels that allow the
DTMO to stay
viable during transport. The remaining DTMOs are optionally maintained in
vitro for future
use. This can be at warm incubator conditions (30-37 C), in conditions as
described above
at cool incubator conditions (4 C), which may prolong its viability in vitro,
or under
cryogenic conditions.
[0097] At block 224, a subset of the DTMOs is implanted into the subject. An
exemplary
embodiment of a method of implantation is described below. Other methods of
doing so
will occur to persons of skill in the art and are primarily dependent on the
specific geometry
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of the micro-organ being used. Animal studies have shown that the DMOs and
DTMOs
remain viable in vivo, in the sense that the DTMO continues to produce and
secrete the
therapeutic agent for a period of weeks and months following implantation. In
animal
studies, therapeutic amounts are produced for periods up to 160 days (or
longer). While the
[0098] The in vivo performance of the DTMO is optionally determined (block
228). Based
[0099] Genetic alteration may generally include genetically engineering a
selected gene or
[00100] According to some exemplary embodiments of the invention, a method of
[00101] Reference is now made to FIG. 2, which schematically illustrates a
flowchart of a
method of harvesting a DMO according to some exemplary embodiments of the
invention,
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and to FIGS. 3A-3G, which schematically illustrate exemplary stages of
harvesting a DMO
3024 located within a skin-related tissue structure.
[00102] As indicated at block 2002, the method may optionally include locally
administering an anesthetic, e.g., as is known in the art, to the vicinity of
the DMO to be
harvested.
[00103] Use of DTMOs for protein or RNA based therapy may, in certain
circumstances,
necessitate use of multiple DTMOs. As described throughout, DMOs and DTMOs may
be
maintained in vitro for extended time periods or stored cryogenically or
otherwise, for later
use. Therefore, in some instances, multiple DMOs may be harvested
consecutively during a
to single procedural time period from the same subject. In this way,
multiple DMOs may be
harvested from the subject for later use, without the subject undergoing
separate harvesting
procedures on separate days for each DMO needed. In one embodiment, a
harvester marker
template may be used prior to positioning a harvester apparatus on a subject's
epidermal
surface (step 2004), in order to mark multiple sites for harvesting. In one
embodiment, a
harvester marker template is positioned on the epidermal surface of a subject,
and the
epidermal surface is then marked to indicate, for example, area for
application of local
anesthesia, alignment lines and harvesting lines. In one embodiment, the
surface is marked
using a surgical pen or marker. In one embodiment, the surface is marked using
a non-
permanent dye or ink.
[00104] As indicated at block 2004, the method may further include positioning
an
apparatus including a support structure, (e.g., FIG. 4C; FIG. 5A; FIG. 6; FIG.
11), at a given
harvest site so that the support structure, or a portion thereof, is in
contact with an
epidermal surface of the subject. In some embodiments, a contact between a
support
structure of this invention and the epidermal surface of a subject must be air-
tight so that a
vacuum seal may be formed at a later step. In one embodiment, a harvest site
is on a
subject's back. In another embodiment, a harvest site is on a subject's
abdomen. In yet
another embodiment, a harvest site may be at another location on a subject's
body.
[00105] As indicated at block 2006 and FIG. 3A, a support structure (e.g.,
FIG. 4C; FIG.
13A; FIG. 6; FIG. 11), which may include a vacuum chamber (FIG. 11 1130) and
guide
channel 2008 (FIG. 11 1108), under vacuum conditions may be used to hold and
support
the skin-related tissue structure including epidermal (3000), dermal (3002)
and fat (3004)
tissue layers, in place for proper harvesting of a DMO. For example,
application of vacuum
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conditions causes a vacuum to be formed within the vacuum chamber thereby
drawing the
epidermal surface of the skin-related structure into the interior of the
support structure,
wherein a central channel may support epidermal and dermal skin layers of the
skin-related
structure.
[00106] As used herein, the term "guide channel" may also be referred to
herein as a
"needle guide channel".
[00107] Under vacuum conditions a central channel may provide support for the
skin-
related tissue structure to be shaped so that dermal tissue is within the
central channel. In
some exemplary embodiments, the vacuum chamber includes one elevated
protrusion. In
to other exemplary embodiments, the vacuum chamber includes two elevated
protrusions. In
instances where a support structure that includes one or two elevated
protrusions is used,
the elevated protrusions additionally may support epidermal and dermal skin
layers of the
skin-related structure.
[00108] In certain instances, application of vacuum conditions using a vacuum
chamber
with two elevated protrusions may create a precise geometry of the skin-
related structure
such that dermal tissue is harvested and a plug of epidermal tissue is not
harvested.
[00109] In exemplary embodiments, a vacuum condition may cause the skin-
related
structure to be held at an inner support surface of the vacuum chamber,
including within a
central channel and elevated protrusions if present. The guide channel 3008,
which in one
embodiment may be tubular in shape, may provide guidance and/or stability for
inserting
and/or using a cutting tool to ensure proper cutting along a cutting axis. In
some
embodiments, the cutting axis is coaxial with the guide channel. While the
coring tube is
coaxial with the guide channel, the coring needle may not always be in the
vertical center of
the central channel. In one embodiment, the coring tube is in the horizontal
center of the
central channel.
[00110] In certain embodiments, the inner dimensions of a support structure
including a
vacuum chamber may be between 3.0 -8.0 mm. In one embodiment, the dimension
may be,
for example 3.0 mm in diameter. In another embodiment, the dimension may be,
for
example 3.5 mm in diameter. In a yet another embodiment, the dimension may be,
for
example 4.0 mm in diameter. In another embodiment, the dimension may be, for
example
4.5 mm in diameter. In another embodiment, the dimension may be, for example
5.0 mm in
diameter. In still another embodiment, the dimension may be, for example 5.5
mm in
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diameter. In yet another embodiment, the dimension may be, for example 6.0 mm
in
diameter In a further embodiment, the dimension may be, for example 6.5 mm in
diameter.
In another embodiment, the dimension may be, for example 7.0 mm in diameter.
In yet
another embodiment, the dimension may be, for example 7.5 mm in diameter. In
another
embodiment, the dimension may be, for example 8.0 mm in diameter.
[00111] In one embodiment, the appropriate sized support structure having
particular
inner dimensions of a vacuum chamber is pre-determined prior to actual
harvesting.
[00112]
[00113] As indicated at block 2008 and FIG. 3B, an introducer (e.g., FIG. 4D),
including
to for instance, an inner needle (3010; 4006) and an outer sleeve (3012;
4008), may then be
used to puncture the skin-related tissue by inserting the introducer through
the guide
channel of the support structure, 3006, and into the skin-related tissue
structure at a point of
penetration. This single puncture site may be used for all further entry and
egress into and
out of the skin-related structure. In this way damage and scarring to the
subject is limited.
Introduction of the cutting tool through the outer sleeve of the introducer
prevents or
minimizes harvesting of epidermal tissue.
[00114] An introducer may be composed of tubular elements, for instance an
inner needle
and an outer sleeve. In some embodiments, the outer sleeve is fitted over the
inner needle.
Together they may be inserted into the skin-related structure. In certain
embodiments,
where a support structure with a vacuum chamber including a proximal elevated
protrusion
is used the configuration of the skin-related tissue is such that insertion of
the introducer is
generally perpendicular to the skin surface at the point of penetration. In
one embodiment
where a support structure with a vacuum chamber including a proximal elevated
protrusion
is used, an introducer may be inserted into the skin-related structure such
that the tip of the
inner needle extends into the region of tissue in the area of the first, i.e.,
proximal elevated
protrusion, 3007, and the distal end of the outer sleeve extends to about mid-
way under this
first elevated protrusion. In one embodiment, following insertion of the
introducer, the
distal end of the outer sleeve transverses all of the layers of the skin at
the puncture site and
is located in the underlying fat layer.
[00115] In the absences of the step at block 2008, using an introducer to
puncture the
skin, the step at block 2012 below would result in the harvesting of a plug of
full thickness
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skin prior to the harvest of the dermal micro-organ, which would necessitate
additional
processing of the tissue to remove the full thickness skin plug in order to
produce a DMO.
[00116] In one embodiment, the inner needle is beveled. In another embodiment,
the
inner needle is not beveled. In one embodiment, insertion of the introducer is
with the bevel
of the inner needle pointed downward. In another embodiment, insertion of the
introducer is
with the bevel of the inner needle pointed upward. In yet another embodiment,
insertion of
the introducer is with the bevel of the inner needle at an intermediate angle
between upward
and downward.
[00117] The exemplary embodiment described below (block 2010) is based on the
use of
to a support structure including, at least, a vacuum chamber including
proximal (3007) and
distal (3009) elevated protrusions.
