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WO2019066663A1 - Procédé d'obtention de cellules à partir d'un dispositif de digestion de peau entière - Google Patents

Procédé d'obtention de cellules à partir d'un dispositif de digestion de peau entière Download PDF

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Publication number
WO2019066663A1
WO2019066663A1 PCT/NZ2018/050131 NZ2018050131W WO2019066663A1 WO 2019066663 A1 WO2019066663 A1 WO 2019066663A1 NZ 2018050131 W NZ2018050131 W NZ 2018050131W WO 2019066663 A1 WO2019066663 A1 WO 2019066663A1
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Prior art keywords
cells
skin
vitro method
keratinocytes
tissue
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Inventor
Peter Roderick Dunbar
Vaughan John Feisst
Elliott Thomas James DUNN
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Auckland Uniservices Ltd
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Auckland Uniservices Ltd
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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases [EC 2.]
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • 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
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts

Definitions

  • the present invention relates to a method of isolating cells, for example epithelial cells and/or connective tissue cells, such as keratinocytes and/or fibroblast cells from a skin sample, such as a human skin sample, comprising the step of digesting the sample in the presence of a protease.
  • the disclosure also extends to cells obtainable from said digest, a method of culturing the cells obtained from the digest, in particular to prepare a fully differentiated tissue and use of said tissue in treatment, in particular in the treatment of a condition, disease or infection disclosed herein BACKGROUND
  • Engineered tissues comprising epithelial cells have a wide range of uses. For example, such tissues are used in skin grafts for patients with burns or chronic wounds or in the development and testing of pharmaceutical, cosmetic and other topical products. While engineered tissues comprising only epithelial cells have utility, for some applications it is preferable to also have an underlying dermis layer.
  • the treatment of severe burns generally requires full-thickness skin comprising both an epidermal layer containing epithelial cells (keratinocytes] and an underlying dermal layer containing fibroblastic cells. This may be obtained by taking the patient's own healthy skin and grafting it onto the wound. This leaves a wound in the patient where the graft was taken from. In addition, if the graft is a large area it is difficult to take enough healthy skin from the patient for an adequate graft.
  • Ex vivo grown skin products (sometimes referred to as engineered skin] are an alternative to the whole graft coming directly from the patient's body. These engineered skin products can be an epidermal layer of keratinocytes or full -thickness products comprising a dermis and epidermis.
  • fibroblastic feeder cells To grow keratinocytes need to be co-cultured with fibroblastic feeder cells.
  • the fibroblasts employed are usually irradiated xenogeneic murine fibroblast feeder cells (MEF- murine embryonic feeder cells]. This is because unirradiated fibroblasts outgrow the keratinocytes and swamp the culture.
  • MEF- murine embryonic feeder cells irradiated xenogeneic murine fibroblast feeder cells
  • the sample processing may employ physical separation with a scalpel and enzymes, such as dispase the activity of which results in the epidermis and dermis being detached. Usually a combination of treatment with dispase and physical separation is employed.
  • production of either epidermis or synthesis of full-thickness skin in the art employs human keratinocytes co-cultured with irradiated mouse embryonic fibroblast feeder cells (xenogeneic cells].
  • xenogeneic cells irradiated mouse embryonic fibroblast feeder cells
  • This provides either a partial-thickness or a full-thickness skin product, which is potentially immunogenic because it is not fully human.
  • This carries a higher risk of an adverse immune response and graft rejection when such a tissue is transplanted into a human patient, in comparison to fully human skin products.
  • the inclusion of xenogeneic cells in the cell cultures also increases the risk of transmission of infectious disease from animals to humans.
  • the present inventors have identified that a mixture of keratinocyte and unirradiated fibroblasts, for example derived from a human tissue sample can be employed to grow skin products comprising a fully-human epidermis (including fully human full-thickness skin].
  • the present disclosure provides an efficient method of isolating the mixture of cells.
  • the method of the present disclosure may also provide increased numbers of cells (yield] in comparison to prior art methods.
  • An in vitro method suitable for obtaining cells for example epithelial cells and/or connective tissue cells (such as epithelial cells], in particular keratinocytes and/or fibroblast cells from a whole skin sample comprising a dermis and epidermis, by treating said sample with a digest capable of digesting the epidermis and dermis comprising at least one protease, for example a protease capable of digesting the epidermis and dermis.
  • a digest capable of digesting the epidermis and dermis comprising at least one protease, for example a protease capable of digesting the epidermis and dermis.
  • the skin sample is human, for example the sample is from any suitable location on a human body, such as: shoulder, arm, thigh, calf, breast, abdomen, foreskin and buttocks.
  • epithelial cells are human keratinocytes, for example selected from skin keratinocytes, foreskin keratinocytes, vaginal keratinocytes, placenta and cervical keratinocytes.
  • human keratinocytes for example selected from skin keratinocytes, foreskin keratinocytes, vaginal keratinocytes, placenta and cervical keratinocytes.
  • An in vitro method further comprising a first culture step of culturing the cells obtained in suitable media in the presence of an effective amount of a ROCK inhibitor, such as a small molecule ROCK inhibitor or an antibody (in particular where the first culture step is to expand the number of keratinocytes, for example without differentiation].
  • a ROCK inhibitor such as a small molecule ROCK inhibitor or an antibody
  • ROCK inhibitor is selected from the group consisting of: Y-27632, SB772077B, Fasudil, Ripasudil, Y39983, Wf-536, SLx-2119, an azabenimidazole-aminofurazan, DE-104, H-1152, ROKa inhibitor, XD-4000, HMN-1152, 4-(l-aminoalkyl]-N-(4-pyridyl]cyclohexane-carboxamide, rhostatin, BA-210, BA-207, BA- 215, BA-285, BA-1037, Ki-23095, VAS-012, RKI-1447, GSK429286A, Y-30141, HA-100, H-7, iso H-7, H-89, HA-1004, HA-1077, H-8, H-9, KN-62, GSK269962, quinazoline and combinations of two or more of the same.
  • the concentration of ROCK inhibitor is in the range 0.1 to ⁇ , for example 0.2 to 50 ⁇ or 0.3 to 25 ⁇ or 0.1 to 0.95 ⁇ , such as 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95 ⁇ , in particular 0.4 ⁇ .
  • antibiotics and/or antimycotics are independently selected from the group consisting of: Penicillin, Streptomycin, Amphotericin B, Nystatin, Actinomycin D, Ampicillin, Carbenicillin, Cefotaxime, Fosmidomycin, Gentamicin, Kanamycin, Neomycin, Polymyxin, Blasticidin, Geneticin, Hygromycin B, Mycophenolic acid, Puromycin and Zeocin, for example independently selected from Penicillin, Streptomycin, Gentamicin and Amphotericin B, in particular Gentamicin and Amphotericin B.
  • the media comprises or consists of: DMEM High glucose, Ham's F12, foetal bovine serum, and at least one antibiotic (for example 1, 2 or 3 antibiotics selected independently selected from penicillin, streptomycin, amphotericin B, such as two or three] and keratinocyte growth factor (KGF).
  • antibiotic for example 1, 2 or 3 antibiotics selected independently selected from penicillin, streptomycin, amphotericin B, such as two or three] and keratinocyte growth factor (KGF).
  • the media (Kelch's medium] consists of: DMEM High glucose:Ham's F12 (3:1], 10% foetal bovine serum, penicillin, streptomycin, 0.625 ⁇ g/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF]
  • Green's media DMEM High glucose:Hams F12 3:1, 10% foetal bovine serum, lOng/ml EGF, O.lnM choleratoxin, 0 ⁇ g/ml hydrocortisone, 180 ⁇ adenine, 5ug/ml insulin, 5 ⁇ g/ml apotransferrin, 2nM 3,3,5,-tri-idothyronine, 2mM glutamine, gentamicin, 0.625 ⁇ g/ml Amphotercin B]
  • An in vitro method according to any one of paragraphs 18 to 32, wherein the method comprises a second step of culturing the keratinocytes and fibroblasts in the absence of a ROCK inhibitor, for example wherein the second culture step is to form a differentiated epidermis.
  • an in vitro method wherein the keratinocytes in the second step are cultured, for a second phase in contact with a gas permeable membrane (interface], for example where the keratinocytes are in contact with the gas permeable membrane following a period of culturing without contacting a gas permeable layer, such as where contact with gas permeable layer is a distance of 2cm or less, such as 1cm or less, in particular 0.5, 0.4, 0.3, 0.2, 0.1 or 0.05cm.
  • a gas permeable membrane interface
  • an in vitro method according to any one of claims 33 to 35, wherein the keratinocytes are deposited on a substrate, for example a matrix, for example a spun matrix or mesh.
  • a substrate for example a matrix, for example a spun matrix or mesh.
  • the substrate (such as a matrix] comprises a biocompatible biodegradable polymer.
  • electrospun fibres from said electrospinning are about 0.3 ⁇ to about 5 ⁇ in diameter or 2 to 5 ⁇ in diameter, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 ⁇ .
  • fibres are spun from a polymer which is synthetic, naturally occurring or a combination thereof, for example selected from the group consisting of PLGA, PLA, PCL, PHBV, PDO, PGA, PLCL, PLLA-DLA, PEUU, cellulose- acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP, blended PLA/PCL, gelatin-PVA, PCT/collagen, sodium aliginate/PEO, chitosan/PEO, chitosan/PVA, gelatin/elastin/PLGA, silk/PEO, silk fibroin/chitosan, PDO/elastin, PHBV/collagen, hyaluronic acid/gelatin, collagen/chondroitin sulfate, collagen/chitosan, PDLA/HA, PLLA/HA, gelatin/HA, gelatin/siloxane, PLLA/MWNTs
  • the polymer is synthetic, for example PLGA, PLA, PCL, PHBV, PDO, PGA, PLCL, PLLA-DLA, PEUU, cellulose-acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP, blended PLA/PCL, PDLA/HA, PLLA/HA, PLLA/MWNTs/HA, PLGA/HA, 100 dioxanone linear homopolyer and combinations of two or more of the same.
  • synthetic for example PLGA, PLA, PCL, PHBV, PDO, PGA, PLCL, PLLA-DLA, PEUU, cellulose-acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP, blended PLA/PCL, PDLA/HA, PLLA/HA, PLLA/MWNTs/HA, PLGA/HA, 100 dioxanone linear homopolyer and combinations of two or more of the same.
  • polymer is poly(lactic-co-glycolic acid] (PLGA].
  • biocompatible biodegradable polymer is 10 to 40%w/v, for example 26% to 40% w/v, for example 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39% w/v, in particular 15, 16, 17, 18, 19, 20 or 21% w/v.
  • an in vitro method wherein the protein is selected from an extracellular matrix protein (such as collagen, laminin and other extracellular matrix proteins] or peptide thereof, for example a synthetic peptide.
