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Title:
METHOD OF GENERATING HUMAN EPIDERMIS
Document Type and Number:
WIPO Patent Application WO/2019/066662
Kind Code:
A1
Abstract:
An in vitro method of generating a fully human epidermis (for example fully human full- thickness skin comprising a dermis and an epidermis] comprising a first step of culturing human keratinocytes and human fibroblast feeder cells in media comprising an effective amount of a ROCK inhibitor. Also provided is a skin product comprising a fully human epidermis produced using the disclosed method, including the use of the skin product for treatment.

Inventors:
DUNBAR PETER RODERICK (NZ)
FEISST VAUGHAN JOHN (NZ)
Application Number:
NZ2018/050130
Publication Date:
April 04, 2019
Filing Date:
September 28, 2018
Export Citation:
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Assignee:
AUCKLAND UNISERVICES LTD (NZ)
International Classes:
C12N5/071
Domestic Patent References:
WO2018124887A12018-07-05
WO2016209089A12016-12-29
Foreign References:
US4045418A1977-08-30
US4057537A1977-11-08
US5468253A1995-11-21
Other References:
V. D. GANDHAM ET AL: "Effects of Y27632 on keratinocyte procurement and wound healing", CLINICAL AND EXPERIMENTAL DERMATOLOGY, 1 May 2013 (2013-05-01), GB, pages n/a - n/a, XP055529725, ISSN: 0307-6938, DOI: 10.1111/ced.12067
XANTHE L. STRUDWICK ET AL: "Combination of Low Calcium with Y-27632 Rock Inhibitor Increases the Proliferative Capacity, Expansion Potential and Lifespan of Primary Human Keratinocytes while Retaining Their Capacity to Differentiate into Stratified Epidermis in a 3D Skin Model", PLOS ONE, vol. 10, no. 4, 13 April 2015 (2015-04-13), pages e0123651, XP055315198, DOI: 10.1371/journal.pone.0123651
SANDRA CHAPMAN ET AL: "The effect of Rho kinase inhibition on long-term keratinocyte proliferation is rapid and conditional", STEM CELL RESEARCH & THERAPY, BIOMED CENTRAL LTD, LONDON, UK, vol. 5, no. 2, 28 April 2014 (2014-04-28), pages 60, XP021187205, ISSN: 1757-6512, DOI: 10.1186/SCRT449
XUEFENG LIU ET AL: "ROCK Inhibitor and Feeder Cells Induce the Conditional Reprogramming of Epithelial Cells", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 180, no. 2, 1 February 2012 (2012-02-01), pages 599 - 607, XP055131196, ISSN: 0002-9440, DOI: 10.1016/j.ajpath.2011.10.036
SANDRA CHAPMAN ET AL: "Human keratinocytes are efficiently immortalized by a Rho kinase inhibitor", JOURNAL OF CLINICAL INVESTIGATION, vol. 120, no. 7, 1 July 2010 (2010-07-01), pages 2619 - 2626, XP055123778, ISSN: 0021-9738, DOI: 10.1172/JCI42297
REBECCA LAMB ET AL: "Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model", PLOS ONE, vol. 8, no. 1, 11 January 2013 (2013-01-11), pages e52494, XP055315202, DOI: 10.1371/journal.pone.0052494
ERIK D. ANDERSON ET AL: "Prolonging culture of primary human keratinocytes isolated from suction blisters with the Rho kinase inhibitor Y-27632", PLOS ONE, vol. 13, no. 9, 12 September 2018 (2018-09-12), pages e0198862, XP055530086, DOI: 10.1371/journal.pone.0198862
HOLLIGER; HUDSON, NATURE BIOTECH., vol. 23, no. 9, 2005, pages 1126 - 1136
ADAIR; LAWSON, DRUG DESIGN REVIEWS - ONLINE, vol. 2, no. 3, 2005, pages 209 - 217
CHRISTENSON L; MIKOS A G; GIBBONS D F ET AL.: "Biomaterials for tissue engineering: summary", TISSUE ENG., vol. 3, no. 1, pages 71 - 73
Attorney, Agent or Firm:
CATALYST INTELLECTUAL PROPERTY (NZ)
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Claims:
An in vitro method of generating a fully human epidermis (for example fully human full- thickness skin comprising a dermis and an epidermis] comprising a first step of culturing human keratinocytes and human fibroblast feeder cells in media comprising an effective amount of a ROCK inhibitor.

