Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B17/322—Skin grafting apparatus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B2017/0023—Surgical instruments, devices or methods, e.g. tourniquets disposable
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B2017/00969—Surgical instruments, devices or methods, e.g. tourniquets used for transplantation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B2017/320052—Guides for cutting instruments
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B2017/320064—Surgical cutting instruments with tissue or sample retaining means
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
  • A61B17/00—Surgical instruments, devices or methods, e.g. tourniquets
  • A61B17/32—Surgical cutting instruments
  • A61B17/322—Skin grafting apparatus
  • A61B2017/3225—Skin grafting apparatus with processing of harvested tissue
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells

Definitions

• 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.
• therapeutic agents can be delivered orally, transdermally, by inhalation, by injection and by depot with slow release.
• 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.
• the amount of therapeutic agent varies between administrations.
• a dermal micro-organ 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 (DTMOs).
• Skin micro-organs including layers of epidermal and dermal tissues, for example; as outlined in PCT/IL02/0880, 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.
• 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.
• 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.
• Another conventional apparatus for harvesting dermis 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.
• 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.
• the resulting harvested dermis is generally not uniform in dimensions and includes "plugs" of epidermis at either end of the dermal core.
• 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.
• 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.
• 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 tissue and a surrounding matrix.
• the DMO 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.
• the DMO of the invention does not produce keratin or produces negligible amounts of keratin.
• the DMO does not produce keratin and/or keratin cysts following subcutaneous or deeper implantation in a body.
• 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.
• the DMO contains hair follicles and sebaceous glands.
• 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.
• 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.
• the support structure includes one or more vacuum channels to fiuidically connect the vacuum chamber with at least one vacuum source.
• 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.
• 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.
• 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.
• 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.
• an apparatus in yet another embodiment, includes a third tubular element, for instance a cutting tool that inserts within and through the fourth tubular element.
• 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.
• the coring tube is a rotatable coring tube attached to a power source.
• 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.
• release of a vacuum condition may for example include removal of the finger from over the vacuum hole.
• any covering or uncovering of the vacuum hole may be used to control vacuum and release conditions, respectfully.
• a vacuum control mechanism relies on clamping and unclamping the vacuum line or opening and closing a valve in the vacuum control line.
• 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 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.
• a harvesting method includes making only a single puncture point in the skin-related structure.
• a method of harvesting includes the use of a vacuum to recover the DMO from the coring tube into a closed container.
• the closed container is a syringe body.
• the syringe may have an attached septum.
• the DMO remains within the coring tube after retraction from the harvest site and recovering the DMO comprises flushing the DMO from the coring tube.
• exemplary embodiments of the invention provide a method of and apparatus for implanting a dermal micro-organ.
• 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).
• DTMO dermal therapeutic micro-organ
• 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.
• 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 inner needle of the introducer and advancing the implanting tool into the skin-related tissue structure along
• a stopper tool is used to assist in maintaining the position of a DTMO during retraction of the implanting tool.
• 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.
• 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 another anatomical location, if linear implantation is not important.
• the at least one recombinant gene product is an at least one recombinant protein.
• the genetically modified DMO of the invention produces substantially no keratin.
• the invention provides a method of delivering to a recipient a recombinant gene product produced by the DMO.
• the invention provides a method of inducing a local or systemic physiological effect by implanting a DMO in a recipient.
• 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.
• the invention provides a method of implanting a DTMO so as to avoid or to reduce keratin cyst formation.
• the invention provides a method for removal of an implanted DTMO. BRIEF DESCRIPTION OF THE DRAWINGS
• FIG. 1 is a schematic block diagram of an exemplary method of producing and utilizing dermal therapeutic micro-organs (DTMOs), in accordance with an exemplary embodiment of the invention
• FIG. 2 is a schematic flowchart illustrating a method of harvesting a DMO according to some exemplary embodiments of the invention
• FIGS. 3A-3G are schematic illustrations of exemplary stages of harvesting a DMO in accordance with a method of FIG. 2;
• 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).
• 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 supporting a skin-related tissue structure from which a dermal micro-organ may be harvested at a desired position;
• FIG. 6 is a schematic illustration of some components of a dermal harvesting apparatus according to yet another exemplary embodiment of the invention.
• FIG. 7 is a flow chart illustrating a DTMO implanting method, according to some exemplary embodiments of the present invention.