[00118] As indicated at block 2010 and FIG. 3C, the outer sleeve of the
introducer, 3012,
is positioned by inserting the introducer through the inner guide channel,
through the
epidermal 3000 and dermal 3004 layers of the skin-related structure and into
the fat layer
3002 located under the proximal protrusion. Following insertion of the
introducer, the
inner needle is withdrawn from the skin-related structure and the outer sleeve
remains in
place, such that the distal end of the outer sleeve resides about mid-way
under the proximal
protrusion. The result of this action is that the outer sleeve is positioned
coaxially with the
inner guide channel, which may also be the cutting axis, and the tip of the
outer sleeve
extends into the fat (3002) of the skin-related tissue structure supported by
the proximal
protrusion 3007.
[00119] The outer sleeve, 3012, may be made up of a thin tube, a hollow rod,
or any other
suitable thin, generally straight, object able to be placed around the inner
needle and able to
penetrate the necessary skin layers. For example, in one embodiment, an outer
sleeve may
have an inner diameter which corresponds with a needle of size 12-19 GA for
example,
about 14 GA. In one embodiment, an outer sleeve may include a plastic tube of
an
appropriate length. In one embodiment, the outer sleeve includes high-density
polyethylene
(HPDE) tubing. In another embodiment, the outer sleeve includes
polytetrafluoroethylene
(PTFE) tubing. In another embodiment, the outer sleeve includes fluorinated
ethylene
propylene (FEP) tubing. In one embodiment the length of the outer sleeve is
approximately
10-100 mm. In one embodiment, the length of the outer sleeve is approximately
40 mm. In
one embodiment, approximately 5-20% of the length of the outer sleeve enters
the skin-
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related structure beyond the puncture site. In one embodiment, approximately
10-15% of
the length of the outer sleeve enters the skin related structure beyond the
puncture site. In
one embodiment, approximately 12.5% of the length of the outer sleeve enters
the skin-
related structure beyond the puncture site.
[00120] The embodiments above describe penetration of an outer sleeve into a
fat layer.
In another embodiment, an outer sleeve may be inserted into dermis tissue. In
yet another
embodiment, an outer sleeve may be inserted into a subcutaneous space.
[00121] The length of penetration of the outer sleeve 3012 through the skin
into dermis or
fat or a subcutaneous space may generally correspond to 1 to 15 mm, or in one
embodiment
about 5 mm. For example, an outer sleeve may be inserted manually as part of
the
introducer, and guided to a desired depth within the dermis, fat or
subcutaneous tissue
under the proximal protrusion. The outer sleeve is then co-axial with the
cutting axis
within the central channel, so that dermis may be harvested by the cutting
tool.
[00122] In one embodiment, use of an outer sleeve protects an entry puncture
site from
exposure to the rotational and forward motion of a cutting tool, thereby
preventing
additional trauma to the skin at the site of entry.
[00123] As indicated at block 2012 and FIGS. 3D and 3E, a DMO may now be cut
from
the skin-related tissue. A cutting tool, 3014, may be inserted coaxially
through the guide
channel 3008 and outer sleeve 3012, such that the outer sleeve guides the
cutting tool along
a cutting axis (FIG. 5A and FIG. 6) into the skin-related structure. In one
embodiment,
guiding a cutting tool through the outer sleeve may allow the cutting tool to
directly enter
dermal tissue (3004) of the skin-related structure (3E). As an aide to
cutting, the cutting tool
may be rotated as the tube advances towards the distal end of the apparatus. A
motor may
be used to rotate the cutting tool. A motor may be, for example, a pneumatic
motor drill or
an electric motor drill. In some embodiments, a medical drill may be used to
rotate the
cutting tool. In one embodiment, the cutting tool attaches at one end to a
drill collet (3016),
as is known in the art
[00124] In one embodiment the method may include rotating the cutting tool
while
advancing the cutting tool, e.g., towards the distal end of the support
structure. For
example, a medical drill or other suitable tool or rotation mechanism may be
used to rotate
a coring tube 3014 while it is advanced manually or automatically, thereby
more smoothly
cutting dermal tissue for a DMO. For example, a proximal end of coring tube
may be
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connected to a medical drill using a drill collet, 3016. Examples of medical
drills include
the Aesculap Micro Speed drill manufactured by Aesculap AG & Co. KG, Am
Aesculap
Platz, D-78532 Tuttlingen, Germany, which may include a control unit, a motor,
a
connection cord, a hand piece and/or a foot switch, catalogue numbers GD650,
GD658,
GB661, GB166 and GB660, respectively; and a Nouvag medical drill, TCM-3000-BL,
and
hand piece, catalogue numbers 3285 and 1710, respectively. Such a drill, or
any other
suitable drill or rotation mechanism, may be used to rotate the cutting edge
of the cutting
tool at a rotational speed appropriate for cutting of the dermal tissue, for
example, a
relatively high rotational speed, for example, a speed higher than 1,000 RPM,
e.g., between
to 1,000 RPM and 10,000 RPM. For example, tube 3014 may be rotated at a
rotational speed
higher than 2,000 RPM, e.g., approximately 7,000 RPM. Alternatively, a
relatively low
rotational speed of less than 1000 RPM may be used, or no rotation at all, as
described
below. Optionally, the rotational speed of the drill may vary in an
oscillatory manner, i.e.,
the direction of rotation may vary periodically between "clockwise" and
"counterclockwise"
directions. While rotated by a drill, a coring tube may be manually or
automatically
advanced, e.g., towards the distal end of the support structure, e.g., towards
the distal
elevated protrusion 3009.
[00125] In one embodiment, a method of cutting a dermal micro-organ may
include
stopping the forward motion of the coring tube at a particular location. In
one embodiment,
the meeting of the collet of the drill (3016) with the proximal end of the
introducer sleeve
(3012) at the outer surface of a support structure (3006) may act as a hard
stop, preventing
further forward motion of a coring tube. In another embodiment, the meeting of
or an
element placed on the external distal portion of the coring tube, e.g., a cap
encircling the
coring tube (FIG. 13; 1302), with the outer surface of a support structure may
act as a hard
stop, preventing further forward motion of a coring tube.
[00126] In certain exemplary embodiments, a method of cutting a DMO using a
support
structure including a vacuum chamber with at least a distal elevated
protrusion may include
stopping the forward motion of coring tube, for example, at a position such
that the tip of
the coring tube (FIG. 3E) has been advanced to reside within the region of the
skin-related
structure positioned under the distal elevated protrusion 3009. In one
embodiment, the
meeting of the collet of the drill with the introducer sleeve at the outer
surface of a support
structure may position the distal tip of the coring tube in fat. In one
embodiment, the
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meeting of the collet of the drill with the introducer sleeve at the outer
surface of a support
structure may position the distal tip of the coring tube to reside under the
distal elevated
protrusion such that the tip enters the fat layer.
[00127] In one embodiment, the geometry of the skin-related structure created
within a
vacuum chamber with at least a distal elevated protrusion ensures that at
termination of
forward movement the distal tip of a coring tube will have crossed the
dermis/fat interface,
so that the tip resides in fat. The dermis/fat interface has a weak
connection. In one
embodiment, the weak connection between dermis and fat may ensure that the
dermal tissue
sample can be separated from the body fat during recovery of the DMO.
to [00128] The cutting tool may include any suitable cutting tool, for
example, a coring tube
(e.g., Figure 4E; 4010). Coring tube may include a generally symmetrically
sharpened
tubular tool, e.g., a hypodermic tube processed to have sharpened cutting edge
with a
desired shape. A coring tube may include, for example, a standard medical
grade tube,
having a thin wall, e.g., having a thickness of between 0.05 mm and 0.3 mm. A
coring tube
may have an inner diameter, for example, between 0.5 mm-4 mm. In one
embodiment, an
inner diameter may be between 1-2 mm. In another embodiment, an inner diameter
may be
between 1-3 mm. In yet another embodiment, an inner diameter may be between 2-
4 mm.
In still another embodiment, an inner diameter may be between 0.5-1.5 mm. In
one
embodiment, an inner diameter may be about 1.21 mm. In another embodiment, an
inner
diameter may be about 1.5 mm. In still another embodiment, an inner diameter
may be
about 1.71 mm. In yet another embodiment, an inner diameter may be about 2 mm.
In one
embodiment, the coring tube has about the dimensions of a 14 GA needle. In
another
embodiment, the coring tube has about the dimensions of a 12 GA, 13 GA, 15 GA,
16 GA,
17 GA, 18 GA or 19 GA needle. The dimensions, e.g., the diameter, of coring
tube and/or
the dimensions of introducer may be predetermined based on the volume and/or
dimensions
of the DMO intended to be harvested. A coring tube may have a sharpened end
("tip")
adapted to serve as a cutting edge. In one embodiment, the sharpened edge is
sharpened on
the outer diameter. In another embodiment, the sharpened edge is sharpened on
the inner
diameter. A coring tube may be inserted through the outer sleeve and into the
skin-related
tissue structure in order to prevent harvesting of epidermal tissue. In one
embodiment, use
of a support structure with at least a proximal elevated protrusion creates a
precise geometry
of the skin-related structure when under vacuum conditions such that an
epidermal layer
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and/or a plug of epidermis is not harvested.