  • an in vitro method wherein the extracellular matrix protein is selected from the group consisting of collagen IV, collagen I, laminin and fibronectin, or a combination thereof, in particular collagen IV.
  • An in vitro method wherein the substrate is moveable, for example by rotation or sliding, between the position where one face is contact with the gas permeable lay and the position where said one face is not in contact (removed from contact] with said gas permeable layer.
  • the culture device comprises: a container comprising a first endwall (bottom], and at least one sidewall, a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to reside upon,
  • At least a part of at least one of the first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • the device is configurable between (a] a first mode in which the substrate is not disposed in gaseous communication with a gas permeable material, and (b] a second mode in which the substrate is moved to be disposed in gaseous communication with a gas permeable material.
  • the scaffold comprises a frame defining an interior perimeter and an exterior perimeter, said frame comprising a substantially planar upper surface, a substrate for cells to reside upon held in a substantially planar arrangement across the interior perimeter of the frame, wherein the scaffold is configured to bring substantially all of the substrate or the cells or tissues present on the substrate (in particular one face of the substrate] into contact with a gas permeable interface when the scaffold is placed in a culture device comprising at least one gas permeable interface.
  • a fully human skin product comprising an epidermis, for example comprising both an epidermis and a dermis (i.e. a fully human full -thickness skin], obtainable by the method of any one of paragraphs 18 to 54.
  • a human skin product according to paragraphs 55 for use in treatment.
  • a human skin product for use according to paragraph 56 wherein the treatment is for a condition or disease selected from the group consisting of: tissue damage, for example cuts, lacerations, abrasions (such as excoriation], shearing force damage, bites (including animal bites such as dog bites and insect bites]; skin regeneration (for example with nerves & organelles]; wound healing, for example promoting/enhancing wound healing, including erosions, ulcers (such as diabetic ulcers] wounds from leprosy, wounds from dystrophic epidermolysis, wounds from hidradentitis suppurativa, wounds from mucous membrane pemphigoid, wounds from pemphigoid, wounds from perphigus vulgaris, wounds from pyoderma gangrenosum, wounds from shingles; burn healing including radiation burns, sunburn, chemical burns (such as acid burns and alkali burns], a thermal burn; skin regeneration and repair, for example atrophy, or after excision of tissue,
  • the condition or disease is selected from: tissue damage; skin regeneration with nerves & organelles; wound healing, for example promoting/enhancing wound healing, including ulcers such as diabetic ulcers; burn healing; skin regeneration and repair; epidermolysis bulosa; enhance skin quality or appearance; prevention or remediation of skin disorders; diminishment or abolishment of scar tissues; breast skin regeneration (after surgery]; cosmetic applications, e.g. anti-aging; dermal regeneration for wrinkles and other skin defects; promotion of hair follicle growth, nerve and other organelle regeneration; healing without scarring, or re-healing to diminish scarring.
  • tissue damage for example cuts, lacerations, abrasions (such as excoriation], shearing force damage, bites (including animal bites such as dog bites and insect bites]; skin regeneration (for example with nerves & organelles]; wound healing, for example promoting/enhancing wound healing, including erosions, ulcers (such as diabetic ulcers] wounds from leprosy, wounds from dystrophic epidermolysis, wounds from hidradentitis suppurativa, wounds from mucous membrane pemphigoid, wounds from pemphigoid, wounds from perphigus vulgaris, wounds from pyoderma gangrenosum, wounds from shingles; burn healing including radiation burns, sunburn, chemical burns (such as acid burns and alkali burns], a thermal burn ; skin regeneration and repair, for example atrophy, or after
  • a method of treatment comprising suturing a fully human skin product (comprising an epidermis] according to paragraphs 55 to a patient in need thereof.
  • tissue damage for example cuts, lacerations, abrasions (such as excoriation], shearing force damage, bites (including animal bites such as dog bites and insect bites]; skin regeneration (for example with nerves & organelles]; wound healing, for example promoting/enhancing wound healing, including erosions, ulcers (such as diabetic ulcers] wounds from leprosy, wounds from dystrophic epidermolysis, wounds from hidradentitis suppurativa, wounds from mucous membrane pemphigoid, wounds from pemphigoid, wounds from perphigus vulgaris, wounds from pyoderma gangrenosum, wounds from shingles; burn healing including radiation burns, sunburn, chemical burns (such as acid burns and alkali burns], a thermal burn ; skin regeneration and repair, for example atrophy, or after excision of
  • the presently disclosed method effectively eliminates the need for irradiated xenogeneic feeder cells, eliminates the requirement for separating the dermis from epidermis when digesting skin samples, and makes it possible for the cells from the dermis and epidermis to be grown together as a single culture.
  • the presently disclosed method is faster, cheaper, more convenient, involves less handling and therefore has a lower risk of contamination, and importantly, results in a less immunogenic and less "infectious" product compared to the prior art methods.
  • the present disclosure provides cells (in particular a population of keratinocytes and fibroblasts] obtainable from a method disclosed herein, for example according to any one of paragraphs 1 to 32.
  • the digest according to the present disclosure is performed a biologically relevant temperature, for example 35 to 40°C, such as 36, 37, 38 or 39°C, in particular 37°C.
  • the method of the present disclosure is suitable for digesting large samples of skin, for example 100cm 2 in size or larger.
  • the skin sample employed in the method of the present disclosure comprises segments 1cm 2 to about 150cm 2 .
  • both keratinocytes and fibroblast cells are isolated from the same skin sample in a single digestion reaction.
  • the disclosed method allows keratinocyte cells to be isolated from the epidermis and fibroblast cells to be isolated from the dermis using a single digestion reaction. This eliminates the requirement for the epidermis and dermis to be separated and then digested in two separate digest reactions, thereby saving time, simplifying the process, and reducing the potential for errors such as the inadvertent introduction of microbial contamination.
  • the human fibroblast feeder cells are employed in an unirradiated form.
  • the dermis will not be physically separated from the epidermis in the present disclosure.
  • Physically separated as employed herein refers to, for example use of scalpel to cut or divide the two layers. Application of forces such as shearing forces are also considered physical separation.
  • a mixture of keratinocytes and fibroblasts are isolated. That is to say that the keratinocytes and fibroblasts from the digest are not separated from each other.
  • the mixture of cells obtained from the digested skin sample may be cultured in the absence of a ROCK inhibitor. This allows the fibroblast to expand faster than the keratinocytes and so the sample becomes enriched with fibroblasts.
  • the sample is digested in the presence of both trypsin and collagenase.
  • the advantage of digesting in the presence of collagenase is that the collagenase helps to digest the dermis and basement membrane in order to release more cells from the skin tissue. This results in even higher cell yields compared to when trypsin alone is used.
  • keratinocytes are broken down into single cells from a whole skin sample employing a digest comprising trypsin, in particular a digest comprising trypsin and collagenase.
  • fibroblasts are broken down into single cells from a whole skin sample employing a digest comprising trypsin, in particular a digest comprising trypsin and collagenase.
  • the yields of fibroblasts isolated by this method may be greater than the yields than prior are methods.
  • Fibroblast can also be broken down into single cells employing a sequential digest of dispase followed by a digest with collagenase (on whole skin or separated dermis]. This method may be advantages because it may be quicker than to traditional methods to establish the fibroblast culture.
  • the first culture step in the present disclosure is to increase the number keratinocytes (which requires the presence of fibroblast feeder cells].
  • fibroblasts are added to the in the second culture step (for example to boost the number of fibroblasts] where the keratinocytes and fibroblasts stratify and differentiate to form a dermis and an epidermis.
  • keratinocytes and/or fibroblasts isolated by digesting skin according to the present disclosure may proliferate faster when grown in culture compared to keratinocytes and/or fibroblasts isolated from dispase digested epidermis and/or collagenase digested dermis.
  • the collagenase is type I collagenase.
  • type I collagenase has collagenase, caseinase, clostripain and tryptic activities, making it well suited for the digestion of skin sample which comprise a range of different cell types.
  • the method of the present disclosure does not employ dispase to isolate keratinocytes.
  • the method disclosed herein further comprises the step of culturing the isolated human keratinocytes to generate a skin product comprising a fully human epidermis.
  • the isolated human keratinocytes are cultured in media comprising human fibroblast cells and an effective amount of a ROCK inhibitor.
  • a ROCK inhibitor used to accelerate the growth of the keratinocytes to a sufficient rate, such that in co-culture with unirradiated human fibroblasts, the keratinocyte are not outgrown/overgrown by the fibroblasts.
  • human fibroblasts can be used as keratinocyte feeder cells instead of irradiated xenogeneic feeder cells. It is advantageous to be able to grow human keratinocytes with unirradiated human fibroblasts, since this eliminates the need to irradiate one of the cellular components, and also eliminates the need to use xenogeneic mouse embryonic fibroblasts in engineered tissue destined for therapeutic use in human patients. This is turn allows a less immunogeneic skin tissue to be prepared with lower risk of transmission of infectious agents from animals.
  • radiation can cause undesirable mutations in cells, for example that predispose the cells to become mutated and damaged, which may for example turn cancerous.
  • avoiding irradiating the feeder cells is beneficial.
  • the present method means that there is no longer a need to separate the epidermis from the dermis when digesting the skin samples because it is possible to digest both layers of skin together and, for example then grow the cells together as a single culture.
  • the ROCK inhibitor is Y-27632, SB772077B, or a combination of both, in particular SB772077B.
  • the present inventors have established that both of these 2 ROCK inhibitors are particularly suitable for enhancing keratinocyte growth rates.
  • SB772077B however has the advantage of having a greater potency and specificity of binding compared to Y27632, which means it can be used at lower concentrations vs Y27632 to achieve the same effect ( ⁇ 400 nm vs ⁇ ].
  • the human keratinocytes are not cultured in the presence of xenogeneic feeder cells. This has the advantage of reducing the immunogenicity of the cultured skin tissue.
  • the human fibroblast cells are not irradiated.
  • the advantage of this is that the fibroblast cells are still active and are able to function as feeder cells in the first culture stage and then differentiate into the dermis in the second culture phase.
  • the human fibroblasts are allogeneic to a human patient.
  • the human keratinocytes are allogeneic to a human patient.
  • the human fibroblast cells are sex matched to the keratinocytes (and/or a patient].
  • the human fibroblast cells are HLA matched to the keratinocytes (and/or a patient].
  • the human fibroblasts and keratinocytes are from the same donor (and for example HLA matched to a patient].
  • the human fibroblasts are autologous to a human patient.
  • the human keratinocytes are autologous to a human patient.
  • the human keratinocytes and fibroblasts are autologous to a human patient.
  • the media comprises keratinocyte growth factor (KGF].