• 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
• 8E is an embodiment of a stopper.
• FIG. 9A-E are schematic illustrations of exemplary stages of implanting a DTMO in accordance with a method of FIG. 7;
• 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;
• FIG. 11 shows a schematic illustration of an embodiment of a support structure.
• FIG. 12 is a flow chart illustrating a method of removing a previously implanted DTMO
• 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)
• 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.
• explant 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, wherein an “explant” may for example be a dermal micro-organ.
• DMO skin micro-organ
• DMOs 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 from which it is obtained.
• DMOs may include plurality of dermal components that retain the micro-architecture of the tissue or organ from which they were derived, and three dimensional structure of the dermal tissue from which they are derived, having dimensions selected so as to allow passive diffusion of adequate nutrients and gases to cells within the DMO and diffusion of cellular waste out of the cells of the DMO so as to minimize cellular toxicity and concomitant death due to insufficient nutrition and accumulation of waste.
• DMOs may consist essentially of a plurality of dermis components (tissue components of the skin located below the epidermis). These components may contain skin fibroblast, 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.
• 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 functional contact with at least one cell or non-cellular substance with which they were in physical and/or functional contact in vivo.
• the cells of the explant maintain at least one biological activity of the organ or tissue from which they are isolated.
• the donor is a human subject.
• the donor is a non-human mammalian subject.
• TMO therapeutic micro-organ
• DMO dermal micro-organ
• the therapeutic agent may or may not be a naturally occurring body substance.
• DTMO therapeutic Dermal MO which may be in some embodiments of the invention genetically modified.
• implantation 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.
• a DTMO is derived from tissue of the recipient.
• a DTMO is implanted substantially into the dermal layer of skin tissue.
• a DTMO is implanted between the dermal and fat layer of skin tissue.
• 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.
• the recipient is a human subject.
• the recipient is a non-human mammalian subject.
• a recipient receives one or more autologous TMOs or DTMO.
• 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.
• a coring needle of this invention is for single use.
• 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.
• an implanting tool of this invention is for single use.
• tubular element 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.
• a tubular element is an element having the form of a hollow elongated cylinder.
• a tubular element is an element having the form of a cylindrical channel, e.g., a tunnel or channel cut through a solid mass.
• a tubular element is open at both ends.
• a tubular element is open at one end.
• a tubular element comprises a beveled needle tip at one end.
• a tubular element comprises at least one blunt end that is sharpened.
• a tubular element is an element having the form of a solid, non-hollow elongated cylinder, for instance a rod.
• a tubular element may include a guide channel.
• rod 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.
• skin-related tissue structure refers to a structure of tissue components that may be stabilized and/or supported by apparatuses defined herein to enable 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.
• the skin-related tissue structure may include fat tissue and/or muscle tissue in the vicinity of the dermal tissue.
• a skin-related tissue structure of the present invention includes the skin tissue components drawn into the central channel under vacuum conditions.
• a skin-related structure includes epidermal, dermal and fat tissue.
• a skin-related structure includes epidermal, dermal, fat and muscle tissue.
• central channel as used herein may in some embodiments of the invention be used interchangeable with the term “vacuum chamber”.
• tubular elements may be positions approximately equidistant from a common axis.
• a cutting tool is aligned approximately equidistant from a common axis presented by a guide channel.
• a cutting tool is aligned approximately equidistant from a common axis presented by a central channel.
• 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.
• one tubular element is contained within another tubular element, but the central axis of both tubes need not be aligned.
• 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.
• 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.
• the biological activity for example, secretion of a therapeutic protein, e.g., erythropoietin and elevation of hematocrit as a result
• a DTMO may be comparable to or even higher than split thickness skin derived TMO.
• 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.
• a desired agent for example proteins
• 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.
• the DTMO can be implanted into or under the skin or within the body of the subject to produce the agent/protein in vivo.
• a DTMO is not encapsulated in an immunoprotective capsule or sheath.
• the DMO may contain tissue of a basal epidermal layer and, optionally, other epidermal layers of the skin.
• the dermal micro-organ does not include basal epidermal layer tissue.
• the DMO does not include epidermal layers.
• the DMO may contain a few layers of epidermal tissue.
• the dermal micro-organ may lack a complete epidermal layer.
• a DMO may include invaginations of the epidermis within the dermal tissue layers, while still lacking a complete epidermal layer.
• the DMO includes the entire cross-section of the dermis.