An in vitro method according to claim 1, wherein the human keratinocytes are selected from skin (comprising epidermis, such as a sample of dermis and epidermis] keratinocytes, foreskin keratinocytes, vaginal keratinocytes, placenta and cervical keratinocytes.

An in vitro method according to claim 1 or 2, wherein the human fibroblast cells are not irradiated.

An in vitro method according to any one of claims 1 to 3, wherein the human fibroblast cells are sex matched to the keratinocytes.

An in vitro method according to any one of claims 1 to 4, wherein the human fibroblast cells are sex matched to a patient.

An in vitro method according to any one of claims 1 to 5, wherein the human fibroblast cells are HLA matched to the keratinocytes.

An in vitro method according to any one of claims 1 to 6, wherein the human fibroblast cells are HLA matched to a patient.

An in vitro method according to any one of claims 1 to 7, wherein the human fibroblasts and keratinocytes are from the same donor.

An in vitro method according to any one of claims 1 to 8, wherein the human fibroblasts are autologous to a human patient.

An in vitro method according to any one of claims 1 to 9, wherein the human keratinocytes are autologous to a human patient.

An in vitro method according to any one of claims 1 to 10, wherein the media comprises or consists of: DMEM High glucose, Ham's F12, foetal bovine serum, keratinocyte growth factor (KGF] and optionally one or more antibiotics (for example 1, 2, or 3 antibiotics, for example selected from penicillin, streptomycin, amphotericin B and a combination of two or three of the same]

An in vitro method according to claim 11, wherein the ratio of DMEM High glucose:Ham's F12 is 3: l.

An in vitro method according to claims 11 or 12, wherein the foetal bovine serum has a concentration of 10%.

A cell culture medium according to any one of claims 11 to 13, wherein the amphotericin B has a concentration of 0.625 μg/ml.

An in vitro method according to any one of claims 11 to 14, wherein the media (Kelch's medium] consists of: DMEM High glucose:Ham's F12 (3 :1], 10% foetal bovine serum, penicillin, streptomycin, 0.625μg/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF].

An in vitro method according to any one claims 1 to 11, wherein the media is Green's media. An in vitro method according to any one of claims 1 to 11, wherein the media is serum free.

18. An in vitro method according to any one of claims 1 to 17, wherein ROCK inhibitor is a small molecule inhibitor or an antibody inhibitor, such as a small molecule inhibitor.

19. An in vitro method according claim 18, wherein the ROCK inhibitor is selected from the group comprising: Y-27632, SB 772077B, 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.

20. An in vitro method according to claim 19, wherein the ROCK inhibitor is Y-27632, SB 772077B, or a combination of both, in particular SB 772077B.

21. An in vitro method according to any one of claims 1 to 20, wherein the concentration of ROCK inhibitor is in the range 0.1 to ΙΟΟμΜ, for example 0.2 to 50μΜ or 0.3 to 25μΜ or 0.1 to 0.95μΜ, 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 0.95μΜ, in particular 0.4μΜ.

22. An in vitro method according to any one of claims 1 to 21, wherein the method comprises a second step of culturing the keratinocytes and fibroblasts in the absence of a ROCK inhibitor.

23. An in vitro method according to any one of claims 1 to 19, wherein the second culture step has a first phase of culturing the keratinocytes when they are not in contact with a gas permeable membrane (interface] and are not at the liquid interface.