  • KGF keratinocyte growth factor
  • the media comprises DMEM High glucose, Ham's F12, foetal bovine serum, Penicillin, Streptomycin, Amphotericin B and KGF.
  • the media (Kelch's medium] consists of: DMEM High glucose:Ham's F12
  • the disclosed cell culture medium does not contain choleratoxin.
  • KGF can be successfully used as a substitute for choleratoxin and when included in a base medium which lacks choleratoxin, is able to provide similar keratinocyte growth kinetics as Green's medium.
  • the cell culture medium is a minimal medium suitable for supporting the growth of keratinocytes, for example human keratinocytes, which strips out all of the unnecessary components normally present in Green's medium.
  • the disclosed culture medium produces similar keratinocyte growth in the absence of mouse embryonic feeder cells (MEFs] as Green's medium with MEFs.
  • the presently disclosed cell culture medium can be used in place of Green's medium and without MEFs, thereby eliminating the need for both choleratoxin and MEFs. Furthermore, it is more convenient and easier to prepare.
  • the media further comprises a keratinocyte growth accelerator, such as a ROCK inhibitor.
  • a keratinocyte growth accelerator such as a ROCK inhibitor.
  • Kelch's medium consists of DMEM High glucose:Ham's F12 (3:1], 10% foetal bovine serum, penicillin, streptomycin, 0.625 ⁇ g/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF] and a ROCK inhibitor.
  • the media is Green's media.
  • the media does not employ an antibiotic associated with allergic reactions in some patients, such as penicillin.
  • the keratinocytes are deposited on a substrate, for example a matrix.
  • the substrate is coated with an extracellular matrix protein or peptide thereof, for example a synthetic peptide (such as peptides representing the partial amino acid sequences of collagen, laminin and other extracellular matrix proteins].
  • an extracellular matrix protein or peptide thereof for example a synthetic peptide (such as peptides representing the partial amino acid sequences of collagen, laminin and other extracellular matrix proteins].
  • the presence of the coating produces a second cellular signal (the first signal being growing the skin tissue at an air-liquid or gas permeable interface], which enhances the proper stratification of the skin tissue.
  • the extracellular matrix protein is selected from the group consisting of collagen IV, collagen I, laminin and fibronectin, or a combination thereof, in particular collagen IV.
  • collagen IV is that it was found to consistently produce the best epidermal stratification compared to collagen I, laminin and fibronectin.
  • an epidermis cultured (in particular a human epidermis] by the method as described herein, for example for use in treatment.
  • a full thickness skin in particular human or fully human full thickness skin], for example comprising both an epidermis and a dermis, cultured by the method as described herein, for example for use in treatment.
  • “Whole skin sample” as employed herein refers to an ex vivo tissue comprising a dermis and epidermis.
  • the dermis and epidermis can be considered to be two separate organs. In a whole skin sample these two organs are still associated (i.e. are not separated].
  • the whole skin sample may be cut into small pieces. However, generally each of the pieces will comprise a dermis and epidermis.
  • Underside of the sample as employed herein refers to the part of the sample that was a adjacent to subcutaneous tissue.
  • Digest as employed herein refers to the process of breaking down of the structure of the skin sample. However, in other contexts is refers to the components, such as enzymes employed to breakdown the tissue. The meaning will be apparent to the skilled person form the context.
  • Single step digestion refers to wherein one or more enzymes employed in the digest are used in one period of time to disintegrate the skin tissue structure, in particular to provide single cells. Thus, in a single digest step if two or more enzymes are employed they are employed concomitantly. One digestion step followed by a second digestion step as employed in the prior art is not a single step digestion within the meaning of the present specification.
  • Skin product refers to engineered tissue comprising an epidermis and/or dermis.
  • skin tissue refers to any tissue, including epidermis, and/or dermis and optionally basement membrane tissue, for example full thickness skin, including engineered tissue or native tissue (from a donor or patient].
  • Engineered refers to tissue generated in vitro, using cell-based technology.
  • epidermis refers to the outer of the two layers which make up the skin, the inner layer being the “dermis”.
  • Epidermis is primarily composed of differentiated keratinocytes.
  • a large component of dermis are fibroblasts.
  • Dermis as employed herein refers to native dermis or dermal substitutes.
  • Dermal substitutes as employed herein refers engineered dermis grown in vitro to approximate native dermis and for example where components cells are predominantly fibroblasts.
  • epidermal and epipithelium refer to the cellular covering of internal and external body surfaces (cutaneous, mucous and serous membrane], including the glands and other structures derived therefrom, e.g., corneal, oesophageal, laryngeal, epidermal, hair follicle and urethral epithelial cells.
  • the epithelial cells employed are skin cells, such as human skin cells, for example cells which form an epidermis and dermis, such as keratinocytes and fibroblasts respectively.
  • epithelial tissues include: olfactory epithelium, which is the pseudostratified epithelium lining the olfactory region of the nasal cavity, and containing the receptors for the sense of smell; glandular epithelium, which refers to epithelium composed of secreting cells; squamous epithelium, which refers to epithelium composed of flattened plate -like cells.
  • tissue is used to refer to an aggregation of similarly specialized cells united in the performance of a particular function. Tissue is intended to encompass all types of biological tissue including both hard and soft tissue.
  • a "tissue” is a collection or aggregation of particular cells embedded within its natural matrix, wherein the natural matrix is produced by the particular living cells.
  • the term may also refer to ex vivo aggregations of similarly specialized cells which are expanded in vitro such as in artificial organs. Dermis can be considered a different organ to epidermis.
  • epidermis as employed herein is a tissue
  • dermis as employed herein is also a tissue.
  • the method according to the present disclosure is capable of generating full thickness human skin, which is made up of two tissue-specific layers, namely a dermis equivalent and an epidermis equivalent.
  • the skin tissue substantially corresponds to native skin both histologically and functionally, even though it has been grown in vitro.
  • fully human refers to a tissue which does not comprise any non-human cells, proteins or antigens, for example xenogeneic feeder cells, such as mouse embryonic fibroblasts.
  • xenogeneic feeder cells such as mouse embryonic fibroblasts.
  • fully human refers to skin products prepared without employing non-human materials, such as xenogenic cells.
  • the cells and tissue according to the present disclosure do not contain any non-human components because they are from fully human origin.
  • media employed to culture the cells may contain, for example foetal calf serum. This serum does not render the cells and tissue of the present disclosure non-human.
  • fibroblasts are understood to be naturally occurring fibroblasts or their precursor cells, for example, adipose-derived stromal cells, more particularly fibroblasts occurring in the dermis, genetically modified fibroblasts or fibroblasts emanating from spontaneous mutations or precursors thereof.
  • the fibroblasts are differentiated (for example have surface markers indicative of differentiated fibroblasts].
  • the epithelium cells include squamous epithelial cells, columnar epithelial cells, cuboidal epithelial cells and combinations of two of more of the same.
  • keratinocytes are understood to be cells of the epidermis which form keratinizing plate epithelium, genetically modified keratinocytes or keratinocytes emanating from spontaneous mutations or precursors of such keratinocytes of human origin.
  • mucous membrane keratinocytes or intestinal epithelial cells may be applied to the matrix.
  • pre-cultivated cells for example keratinocytes in the first or in the second cell passage, although cells from higher passages may also be used.
  • the cells such as keratinocytes according to the present disclosure, may also comprise one or more of the following cell types selected from melanocytes, Langerhan cells, Merkel cells and a combination of two or more the same.
  • the fibroblasts and keratinocytes are obtained and cultivated by methods disclosed herein.
  • the methods may be adapted to give the required properties of the skin tissue desired.
  • other cell types and/or other cells of other tissue types for example, melanocytes, macrophages, monocytes, leukocytes, plasma cells, neuronal cells, adipocytes, induced and non-induced precursor cells of Langerhans cells, Langerhans cells and other immune cells, endothelial cells, cells from tumors of the skin or skin-associated cells, more particularly sebocytes or sebaceous gland tissue or sebaceous gland explantates, cells of the sweat glands or sweat gland tissue or sweat gland explantates, hair follicle cells or hair follicle explantates; and cells from tumors of other organs or from metastases, may be cultured together with the human keratinocytes.
  • melanocytes for example, melanocytes, macrophages, monocytes, leukocytes, plasma cells, neuronal cells, adipocytes, induced and non-induced precursor cells of Langerhans cells, Langerhans cells and other immune cells, endothelial cells, cells
  • the cells mentioned may be of human and animal origin but unless mentioned otherwise, will be human in order to produce a fully human epidermis.
  • Stem cells of various origins, tissue-specific stem cells, embryonal and/or adult stem cells may also be incorporated in the method and skin product of the present disclosure.
  • Trypsin as used herein (EC number 3.4.21.4] is a serine protease from the PA clan superfamily, found in the digestive system, such as in the pancreas of many vertebrates where it hydrolyses proteins. Trypsin cleaves peptide chains primarily at the carboxyl side of the amino acids lysine or arginine. The rate of hydrolysis is slower if an acidic residue is on either side of the cleavage site and no cleavage occurs if a proline residue is on the carboxyl side of the cleavage site. As used in the presently disclosed method, trypsin is able to digest both the epidermis and dermis layers of skin samples.
  • Collagenase refers to a group of enzymes which break down the native collagen that holds animal tissues together. Collagenases are made by a variety of different microorganisms and by many different animal cells. Crude collagenase preparations contain several isoforms of two different collagenases, a sulfhydryl protease, clostripain, a trypsin-like enzyme, and an aminopeptidase. This combination of collagenolytic and proteolytic activities is effective at breaking down intercellular matrices, the essential part of tissue dissociation.
  • One component of the complex is a hydrolytic enzyme which degrades the helical regions in native collagen preferentially at the Y- Gly bond in the sequence Pro-Y-Gly-Pro, where Y is most frequently a neutral amino acid. This cleavage yields products susceptible to further peptidase digestion. Crude collagenase is inhibited by metal chelating agents such as cysteine, EDTA or o-phenanthroline but not DFP. It is also inhibited by a2-macroglobulin, a large plasma glycoprotein. Ca 2+ is required for enzyme activity. 4 main types of collagenase are typically used depending on the requirements:
  • Type 1 crude collagenase has the original balance of collagenase, caseinase, clostripain and tryptic activities.
  • Type 2 contains higher relative levels of protease activity, particularly clostripain.
  • Type 3 contains lowest levels of secondary proteases.
  • Type 4 is designed to be especially low in tryptic activity to limit damage to membrane proteins and receptors.
  • Type 1 collagenase is employed in the methods of the present disclosure.