• the dermal micro-organ includes part of the cross-section of the dermis.
• 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.
• the DMO includes primarily dermal tissue, but may also include fat tissue.
• 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. //.
• the DMO to be harvested can be removed from the body by any means of 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.
• the DMO may be obtained by direct biopsy and then be cut to the required size.
• a tissue sample may be obtained by direct biopsy, in which the desired size of the dermal micro-organ is obtained.
• non-desired tissue may be cut from the harvested biopsy or directly harvested micro-organ.
• a DMO may be obtained by direct biopsy and then processed to become a DTMO by genetic modification of the DMO in vitro.
• 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.
• the dermal micro-organ is directly harvested from the body.
• 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.
• the dimension may be, for example, 1.71 mm.
• the dimension may be 1.21 mm.
• the dimension may be, for example, 1-3 mm.
• the dimension may be, for example, 2-4 mm.
• the dimension may be, for example, 1-2 mm.
• the dimension may be 0.5-1.5 mm.
• the dimension may be, for example, about 1.5 mm.
• the dimension may be, for example, about 2 mm.
• the cutting tool has dimensions corresponding to needle size dimensions.
• the cutting tool is, for example, a 14GA needle.
• the cutting tool is a 15GA needle.
• the cutting tool is a 16GA needle.
• the cutting tool is a 17GA needle.
• the cutting tool is an 18GA needle.
• the cutting tool is a 19GA needle.
• the cutting tool is a 12GA needle.
• 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.
• 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.
• 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.
• 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.
• the tip may be sharpened by removing material from the outer or inner surface of the tip.
• 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.
• at least one dimension may be 0.5-3.5 mm and at least one dimension may be 1.5-10 mm.
• 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.
• 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 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.
• a DMO When a DMO has the above listed dimensions, it maybe maintained in vitro, e.g., in 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.
• the growth media may include growth factors, fetal calf serum (FCS), human serum, or Synthetic Serum Substitute (SSS).
• FCS fetal calf serum
• SSS Synthetic Serum Substitute
• the growth media may include serum either from the donor or the recipient subject.
• the growth media may include autologous serum. In another embodiment, no serum is added to the media.
• 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.
• a DMO is stored, for example cryogenically or otherwise, prior to genetic modification processing.
• a DMO is stored, for example cryogenically or otherwise, following genetic modification processing.
• 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.
• a DMO may be processed independently into a DTMO, as described herein.
• 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 the presence of the therapeutic agent in the growth media of the cells.
• 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.
• 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.
• 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.
• a DTMO may be produced by following the steps described independent of a bioreactor.
• a DMO is harvested from a subject.
• the DMO is harvested from the same subject to which therapy will later be applied.
• the DMO is from dermal tissue.
• 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.
• the DMO or DTMO can be cryogenically stored for later use (i.e., introduction at the same stage of the process).
• the DMO can be implanted directly back into the patient from which it was harvested to produce a therapeutic, cosmetic, or other physiological affect.
• a DMO 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.
• 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, 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.
• 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
• the DMO is genetically altered.
• many methods of genetic alteration are known and may be used in conjunction with the present invention.
• 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 as to the particular methodology and apparatus for introducing the virus to the DMO.
• the genetically altered DTMO is optionally tested for production and secretion rates of the therapeutic agent.
• the quantity of secretion for example, ELISA, other immunoassays, spectral analysis, etc.
• 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.
• the amount of DTMO required for producing a desired therapeutic effect is determined.
• 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.
• the desired number of the DTMOs are selected.
• a DTMO is loaded into an implantation tool. Exemplary implementation tools are described below.
• 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.
• 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 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).
• the tissue of the DMO or DTMO appears to be integrated or well taken into the tissue of the subject into which it is implanted (especially if the tissue is implanted in a tissue of the same kind from which it was harvested), the cells included within the DMO or the DTMO continue to produce and secrete the therapeutic agent.
• the in vivo performance of the DTMO is optionally determined (block 228). Based on this evaluation for example, and/or on past patient data (block 226), patient dosage may then be adjusted (block 230) by increasing the amount of the implant or removing some of the implant, as described below. As the efficacy of the implant changes, additional DTMOs can be implanted.
• Genetic alteration may generally include genetically engineering a selected gene or genes into cells that causes the cells to produce and optionally to secrete a desired therapeutic agent such as a protein.