[00129] According to some exemplary embodiments of the invention, at least
part of an
inner surface and/or an outer surface of tube may be coated with a low
friction material,
e.g., Teflon , Parylene or any other suitable coating material, e.g., to ease
the separation of
the harvested tissue from the inner surface of the cutting tool in a
subsequent action and/or
to reduce any forces acting on the tissue during the cutting action, as
described below.
[00130] As indicated at block 2014 and FIGS. 3F and 3G, the method of
harvesting
includes recovery of the DMO 3024. After a coring tube has been advanced to a
hard-stop
and the distal tip of the coring tube is located within the skin-related
tissue structure
to positioned under the distal elevated protrusion (e.g., FIG. 6), the
collet of the drill 3016
(FIG. 4H) is opened and while holding the coring tube in place, the drill is
removed. In one
embodiment, an external cap, for example a luercap, may be use to hold the
coring tube in
place. Then the collet 3020/septum 3021 of the syringe 3022 assembly is
attached (FIGS.
4B, 4A( 4004) and 4 (4012), respectively) to the coring tube.
[00131] In one embodiment, a male luercap (1302) is attached, e.g., glued, to
the outer
surface of a coring needle (1304). The ability to hold the glued cap in place
while removing
the drill and connecting the syringe, prevents movement of the coring needle
during the final
steps of harvesting of a DMO. This may prevent potential loss of a DMO.
[00132] First the collet 3020 is attached. This is a stand-alone collet, not
that of the
medical drill. The collet may be slipped over the coring tube when in the open
position.
After the collet is in place and put in the closed position, the septum 3021
is pushed over the
needle and attached to the collet. In one embodiment, the septum includes a
needleless
valve. In one embodiment, the needleless valve provides an airtight seal. The
collet and
septum together are referred to as a needleless valve assembly. The collet
prevents the
septum from pushing the coring tube forward during attachment of the septum.
Once the
septum is punctured by the coring tube and attached (e.g., by screwing) to the
collet, the
syringe 3022 can be attached to the needless valve assembly. When the syringe
plunger is
withdrawn there is creation of a vacuum condition within the coring tube. At
this point, the
entire assembly with the coring tube may be retracted (FIGS. 3F and 3G), which
causes the
cut tissue to be drawn into the syringe.
[00133] In one embodiment, a vacuum condition is applied at the same time as
the coring
tube is withdrawn from the skin-related tissue structure, and the DMO is
collected for
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example, into the syringe body. In another embodiment, the coring tube is
withdrawn from
the skin-related tissue structure and then a vacuum condition is applied to
the coring tube
resulting in recovery of the DMO, for example, into the syringe body. In yet
another
embodiment, a vacuum condition is applied to the coring tube while the coring
tube remains
in the skin-related tissue structure and the DMO is recovered, for example,
into the syringe
body. In still another embodiment, a drill is disconnected from a coring
needle (1304) while
holding a glued cap (1302) in place. (FIG. 13) Then a female luer syringe
(1306) is attached
directly to the clued cap. When the plunger of the syringe is withdrawn and
the
syringe/coring needle assembly retracted from the skin-related tissue
structure, the DMO is
to suctioned directly into the syringe body. Connection of the coring tube
with the syringe
using a leurcap eliminates the need for a collet and needleless valve
assembly.
[00134] In one embodiment, the syringe is partially filled with saline or
other suitable
liquid, such that the tissue sample is withdrawn into a fluid environment
which supports
tissue viability.
[00135] The septum 3021 is needed to make sure that there is an air-tight
connection
between the syringe 3022 and the interior of the coring needle. If the syringe
is attached to
the collet directly without a septum, withdrawal of the plunger of the syringe
would not
cause a vacuum condition in the coring tube since a collet is in general not
airtight. FIG. 4A
(4012) shows one embodiment of a septum which may in certain embodiments be
used in a
method of harvesting a DMO of this invention.
[00136] In still another embodiment, recovery of a DMO is achieved by
withdrawing the
cutting tool, from the skin-related structure, wherein the DMO is retained
within the cutting
tool. The DMO 3024 may then be recovered from the cutting tool using positive
pressure,
e.g., the proximal end of the cutting tool may be attached to a syringe and
positive pressure
applied by a syringe plunger so that a DMO is "pushed" from the distal end of
the cutting
tool. In addition, suitable fluids, such as sterile fluids, may be used to
assist in removing the
DMO from cutting tool 3014.
[00137] In yet another embodiment, a DMO may be, for example, carefully
removed
from a cutting tool using mechanical means, such as tweezers or similar tools,
which can be
used to grasp the distal end of the DMO located at the distal end of the
cutting tool.
[00138] As indicated at block 2016, the apparatus may then be removed from the
harvest
site. At this time, the outer sleeve may be removed manually from the skin-
related tissue.
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[00139] It will be appreciated by those skilled in the art that any
combination of the
above actions may be implemented to perform harvesting according to
embodiments of the
invention. Further, other actions or series of actions may be used.
[00140] Reference is also made to FIGS. 4C-E, which present exemplary
embodiments of
a harvesting apparatus, wherein FIG. 4C presents an example of a support
structure attached
to a vacuum source; FIG. 4D presents an example of an introducer, inner guide
needle
(4006) and outer sleeve (4008-white tube); FIG. 4E (4010) presents an example
of a coring
tube; and 4014 presents an example of a medical drill useful for rotating a
cutting tool, and
4012 presents an example of a drill collet for attaching the coring tube.
FIGS. 4A-B present
to exemplary embodiments of a syringe (FIG. 4A (4012)), septum (FIG. 4A
4004) and syringe
collet (FIG. 4B) for use in the recovery of a dermal micro-organ.
[00141] According to some embodiments of the present invention, the above
described
manual procedures may be facilitated by an integrated apparatus (not shown)
configured to
perform some or all of the above procedures for harvesting the DMO. For
example, in
regard to one harvesting method embodiment, the integrated apparatus may be
configured
to enable positioning and guiding the insertion of an introducer FIG. 4D,
guiding the
insertion of coring tube FIG. 4E 4010 and controlling its rotational and
forward movement
during the cutting process, and/or removing DMO from a coring tube. Such an
apparatus
may enable relatively simple operation when performing a harvesting procedure.
[00142] Reference is now made to FIG. 5A, which schematically illustrates a
dermal
harvesting apparatus 5000 according to another exemplary embodiment of the
invention. As
used herein, the term "support structure" refers to the body of the apparatus
used to support
a skin-related tissue structure. The term "support structure" may also be
referred to herein as
an "apparatus". In this context an "apparatus" has all the qualities and
properties of a
"support structure".
[00143] Apparatus 5000 may include a guide channel 5003 and a vacuum chamber
5001
including an elevated protrusion 5006. Elevated protrusion 5006 may have a
predetermined
size and/or shape adapted, for example, to enable the creation of a "plateau"
of a single
layer of skin tissue in a generally flat orientation, elevated above the
trajectory of a coring
tube 5016. For example, section 5006 may be higher than other sections of
chamber 5001,
such that a fat layer 5018 may be drawn into section 5006 and supported along
the
trajectory of coring tube 5016. As a result, after harvesting a DMO of a
predetermined
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length, coring tube 5016 may be advanced into fat layer 5018, thus separating
the harvested
DMO from tissue surrounding the DMO. The harvested DMO may remain within
coring
tube 5016 as it is withdrawn from the body, or a vacuum condition may be
applied to the
proximal end of the coring tube to suction the DMO from the coring tube. The
configuration of Apparatus 5000 may eliminate the need for forming an "exit"
incision in
the skin, thus enabling the harvesting of a DMO with only a single incision.
[00144] According to some exemplary embodiments of the invention, apparatus
5000
may also include a drill stopper 5008 to enable manually advancing coring tube
5016 for a
predetermined distance along chamber 5001, e.g., to a position in which coring
tube 5016
has advanced into fat tissue 5018.
[00145] According to some exemplary embodiment of the invention, apparatus
5000 may
also include a vacuum conduit 5004 which is connected to the vacuum chamber
5001 and a
vacuum source 5002.
[00146] Reference is now made to FIG. 5B, which schematically illustrates a
cross-
sectional side view of a harvesting apparatus 5050 being implemented for
externally
supporting a skin-related tissue structure at a desired position according to
another
exemplary embodiment of the invention.
[00147] In some embodiments, apparatus 5050 may include two channels 5064
located at
least partially along two sides of chamber 5001, respectively, to allow
clamping epidermis
layer 5020. Channels 5064 may be positioned, e.g., centered, at a desired
height, for
example, at approximately the same height as where the center of the DMO is to
be
harvested. In one embodiment, the central channel may be positioned at a
height of about 2
mm below upper surface of the vacuum chamber. so that the clamping may
stabilize and/or
support the tissue being cut. According to these exemplary embodiments,
apparatus 5050
may also include two flexible membrane elements 5058, on either the inner
surface or outer
surface of channels 5064, so as to allow external clamping of the tissue
without substantially
affecting the vacuum condition applied to chamber 5001. According to other
embodiments
of the invention, apparatus 5050 may not include elements 5058 and/or channels
5064.