24. An in vitro method according to claim 20, wherein the keratinocytes in the second step are cultured, for a second phase in contact with a gas permeable membrane (interface), for example the period of culture where the keratinocytes are in contact with the gas permeable membrane follows the period of culturing without contacting a gas permeable layer.

25. An in vitro method according to any one of claims 1 to 21, wherein the keratinocytes are deposited on a substrate, for example a matrix, for example a spun matrix or mesh.

26. An in vitro method according to claim 22, wherein the substrate (such as a matrix] comprises a biocompatible biodegradable polymer.

27. An in vitro method according to claim 22 or 23, wherein the substrate is prepared by electrospinning.

Description:
METHOD OF GENERATING HUMAN EPIDERMIS

The present invention relates to a method of generating an epidermis, such as a fully human epidermis by culturing keratinocytes with human fibroblast feeder cells together with a ROCK inhibitor, for example SB 772077B or Y-27632.

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, some applications, for example treatment of burns, may require full-thickness skin comprising both an epidermal layer containing epithelial cells (keratinocytes) and an underlying dermal layer containing fibroblastic cells.

Isolated keratinocytes need to be co-cultured with fibroblastic feeder cells for survival and proliferation. Production of either epidermis or synthesis of full-thickness skin in the art employs human keratinocytes that have been co-cultured with irradiated mouse embryonic fibroblast feeder cells (xenogeneic 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. The inclusion of xenogeneic cells in the cell cultures also increases the risk of transmission of infectious disease from animals to humans.

The reason human keratinocytes are grown with irradiated mouse fibroblast (MEF) feeder cells is that human fibroblasts outgrow and effectively "swamp" or "overrun" the keratinocytes.

It would be useful to make single layer epidermis and full thickness skin consisting of dermis and epidermis without employing xenogeneic material.

The present inventors have devised a method that allows the preparation of this very beneficial skin product in an efficient and rapid manner and, for example the product can be available to patients in two to three weeks. SUMMARY OF INVENTION

The invention is summarised in the following paragraphs:

1. An in vitro method of generating a fully human epidermis (for example fully human full- thickness skin comprising a dermis and an epidermis) comprising a first step of culturing human keratinocytes and human fibroblast feeder cells in media comprising an effective amount of a ROCK inhibitor.

2. An in vitro method according to paragraph 1, wherein the human keratinocytes are selected from skin (comprising epidermis, such as a sample of dermis and epidermis) keratinocytes, foreskin keratinocytes, vaginal keratinocytes, placenta and cervical keratinocytes.

3. An in vitro method according to paragraph 1 or 2, wherein the human fibroblast cells are not irradiated.

4. An in vitro method according to any one of paragraphs 1 to 3, wherein the human fibroblast cells are sex matched to the keratinocytes. An in vitro method according to any one of paragraphs 1 to 4, wherein the human fibroblast cells are sex matched to a patient.

An in vitro method according to any one of paragraphs 1 to 5, wherein the human fibroblast cells are HLA matched to the keratinocytes.

An in vitro method according to any one of paragraphs 1 to 6, wherein the human fibroblast cells are HLA matched to a patient.

An in vitro method according to any one of paragraphs 1 to 7, wherein the human fibroblasts and keratinocytes are from the same donor.

An in vitro method according to any one of paragraphs 1 to 8, wherein the human fibroblasts are autologous or allogeneic (such as autologous) to a human patient.

An in vitro method according to any one of paragraphs 1 to 9, wherein the human keratinocytes are autologous or allogeneic (such as autologous) to a human patient.

An in vitro method according to any one of paragraphs 1 to 10, wherein the media does not comprise an antibiotic, an antimycotic and a combination of two or more of the same.

An in vitro method according to any one of paragraphs 1 to 10, wherein the media comprises one or more antibiotics and/or one or more antimycotics.

An in vitro method according to paragraph 12, wherein the antibiotics and/or antimycotics are 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 Penicillin, Streptomycin, Gentamicin and Amphotericin B, in particular Gentamicin and Amphotericin B.