• a desired therapeutic agent such as a protein.
• 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.
• a method of harvesting a DMO from a subject may include generating and/or maintaining a skin-related tissue structure associated with the DMO, e.g., located generally at a targeted harvest site for harvesting the DMO, at a desired shape and position such that the cutting tool may be able to separate the DMO from tissue in the vicinity of the DMO.
• a skin-related tissue structure associated with the DMO e.g., located generally at a targeted harvest site for harvesting the DMO, at a desired shape and position such that the cutting tool may be able to separate the DMO from tissue in the vicinity of the DMO.
• an epidermis portion in the vicinity of the targeted harvest site may be lifted, e.g., by attaching at least part of the epidermis portion to a predefined surface such that at least part of the skin-related tissue structure may be lifted and maintained at the desired shape and/or position.
• attaching the epidermis to the predefined surface may include applying a vacuum condition, e.g., as described below.
• attaching the epidermis to the predefined surface may include applying an adhesive to the surface.
• FIG. 2 schematically illustrates a flowchart of a method of harvesting a DMO according to some exemplary embodiments of the invention
• FIGS. 3A-3G schematically illustrate exemplary stages of harvesting a DMO 3024 located within a skin-related tissue structure.
• 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.
• an anesthetic e.g., as is known in the art
• DTMOs for protein or RNA based therapy may, in certain circumstances, necessitate use of multiple DTMOs.
• 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 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.
• 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.
• 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.
• the surface is marked using a surgical pen or marker.
• the surface is marked using a non- permanent dye or ink.
• 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.
• 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.
• a harvest site is on a subject's back.
• a harvest site is on a subject's abdomen.
• a harvest site may be at another location on a subject's body.
• a support structure e.g., FIG. 4C; FIG. 13A; FIG. 6; FIG. 11
• a vacuum chamber FIG. 11 1130
• 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.
• 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 of the skin-related structure.
• guide channel may also be referred to herein as a “needle guide channel”.
• 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.
• 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.
• 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.
• the inner dimensions of a support structure including a vacuum chamber may be between 3.0 -8.0 mm.
• the dimension may be, for example 3.0 mm in diameter.
• the dimension may be, for example 3.5 mm in diameter.
• the dimension may be, for example 4.0 mm in diameter.
• the dimension may be, for example 4.5 mm in diameter.
• the dimension may be, for example 5.0 mm in diameter.
• the dimension may be, for example 5.5 mm in diameter.
• the dimension may be, for example 6.0 mm in diameter
• the dimension may be, for example 6.5 mm in diameter.
• the dimension may be, for example 7.0 mm in diameter.
• the dimension may be, for example 7.5 mm in diameter.
• the dimension may be, for example 8.0 mm in diameter.
• the appropriate sized support structure having particular inner dimensions of a vacuum chamber is pre-determined prior to actual harvesting.
• an introducer e.g., FIG. 4D
• an inner needle (3010; 4006) and an outer sleeve (3012; 4008)
• an outer sleeve (3012; 4008)
• 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.
• An introducer may be composed of tubular elements, for instance an inner needle and an outer sleeve.
• the outer sleeve is fitted over the inner needle. Together they may be inserted into the skin-related structure.
• 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.
• 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.
• 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.
• step at block 2012 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 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.
• 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.
• a support structure including, at least, a vacuum chamber including proximal (3007) and distal (3009) elevated protrusions.
• 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.
• 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 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.
• 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.
• an outer sleeve may have an inner diameter which corresponds with a needle of size 12-19 GA for example, about 14 GA.
• an outer sleeve may include a plastic tube of an appropriate length.
• the outer sleeve includes high-density polyethylene (HPDE) tubing.
• the outer sleeve includes polytetrafiuoroethylene (PTFE) tubing.
• the outer sleeve includes fiuorinated ethylene propylene (FEP) tubing.
• FEP fiuorinated ethylene propylene
• 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- 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.
• FEP fiuorinated ethylene propylene
• an outer sleeve may be inserted into dermis tissue.
• an outer sleeve may be inserted into a subcutaneous space.
• 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.
• 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.
• 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.
• 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.
• 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).
• 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.
• a medical drill may be used to rotate the cutting tool.
• the cutting tool attaches at one end to a drill collet (3016), as is known in the art
• the method may include rotating the cutting tool while advancing the cutting tool, e.g., towards the distal end of the support structure.