[00148] According to the exemplary embodiment of FIG. 5B, improved
stabilization of
dermis 5014 and/or improved prevention of recruitment of fat 5018 into vacuum
chamber
5001 may be accomplished by external clamping of the skin-related tissue
structure
supported within the vacuum chamber. For example, a clamping tool 5070, may be
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implemented to "pinch" the skin-related tissue structure supported inside
vacuum chamber
5001, e.g., symmetrically. Two clamping ends 5054 of clamping tool 5070 may be
inserted
into channels 5056, respectively. Tool 5070 may be closed such that clamping
ends 5054
may press down against flexible elements 5058. Thus, the skin-related tissue
structure in
chamber 5001 may be clamped from the sides without substantially affecting the
vacuum
condition in chamber 5001. A clamping force applied by clamping ends 5054 may
correspond, for example, to a constant or variable force of a spring 5062 or
other suitable
device.
[00149] Reference is now made to FIG. 6 and FIG. 11, which schematically
illustrate
to embodiments of harvesting apparatuses according to some exemplary
embodiment of the
invention.
[00150] Apparatus 6000 may include a guide channel 6003 (FIG. 11, 1108) and a
vacuum
chamber 6001 (FIG. 11, 2030) including two elevated protrusions 6007
(proximal) and
6006 (distal) (FIG. 11, 1107 and 1109, respectively), and a central channel
between the two
protrusions. The apparatus 6000 may further include one or more vacuum
channels and a
vacuum conduit 6004 (FIG. 11, 1126) to fluidically connect the vacuum chamber
with at
least one vacuum source 6002. Elevated protrusions 6006 and 6007 may have a
predetermined size and/or shape adapted, for example, to enable the creation
of a "plateau"
of a single layer of skin tissue in a generally flat orientation, elevated
above the trajectory of
a coring tube 6016. Elevated protrusions 6006 and 6007 may or may not have the
same size
and shape. For example, section 6006 and 6007 may be higher than other
sections of
chamber 6001, such that epidermal 6020, dermal 6060 and fat layers 6018 may be
drawn
into sections 6006 and 6007, respectively, such that in some embodiments,
dermal tissue
layers 6060 are supported within the central channel within the trajectory of
the coring tube
6016.
[00151] Application of a vacuum condition to an apparatus including a vacuum
chamber
including two elevated protrusions, for example, creates a precise geometry of
the skin-
related tissue structure so that dermal tissue is harvested and a complete
epidermal skin
layer is not harvested. The presence of the proximal elevated protrusion in
combination
with use of the introducer avoids the harvesting of a plug of epidermal tissue
at the
proximal end of the DMO. After harvesting a DMO of a predetermined length, a
coring
tube 6016 may be advanced into a fat layer 6018, thus allowing separation of
the harvested
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DMO from tissue surrounding the DMO. The harvested DMO may remain within
coring
tube 6016 as it is withdrawn from the body, or a vacuum condition may be
applied to the
proximal end of the coring tube to suction the DMO from the coring tube.
[00152] The configuration of Apparatus 60000 may eliminate the need for
forming an
"exit" incision in the skin, thus enabling the harvesting of a DMO with only a
single
puncture site.
[00153] According to some embodiments of the invention, the internal width of
a vacuum
chamber of apparatus 5000 and/or 6000 is about 3.5 mm. In one embodiment, a
central
channel may have a width of, for example, about 4 mm. In another embodiment, a
central
to channel may have a width of, for example, 3.0 mm. Furthermore, in some
embodiments, a
central channel may have a height, excepting of the protrusions, of, for
example, about 5
mm. In other embodiments, other ranges, such as for example, 3-25 mm, may also
be used
for the width and/or height of central channel, for example, any desired
dimensions in the
range of 3-25 mm may be used in some embodiments of the invention. The length
of
central channel may be generally similar to the length of the DMO being
harvested, for
example, approximately 30 mm in length; however, other ranges, for example, in
the range
of 5-100 mm. In another embodiment, the dimensions of the channel length may
be, for
example, about 10-60 mm in length. In another embodiment, the dimensions of
the channel
length may be, for example, about 20-60 mm in length. In another embodiment,
the
dimensions of the channel length may be, for example, about 20-50 mm in
length. In
another embodiment, the dimensions of the channel length may be, for example,
about 20-
40 mm in length. In another embodiment, the dimensions of the channel length
may be, for
example, about 20-100 mm in length. In another embodiment, the dimensions of
the
channel length may be, for example, about 30-100 mm in length. In another
embodiment,
the dimensions of the channel length may be, for example, about 40-100 mm in
length. In
another embodiment, the dimensions of channel length may be, for example,
about 50-100
mm in length. In another embodiment, the dimensions of the channel length may
be, for
example, about 60-100 mm in length. In another embodiment, the dimensions of
the
channel length may be, for example, about 70-100 mm in length. In another
embodiment,
the dimensions of the channel length may be, for example, about 80-100 mm in
length. In
another embodiment, the dimensions of the channel length may be, for example,
about 90-
100 mm in length. In another embodiment the length may be around 20 mm. In
another
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embodiment, the length may be about 30 mm. In another embodiment, the length
may be
about 40 mm.
[00154] Prior to actual harvesting of the DMO, an apparatus of 6000 may be
used in
conjunction with an introducer that includes, for instance, an inner needle
and an outer
sleeve (FIG. 4D, 4008) for puncturing the skin and inserting a portion of an
outer sleeve
through the skin at the puncture site and into fat tissue to prevent
harvesting of epidermal
tissue. Such an introducer may be inserted through a guide channel 6003 of
apparatus 6000,
wherein the tip of the outer sleeve reaches the area of proximal elevated
protrusion 6006
and the tip of the needle extends beyond the distal end of the outer sleeve.
In one
to embodiment, the tip of the outer sleeve passes through the skin layers
and into the fat layer.
In one embodiment, the inner needle is a 14GA needle. In another embodiment,
the inner
needle is a 12GA needle. In another embodiment, the inner needle is a 13GA
needle. In
another embodiment, the inner needle is a 15GA needle. In yet another
embodiment, the
inner needle is a 16GA needle. In still another embodiment, the inner needle
is a 17GA
needle. In another embodiment, the inner needle is an 18GA needle. In another
embodiment, the inner needle is a 19GA needle.
[00155] As a result of withdrawing the inner needle, the outer sleeve may
reside and
extend from within the guide channel 6003 into dermal tissue 6060.
Alternatively, the outer
sleeve may reside and extend from within the guide channel 6003 into fat
tissue 6018. In
one embodiment, an outer sleeve may be located from approximately the site of
insertion to
approximately the center of the proximal elevated protrusion 6007. The outer
sleeve may
act as a "sleeve" through which the coring needle may be introduced directly
into the fat and
immediately in front of the dermal tissue which is to be harvested. The outer
sleeve thereby
prevents harvesting of epidermal tissue. In another embodiment, the outer
sleeve may act as
a "sleeve" through which the coring needle may be introduced directly into the
dermis,
which is to be harvested. In addition, the outer sleeve protects the puncture
site from the
rotation and forward movement of the coring needle to prevent additional
trauma to the
puncture site. In certain instances, the outer sleeve may have a low
coefficient of friction to
prevent resistance to the rotational and forward motion of the coring needle,
thereby
preventing unwanted heat generation.
[00156] According to some exemplary embodiments of the invention, apparatus
6000
may also include a drill collet 6008 or glued cap to act as a hard stop
enabling manual
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advancing of a coring tube 6016 for a predetermined distance along chamber
6001, e.g., to a
position in which coring tube 6016 has advanced into fat tissue 6018 within
distal elevated
protrusion 6006.
[00157] Tool 6016 may be connected to a motor, e.g., as described above, to
rotate tool
6016 at a rotational speed appropriate for cutting of the dermal tissue, for
example, a
relatively high rotational speed, for example, a speed higher than 1,000 RPM,
e.g., between
1,000 RPM and 10,000 RPM. For example, tool 6016 may be rotated at a
rotational speed
higher than 2,000 RPM, e.g., approximately 7,000 RPM. When complete, the
forward and
rotational movements of tool 6016 may be stopped, and cutting tool 6016 may be
retracted
to with harvested DMO within it, thereby removing the cutting tool from the
harvest site.
DMO may be removed from cutting tool 6016, e.g., using a syringe to flush
sterile fluid, for
example saline, through tool, or a vacuum source to draw out DMO from a back
end (not
shown) of cutting tool 6016, as described above.
[00158] It will be appreciated by those skilled in the art that apparatus 6000
may enable
harvesting of the DMO by forming only one incision or puncture point.