An in vitro method according to any one of paragraphs 1 to 10, 12 or 13, wherein 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 and two or three) and keratinocyte growth factor (KGF).

An in vitro method according to paragraph 14, wherein the ratio of DMEM High glucose:Ham's F12 is 3: 1.

An in vitro method according to paragraphs 14 or 15, wherein the foetal bovine serum has a concentration of 10%.

An in vitro method according to any one of paragraphs 14 to 16, wherein the amphotericin B has a concentration of 0.625 μg/ml.

An in vitro method according to any one of paragraphs 14 to 17, wherein the media (Kelch's medium) consists of: DMEM High glucose:Ham's F12 (3: 1), 10% foetal bovine serum, penicillin, streptomycin, 0.625μg/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF). An in vitro method according to any one paragraphs 1 to 10 and 12 to 18, wherein the media is Green's media.

An in vitro method according to any one of paragraphs 1 to 13, wherein the media is serum free. An in vitro method according to any one of paragraphs 1 to 20, wherein ROCK inhibitor is a small molecule inhibitor or an antibody inhibitor, such as a small molecule inhibitor.

An in vitro method according paragraph 15, wherein the ROCK inhibitor is selected from the group comprising: Y-27632, SB 772077B, 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.

An in vitro method according to paragraph 22, wherein the ROCK inhibitor is Y-27632, SB 772077B, or a combination of both, in particular SB 772077B.

An in vitro method according to any one of paragraphs 1 to 23, wherein the concentration of ROCK inhibitor is in the range 0.1 to ΙΟΟμΜ, for example 0.2 to 50μΜ or 0.3 to 25μΜ or 0.1 to 10μΜ or 0.1 to 0.95μΜ, 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 0.95μΜ, in particular 0.4μΜ.

An in vitro method according to any one of paragraphs 1 to 24, 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 step is performed after the first step of culturing in the presence of a ROCK inhibitor].

An in vitro method according to any one of paragraphs 1 to 19, wherein the second culture step has a first phase of culturing the cells (such as keratinocytes) when they are not in contact with a gas permeable membrane (interface] and are not at the liquid interface.

An in vitro method according to paragraph 26, 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 follows the period of culturing without contacting a gas permeable layer.

An in vitro method according to any one of paragraphs 1 to 27, wherein the keratinocytes are deposited on a substrate, for example a matrix, for example a spun matrix or mesh, for example as disclosed in WO2018/124887 incorporated herein by reference.

An in vitro method according to paragraph 28, wherein the substrate (such as a matrix) comprises a biocompatible biodegradable polymer.

An in vitro method according to paragraphs 28 or 29, wherein the substrate is prepared by electrospinning.

An in vitro method according to paragraph 30, wherein electrospun fibres from said electrospinning are about 0.3 μιη to about 5 μιη in diameter or 2 to 5 μιη, such as 1, 1.5, 2, 2.5, 3, 3.5, 4, or 4.5 μιη.

An in vitro method according to any one of paragraphs 23 to 25, 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/HA, PLGA/HA, dioxanone linear homopolymer (such as 100 dioxanone linear homopolymer) and combinations of two or more of the same. In one embodiment 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.

An in vitro method according to paragraph 32, wherein the polymer is poly(lactic-co-glycolic acid] (PLGA).

An in vitro method according to any one of paragraphs 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 according to any one of paragraphs 28 to 34, wherein the substrate has a thickness of 10 to 100 μπι.

An in vitro method according to any one of paragraphs 28 to 35, wherein substrate is three dimensional, and is substantially planar, with two faces.

An in vitro method according to any one of paragraphs 36, wherein at least one face of the substrate is micropatterned, undulating and/or dimpled.

An in vitro method according to claim any one of paragraphs 28 to 37, wherein the substrate is coated with protein, polypeptide or peptide to assist the keratinocytes adhering to the substrate and/or stratification of the cells.