• 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.
• a proximal end of coring tube may be connected to a medical drill using a drill collet, 3016.
• 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.
• 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 1,000 RPM and 10,000 RPM.
• tube 3014 may be rotated at a rotational speed higher than 2,000 RPM, e.g., approximately 7,000 RPM.
• a relatively low rotational speed of less than 1000 RPM may be used, or no rotation at all, as described below.
• 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.
• a coring tube 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.
• a method of cutting a dermal micro-organ may include stopping the forward motion of the coring tube at a particular location.
• 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.
• 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.
• 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.
• 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.
• 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 to reside under the distal elevated protrusion such that the tip enters the fat layer.
• 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.
• 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.
• 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.
• 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.
• 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.
• the sharpened edge is sharpened on the outer diameter.
• 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.
• 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 and/or a plug of epidermis is not harvested.
• 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.
• a low friction material e.g., Teflon ® , Parylene or any other suitable coating material
• the method of harvesting includes recovery of the DMO 3024.
• 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 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.
• an external cap for example a luercap, may be use to hold the coring tube in place.
• the collet 3020/septum 3021 of the syringe 3022 assembly is attached (FIGS. 4B, 4A( 4004) and 4 (4012), respectively) to the coring tube.
• a male luercap (1302) is attached, e.g., glued, to the outer surface of a coring needle (1304).
• 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.
• the septum 3021 is pushed over the needle and attached to the collet.
• the septum includes a needleless valve.
• 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.
• the syringe 3022 can be attached to the needless valve assembly.
• the syringe plunger is withdrawn there is creation of a vacuum condition within the coring tube.
• 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.
• 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 example, into the syringe body.
• 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.
• 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.
• a drill is disconnected from a coring needle (1304) while holding a glued cap (1302) in place. (FIG.
• a female luer syringe (1306) is attached directly to the clued cap.
• the DMO is 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.
• 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.
• FIG. 4A shows one embodiment of a septum which may in certain embodiments be used in a method of harvesting a DMO of this invention.
• 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.
• suitable fluids such as sterile fluids, may be used to assist in removing the DMO from cutting tool 3014.
• 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.
• the apparatus may then be removed from the harvest site.
• the outer sleeve may be removed manually from the skin-related tissue.
• FIGS. 4C-E present exemplary embodiments of a harvesting apparatus
• 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
• 4014 presents an example of a medical drill useful for rotating a cutting tool
• 4012 presents an example of a drill collet for attaching the coring tube.
• FIGS. 4A-B present 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.
• FIGS. 4A-B present exemplary embodiments of a syringe (FIG. 4A (4012)), septum (FIG. 4A 4004) and syringe colle
• 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.
• 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.
• FIG. 5A schematically illustrates a dermal harvesting apparatus 5000 according to another exemplary embodiment of the invention.
• support structure refers to the body of the apparatus used to support a skin-related tissue structure.
• 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”.
• 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.
• 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.
• 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.
• 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.
• 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.
• apparatus 5000 may also include a vacuum conduit 5004 which is connected to the vacuum chamber 5001 and a vacuum source 5002.
• FIG. 5B 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.
• 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.
• 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.
• 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.
• apparatus 5050 may not include elements 5058 and/or channels 5064.
• 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.
• a clamping tool 5070 may be 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.
• 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.
• FIG. 6 and FIG. 11 schematically illustrate embodiments of harvesting apparatuses according to some exemplary embodiment of the invention.
• 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 fiuidically 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.
• 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.
• a coring tube 6016 may be advanced into a fat layer 6018, thus allowing separation of the harvested 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.
• 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.
• the internal width of a vacuum chamber of apparatus 5000 and/or 6000 is about 3.5 mm.
• a central channel may have a width of, for example, about 4 mm.
• a central channel may have a width of, for example, 3.0 mm.
• a central channel may have a height, excepting of the protrusions, of, for example, about 5 mm.
• 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.
• the dimensions of the channel length may be, for example, about 10-60 mm in length.
• the dimensions of the channel length may be, for example, about 20-60 mm in length.
• the dimensions of the channel length may be, for example, about 20-50 mm in length.
• the dimensions of the channel length may be, for example, about 20- 40 mm in length.
• the dimensions of the channel length may be, for example, about 20-100 mm in length.
• 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 embodiment, the length may be about 30 mm. In another embodiment, the length may be about 40 mm.
• 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.