Furthermore,
apparatus 6000 may be efficiently applied for harvesting a DMO from areas
having
relatively thick skin, e.g., from a region of the donor's back.
[00159] In some embodiments, elements of harvesting apparatus may be single
use items.
[00160] Reference is now made to FIGS. 10A and 10B, which show embodiments of
a
syringe with a septum and collet. FIG. 10A shows a syringe with a septum and
collet
attached to the back end of a cutting tool which is inserted through a support
structure guide
channel and an outer sleeve, wherein the support structure is connected to a
vacuum source.
FIG. 10B shows an embodiment of a syringe with a collet and needless valve
attached to
the back end of a coring needle.
[00161] FIGS. 10A and 10B illustrate an embodiment of the invention, wherein a
female
luer syringe is attached to the back end of the coring needle via a collet and
needleless
valve. FIGS. 10A and 10B illustrate embodiments that may be used in order to
suction the
DMO out of the cutting tool and into the syringe body.
[00162] It will be appreciated by those skilled in the art that the harvesting
methods
and/or apparatuses according to embodiments of the invention, e.g., as
described above,
may include introducing thin tissue cutting devices within the dermis. Thus,
the harvesting
methods and/or apparatuses according to embodiments of the invention may
enable
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harvesting the DMO with relatively minimal damage to the outer skin surface,
and therefore
may provide a minimally invasive method of harvesting the desired tissues.
[00163] Although some embodiments of the invention described herein may refer
to
methods and/or apparatuses for harvesting a DMO, it will be appreciated by
those skilled in
the art that according to other embodiments of the invention at least some of
the methods
and/or apparatuses may be implemented for any other procedures, e.g., plastic
surgical
procedures, dermatological procedures, or any other procedures including
harvesting of
tissues. For example, the methods and/or apparatuses according to embodiments
of the
invention may be implemented for harvesting dermal tissue to be used, e.g., in
a subsequent
to implantation, as filler material.
[00164] According to some embodiments of the present invention, a system and
method
are provided for ex-vivo ("in vitro") handling or processing of dermal micro-
organs. In
some embodiments, the dermal MOs may be directly placed into tissue culture
wells or
transduction chambers of a bioreactor, for further processing. In some
embodiments, e.g., if
the DMO remains in the coring tube as it is withdrawn from the skin, the DMO
may be
flushed out of the coring tube by the use of biologically compatible fluid,
e.g., saline or
growth medium, applied to the back end of the coring tube. The flushing of the
DMO may
be such that it is flushed directly into a chamber of a bioreactor.
Alternatively, vacuum may
be applied to a back end of the coring tube to "draw out" the DMO, e.g.,
directly into a
chamber of a bioreactor.
H. Methods and Apparatuses for Implanting a DMO/DTMO
[00165] According to some embodiments of the present invention, a system and
method
are provided for implantation of DTMOs. After producing and/or processing of a
DMO, for
example, by genetically modifying the DMO, the modified DMO or DTMO may be
implanted back into the patient, for example, for protein or RNA based
therapy. The
number of full or partial DTMOs that are implanted may be determined by the
desired
therapeutic dosing of the secreted protein. DTMOs may be implanted
subcutaneously or
into dermal tissue or at other locations within the body. Subcutaneous
implantation by use
of an implantation needle, for example, may enable the DTMO to remain in a
linear form in
a subcutaneous space. The linear form of implantation may help facilitate
localization in
case later removal or in-situ ablation of the DTMO is required, for example,
in order to stop
treatment or reduce the dose of therapeutic protein. Other known geometrical
implantation
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patterns could be used. The linear implantation may also assist in the
integration of the
dermal tissue to the surrounding tissue.
[00166] Reference is now made to FIGS. 7, 8A-E and 9A-E. FIG. 7 schematically
illustrates a flowchart of a method of implanting a DMO/DTMO according to some
exemplary embodiments of the invention, and to FIGS.8A-E, which present some
embodiments of elements of an implanting apparatus. The methods of
implantation
presented herein refer to implantation of either a DMO or a DTMO, and the
terms may be
used interchangeable in describing the methods and apparatus for implantation.
For ease of
reading only, a DTMO is recited in the description below wherein it is
recognized that the
to term "DMO" is interchangeable with the term "DTMO" in the following
description.
[00167] As indicated at block 7002, a DTMO may be loaded into a syringe (FIG.
8A).
For instance a DTMO may be aspirated into a loading syringe. Loading may
entail drawing
up biologically compatible fluid, e.g., saline or growth medium. Following
loading of the
DMTO in the syringe, an implanting tool, for example an implantation needle
(FIG. 8B),
may be attached to the syringe.
[00168] As indicated at block 7004, according to some exemplary embodiments of
the
invention, the DTMO, optionally together with surrounding sterile saline fluid
may be
loaded into an implanting tool (FIG. 8B) by connecting a loading syringe
containing the
DTMO with the proximal end of an implantation needle, and then gently loading
the
DTMO into the needle using positive pressure. Alternatively, a DTMO may be
aspirated
directly into an implantation needle, for example, through the distal end of
an implantation
needle by withdrawing a plunger of a syringe attached to the proximal end of
an
implantation needle. Optionally, a tip of the needle may have a removable
short extension
of silicon tubing, or the like, affixed to it, to facilitate the aspiration of
the DTMO into the
needle cannula through the distal end while retracting the plunger of the
syringe.
[00169] An implantation needle may have any suitable diameter, for example,
between
17GA and 8GA. In one embodiment, the diameter of an implanting tool is about
that of a
10 GA needle. In some embodiments, the tip of an implanting tool has a beveled
edge. In
other embodiments, the tip of an implanting tool does not have a beveled edge.
[00170] In some embodiments, after loading the DTMO into the implantation
needle, the
proximal (back) end of the implanting needle is plugged to prevent the DTMO
from
coming out the back end of the tube. In other embodiments, an adjustment of
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positive/negative pressure using the plunger of the loading syringe is used to
keep the
DTMO within the implanting needle.
[00171] As indicated at block 7006 a local anesthetic may be optionally
administered,
e.g., as is known in the art, to the vicinity of an intended implantation
site.
[00172] As indicated at block 7008, the method may further include positioning
an
apparatus including a support structure (FIG. 8D), at a given implantation
site so that the
support structure is in contact with an epidermal surface of the subject. In
some
embodiments, contact between a support structure of this invention and the
epidermal
surface of a subject must be air-tight so that a vacuum seal may be formed at
a later step. In
to one embodiment, an implantation site is on a subject's abdomen. In
another embodiment, an
implantation site is on a subject's back. Alternatively, the implantation site
may be at
another location on the subject's body.
[00173] In some instances, dosage may be adjusted based on the
number/size/efficacy of
DTMOs to be implanted. For example, multiple DTMOs may be implanted
consecutively
during a single procedural time period in order to reach a target dose. In one
embodiment,
an implant marker template may be used prior to positioning an implanting
apparatus on a
subject's epidermal surface (step 7008), in order to mark multiple sites for
implanting. In
one embodiment, an implanting marker template is positioned on the epidermal
surface of a
subject, and the epidermal surface is then marked to indicate, for example,
anesthesia lines
and alignment lines for later placement of the support structure 9006. In one
embodiment,
the surface is marked using a surgical pen or marker. In one embodiment, the
surface is
marked using a non-permanent dye or ink.
[00174] As indicated at block 7010, a support structure FIG. 8D, which may
include a
vacuum chamber with at least one elevated protrusion and guide channel 9008,
wherein the
elevated protrusion, 9007, is adjacent to the guide channel. For instance a
support structure
of FIG. 5A or 5B may be used to hold and support the skin-related tissue
structure in place
for proper implanting of a DTMO, for example, to create a unique geometry of
the skin,
such that the path of implantation is precisely controlled. For example,
application of
vacuum conditions causes a vacuum to be formed within the vacuum chamber
thereby
drawing the epidermal surface of the skin-related structure into the interior
of the support
structure, wherein a central channel may support epidermal and dermal skin
layers, and
possibly fat layers, of the skin-related structure. In exemplary embodiments,
a vacuum
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condition may cause the skin-related structure to be held at an inner support
surface of the
vacuum chamber. The guide channel may provide guidance and/or stability for an
implanting tool to ensure proper implanting along a linear implanting axis,
which in one
embodiment is in the subcutaneous space, such as between the dermis and fat
layers. In
some embodiments, implanting results in a DTMO remaining substantially linear
after
implantation. In some embodiments, the implanting axis is coaxial with the
needle guide
channel and central channel. In another embodiment, a DTMO is implanting
without
ensuring linearity.
[00175] As indicated at block 7012 and FIGS. 9A and 9B, and similar to the use
for
methods of harvesting described above, an introducer, FIG. 8C, may then be
used to
puncture the skin-related tissue by inserting the introducer through the guide
channel of the
support structure and into the skin-related structure along the implanting
axis. This single
puncture site may be used for all further entry into the skin-related
structure. In this way
damage and scarring to the subject is limited. In addition, use of an
introducer eliminates
exposure of the loaded DTMO within an implantation tool to a vacuum condition
while
implantation tool is penetrating the skin-related structure. Exposure of the
DTMO loaded in
the implantation needle to vacuum conditions present within the support
structure may lead
to a risk of the DTMO being suctioned into the vacuum line.