  • ROCK inhibitors include but are not limited to: SB772077B, Y-27632, Fasudil, Ripasudil, Y39983, Wf-536, SLx-2119, an azabenimidazole-aminofurazan, DE-104, H-1152, ROKa inhibitor, XD-4000, HMN-1152, 4-(l-aminoalkyl]-N-(4-pyridyl]cyclohexane-carboxamide, rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, RKI-1447, GSK429286A, Y-30141, HA-100, H-7, iso H-7, H-89, HA-1004, HA-1077, H-8, H-9, KN-62, GSK269962, and quinazoline.
  • Feeder cells refers to a population of cells, typically connective tissue cells that are used to nourish cultured tissue and cells, in particular used to feed the human keratinocytes as described herein.
  • the feeder cells supply metabolites and other nutrients to the cells they support.
  • Feeder cells typically do not grow or divide and are usually inactivated by irradiation, for example gamma irradiation. However, in the present disclosure the feeder cells are unirradiated.
  • the sowing of the skin cells on the matrix takes place in the presence of a physiological solution.
  • physiological solution refers to a solution that is similar or identical to one or more physiological condition(s] or that can change the physiological state of a certain physiological environment.
  • physiological solution as used herein also refers to a solution that is capable of supporting growth of cells (including, but not limited to, mammalian, vertebrate, and/or other cells].
  • a physiological solution comprises a defined culture medium, in which the concentration of each of the medium components is known and/or controlled.
  • defined media typically contain all the nutrients necessary to support cell growth, including, but not limited to, salts, amino acid, vitamins, lipids, trace elements, and energy sources (such as carbohydrates.]
  • Non- limiting examples of defined media include DMEM, Basal Media Eagle (BME], Medium 199; F- 12 (Ham] Nutrient Mixture; F-IO (Ham] Nutrient Mixture; Minimal Essential Media (MEM], Williams' Media E, and RPMI 1640.
  • DMEM or "Dulbecco's Modified Eagle Medium” is a modification of Basal Medium Eagle which contains a four-fold higher concentration of amino acids and vitamins, together with additional components. DMEM normally contains about 1000 mg/L of glucose. "DMEM high glucose” refers to a version of DMEM which contains 4500 mg/L of glucose instead of the usual 1000 mg/L.
  • Ham's F12 is a medium designed for low density, serum-free growth of Chinse Hamster Ovary (CHO] cells.
  • Ham's F12 is based on Ham's F10 medium but with increased concentrations of choline, inositol, putrescine and other amino acids.
  • the ratio of DMEM:Ham's F12 in the cell culture medium may be 1 :1, 2: 1, 3:1, 4: 1, 5:1. In one embodiment, the ratio is 3:1.
  • Green's media as employed herien is DMEM:Hams F12 (Life Technologies 31765-035] at a ratio of 3:1.
  • Fetal calf serum (FCS] is preferably used as the serum, although NCS and serum substitute products are also suitable, while Hepes buffer, for example, is used as the buffer.
  • the pH value of the solution of cell culture medium, buffer and serum is usually in the range from 6.0 to 8.0, for example, from 6.5 to 7.5 and, more particularly, 7.0.
  • FBS Fetal bovine serum
  • BSA bovine serum albumin
  • ICS iron-supplemented bovine calf serum
  • 10% serum is typically used but other concentrations may also be used d, for example 0.1% to 20%, such as 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% and 20% serum may be employed.
  • Penicillin refers to a group of ⁇ -lactam antibiotics, including penicillin G, penicillin V, procaine penicillin and benzathine penicillin. Penicillin acts by inhibiting the formation of peptidoglycan cross-links in the bacterial cell wall. This weakens the cell walls of dividing bacterial, eventually causing the cell walls to burst and the bacteria to die because of osmostic pressure. Gram-positive bacteria have thick cell walls containing high levels of peptidoglycan, whereas gram-negative bacteria are characterised by thinner cell walls with low levels of peptidoglycan. Thus, penicillin is most effective against gram-positive bacteria.
  • Streptomycin is an antibiotic that was originally purified from Streptomyces griseus. It acts by binding to the 30S subunit of the bacterial ribosome, leading to inhibition of protein synthesis and death in susceptible bacteria. Streptomycin is able to cross the outer cell wall of negative organisms by passive diffusion through aqueous channels. Conversely, the thicker cell walls of gram-positive bacteria inhibits transport of streptomycin. Accordingly, streptomycin works better on gram-negative bacteria.
  • the medium does not employ an antibiotic which is associated with allergic reactions in patients, such as penicillin and/or streptomycin.
  • the cell culture medium contains both penicillin and streptomycin, thereby helping to protect the cells grown in the culture from both gram-positive and gram-negative bacteria.
  • Gentamicin is an antibiotic comprising a complex of three different closely rated aminoglycoside sulfates, Gentamicins CI, C2 and Cla, obtained from Micromonospora purpurea and related species.
  • Gentamicin is a broad spectrum antibiotic typically used for serious infections of the following microorganisms: P. aeruginosa, Proteus species (indole-positive and indole-negative], E. coli, Klebsiella-Enterobactor-Serratia species, Citrobacter species and Staphylococcus species (coagulase-positive and coagulase-negative].
  • the cell culture medium contains gentamicin.
  • Amphotericin B is an anti-fungal medication used for serious fungal infections and leishmaniasis. It functions by binding with ergosterol, a component of fungal cell membranes, forming pores that case rapid leakage of monovalent ions (eg. K + , Na + , H + and CI ], which leads to fungal cell death.
  • monovalent ions eg. K + , Na + , H + and CI
  • the cell culture medium contains an antibiotic which targets gram- positive bacteria, such as penicillin, an antibiotic which targets gram-negative bacteria, such as streptomycin, and an anti-fungal medication.
  • KGF Keratinocyte growth factor
  • FGFR2b fibroblast growth factor receptor 2b
  • the culture medium is DMEM (Dulbecco's Modified Eagle Medium], M199, Ham's F12 Medium, or a combination thereof.
  • DMEM Dulbecco's Modified Eagle Medium
  • M199 M199
  • Ham's F12 Medium or a combination thereof.
  • any other cell culture medium which allows the cultivation of keratinocytes and fibroblasts may also be used.
  • Greens medium is employed.
  • the cell culture medium consists of DMEM High glucose, Ham's F12, foetal bovine serum, penicillin, streptomycin, amphotericin B and keratinocyte growth factor (KGF].
  • the ratio of DMEM High glucose:Ham's F12 is 3:1.
  • the foetal bovine serum has a concentration of 10%.
  • the amphotericin B has a concentration of 0.625 ⁇ g/ml.
  • the KGF has a concentration of 20ng/ml.
  • the cell culture medium consists of: DMEM High glucose:Ham's F12 (3:1], 10% foetal bovine serum, penicillin, streptomycin, 0.625 ⁇ g/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF].
  • KGF keratinocyte growth factor
  • the medium further comprises a epithelial cell growth accelerator (for example a keratinocyte growth accelerator], such as a ROCK inhibitor.
  • the cell culture medium consists of DMEM High glucose:Ham's F12 (3:1], 10% foetal bovine serum, penicillin, streptomycin, 0.625 ⁇ g/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF] and a ROCK inhibitor (for example a ROCK inhibitor disclosed herein].
  • the media may contain other factors, for example, hormones, growth factors, adhesion proteins, antibiotics, selection factors, enzymes and enzyme inhibitors and the like. Growth factors for example may help to enhance the proliferation of the seeded cells.
  • Antibody refers to a full-length antibody, a binding fragment thereof, or an antibody molecule comprising any one of the same.
  • antibody binding fragments include Fab, modified Fab, Fab', modified Fab', F(ab']2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH], scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9]:1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3], 209-217].
  • a peptide as employed herein is a sequence of 2 to 50 amino acids.
  • a synthetic peptide as employed herein refers to a peptide prepared by synthetic chemistry techniques (as opposed to peptides expressed recombinantly].
  • Substantially planar as employed herein refers to having a surface substantially (a major portion of which is] lying in one plane.
  • matrix refers to any physical structure including but not limited to, a solid or semisolid structure, such as a meshwork of fibres with pores suitable for providing:
  • the relevant cells In contact with the gas permeable layer/membrane as employed herein refers to the relevant cells being on the membrane/layer or in the proximity of the membrane/layer, such that the growth and/or in particular differentiation of the cells can occur.
  • proximity will generally mean that there is nothing separating the gas permeable layer/membrane and the relevant cells (the space therebetween will be filled for example with culture media, buffer, C02or a combination of the same, in particular cell culture media].
  • the distance of the relevant cells in proximity to the gas permeable layer is 2cm or less, such as 1cm or less, in particular 0.5, 0.4, 0.3, 0.2, 0.1 or 0.05cm.
  • the outer of cells for differentiation rests on the gas permeable layer/membrane.
  • Cells not in contact with the gas permeable membrane will generally be greater than 2cm away from the layer or will be separated from the membrane by an intervening layer or layers or cells.
  • the matrix will be three dimensional, with a first 2D face and a second face 2D
  • the matrices of the present disclosure may be constructed of natural or synthetic materials.
  • a matrix may be in a particular shape or form so as to influence or delimit a three-dimensional shape or form assumed by a population of proliferating cells.
  • Such shapes or forms include, but are not limited to, films (e.g. a form with two-dimensions substantially greater than the third dimension], ribbons, cords, sheets, flat discs, cylinders, spheres, 3-dimensional amorphous shapes, etc.
  • the matrices comprise only synthetic materials. In another embodiment the matrix comprises a mixture of synthetic and natural materials.
  • synthetic materials for making the matrix of the present invention are both biocompatible and biodegradable (e.g. subject to enzymatic and hydrolytic degradation], such as biodegradable polymers.
  • biocompatible refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal.
  • a biocompatible material when introduced into an individual, is not toxic nor injurious to that individual, nor does it induce immunological rejection of the material in the mammal.
  • biodegradable or “bioabsorbable” as used herein is intended to describe materials that exist for a limited time in a biological environment and degrade under physiological conditions to form a product that can be metabolized or excreted without damage to the subject.
  • the product is metabolized or excreted without permanent damage to the subject.
  • the matrix is completely resorbable by the body of a subject.
  • a bioabsorbable matrix of the present disclosure may exist for days, weeks or months when placed in the context of a biological environment.
  • a bioabsorbable matrix may exist for 1, 2, 3, 4, 5, 6, 7, 8,9 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180 days or more when placed in the context of biological environment.
  • the matrix layer is resorbed by the body of said subject at about a same rate as growth of tissue cells underlying said matrix layer in said area.
  • the cells are epithelial cells.
  • the matrix layer is substantially completely resorbed by said body within about 3 to 12 months after the skin graft is applied. In certain embodiments, the matrix is substantially completely resorbed within about 3 months.
  • Biodegradable materials such as polymers may be hydrolytically degradable, may require cellular and/or enzymatic action to fully degrade, for example hydrolysis, oxidation, enzymatic processes, phagocytosis, or other processes, including a combination of the foregoing.