• 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.
• the tip of the outer sleeve passes through the skin layers and into the fat layer.
• 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.
• the outer sleeve may reside and extend from within the guide channel 6003 into dermal tissue 6060.
• the outer sleeve may reside and extend from within the guide channel 6003 into fat tissue 6018.
• 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.
• 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.
• 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.
• 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.
• apparatus 6000 may also include a drill collet 6008 or glued cap to act as a hard stop enabling manual 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.
• a drill collet 6008 or glued cap to act as a hard stop enabling manual 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.
• 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.
• a relatively high rotational speed for example, a speed higher than 1,000 RPM, e.g., between 1,000 RPM and 10,000 RPM.
• tool 6016 may be rotated at a rotational speed higher than 2,000 RPM, e.g., approximately 7,000 RPM.
• the forward and rotational movements of tool 6016 may be stopped, and cutting tool 6016 may be retracted 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.
• 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.
• 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.
• elements of harvesting apparatus may be single use items.
• FIGS. 10A and 10B 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.
• 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
• the harvesting methods and/or apparatuses according to embodiments of the invention may include introducing thin tissue cutting devices within the dermis.
• the harvesting methods and/or apparatuses according to embodiments of the invention may enable 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.
• a system and method are provided for ex-vivo ("in vitro") handling or processing of dermal micro-organs.
• the dermal MOs may be directly placed into tissue culture wells or transduction chambers of a bioreactor, for further processing.
• 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.
• biologically compatible fluid e.g., saline or growth medium
• the flushing of the DMO may be such that it is flushed directly into a chamber of a bioreactor.
• 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.
• a system and method are provided for implantation of DTMOs.
• 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 patterns could be used.
• the linear implantation may also assist in the integration of the dermal tissue to the surrounding tissue.
• 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.
• a DTMO is recited in the description below wherein it is recognized that the term "DMO” is interchangeable with the term "DTMO” in the following description.
• a DTMO may be loaded into a syringe (FIG. 8A).
• a DTMO may be aspirated into a loading syringe.
• Loading may entail drawing up biologically compatible fluid, e.g., saline or growth medium.
• an implanting tool for example an implantation needle (FIG. 8B)
• FIG. 8B an implanting tool
• 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.
• 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.
• 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.
• 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.
• the proximal (back) end of the implanting needle is plugged to prevent the DTMO from coming out the back end of the tube.
• an adjustment of positive/negative pressure using the plunger of the loading syringe is used to keep the DTMO within the implanting needle.
• a local anesthetic may be optionally administered, e.g., as is known in the art, to the vicinity of an intended implantation site.
• 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.
• 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.
• an implantation site is on a subject's abdomen.
• an implantation site is on a subject's back.
• the implantation site may be at another location on the subject's body.
• 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.
• 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.
• 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.
• the surface is marked using a surgical pen or marker.
• the surface is marked using a non-permanent dye or ink.
• 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.
• 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.
• 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.
• a vacuum 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.
• implanting results in a DTMO remaining substantially linear after implantation.
• the implanting axis is coaxial with the needle guide channel and central channel.
• a DTMO is implanting without ensuring linearity.
• an introducer FIG. 8C
• 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.
• 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.
• 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.
• 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.
• 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.
• the distal edge of the outer sleeve extends into the fat under the elevated protrusion. In other preferred embodiment, the distal edge of the outer sleeve extends into dermis under the elevated protrusion.
• 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.
• 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.
• an outer sleeve may include a plastic tube.
• an outer sleeve includes high-density polyethylene (HDPE) tubing.
• an outer sleeve includes Teflon ® .
• an outer sleeve includes polytetrafluoroethylene (PTFE) tubing.
• an outer sleeve includes fiuorinated ethylene propylene (FEP) tubing.
• 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.
• an inner needle is beveled.
• introduction of the inner needle part of an introducer may be with the bevel side facing down.
• the bevel side is facing upward.
• the bevel side is facing any direction in between upward and downward.
• an inner needle is not beveled.
• 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.
• an implanting tool may be a 14 GA needle.
• the implanting tool is inserted with the bevel up.
• the implanting tool is inserted with the bevel down.
• a stopper tool including a stopper tool body 9018 and a stopper pin 9020 ( FIG. 8E) may be connected with an apparatus such that it is secured relative to the apparatus and a stopper pin inserts within the implanting needle 9016 through the proximal end.