[00176] An introducer composed of an inner needle 9010 and an outer sleeve
9012, fitted
as a sheath over the inner needle, may be inserted into the skin-related
structure such that
the distal edge of the outer sleeve extends into the region of tissue just
under the elevated
protrusion. Under vacuum conditions, a precise geometry of the skin-related
tissue structure
is created such that insertion of the introducer is for example, generally
perpendicular to the
skin surface at the point of penetration. In one embodiment, the inner needle
of the
introducer and the implanting tool have about the same dimensions of diameter.
For
instance, the diameter of an inner needle and of an implanting tool may each
be about those
of a 10 GA needle.
[00177] As indicated at block 7014 and FIG. 9B, the inner needle is withdrawn
from the
skin-related tissue structure while the outer sleeve component 9012 of the
introducer is
positioned and remains in place. The result of this action is that the outer
sleeve is
positioned coaxially with the implanting axis and extends into the skin-
related tissue
structure. In preferred embodiments, the distal edge of the outer sleeve
extends into the fat
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under the elevated protrusion. In other preferred embodiment, the distal edge
of the outer
sleeve extends into dermis under the elevated protrusion.
[00178] The outer sleeve may include a thin needle, tube or any other suitable
thin,
generally straight, object able to be placed inside the dermis or in a
subcutaneous space. For
example, an outer sleeve may include a needle of size 6-18 GA, for example,
about 10 GA
or 14 GA, as is known in the art. Alternatively, an outer sleeve may include a
plastic tube.
In one embodiment, an outer sleeve includes high-density polyethylene (HDPE)
tubing. In
another embodiment, an outer sleeve includes Teflon . In yet another
embodiment, an outer
sleeve includes polytetrafluoroethylene (PTFE) tubing. In still another
embodiment, an
to outer sleeve includes fluorinated ethylene propylene (FEP) tubing.
[00179] An introducer may be inserted into the dermis or subcutaneous space by
being
pushed generally perpendicular to the skin surface at the point of
penetration. In one
embodiment, an inner needle is beveled. In such a circumstance, introduction
of the inner
needle part of an introducer may be with the bevel side facing down. In
another
embodiment, the bevel side is facing upward. In yet another embodiment, the
bevel side is
facing any direction in between upward and downward. In still another
embodiment, an
inner needle is not beveled.
[00180] As indicated at block 7016 and FIGS 9C, an implanting tool 9014 with
the
loaded DTMO 9016 may be inserted through the support structure guide channel
and the
outer sleeve, and advanced along an implanting axis into the desired location,
e.g., in the
subcutaneous space, within a fat layer, at the interface of fat layer and
dermis layer, or into
dermal tissue layer, along a distance approximately equivalent to the length
of the DTMO.
A silicon tubing extension, if used for loading of the DTMO 1716, would be
removed prior
to insertion of the implanting tool through the needle guide channel. An
implanting tool
may be a needle, for instance 6GA ¨ 14GA. In one embodiment, an implanting
tool may be
a 10 GA needle. In another embodiment, an implanting tool may be a 14 GA
needle. In one
embodiment, the implanting tool is inserted with the bevel up. In another
embodiment, the
implanting tool is inserted with the bevel down. Once the implanting tool has
been
advanced for placement of a DTMO at a desired location, the plug at the
proximal end of
the implanting tool, if used, is removed.
[00181] As indicated at block 7018 and FIG. 9D, a stopper tool including a
stopper tool
body 9018 and a stopper pin 9020 ( FIG. 8E) may be connected with an apparatus
such that
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it is secured relative to the apparatus and a stopper pin inserts within the
implanting needle
9016 through the proximal end.
[00182] As indicated at block 7020 and FIG. 9E, the implantation needle may be
retracted through e.g., the subcutaneous space, releasing the DTMO from the
implantation
needle and laying the DTMO linearly along the needle tract. In one embodiment,
in order to
ensure linear placement of a DTMO, a stopper pin, 9020, of a stopper tool
(FIG. 8E) is
inserted within the proximal end of the implanting needle. In another
embodiment,
assistance may be given to help release the DTMO for example by connecting a
syringe to
the proximal end of the implantation needle and gently providing positive
pressure with the
to syringe plunger, possibly during retraction of the implantation needle.
[00183] In exemplary embodiments of the invention, a stopper of FIG. 8E is
attached to
the support structure FIG. 8D, such that the rod of the stopper is internal
and coaxial with
the implanting tool. In one embodiment, the stopper's association with the
support structure
is such that its placement is set, e.g., locked-in-place, relative to other
elements of the
implanting apparatus. For instance, the stopper rod may fit inside the back
end of the
implanting needle such that the rod is brought into close proximity with the
loaded DTMO
within the implanting needle, and the implanting needle may be withdrawn over
the
stationary rod of the stopper. Retraction of the implanting needle over the
rod may, in one
embodiment, be for the full extent of the rod. In another embodiment,
retraction may be
over a part of the rod's length. As the rod is stationary, retraction of the
needle may result in
the rod extending beyond the beveled tip of the implanting needle after
retraction.
Retraction of the implanting needle over the rod of the stopper may in some
instances
prevent the DTMO from being pulled back together with the implanting needle.
In yet
another embodiment, retraction may result in the DTMO being released into the
target site,
e.g. subcutaneous space, in a linear form.
[00184] Linear implantation of a DTMO may provide better exposure of the
implanted
tissue to surrounding environment. For example, linear implantation may
facilitate better
integration of the DTMO. In addition, linear implantation may facilitate
diffusion of a
secreted recombinant product, e.g., a recombinant protein or portion thereof.
Moreover,
linear implantation may facilitate increased angiogenesis in the region of the
DTMO. If, at a
future date, it is required that a DTMO be excised or ablated, linear
positioning provides a
known orientation and location of a given DTMO. In one embodiment,
implantation results
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in a DTMO being placed linearly within a subcutaneous space. In another
embodiment,
implantation results in a DTMO being placed linearly within a tissue of the
same kind as
the DTMO, e.g., dermal tissue. In yet another embodiment, implantation results
in a DTMO
being placed linearly deeper in the body.
[00185] As indicated at block 7022, the apparatus may then be removed from the
implant
site. In addition, the outer sleeve may be removed at this time.
[00186] It will be appreciated by those skilled in the art that any
combination of the
above actions may be implemented to perform implanting according to
embodiments of the
invention. Further, other actions or series of actions may be used.
to [00187] In addition and without repeating the description and all of the
embodiments of
apparatuses 5000 and 6000 (FIGS. 3-7 and FIG. 11) described above as
apparatuses for
harvesting, e.g., support structures for harvesting a DMO, in some embodiments
of the
invention apparatuses 5000 and 6000 may also be used in methods of implanting
a DTMO.
Accordingly, in some embodiments of the invention, apparatuses 5000 or 6000
may also be
used in methods of implanting.
M. Methods and Apparatuses for Excising a DMO/DTMO
[00188] According to some embodiments of the present invention, a system and
method
are provided for in-vivo demarcation and localization of the implanted dermal
micro-
organs. Identification of the location of a subcutaneous implantation or
implantation at any
other location in the body, of processed tissue, such as a DTMO, may be
important, for
example, in the case where it is necessary to stop the protein treatment, or
to decrease the
dosage of the secreted protein. For example, termination or titration of
dosage may be
performed by removing one or more DTMOs entirely and/or by ablating one, a
portion of
one, or more than one of the implanted DTMOs. In order to identify a
subcutaneously
implanted DTMO, according to one embodiment, the DTMO may be colored prior to
implantation by an inert, biocompatible ink or stain containing, for example,
a
chromophore, which may be visible to the naked eye or may require special
illumination
conditions to visualize it. In this way a DTMO may be distinguished from its
surrounding
tissue by visual inspection and/or by use of enhanced imaging means.
[00189] According to one embodiment, at least the peripheral surface of a DTMO
may be
coated with, for example, biocompatible carbon particles, biocompatible tattoo
ink, or other
suitable materials including titanium particles, magnetic particles and/or
microspheres.
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Once implanted subcutaneously, the DTMO may be visible with the naked eye, by
suitable
enhanced imaging device, or other means of detection. Other ways to enhance
the visibility
of an implanted DTMO may include using a strong light source above the skin
surface, or
pinching the skin and directing the light source at the skin from one side,
such that the skin
may appear translucent and the dyed DTMO may be more visible. Alternatively,
the stain
may be fluorescent, visible only when illuminated using UV light, such as
using fluorescent
plastic beads.