An in vitro method according to paragraph 38, wherein the protein is selected from an extracellular matrix protein [such as the amino acid of collagen, laminin and other extracellular matrix proteins] or peptide thereof or a lipopeptide, for example a synthetic peptide.

An in vitro method according to paragraph 39, 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 according to any one of paragraphs 28 to 40, wherein the substrate is located in a culture device comprising a gas permeable membrane, for example such that one face can be orientated to be in contact with the gas permeable layer or said one face is removed from contact with said gas permeable layer.

An in vitro method according to paragraph 41, wherein the substrate is moveable, for example by rotation or sliding, between the position where one face is contact with the gas permeable layer and the position where said one face is removed from contact with said gas permeable layer.

An in vitro method according to any one of paragraphs 1 to 42, further comprising the pre-step of digesting a sample comprising human keratinocytes and human fibroblast feeder cells, such as a skin sample, using a protease digest.

An in vitro method according to paragraph 43, wherein the protease is selected from dispase, trypsin and combinations thereof.

An in vitro method according to paragraph 44, wherein dispase is employed to digest the epidermis.

An in vitro method according to paragraph 44 or 45, wherein trypsin is employed to digest the dermis.

An in vitro method according to any one of paragraphs 43 to 45, wherein the digest is performed in the presence of collagenase. An in vitro method according to any one of paragraphs 43 to 47, wherein the digest is performed without separating the dermis and epidermis (referred to herein as a whole skin digest). An in vitro method according to any one of paragraphs 1 to 48, wherein the human keratinocytes are not cultured in the presence of xenogeneic feeder cells

A fully human skin product comprising a human epidermis cultured by the method of any one of paragraphs 1 to 49, i.e. a fully human skin product obtainable from said method.

A fully human skin product according to paragraph 50, which comprises a differentiated dermis and epidermis.

A fully human epidermis according to paragraphs 50 or 51 for use in treatment

A fully human epidermis according to paragraph 52, 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, such as where the excision is: cancerous cells skin cells (including melanoma, basal cell carcinoma, squamous cell carcinoma (such as Bowen's disease), extra-mammary Paget's disease), breast cancer, tissue with Darrier's disease, a cyst, a wart (including plantar warts), necrotizing fascilitis (such as methicillin-resistant Staphylococcus aureus, necrotic tissue (such as gangrene), tissue exhibiting Hailey-Hailey disease, tissue exhibiting blisters associated with pemphigus vulgaris; epidermolysis bullosa; enhance skin quality (for example to treat ichthyosis) or appearance; prevention or remediation of skin disorders, for example an abscess, dermatitis (including contact dermatitis), atopic dermatitis, acne, actinic keratosis, rosacea, a carbuncle, eczema, psoriasis, cellulitis, kertosis, pilaris, melasma, impetigo or a fissure; diminishment or abolishment of scar tissues (for example to treat keloids); 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. In one embodiment 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

A method of treatment comprising suturing a fully human epidermis according to paragraphs 50 or 51 to a patient in need thereof. Use of a fully human epidermis according to paragraphs 50 or 51 in the manufacture of a medicament 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, such as where the excision is: cancerous cells skin cells (including melanoma, basal cell carcinoma, squamous cell carcinoma (such as Bowen's disease], extra-mammary Paget's disease], breast cancer, tissue with Darrier's disease, a cyst, a wart (including plantar warts], necrotizing fascilitis (such as methicillin-resistant Staphylococcus aureus, necrotic tissue (such as gangrene], tissue exhibiting Hailey-Hailey disease, tissue exhibiting blisters associated with pemphigus vulgaris; epidermolysis bullosa; enhance skin quality (for example to treat ichthyosis] or appearance; prevention or remediation of skin disorders, for example an abscess, dermatitis (including contact dermatitis], atopic dermatitis, acne, actinic keratosis, rosacea, a carbuncle, eczema, psoriasis, cellulitis, kertosis, pilaris, melasma, impetigo or a fissure; diminishment or abolishment of scar tissues (for example to treat keloids]; 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.