  • Biodegradable polymers are known to those of ordinary skill in the art and include, but are not limited to, synthetic polymers, natural polymers, blends of synthetic and natural polymers, inorganic materials, and the like.
  • the matrix incorporates one or more synthetic polymers in its construction.
  • the matrix may be made from heteropolymers, monopolymers, or combinations thereof.
  • polymers suitable for manufacturing cell matrices include, but are not limited to aliphatic polyesters, copoly(ether-esters], polyalkylenes oxalates, polyamides, poly(iminocarbonates], polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides], polyphosphazenes, biomolecules and blends thereof.
  • Suitable aliphatic polyesters include homopolymers, copolymers (random, block, segmented, tappered blocks, graft, triblock, etc.] having a linear, branched or star structure.
  • Suitable monomers for making aliphatic homopolymers and copolymers may be selected from the group consisting of, but are not limited, to lactic acid, lactide (including L-, D-, meso and D,L mixtures], glycolic acid, glycolide, epsilon-caprolactone, p-dioxanone (l,4-dioxan-2-one], trimethylene carbonate (l,3-dioxan-2-one], delta-valerolactone, beta-butyrolactone, epsilon-decalactone, 2,5- diketomorpholine pivalolactone, alpha, alpha-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-
  • Elastomeric copolymers also are particularly useful in the presently disclosed matrices.
  • Suitable bioabsorbable biocompatible elastomers include but are not limited to those selected from the group consisting of elastomeric copolymers of epsilon-caprolactone and glycolide (for example having a mole ratio of epsilon-caprolactone to glycolide from about 35:65 to about 65:35, more preferably from 45:55 to 35:65] elastomeric copolymers of epsilon-caprolactone and lactide, including L-lactide, D-lactide blends thereof or tactic acid copolymers (for example having a mole ratio of epsiton-caprolactone to lactide of from about 35:65 to about 65:35 and more preferably from 45:55 to 30:70 or from about 95: 5 to about 85:15] elastomeric copolymers of p-dioxanone (1
  • bioabsorbable elastomers examples include US4,045,418; US4,057,537 and 5,468,253 all hereby incorporated by reference.
  • These elastomeric polymers will have an inherent viscosity of from about 1.2 dL/g to about 4 dL/g, suitably an inherent viscosity of from about 1.2 dL/g to about 2 dL/g and most suitably an inherent viscosity of from about 1.4 dL/g to about 2 dL /g as determined at 25°C in a 0.1 gram per deciliter (g/dL] solution of polymer in hexafluoroisopropanol (HFIP].
  • HFIP hexafluoroisopropanol
  • Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, polyvinyl alcohols, polylactide, chondroitin sulfate (a proteoglycan component], polyesters, polyethylene glycols, polycarbonates, polyvinyl alcohols, polyacrylamides, polyamides, polyacrylates, polyesters, polyetheresters, polymethacrylates, polyurethanes, polycaprotactone, polyphophazenes, polyorthoesters, polyglycolide, copolymers of lysine and lactic acid, copolymers of lysine-RGD and lactic acid, and the like, and copolymers of the same.
  • Synthetic polymers can further include those selected from the group consisting of aliphatic polyesters, poly(amino acids], poly(propylene fumarate], copoly(ether-esters], polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates], polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides], polyphosphazenes, and blends thereof.
  • the matrix incorporates polylactic acid (PLA].
  • PLA is particularly suited to tissue engineering methods using the cellular matrix as PLA degrades within the human body to form lactic acid, a naturally occurring chemical which is easily removed from the body.
  • the cellular matrix of the invention may also incorporate polyglycolic acid (PGA] and/or polycaprolactone (PCL] as matrix materials.
  • PGA and PCL have similar degradation pathways to PLA, but PGA degrades in the body more quickly than PLA, while PCL has a slower degradation rate than PLA.
  • PGA has been widely used in tissue engineering. PGA matrices can be easily manipulated into various three dimensional structures, and offer an excellent means of support and transportation for cells (Christenson L, Mikos A G, Gibbons D F, et al: Biomaterials for tissue engineering: summary. Tissue Eng. 3 (1]: 71-73; discussion 73-76, 1997]. Matrices manufactured from polyglycolic acid alone, as well as combinations of PGA and other natural and/or synthetic biocompatible materials, are within the scope of the present disclosure.
  • the matrix comprises poly(lactic-co-glycolic acid] (PLGA], such as PLGA microfiber or nanofibres.
  • PLGA poly(lactic-co-glycolic acid]
  • the matrix comprises dioxanone linear homopolymer, such as 100 dioxanone linear homopolymer (e.g. Dioxaprene 100M].
  • the matrix comprises a combination of PLGA and 100 Dioxanone.
  • the term "fibre” is used herein to refer to materials that are in the form of continuous filaments or discrete elongated pieces of material, typically comprising or composed of biodegradeable polymers such as those described above.
  • the fibres of the present disclosure typically have diameters in the micrometer range, such as 0.5 ⁇ to 5 ⁇ , for example 1 ⁇ , 1.5 ⁇ , 2 ⁇ , 2.5 ⁇ , 3 ⁇ , 3.5 ⁇ , 4 ⁇ , 4.5 ⁇ or 5 ⁇ , in particular in the range 1 to 3 ⁇ .
  • fibre matrix is used herein to refer to the arrangement of fibres into a supporting framework, such as in the form of a sheet of fibres that can then be used to support cells or other additional materials (see also definition of "matrix” above].
  • a supporting framework such as in the form of a sheet of fibres that can then be used to support cells or other additional materials (see also definition of "matrix” above].
  • Various methods are known to the skilled person which can be used to produce suitable fibers, include, but are not limited to, interfacial polymerization and electrospinning.
  • a matrix of the present disclosure is formed using electrospinning.
  • electrospinning generally refers to techniques that make use of a high -voltage power supply, a spinneret (e.g., a hypodermic needle], and an electrically conductive collector plate (e.g., aluminum foil or stainless steel].
  • a spinneret e.g., a hypodermic needle
  • an electrically conductive collector plate e.g., aluminum foil or stainless steel.
  • an electrospinning liquid i.e. a melt or solution of the desired materials that will be used to form the fibers
  • the repulsion between the charges immobilized on the surface of the resulting liquid droplet overcomes the confinement of surface tension and induces the ejection of a liquid jet from the orifice.
  • the charged jet then goes through a whipping and stretching process, and subsequently results in the formation of uniform nanofibers.
  • the diameters of the fibres can then be continuously reduced to a desired scale, for example micrometers, or even as small as nanometers and, under the influence of an electrical field, the fibres can subsequently be forced to travel towards a grounded collector, onto which they are typically deposited as a non- woven mat.
  • electrospun fibres can mimic the architecture of the extracellular matrix.
  • Examples of materials used to produce the nanofibers of the present disclosure are selected from those listed in Tables 1 and 2 below.
  • Table 1- Exemplary Materials for producing electrospun fibres (natural polymers).
  • the matrix of the present disclosure is composed of synthetic microfibers or nanofibres, for example using the materials listed in Table 2.
  • the selection of a particular polymer and its use in a specified amount or concentration, or range thereof, provides the ability to control, customize and tailor the degradation rate of the polymer and therefore, the degradation rate of the matrix. This is useful because it is desirable for the matrix to remain as part of the skin graft in order to provide structural support to the grown skin tissue but to eventually degrade and be bioabsorbed by the patient's body once the patient's own cells have assimilated the skin graft, thereby eliminating the requirement for the matrix to be retrieved from the patient's body later on.
  • Various blends of polymers may be used to form the fibres to improve their biocompatibility as well as their mechanical, physical, and chemical properties.
  • two or more fibre matrices of the present disclosure are layered together.
  • the superior and unexpected advantages of each fibre matrix can be combined, and in some cases, result in a synergistic effect.
  • a first matrix may comprise microwells for receiving one or more relevant cells and/or skin tissue, which is then layered on a second matrix having radially-aligned fibres.
  • the first matrix can provide the benefit of increasing the repair of damaged skin by providing relevant cells and/or skin tissue whereas the second matrix can provide the benefit of directing and enhancing cell migration from the periphery to the centre of the layered matrices.
  • Layering two or more matrices may also help to enhance the watertight properties of a matrix. The skilled person would be able to derive various combinations of two or more different matrices in order to achieve desired properties.
  • the matrix of the present disclosure may be further treated via a single procedure or a combination of procedures which reduce the number of microorganisms capable of growing in the matrix under conditions at which the matrix is stored and/or distributed.
  • the matrix is sterilised using gamma radiation.
  • the matrix is sterilised using ethylene oxide (EtO].
  • the matrix is sterilised using Revox which utilises percetic acid.
  • the matrix is sterilized using ionizing radiation such as E-beam irradiation.
  • Electron beam processing has the shortest process cycle of any currently recognized sterilization method. E-beam irradiation, products are exposed to radiation for seconds, with the bulk of the processing time consumed in transporting products into and out of the radiation shielding. Overall process time, including transport time, is 5 to 7 minutes.
  • Electron beam processing involves the use of high energy electrons, typically with energies ranging from 3 to 10 million electron volts (MeV], for the radiation of single use disposable medical products. The electrons are generated by accelerators that operate in both a pulse and continuous beam mode. These high energy levels are required to penetrate a product that is packaged in its final shipping container.
  • the electrons interact with materials and create secondary energetic species, such as electrons, ion pairs, and free radicals. These secondary energetic species are responsible for the inactivation of the microorganisms as they disrupt the DNA chain of the microorganism, thus rendering the product sterile.
  • secondary energetic species such as electrons, ion pairs, and free radicals.
  • the seeding densities of the cellular matrix may vary and the individual layers of the cell matrix may have the same or different seeding densities. Seeding densities may vary according to the particular application for which the cellular matrix is applied. Seeding densities may also vary according to the cell type that is used in manufacturing the cellular matrix.
  • the number and concentration of cells seeded into or onto the matrix can be varied by modifying the concentration of cells in suspension, or by modifying the quantity of suspension that is distributed onto a given area or volume of the matrix.
  • the seeding density is about 150,000 keratinocytes/cm 2 or higher such as 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000 or 600,000 keratinocytes/cm 2 .
  • the seeding density is about 50,000 fibroblasts/cm 2 or higher, such as 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000 or 200,000 fibroblasts/cm 2 .
  • Seeding densities of the individual layers of the matrix will depend on the use for which the matrix is intended. Although one skilled in the art may appreciate particular seeding densities a specific application will require, individual layers of the matrix may be seeded at a variety of seeding densities. One skilled in the art will appreciate that the seeding densities for the individual layers of the matrix may vary according to the use for which the matrix is intended.
  • Seeding the matrix by painting is accomplished by dipping a brush into the inoculum, withdrawing it, and wiping the inoculum -laden brush across the matrix.