[00190] According to another embodiment, the location of a subcutaneously
implanted
DTMO may be identified by co-implanting a biocompatible structure along with
the
to DTMO. An example of such a biocompatible structure is a non-absorbable
single stranded
nylon suture commonly used in many surgical procedures. Such a suture may be
implanted
in the same implantation tract with the DTMO, or may be implanted directly
above the
DTMO in the upper dermis or below the DTMO in the fat, such that the spatial
location of
the DTMO may be determined by the suture location. Further, the depth of the
DTMO may
be known to be at the depth of the subcutaneous space. The suture may be
visible to the
naked eye, observed with the assistance of illumination means, and/or observed
with the aid
of other suitable imaging means, such as ultrasound. Alternatively, the suture
can be
fluorescent, and visible through the skin under appropriate UV illumination.
The suture
may alternatively be of an absorbable material, so that it may enable
determination of
localization for a desired period of time, such as a few months.
[00191] According to another embodiment, the DTMO may be genetically modified
or
engineered to include a gene to express a fluorescent marker or other marker
capable of
being visualized. For example, the DTMO can be modified with the GFP (Green
Fluorescent Protein) gene or Luciferase reported gene, which, for example, may
be
expressed along with the gene for the therapeutic protein. In this manner, the
DTMO may
be visualized non-invasively using appropriate UV or other suitable
illumination and
imaging conditions.
[00192] According to another embodiment, one or more tattoo marks, e.g. small
tattoo
dots, can be applied on the skin in the vicinity of the implantation site. In
a preferred
embodiment, a small tattoo dot is applied to the skin at either end of a
linearly implanted
DTMO. The tattoo ink can be permanent, or temporary, such as ink used for
cosmetic
make-up use.
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[00193] According to some embodiments of the present invention, a system and
method
are provided for removal or ablation of implanted DTMOs. In a case, for
example, where
DTMO-based therapy to a patient must be terminated, or if the protein
secretion must be
decreased, one or more implanted DTMO may be partially or entirely removed, or
partially
or entirely ablated. In one embodiment, the DTMO may be surgically removed.
[00194] According to one embodiment in which small tattoo dots are applied on
the skin
at either end of a linearly implanted DTMO, surgical excision of the DTMO can
be
accomplished by resection of an elliptical tissue sample, including at least
both tattoo dots
and including all of the layers of skin and some subcutaneous tissue to ensure
that the
to DTMO has been removed. The excision site can then be sutured closed.
[00195] In addition and without repeating the description and all of the
embodiments of
apparatuses 6000 and 1100 (FIGS. 3-6 and 11) described above as apparatuses
for
harvesting, e.g., support structures for harvesting a DMO, in some embodiments
of the
invention apparatuses 6000 and 1100 may also be used in methods of excising a
DTMO.
[00196] Reference is now made to FIG. 12, which illustrates an exemplary
embodiment
of excising a DTMO. It will be appreciated by those skilled in the art that
any combination
of the above actions for harvesting may be implemented to perform excising of
an DTMO
according to embodiments of the invention. Further, other actions or series of
actions may
be used.
[00197] Without repeating all of the embodiments for harvesting described in
detail
above, reference is now made to FIG. 12. Briefly, at block 1202 the location
of the
implanted subcutaneous DTMO may be determined. At block 1203, a local
anesthetic may
be optionally administered at the site of DTMO removal. At block 1204 a
support structure
may be positioned over the site of the DTMO to be removed. A support structure
(e.g., FIG.
4C; FIGS. 5A-B, FIG. 6, FIG. 11), which may include a vacuum chamber and a
guide
channel, may be used to hold and support the skin-related tissue structure for
proper
excising of a DTMO and minimal surrounding tissue thereof.
[00198] At block 1206, vacuum conditions are applied and a skin-related tissue
structure
including the DTMO to be excised may be shaped so that the tissue containing
the DTMO
to be excised is within a central channel and is aligned with a cutting axis.
[00199] At block 1208, an introducer including an inner needle and an outer
sleeve may
be used to puncture the skin-related tissue by inserting the introducer
through the guide
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channel of the support structure and into the skin-related tissue structure at
a point of
penetration. The inner needle is then removed and the outer sleeve remains
positioned with
the distal end of the outer sleeve residing in a region proximal to the DTMO
to be excised.
[00200] At block 1210, a core of tissue that includes the DTMO may be
harvested. A
coring needle, of the same or larger diameter than that of the DMO harvesting
needle (for
example, 11 GA or similar), may be inserted through the guide channel and
outer sleeve
along a cutting axis in order to harvest the previously implanted DTMO. In one
embodiment, a DTMO is excised using a coring tube similar to, or larger in
diameter than
that used for direct harvesting of the DMO. In one embodiment, additional
tissue
to surrounding the DTMO being excised is harvested during the excision of
the DTMO. In
one embodiment, the additional tissue includes epidermal tissue. In one
embodiment, the
additional tissue includes dermal tissue not associated with the DTMO. In one
embodiment, the additional tissue includes fat tissue. In one embodiment, such
a coring
approach may be combined with vacuum suction at the proximal end of the
cutting tool to
help remove the cut tissue sample from the body.
[00201] According to an embodiment of the present invention, minimally
invasive or
non-invasive methods of ablating the DTMO in-situ may be used to make the
procedure
less traumatic and less invasive for the patient. In one embodiment,
potentially in
conjunction with the case of the dyed DTMO, a laser, for example, a non-
invasive Yag
laser may be used. The energy of the Yag laser, for example, may be
selectively absorbed
by the chromophore of a dyed DTMO, such that the energy is primarily directed
to the
DTMO, with minimum damage caused to the surrounding tissue. Other light energy
sources may also be used. Alternatively, such a laser approach can be used
with other
means of locating the DTMO other than use of a dye.
[00202] According to another embodiment, the DTMO may be ablated by delivering
destructive energy from a minimally invasive probe inserted into the
subcutaneous space
along the length of the DTMO. Such a probe may enable delivery of a variety of
energy
types, including radio frequency, cryogenic temperatures, microwave, resistive
heat, etc. A
co-implanted structure, such as a suture, may be used to determine the
location of the
DTMO, thereby enabling the probe to be inserted subcutaneously, for example,
along or
directly above or below the suture. In such a case, for example, the
destructive energy may
be delivered while the suture is still in place. Alternatively, the suture may
be removed after
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placement of the probe and before delivery of the destructive energy. The
amount of energy
applied may be either that required to denature the proteins in the tissue
such as during
coagulation by diathermy. Additionally or alternatively, the amount of energy
applied may
be as much as is used in electro-surgical cutting devices, which char tissue.
Of course, other
means of localization and other means of delivering destructive energy may be
used.
Iv. Methods and Apparatuses for Processing a DMO
[00203] After a DMO is harvested, e.g., according to embodiments of the
present
invention, the DMO is optionally genetically altered. Methods and Apparatuses
for
processing a DMO have been described in detail in United States Publication
No. US-
2012/0201793-A1, which is incorporated herein by reference in full.
[00204] In one embodiment, the invention provides a method of delivering a
gene
product of interest into a subject by implanting the genetically modified DMO
of the
invention into a subject.
[00205] The invention contemplates, in one aspect, the use of the genetically
modified
DTMO for transplantation in an organism. As used herein the terms
"administering",
"introducing", "implanting" and "transplanting" may be used interchangeably
and refer to
the placement of the DTMO of the invention into a subject, e.g., an
autologous, allogeneic
or xenogeneic subject, by a method or route which results in localization of
the DTMO at a
desired site. The DTMO is implanted at a desired location in the subject in
such a way that
at least a portion of the cells of the DTMO remain viable. In one embodiment
of this
invention, at least about 5%, in another embodiment of this invention, at
least about 10%, in
another embodiment of this invention, at least about 20%, in another
embodiment of this
invention, at least about 30%, in another embodiment of this invention, at
least about 40%,
and in another embodiment of this invention, at least about 50% or more of the
cells remain
viable after administration to a subject. The period of viability of the cells
after
administration to a subject can be as short as a few hours, e.g., twenty-four
hours, to a few
days, to as long as a few weeks to months or years.
[00206] Alternatively, the DTMO, which includes genetically modified cells can
be kept
in vitro and the therapeutic agent, left in the supernatant medium surrounding
the tissue
sample, can be isolated and injected or applied to the same or a different
subject.
[00207] Alternatively or additionally, a DTMO can be cryogenically preserved
by
methods known in the art, for example, without limitation, gradual freezing (0
C., -20 C.,
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-80 C., -196 C.) in DMEM containing 10% DMSO, immediately after being formed
from
the tissue sample or after genetic alteration.
[00208] In accordance with an aspect of some embodiments of the invention, the
number
of DTMOs to be implanted is determined from one or more of: Corresponding
amounts of
the therapeutic agent of interest routinely administered to such subjects
based on regulatory
guidelines, specific clinical protocols or population statistics for similar
subjects.