• 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.
• a stopper pin, 9020, of a stopper tool (FIG. 8E) is inserted within the proximal end of the implanting needle.
• 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 syringe plunger, possibly during retraction of the implantation needle.
• 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.
• 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.
• 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.
• 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.
• Linear implantation of a DTMO may provide better exposure of the implanted tissue to surrounding environment.
• linear implantation may facilitate better integration of the DTMO.
• linear implantation may facilitate diffusion of a secreted recombinant product, e.g., a recombinant protein or portion thereof.
• 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 in a DTMO being placed linearly within a subcutaneous space.
• 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.
• the apparatus may then be removed from the implant site.
• the outer sleeve may be removed at this time.
• 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.
• 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.
• 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.
• a DTMO may be distinguished from its surrounding tissue by visual inspection and/or by use of enhanced imaging means.
• 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.
• 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.
• the stain may be fluorescent, visible only when illuminated using UV light, such as using fluorescent plastic beads.
• the location of a subcutaneously implanted DTMO may be identified by co-implanting a biocompatible structure along with the DTMO.
• 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.
• 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.
• 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.
• the DTMO may be genetically modified or engineered to include a gene to express a fluorescent marker or other marker capable of being visualized.
• 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.
• GFP Green Fluorescent Protein
• Luciferase reported gene which, for example, may be expressed along with the gene for the therapeutic protein.
• the DTMO may be visualized non-invasively using appropriate UV or other suitable illumination and imaging conditions.
• one or more tattoo marks can be applied on the skin in the vicinity of the implantation site.
• 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.
• a system and method are provided for removal or ablation of implanted DTMOs.
• one or more implanted DTMO may be partially or entirely removed, or partially or entirely ablated.
• the DTMO may be surgically removed.
• 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 DTMO has been removed.
• the excision site can then be sutured closed.
• apparatuses 6000 and 1100 may also be used in methods of excising a DTMO.
• FIG. 12 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.
• FIG. 12 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.
• a support structure e.g., FIG. 4C; FIGS. 5A-B, FIG. 6, FIG. 11
• 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.
• 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 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.
• 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.
• a DTMO is excised using a coring tube similar to, or larger in diameter than that used for direct harvesting of the DMO.
• additional tissue surrounding the DTMO being excised is harvested during the excision of the DTMO.
• the additional tissue includes epidermal tissue.
• the additional tissue includes dermal tissue not associated with the DTMO.
• the additional tissue includes fat tissue.
• 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.
• 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.
• a laser for example, a non-invasive Yag laser may be used.
• the energy of the Yag laser 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.
• such a laser approach can be used with other means of locating the DTMO other than use of a dye.
• 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.
• 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.
• the destructive energy may be delivered while the suture is still in place.
• the suture may be removed after 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.
• 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.
• 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.
• the invention contemplates, in one aspect, the use of the genetically modified DTMO for transplantation in an organism.
• 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.
• 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.
• 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.
• a DTMO can be cryogenically preserved by methods known in the art, for example, without limitation, gradual freezing (0° C, -20° C, -80° C, -196° C.) in DMEM containing 10% DMSO, immediately after being formed from the tissue sample or after genetic alteration.
• 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.
• 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.
• DTMOs 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.
• 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.
• the method includes periodically repeating (a)-(d).
• ablating or removing consists of ablating or removing a portion of one or more of the implanted DTMOs.
• removing includes surgical removal.
• ablating includes killing a portion of the implanted DTMO.
• 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.
• Dermal micro-organs were harvested from a human subject under sterile conditions.
• 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.
• a finger was placed over the vacuum control hole causing a vacuum to raise the skin-related tissue structure into the vacuum chamber.
• 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).
• FIG. 14A An isolated harvested DMO is shown in comparison to a toothpick, wherein the DMO is approximately 30 mm in length. As shown in FIG. 14B (1402), there was only minimal scarring of the skin tissue at the harvest sites.
• Dermal micro-organs were implanted into a human subject under sterile conditions.
• a finger was placed over the vacuum control hole causing a vacuum to raise the skin-related tissue structure into the vacuum chamber.
• 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.
• 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 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.
• 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.
• the term “comprising” is intended to mean that the system includes the recited elements, but not excluding others which may be optional.
• 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.
• 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.
• 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.
• 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.
• 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.
• "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.
• 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.
• “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.

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• 2012
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