  • This method suffers the disadvantage that substantial numbers of cells may cling to the brush, and not be applied to the lattice. However, it may nevertheless be useful, especially in situations where it is desired to carefully control the pattern or area of lattice over which the inoculum is distributed.
  • Seeding the matrix by spraying generally involves forcing the inoculum through any type of nozzle that transforms liquid into small airborne droplets. This embodiment is subject to two constraints.
  • nozzles that are commonly available satisfy both constraints. Such nozzles may be connected in any conventional way to a reservoir that contains an inoculum of epithelial stem cells.
  • Seeding the matrix by pipetting is accomplished using pipettes, common "eye-droppers,” or other similar devices capable of placing small quantities of the inoculum on the surface of the matrix of the present disclosure.
  • the aqueous liquid will permeate through the porous matrix.
  • the cells in suspension tend to become enmeshed at the surface of the matrix and are thereby retained upon the matrix surface.
  • an inoculum of cells may be seeded by means of a hypodermic syringe equipped with a hollow needle or other conduit.
  • a suspension of cells is administered into the cylinder of the syringe, and the needle is inserted into the matrix.
  • the plunger of the syringe is depressed to eject a quantity of solution out of the cylinder, through the needle, and into the scaffold.
  • An important advantage of utilizing an aqueous suspension of cells is that it can be used to greatly expand the area of matrix on which an effective inoculum is distributed. This provides two distinct advantages. First, if a very limited amount of intact tissue is available for autografting, then the various suspension methods may be used to dramatically increase the area or volume of a matrix that may be seeded with the limited number of available cells. Second, if a given area or volume of a matrix needs to be seeded with cells, then the amount of intact tissue that needs to be harvested from a donor site may be greatly reduced. The optimal seeding densities for specific applications may be determined through routine experimentation by persons skilled in the art.
  • the dimensions of the matrix should be substantially planar and of a thickness that gives seeded cells sufficient access to a nutrient medium.
  • the cell matrix When implanted, the cell matrix must have sufficient access to body fluids for nutrition and waste removal.
  • the thickness of the matrix may be varied by changes in the matrix's porosity. Thus, increases in matrix porosity may permit matrices to take on greater thickness as larger pore sizes improve access to external medium and body fluids.
  • the matrix has a thickness of 100 ⁇ or less, for example 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 ⁇ . By keeping the matrix 100 ⁇ or less in thickness, this allows the seeded keratinocytes to receive nutrients and remove waste by diffusion alone, without requiring a vasculature system in order to survive.
  • Seeding the layered matrix involves introducing one or more desired cell populations to a selected substrate material, and subsequently joining the materials to create a layered matrix.
  • the matrices may be pre-joined, and the selected populations] of cells introduced at a selected location. Seeding is distinct from the spontaneous infiltration and migration of cells into the matrix from a wound site when the matrix is placed at the wound site.
  • the matrices are seeded on at least one surface before the respective cell-seeded surfaces are opposed to each other to form a layered arrangement.
  • additional materials and/or biological molecules can be attached to the matrices of the present disclosure.
  • the term "attached" includes, but is not limited to, coating, embedding or incorporating by any means the additional materials and/or biological molecules, and attached can refer to incorporating such components on the entire matrix or only a portion thereof.
  • cell factors are coated/attached to the matrix of the present disclosure.
  • cell factors refers to substances that are synthesized by living cells (e.g. stem cells] and which produce a beneficial effect in the body (e.g. mammalian or human body].
  • Cell factors include, but are in no way limited to, growth factors, regulatory factors, hormones, enzymes, lymphokines, peptides and combinations thereof.
  • Cell factors may have varying effects including, but not limited to, influencing the growth, proliferation, commitment, and/or differentiation of cells (e.g. stem cells] either in vivo or in vitro.
  • cell factors include, but are not limited to, cytokines (e.g. common beta chain, common gamma chain, and IL-6 cytokine families], vascular endothelial growth factor (e.g. VEGF-A, -B, -C, -D, and -E], adrenomedullin, insulin-like growth factor, epidermal growth factor EGF, fibroblast growth factor FGF, autocrin motility factor, GDF, IGF, PDGF, growth differentiation factor 9, erythropoietin, activins, TGF-a, TGF- ⁇ , bone morphogenetic proteins (BMPs], Hedgehog molecules, Wnt-related molecules, and combinations thereof.
  • cytokines e.g. common beta chain, common gamma chain, and IL-6 cytokine families
  • vascular endothelial growth factor e.g. VEGF-A, -B, -C, -D, and -E
  • a growth factor such as EGF (Epidermal Growth Factor], IGF-I (Insulinlike Growth Factor], a member of Fibroblast Growth Factor family (FGF], Keratinocyte Growth Factor (KGF), PDGF (Platelet-derived Growth Factor AA, AB, BB], TGF- ⁇ (Transforming Growth Factor family - ⁇ , ⁇ 2, ⁇ 3], CIF (Cartilage Inducing Factor], at least one of BMP's 1-14 (Bone Morphogenic Proteins], Granulocyte-macrophage colony- stimulating factor (GM-CSF], or combinations thereof, which may promote tissue regeneration, can be attached to or coated to the matrices of the present disclosure.
  • EGF Epidermal Growth Factor
  • IGF-I Insulinlike Growth Factor
  • FGF Fibroblast Growth Factor family
  • KGF Keratinocyte Growth Factor
  • PDGF Platert-derived Growth Factor AA, AB, BB]
  • TGF- ⁇ Trans
  • the growth factor is VEGF. In another embodiment, the growth factor is
  • PDGF PDGF-like growth factor
  • the skilled addressee is aware of various other materials and biological molecules which may be attached to or used to coat a matrix of the presently-disclosed subject matter, and can be selected for a particular application based on the tissue to which they are to be applied.
  • an extracellular matrix protein such as, fibronectin, laminin, and/or collagen
  • the matrix is coated with collagen IV, collagen I, laminin and fibronectin, or a combination thereof.
  • the present inventors have discovered that these proteins help provide a secondary cellular signal which in conjunction with growth as an air liquid interface or on a gas permeable membrane, causes proper stratification of skin cells grown using the matrix.
  • collagen IV is used.
  • Collagen IV was shown to be particularly effective at producing proper epidermal stratification.
  • the extracellular matrix proteins may be in the form of full length proteins or peptides thereof, for example synthetic peptides.
  • a therapeutic agent is further attached to the matrix.
  • therapeutic agent refers to any of a variety of agents that exhibit one or more beneficial therapeutic effects when used in conjunction with methods, matrices and/or skin tissues of the present disclosure.
  • therapeutic agents include, without limitation, proteins, peptides, drugs, cytokines, extracellular matrix molecules, and/or growth factors.
  • proteins, peptides, drugs, cytokines, extracellular matrix molecules, and/or growth factors include, without limitation, proteins, peptides, drugs, cytokines, extracellular matrix molecules, and/or growth factors.
  • the therapeutic agent is an anti-inflammatory agent or an antibiotic.
  • anti-inflammatory agents that can be incorporated into the matrices include, but are not limited to, steroidal anti-inflammatory agents such as betamethasone, triamcinolone dexamethasone, prednisone, mometasone, fluticasone, beclomethasone, flunisolide, and budesonide; and non-steroidal anti-inflammatory agents, such as fenoprofen, flurbiprofen, ibuprofen, ketoprofen, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, ketorolac, nabumetone, sulindac tolmetin meclofenamate, mefenamic acid, piroxicam, and suprofen.
  • steroidal anti-inflammatory agents such as betamethasone, triamcinolone dexamethasone, prednisone, momet
  • Non-limiting examples include aminoglycosides, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, or tobramycin; carbapenems, such as ertapenem, imipenem, meropenem; chloramphenicol; fluoroquinolones, such as ciprofloxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, sparfloxacin, or trovafloxacin; glycopeptides, such as vancomycin; lincosamides, such as clindamycin; macrolides/ketolides, such as azithromycin, clarithromycin, dirithromycin, erythromycin, or tel
  • analgesic refers to agents used to relieve pain and, in some embodiments, can be used interchangeably with the term "anti- inflammatory agent” such that the term analgesics can be inclusive of the exemplary antiinflammatory agents described herein.
  • exemplary analgesic include, but are not limited to: paracetamol and non-steroidal anti-inflammatory agents, COX-2 inhibitors, and opiates, such as morphine, and morphinomimetics.
  • anesthetic refers to agents used to cause a reversible loss of sensation in subject and can thereby be used to relieve pain.
  • exemplary anesthetics that can be used in accordance with the presently-disclosed subject matter include, but are not limited to, local anesthetics, such as procaine, amethocaine, cocaine, lidocaine, prilocaine, bupivicaine, levobupivicaine, ropivacaine, mepivacaine, and dibucaine.
  • the methods of the present disclosure may be carried out using any cell culture device suitable for the production of fully human epidermis.
  • WO2016/209089 the contents of which are incorporated by reference, describes such devices.
  • the skilled person will be aware of other alternative culture devices.
  • the device comprises: a container comprising a first endwall (bottom], and at least one sidewall, a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to reside upon, wherein at least a part of at least one of the first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; and wherein the apparatus is configurable between (a] a first mode in which the substrate is not disposed in gaseous communication with a gas permeable material, and (b] a second mode in which the substrate is moved to be disposed in gaseous communication with a gas permeable material.
  • This particular device has the advantage of 2 modes of use, wherein the first mode allows the human keratinocytes to be fully submerged in culture media during the initial growth phase and wherein the second mode allows the keratinocytes to be exposed to the atmosphere which triggers proper differentiation of the skin cells. This eliminates the need for the substrate to be moved from one device to another in order to expose the skin cells to the atmosphere.
  • At least a part of the bottom comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; wherein at least a part of the second endwall (top] or at least a part of the at least one sidewall comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; and wherein the scaffold engages with the at least one sidewall to (a] allow substantially linear movement of the scaffold at least partway between the first endwall (bottom] of the chamber and the second endwall (top], and restrict rotation or inversion of the scaffold about an axis perpendicular to the at least one sidewall, or (b] allow rotational movement of the scaffold about an axis perpendicular to the at least one sidewall.
  • At least a part of the bottom comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; wherein at least a part of the top comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; wherein the scaffold engages with the at least one sidewall to (a] allow substantially linear movement of the scaffold at least partway between the bottom of the chamber and the top, and (b] restrict rotation or inversion of the scaffold about an axis perpendicular to the at least one sidewall.
  • a gas permeable material is present in both the bottom and the top, and the chamber is liquidly sealable from, but in gaseous communication with, the environment.
  • the gas permeable material is a gas permeable membrane.
  • the gas permeable material is polydimethylsiloxane.
  • the scaffold receives the substrate to present culturing surfaces on opposite planar sides of the substrate.