Corresponding amounts of the therapeutic agent such as protein of interest
specifically to
that same subject in the case that he/she has received it via injections or
other routes
previously. Subject data such as weight, age, physical condition, clinical
status.
to Pharmacokinetic data from previous tissue sample which includes a
genetically modified
cell administration to other similar subjects. Response to previous DTMO
administration to
that subject.
[00209] In accordance with an aspect of some embodiments of the invention,
only some
of the DTMOs are used in a given treatment session. The remaining DTMOs may be
returned to maintenance (or stored cryogenically or otherwise), for later use.
[00210] There is also provided in accordance with an embodiment of the
invention,
method of adjusting the dosage of a therapeutic agent produced by a DTMO
implanted in a
subject and excreting a therapeutic agent, including (a) monitoring level of
therapeutic
agent in the subject; (b) comparing the level of agent to a desired level; (c)
if the level is
lower than a minimum level, then implanting additional DTMO; (d) and if the
level is
higher than a maximum level, then ablating or removing one or more of the
implanted
DTMOs. Optionally, the method includes periodically repeating (a)-(d).
Alternatively or
additionally, ablating or removing consists of ablating or removing a portion
of one or more
of the implanted DTMOs. Optionally, removing includes surgical removal.
Optionally,
ablating includes killing a portion of the implanted DTMO.
[00211] As described above with reference to FIG. 1, at least part of the
process of
sustaining the DMO during the genetic alteration, as well as the genetic
alteration itself,
may be performed in a bioreactor.
EXAMPLES
Example 1
Harvesting of a Dermal Micro-organ
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[00212] Dermal micro-organs were harvested from a human subject under sterile
conditions.
Experimental Procedure
[00213] With the subject prone, a harvest site on the lower abdomen was
selected,
disinfected, marked with guidelines, and injected with local anesthesia. The
harvest site was
in an area of healthy skin, free of stretch marks or other obvious skin
abnormalities. A
sterile harvesting support (Fig. 4C) structure containing a vacuum control
hole was
connected to a vacuum source, the vacuum turned on and the support structure
placed on the
subject's epidermis at the selected harvest site with the vacuum control hole
uncovered.
to [00214] A finger was placed over the vacuum control hole causing a
vacuum to raise the
skin-related tissue structure into the vacuum chamber.
[00215] With the sharpened bevel point of the Introducer inner needle facing
down (FIG.
4D, 4006), the Introducer (4006 and 4008) was inserted into the needle guide
of the support
structure, quickly and to the full stop. The Introducer's inner needle 4006
was then removed,
leaving behind the Introducer sleeve 4008.
[00216] Next the sharpened tip of the coring needle attached to the medical
drill was
inserted into the Introducer sleeve and gently pushed forward until the tip
reached the distal
end of the sleeve. The drill was then activated and pushed forward to the full
stop, pushing
the tip of the coring needle through the dermal tissue and into fat tissue. At
this point the
vacuum was deactivated by removing the finger from the vacuum control hole.
[00217] The drill was then disconnected from the coring needle and the
needleless valve
syringe assembly was connected to the coring needle by slipping the collet
onto the needle
and tightening it onto the needle, and then by piercing the septum with the
exposed end of
the coring needle and connecting that to the collet. The syringe was connected
to the septum
and the plunger of the syringe was withdrawn to create a vacuum, while the
syringe was
retracted together with the coring needle. The DMO was suctioned into the
syringe body
during this withdrawal process (FIG. 3G).
Experimental Results
[00218] Multiple DMOs were harvested. An isolated harvested DMO is shown in
FIG.
14A in comparison to a toothpick, wherein the DMO is approximately 30 mm in
length. As
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shown in FIG. 14B (1402), there was only minimal scarring of the skin tissue
at the harvest
sites.
Example 2
Implanting of a Dermal Micro-organ
[00219] Dermal micro-organs were implanted into a human subject under sterile
conditions.
Experimental Procedure
[00220] Similar to preparations for harvesting, the implantation of dermal
micro-organs
into a human subject began with the subject prone, with implant sites selected
on the lower
to abdomen, disinfected, marked with guidelines to align the support
structure for implanting,
and the sites injected with local anesthesia. A sterile implanting support
structure (Fig. 8D)
containing a vacuum control hole was connected to a vacuum source, the vacuum
turned on
and the support structure placed on the subject's epidermis at the marked
implantation sites
with the vacuum control hole uncovered.
[00221] A finger was placed over the vacuum control hole causing a vacuum to
raise the
skin-related tissue structure into the vacuum chamber.
[00222] With the sharpened bevel point of the Introducer inner needle facing
down (FIG.
8C), the Introducer was inserted into the needle guide of the support
structure, quickly and
to the full stop. The Introducer's inner needle was then removed, leaving
behind the
Introducer sleeve.
[00223] Next the implantation needle loaded with a DMO at the distal end was
inserted
into the Introducer sleeve, and pushed forward to the full stop. The stopper
element was
than connected to the implanting apparatus by inserting the stopper pin within
the inner
lumen of the implantation needle. The stopper pin was moved forward and
brought into
close proximity to loaded DMO within the implantation needle. The stopper body
was
affixed to the implanting support structure so that the stopper pin remains
stationary while
the implantation needle was retracted over the stopper pin and the DMO was
linearly
implanting within the subcutaneous space at the implantation site.
[00224] The implanting tools were carefully removed from the implant site and
the
vacuum was removed by removing the finger from the vacuum hole. A tattoo dot
was made
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with semi-permanent ink on the surface of the skin at either end of the
linearly implanted
DMO to demark the location of the implantation site.
Experimental Results
[00225] Multiple DMOs were implanted. As shown in FIG. 14B, DMOs were
implanted
in the lower abdomen (1404), wherein tattoo dots identify their site of
implantation. There
was only minimal scarring of the skin tissue at the implantation sites.
[00226] It will thus be clear, the present invention has been described using
non-limiting
detailed descriptions of embodiments thereof that are provided by way of
example and that
are not intended to limit the scope of the invention. For example, only a
limited number of
to genetic changes have been shown. However, based on the methodology
described herein in
which live tissue is replanted in the body of the patient, and the viability
of that tissue in the
body after implantation, it is clear that virtually any genetic change in the
tissue, induced by
virtually any known method will result in secretions of target proteins or
other therapeutic
agents in the patient.
[00227] Variations of embodiments of the invention, including combinations of
features
from the various embodiments will occur to persons of the art. The scope of
the invention is
thus limited only by the scope of the claims. Furthermore, to avoid any
question regarding
the scope of the claims, where the terms "comprise" "include," or "have" and
their
conjugates, are used in the claims, they mean "including but not necessarily
limited to".
[00228] Further, as used herein, the term "comprising" is intended to mean
that the
system includes the recited elements, but not excluding others which may be
optional. By
the phrase "consisting essentially of' it is meant a method that includes the
recited
elements but exclude other elements that may have an essential significant
effect on the
performance of the method. "Consisting of' shall thus mean excluding more than
traces
of other elements. Embodiments defined by each of these transition terms are
within the
scope of this invention.
[00229] Further, as used herein, the term "about", refers to a deviance of
between 0.0001-
5% from the indicated number or range of numbers. In one embodiment, the term
"about",
refers to a deviance of between 1 -10% from the indicated number or range of
numbers. In
one embodiment, the term "about", refers to a deviance of up to 25% from the
indicated
number or range of numbers.
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[00230] Further, as used herein, the term "a" or "one" or "an" refers to at
least one. In one
embodiment the phrase "two or more" may be of any denomination, which will
suit a
particular purpose.
[00231] Further, as used herein, the term "treatment" refers to both
therapeutic treatment
and prophylactic or preventative measures, wherein the object is to prevent or
lessen a
targeted pathologic condition or disorder. Thus, in one embodiment, treating
may include
directly affecting or curing, suppressing, inhibiting, preventing, reducing
the severity of,
delaying the onset of, reducing symptoms associated with a disease, disorder
or condition,
or a combination thereof. Thus, in one embodiment, "treating" refers inter
alia to delaying
progression, expediting remission, inducing remission, augmenting remission,
speeding
recovery, increasing efficacy of or decreasing resistance to alternative
therapeutics, or a
combination thereof. In one embodiment, "preventing" refers, inter alia, to
delaying the
onset of symptoms, preventing relapse to a disease, decreasing the number or
frequency of
relapse episodes, increasing latency between symptomatic episodes, or a
combination
thereof. In one embodiment, "suppressing" or "inhibiting", refers inter alia
to reducing the
severity of symptoms, reducing the severity of an acute episode, reducing the
number of
symptoms, reducing the incidence of disease-related symptoms, reducing the
latency of
symptoms, ameliorating symptoms, reducing secondary symptoms, reducing
secondary
infections, prolonging patient survival, or a combination thereof.
[00232] While certain features of the invention have been illustrated and
described
herein, many modifications, substitutions, changes, and equivalents will now
occur to those
of ordinary skill in the art. It is, therefore, to be understood that the
appended claims are
intended to cover all such modifications and changes as fall within the true
spirit of the
invention.