A use according to paragraph 55, 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, such as where the excision is: cancerous cells skin cells (including melanoma, basal cell carcinoma, squamous cell carcinoma (such as Bowen's disease], extra-mammary Paget's disease], breast cancer, tissue with Darrier's disease, a cyst, a wart (including plantar warts], necrotizing fascilitis (such as methicillin-resistant Staphylococcus aureus, necrotic tissue (such as gangrene], tissue exhibiting Hailey-Hailey disease, tissue exhibiting blisters associated with pemphigus vulgaris; epidermolysis bullosa; enhance skin quality (for example to treat ichthyosis] or appearance; prevention or remediation of skin disorders, for example an abscess, dermatitis (including contact dermatitis], atopic dermatitis, acne, actinic keratosis, rosacea, a carbuncle, eczema, psoriasis, cellulitis, kertosis, pilaris, melasma, impetigo or a fissure; diminishment or abolishment of scar tissues (for example to treat keloids); 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.

Therefore, in one aspect, there is provided an in vitro method of generating a fully human epidermis (for example fully human full -thickness skin comprising a dermis and an epidermis) comprising a first step of culturing human keratinocytes and human fibroblast feeder cells in media comprising an effective amount of a ROCK inhibitor.

Surprisingly in the presence of the ROCK inhibitor, especially low concentrations of ROCK inhibitor, the human keratinocytes grow at a sufficient rate in co-culture with unirradiated human fibroblasts, without being outgrown/overgrown by the fibroblasts. Thus, the presence of the ROCK inhibitor seems to accelerate the growth of the keratinocytes. Advantageously this means that, for example autologous fibroblasts from patient skin can be used as keratinocyte feeder cells instead of irradiated xenogeneic feeder cells, thereby allowing a less immunogeneic skin tissue to be prepared with lower risk of transmission of infectious agents from animals.

The ability to culture these human cells together means that there is no longer a need to separate the epidermis from the dermis when digesting the skin samples. Thus, it is possible to digest both layers of skin together and then grow the cells together as a single culture. This improves the efficiency of the process according to the present disclosure.

Hence, 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. Thus, the presently disclosed method is faster, cheaper, more convenient, involves less handling and therefore lower risk of contamination, and importantly, results in a less immunogenic and less infectious product compared to the prior art methods.

In one embodiment the first culture step is performed for a period of 3 to 14 days, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

In one embodiment the second culture step is performed for a period of 3 to 14 days, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days.

In one embodiment the skin product is obtained in 3 weeks or less, for example 21 days or less, 20 days or less, 19 or less, or 18 days or less.

As discussed above in one embodiment the substrate is located in a culture device comprising a gas permeable membrane, for example the substrate is moveable to a position where cells deposited thereon are in contact with a gas permeable layer, or where said cells are not in contact with a gas permeable layer. A suitable device is disclosed in WO2016/209089, incorporated herein by reference. 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, 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 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.

In one embodiment in 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.

In one embodiment 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 device comprising at least one gas permeable interface.

In one embodiment, the period of culture where the keratinocytes are in contact with the gas permeable membrane follows the period of culturing without contacting a gas permeable layer, for example 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.

Advantageously, the ability to move the substrate between the two modes allows the cells to be submerged in media during the initial phase of growth and then easily put in contact with the gas permeable layer for proper differentiation, for example into full thickness skin.

Accordingly, in one aspect, there is provided an in vitro method of generating a fully human epidermis comprising the step of culturing human keratinocytes and human fibroblast feeder cells in media comprising an effective amount of a ROCK inhibitor.

In one embodiment, the method further comprises the step of digesting a sample comprising human keratinocytes and human fibroblast feeder cells, such as a skin sample, in the presence of trypsin. Advantageously, trypsin allows the dermis and epidermis layers to be digested simultaneously in a single digestion reaction, thereby yielding both keratinocytes and fibroblast cells. This eliminates the requirement for the epidermis and dermis to be digested in two separate reactions, thereby saving time and simplifying the process.