  • the substrate is a substrate as disclosed herein.
  • the scaffold comprises: a frame defining an interior perimeter and an exterior perimeter, said frame comprising a substantially planar upper surface, a substrate for cells to reside upon held in a substantially planar arrangement across the interior perimeter of the frame, wherein the scaffold is configured to bring substantially all of the substrate or the cells or tissues present on the substrate into contact with a gas permeable interface when the scaffold is placed in a culture apparatus comprising at least one gas permeable interface.
  • the scaffold comprises: a first frame defining an interior perimeter and an exterior perimeter, said first frame comprising a substantially planar upper surface, a second frame defining an interior perimeter and an exterior perimeter, said second frame comprising a substantially planar upper surface, wherein the first frame and the second frame detachably engage around at least a part of their perimeters to define an interface to receive and hold the substrate, wherein when held the substrate is held in a substantially planar arrangement across the interior perimeter of the first frame, and wherein when engaged, the upper surface of the first frame and the upper surface of the second frame are substantially co-planar.
  • the dimensions of the interior perimeter of the second frame at its upper surface are greater than the dimensions of the exterior perimeter of the first frame at its upper surface, such that the second frame engages around the exterior perimeter of the first frame at at least the upper surface of the first frame.
  • the substrate is held in a substantially planar arrangement across the interior perimeter of the scaffold at the upper surface of the first frame, thereby to allow direct contact of the upper surface of the substrate with, for example, a gas permeable interface.
  • the substrate is held between the first frame and the second frame by friction fit engagement of the first frame to the second frame.
  • the friction fit engagement is such that it rigidly clamps the substrate maintain the substrate in a substantially planar arrangement across the interior perimeter of the scaffold.
  • the substrate is held between the first frame and the second frame at least in part by one or more protrusions extending between the first frame and the second frame.
  • the lower surface of the scaffold comprises at least one section spanning the exterior perimeter of the scaffold and the adjacent interior perimeter, said section having a lower surface which is raised towards the upper surface of the scaffold, wherein said section defines a void when the scaffold is placed on a flat surface.
  • the section defines a recess provided to allow for easy removal of air bubbles when the scaffold is in submerged culture.
  • the dimensions of the interior perimeter of the scaffold at its upper surface are greater than the dimensions of one or more of the gas permeable interfaces into contact with which the substrate or the cells or tissues present on the substrate held by the scaffold is/are to be brought, such that substantially all of the substrate or the cells or tissues present on the substrate is/are capable of contacting the gas permeable interface.
  • the scaffold comprises one or more transverse members spanning the interior perimeter to provide support for the substrate.
  • tissue such as epithelium, epidermis, stratified epithelium, stratified epidermis and dermis, split thickness skin or full thickness skin, prepared using a method described herein, for example using a matrix of the present disclosure, for the treatment of tissue damage in subject in need thereof.
  • subject is used herein to refer to both human and animal subjects but is generally intended to refer to a human patient in need of treatment.
  • treatment include, but are not limited to, inhibiting the progression of damage to a tissue, arresting the development of damage to a tissue, reducing the severity of damage to a tissue, ameliorating or relieving symptoms associated with damage to a tissue, and repairing, regenerating, and/or causing a regression of damaged tissue or one or more of the symptoms associated with a damaged tissue.
  • the present disclosure provides to a method of treating tissue damage in a subject in need thereof comprising:
  • a skin tissue such as epithelium, stratified epithelium, epidermis, stratified epidermis, stratified epidermis and dermis, split thickness skin or full thickness skin, which has been grown, for example in a method as herein described, such as a method employing a fully human epidermis produced using the method described herein,
  • the tissue damage is a wound, a chronic wound, a surgical wound, an ulcer, a non-healing wound, a scar, a surgical scar, a scald or a burn.
  • the burn is a first degree burn, a second degree burn, a third degree burn, a deep dermal burn or a full thickness burn.
  • the tissue damage is epithelium located on a mucosal surface.
  • the epithelium is located on or in skin, the lungs, the gastrointestinal tract (for example, the oesophagus or mouth], reproductive tract, or the urinary tract (for example, the urethra].
  • treatments include but are not limited to: skin regeneration with nerves & organelles; wound healing, for example promoting/enhancing wound healing, including ulcers such as diabetic ulcers; burn healing; skin regeneration and repair; epidermolysis bullosa; enhance skin quality or appearance; prevention or remediation of skin disorders; diminishment or abolishment of scar tissues; breast skin regeneration (after surgery]; cosmetic applications, e.g. anti- aging; dermal regeneration for wrinkles and other skin defects; promotion of hair follicle growth, nerve and other organelle regeneration; healing without scarring, or re-healing to diminish scarring.
  • the present disclosure provides a matrix, skin tissue and method useful in the regeneration of damaged, lost and/or degenerated tissue.
  • a matrix, method or skin tissue of the present invention may be employed to initiate, increase, support, promote, and/or direct the regeneration of damaged, lost, and/or degenerated tissue, in particular the regeneration of damaged skin.
  • Regeneration refers to any process or quality that initiates, increases, modulates, promotes, supports, and/or directs the growth, regrowth, repair, functionality, patterning, connectivity, strengthening, vitality, and/or the natural wound healing process of weak, damaged, lost, and/or degenerating tissue.
  • These terms can also refer to any process or quality that initiates, increases, modulates, promotes, supports, and/or directs the growth, strengthening, functionality, vitality, toughness, potency, and/or health of weak, tired, and/or normal tissue.
  • wound is used to refer broadly to injuries to the skin and subcutaneous tissue initiated in different ways (e.g., pressure sores from extended bed rest and wounds induced by trauma] and with varying characteristics. Wounds are generally classified into one of four grades depending on the depth of the wound: Grade I: wounds limited to the epithelium; Grade II: wounds extending into the dermis; Grade III: wounds extending into the subcutaneous tissue; and Grade IV (or full-thickness wounds], which are wounds in which bones are exposed (e.g., a bony pressure point such as the greater trochanter or the sacrum].
  • Grade I wounds limited to the epithelium
  • Grade II wounds extending into the dermis
  • Grade III wounds extending into the subcutaneous tissue
  • Grade IV or full-thickness wounds
  • partial thickness wound refers to wounds that encompass Grades I-III; e.g., burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers.
  • deep wound is used to describe to both Grade III and Grade IV wounds.
  • a skin tissue such as epithelium, epidermis, stratified epithelium, stratified epidermis and dermis, split thickness skin or full thickness skin, prepared using a method described herein for facilitating a skin graft, by covering an area of damaged, injured, wounded, diseased, removed or missing skin tissue of a body of a subject.
  • a "graft” refers to a cell, tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect.
  • a “skin graft” is a skin tissue that may be implanted into an individual, for example sutured to the individual.
  • a graft may further comprise a matrix of the present disclosure, for example wherein the matrix is integrated into the skin graft.
  • the tissue or organ may consist of cells that originate from the same individual; this graft is referred to herein by the following interchangeable terms: "autograft", “autologous transplant", “autologous implant” and “autologous graft”.
  • a graft comprising cells from a genetically different individual of the same species is referred to herein by the following interchangeable terms: "allograft”, “allogeneic transplant”, “allogeneic implant” and “allogeneic graft”.
  • a "xenograft”, “xenogeneic transplant” or “xenogeneic implant” refers to a graft from one individual to another of a different species.
  • the tissue is prepared using cells that are autologous to the subject.
  • the tissue is prepared using fibroblasts, keratinocytes, or fibroblasts and keratinocytes that are autologous to the subject.
  • the tissue is prepared using cells that are heterologous to the subject.
  • the tissue is prepared using a combination of cells, wherein some of the cells are autologous to the subject and some of the cells are heterologous to the subject.
  • cells autologous to the subject may be isolated using any method known in the art.
  • autologous cells may be isolated from a skin sample or skin biopsy taken from the subject by digesting the sample tissue and separating fibroblasts and/or keratinocytes from the digested tissue.
  • the tissue is an autograft, for example, a skin autograft.
  • the tissue is an epidermal autograft, a split thickness skin autograft or a full thickness skin autograft.
  • the tissue is an allogeneic graft.
  • tissue prepared using cells autologous to the patient is highly desirable to reduce or prevent immune rejection of the tissue and to reduce the requirement for ongoing immunotherapy or another ancillary treatments.
  • tissue further comprises the matrix. In another embodiment the tissue is separated from the matrix before application to the patient.
  • the application of tissue to the patient will be by surgery.
  • recovery under sterile or aseptic conditions is during or immediately prior to surgery, for example in the surgical suite.
  • the application of tissue to the patient will be at or adjacent the site of tissue damage.
  • the tissues is applied to at least partially cover the site of tissue damage or to completely cover the site of tissue damage.
  • tissue is applied to temporarily cover the site of tissue damage. In an alternative embodiment the tissue is applied to permanently cover the site of tissue damage.
  • the graft is secured, for example sutured in place.
  • the graft is covered by a dressing, for example to keep it clean and moist after it is secured on the wound.
  • the skin product obtainable from the method of the present disclosure is provided for use in treatment, in particular treatment of condition/disease disclosed herein.
  • the efficacy and safety of topically applied pharmaceutical, nutraceutical or cosmetic products are typically tested using animal skin or live animals, human cadaver skin or synthetic human skin models.
  • cells or tissues such as skin tissue prepared using the device or methods described herein are useful for in vitro testing of pharmaceuticals, nutraceuticals or cosmetic products.
  • cells or tissue prepared using the device or methods described herein are used to test transdermal penetration of a compound, to test the permeation of a compound across the epidermis, dermis or basement membrane, to test the efficacy of an active ingredient for treating or preventing a condition, for example, a skin condition, or to test the toxicity of a compound.
  • the skin tissue produced in accordance with the present disclosure is suitable for testing products, for example, for effectiveness, unwanted side effects, for example, irritation, toxicity and inflammation or allergenic effects, or the compatibility of substances.
  • substances may be substances intended for potential use as medicaments, for example as dermatics, or substances which are constituents of cosmetics or even consumer goods which come into contact with the skin, such as laundry detergents, etc.
  • the skin tissue of the present disclosure may also be used, for example, for studying the absorption, transport and/or penetration of substances. It is also suitable for studying other agents (physical quantities], such as light or heat, radioactivity, sound, electromagnetic radiation, electrical fields, for example, for studying phototoxicity, i.e. the damaging effect of light of different wavelengths on cell structures.
  • the skin tissue may also be used for studying wound healing and is also suitable for studying the effects of gases, aerosols, smoke and dusts on cell structures, metabolism or gene expression.
  • the cells or tissue are used to determine if a compound of interest is a skin irritant, for example, to determine if a compound of interest induces a skin rash, inflammation, or contact dermatitis.
  • the effects of substances or agents on human skin can be determined, for example, from the release of substances, for example, cytokines or mediators, by cells of the human or animal skin model system and the effects on gene expression, metabolism, proliferation, differentiation and reorganization of those cells.
  • substances for example, cytokines or mediators
  • a vital dye such as a tetrazolium derivative
  • the testing of substances or agents using the skin tissue may comprise both histological processes and also immunological and/or molecular-biological processes.
  • test agent as used herein is any substance that is evaluated for its ability to diagnose, cure, mitigate, treat, or prevent disease in a subject, or is intended to alter the structure or function of the body of a subject
  • Test agents include, but are not limited to, chemical compounds, biologic agents, proteins, peptides, nucleic acids, lipids, polysaccharides, supplements, signals, diagnostic agents and immune modulators.
  • Test agents may further include electromagnetic and/or mechanical forces.
  • the skin tissue produced in accordance with the disclosure may be used as a model system for studying skin diseases and for the development of new treatments for skin diseases.
  • cells of patients with a certain genetic or acquired skin disease may be used to establish patient-specific skin model systems which may in turn be used to study and evaluate the effectiveness of certain therapies and/or medicaments.
  • the skin tissue may be populated with microorganisms, more particularly pathogenic microorganisms.
  • population with pathogenic or parasitic microorganisms including, in particular, human-pathogenic microorganisms.
  • "Microorganisms” as used herein generally refers to fungi, bacteria and viruses.
  • the microorganisms are preferably selected from fungi or pathogenic and/or parasitic bacteria known to infect skin. These include but are not limited to species of the genus Candida albicans, Trichophyton mentagrophytes, Malassezia furfur and Staphylococcus aureus.
  • Using a correspondingly populated skin tissue it is possible to study both the process of a microorganism population, more particularly the infection process, by the microorganism itself and the response of the skin to that population.
  • the effect of substances applied before, during or after the population on the population itself or on the effects of the population on the skin tissue can be studied.
  • the cells comprise fibroblasts, keratinocytes or immune cells, or a combination of any two or more thereof. In one embodiment the cells comprise fibroblasts and keratinocytes. In various embodiments the tissue is selected from the group comprising epidermis, stratified epidermis and dermis, stratified epidermis and dermis, split thickness skin or full thickness skin.
  • the compound is a pharmaceutical compound, a cosmetic compound or a nutraceutical compound.
  • the compound for testing is applied to tissue alone or in an admixture with pharmaceutically or cosmetically acceptable carriers, excipients or diluents.
  • the compound for testing is applied topically to the tissue in the form of a sterile cream, gel, pour-on or spot-on formulation, suspension, lotion, ointment, dusting powder, a drench, spray, drug- incorporated dressing, shampoo, collar or skin patch.
  • gas permeable material or "gas permeable membrane” as used herein means a material or membrane through which gas exchange may occur.
  • Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.
  • Figure 1 shows a comparison of cell yields using the different skin digest methods.
  • Pieces of full thickness human skin were digested with trypsin alone, or trypsin combined with collagenase, to generate mixed populations of epidermal and dermal cells; or sequentially with dispase and collagenase to generate separate populations of epidermal cells.
  • Cell yields were normalised to the weight of each tissue sample. Trypsin only (T] and trypsin collagenase (TC] cell yields are all cells isolated from both the epidermis and dermis.
  • Dispase collagenase (DC] sequential digest cell yields are a combination of the individual cell yields of both the epidermis and dermis digests. * P ⁇ 0.05.
  • Figure 2 shows keratinocyte proliferation from skin samples digested with different methods.
  • Pieces of full thickness human skin were digested with trypsin only, or trypsin combined with collagenase, to generate mixed epidermal and dermal cells; or with dispase to first separate the epidermis and then break it up to generate pure epidermal cells.
  • Trypsin only and trypsin collagenase samples with mixed cells from epidermis and dermis were grown in Greens medium with ROCK inhibitor Y27632 and no mouse feeder cells.
  • Dispase-digested epidermal samples were grown in Greens medium with ROCK inhibitor Y27632 and mouse feeder cells ("MEF"]. Samples were passaged 1:4 when they reached 80-90% confluency.
  • Figure 3 shows keratinocyte proliferation from skin samples expanded in keratinocyte medium with ROCK inhibitor Y27632 or SB 772077B.
  • Pieces of full thickness human skin were digested with trypsin combined with collagenase to generate mixed epidermal and dermal cells, or with dispase to first separate the epidermis and then break it up to generate pure epidermal cells.
  • Trypsin collagenase full thickness skin samples with mixed cells from epidermis and dermis were grown in Greens medium with no xenogeneic mouse feeder cells and ROCK inhibitor Y27632 or SB 772077B.
  • Dispase- digested epidermal samples were grown in Greens medium with ROCK inhibitor Y27632 and irradiated xenogeneic mouse feeder cells ("MEF"]. Samples were passaged 1:4 when they reached 80-90% confluency
  • Keratinocyte extraction (dispase digest):
  • the pieces of skin were placed in the wells of a 6 well plate.
  • lOmg/ml dispase with MilliQ water was prepared and the enzyme stock was filtered through a 0.2 ⁇ syringe filter. 600 ⁇ 1 of dispase was added to 5.4ml DO in each 6 well plate.
  • the plate was placed in an incubator at 37°C with 5% C0 2 overnight.
  • the epidermis was separated from the dermis using a scalpel.
  • the epidermis was placed in Green's medium and a scalpel was used to cut the epidermis into small pieces.
  • the epidermis was broken up by passing through a pipette repeatedly. This was performed gently because excessive force will reduce cell viability.
  • the epidermal cell suspension was then passed through a ⁇ cell strainer before the keratinocyte cell suspension was centrifuged at 1800rpm for 10 min. Finally, all supernatant was discarded and the cell pellets were resuspended in Green's medium to assess cell number and viability.
  • 5mg/ml collagenase type I was prepared using MilliQ water. Next, the enzyme stock was filtered through a 0.2um syringe filter. 600ul of enzyme and 5.4ml DO was added to each well of a 6 well plate, mixed and placed in an incubator at 37 Q C with 5% CO2 overnight. The next day, DF10 was placed into a 10cm diameter dish. Then, one piece at a time, the dermis was transferred to the fresh DFIO containing dish and teased apart with a scalpel. To obtain a single cell suspension, both DFIO and the enzyme containing medium were passed through a ⁇ strainer. The mixture was centrifuge at 1800 rpm for lOmin and then as much supernatant was removed as possible without disturbing the cell pellet. Finally, the cell pellets were resuspended in DFIO to assess cell number and viability.
  • the pieces of skin were placed in the wells of a 6 well plate.
  • 0.25% trypsin was diluted to 0.1% in DO, making a total volume of 6ml.
  • the enzyme containing medium was then placed in an incubator at 37°C with 5% CO2 overnight.
  • the skin and enzyme containing medium were transferred to 6ml of DFIO in a 10cm dish and the skin was teased apart with a scalpel.
  • the skin was broken up further by passing the mixture through a pipette repeatedly.
  • the cell mix was passed through a ⁇ strainer before it was centrifuged at 1800rpm for lOmin. As much of the supernatant was removed as possible without disturbing cell pellet before the cell pellets were resuspended in DFIO to assess cell number and viability.
  • the pieces of skin were placed in the wells of a 6 well plate.
  • 5mg/ml collagenase type I
  • the enzyme stock was filtered through a 0.2um syringe filter in hood.
  • 0.25% trypsin was then diluted to 0.1% in DO and 600 ⁇ 1 of collagenase was added, making a total volume of 6ml.
  • the plate was placed in an incubator at 37°C with 5% CO2 overnight.
  • the skin and enzyme containing medium was transferred to 6ml of DF10 in a 10cm dish and the skin teased apart with a scalpel.
  • the skin was next broken up further by passing through a pipette repeatedly.
  • the cell mix was passed through a ⁇ strainer.
  • Figure 1 shows that digesting human skin with trypsin only or trypsin combined with collagenase results in significantly greater cells yields compared to the dispase collagenase sequential digest method.
  • Figure 2 shows the results of keratinocytes grown from trypsin only, trypsin collagenase and dispase collagenase sequentially digested skin samples. All samples were grown in Green's medium containing ROCK inhibitor but only dispase digested epidermal samples had mouse feeder cells added. Keratinocyte growth was superior in trypsin collagenase digested samples with no added feeder cells and comparable in trypsin alone digested samples with no added feeder cells compared to dispase digested epidermis that had feeder cells added to promote keratinocyte growth.
  • human keratinocytes can be grown in the presence of dermal fibroblasts, for example in the presence of a keratinocyte growth accelerator, such as ROCK inhibitor. This obviates both the need to separate human epidermis from dermis during processing of human skin samples, and the need to add xenogeneic feeder cells to ensure optimal keratinocyte growth.
  • a keratinocyte growth accelerator such as ROCK inhibitor.
  • Figure 3 shows the results of fibroblasts grown from trypsin only, trypsin collagenase and dispase collagenase sequentially digested skin samples. All samples were grown in the same fibroblast growth medium. Fibroblast growth was superior in trypsin collagenase digested samples compared to dispase collagenase sequentially digested dermis. These results show that digestion of full thickness skin with trypsin, especially with addition of collagenase, enables superior yields of fibroblasts to be obtained, compared with the traditional method of first separating the dermis from the epidermis using dispase and then digesting the dermis separately with collagenase.

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Abstract

L'invention concerne un procédé in vitro approprié pour obtenir des cellules, par exemple des cellules épithéliales et des cellules de tissu conjonctif (telles que des kératinocytes et/ou des cellules fibroblastiques) à partir d'un échantillon de peau de cellules entières comprenant un derme et un épiderme, par traitement dudit échantillon avec un dispositif de digestion comprenant une protéase capable de digérer l'épiderme et le derme. L'invention concerne également des kératinocytes et/ou des cellules de fibroblastes obtenus à l'aide du procédé selon l'invention.
PCT/NZ2018/050131 2017-09-30 2018-09-28 Procédé d'obtention de cellules à partir d'un dispositif de digestion de peau entière Ceased WO2019066663A1 (fr)

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Cited By (2)

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CN109943520A (zh) * 2019-03-08 2019-06-28 北京达博威迎医药技术有限公司 汗腺细胞的分离和培养获得汗腺类器官的方法及其应用
WO2021048441A1 (fr) * 2019-09-13 2021-03-18 Veritacell Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109943520A (zh) * 2019-03-08 2019-06-28 北京达博威迎医药技术有限公司 汗腺细胞的分离和培养获得汗腺类器官的方法及其应用
WO2021048441A1 (fr) * 2019-09-13 2021-03-18 Veritacell Procédé et appareil pour l'isolement de cellules et cellules isolées pour la cicatrisation

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