In one embodiment, the method further comprises the step of digesting a sample comprising human keratinocytes and human fibroblast feeder cells, such as a skin sample, in the presence of 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.

In another embodiment, the sample is digested in the presence of both trypsin and collagenase. Advantageously, the present inventors have discovered that digesting skin samples with trypsin only or with trypsin and collagenase results in significantly greater cells yields compared to the dispase collagenase sequential digest method. Further advantageously, the present inventors have found that keratinocytes and/or fibroblasts isolated by digesting skin in both trypsin and collagenase may proliferate faster when grown in culture compared to keratinocytes and/or fibroblasts isolated from dispase digested epidermis and collagenase digested dermis. In one embodiment, 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 one embodiment, the keratinocytes and the fibroblasts are from a whole skin digest.

In one embodiment, the media (Kelch's medium) consists of: DMEM High gIucose:Ham's F12 (3:1), 10% foetal bovine serum, penicillin, streptomycin, 0.625μg/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF). Advantageously, the disclosed cell culture medium does not contain choleratoxin. Surprisingly, the inventors have discovered that 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 in the method of the present disclosure. In addition, 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. Further advantageously, the inventors have established that the disclosed culture medium produces similar keratinocyte growth in the absence of mouse embryonic feeder cells (MEFs) as Green's medium with MEFs, in the method of the present disclosure.

Hence, 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, because the cell culture medium is more convenient and easier to prepare reproducibly (i.e. with less batch to batch variability) and also costs less.

In one embodiment, the substrate is coated with an extracellular matrix protein or peptide thereof, for example a synthetic peptide (such as the amino acid of collagen, laminin and other extracellular matrix proteins). Advantageously, 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. Collagen IV is that it was found to consistently produce good epidermal stratification.

In one embodiment, the ROCK inhibitor is Y-27632, SB 772077B, or a combination of both, in particular SB 772077B. The present inventors have discovered that these two ROCK inhibitors are particularly suitable for enhancing keratinocyte growth rates. SB 772077B 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 ΙΟμιη).

DETAILED DESCRIPTION

The terms "epithelia" and "epithelium" refer to the cellular covering of internal and external body surfaces (cutaneous, mucous and serous), including the glands and other structures derived therefrom, e.g., skin, corneal, esophageal, laryngeal, epidermal, hair follicle and urethral epithelial cells.

In one embodiment, the epithelial cells employed are skin cells, such as human skin cells, for example cells which form an epidermis and dermis, such as fibroblasts and keratinocytes.

Other exemplary 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.

As used herein "epidermis" refers to the outer of the two layers which make up the skin, the inner layer being the dermis.

As used herein "fully human" refers to a tissue which does not comprise any non-human cells, for example xenogeneic feeder cells, such as mouse embryonic fibroblasts. Furthermore, non- human cells are not employed to prepare fully-human tissues. The cells and tissue according to the present disclosure do not contain any non-human components because they are from fully human origin. However, 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.

As used herein "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. In one embodiment 50% or more, such as 60, 70, 80, 90, 95% or more of the fibroblast cells employed are differentiated.

As used herein "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. Alternatively, to the normal skin keratinocytes, mucous membrane keratinocytes or intestinal epithelial cells may be applied to the matrix. These are for example pre -cultivated cells and, in one embodiment, keratinocytes in the first or in the second cell passage, although cells from higher passages may also be used.

The fibroblasts and keratinocytes are obtained and cultivated by methods known among skilled addressees, which may be adapted to the required properties of the skin tissue to be produced.

In one embodiment 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. 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 skin model.

Accordingly, 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.

The term "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.

The term "skin tissue," or "skin" as used herein, refers to any tissue, including epidermis, dermis and basement membrane tissue, for example full thickness skin.

Skin product as employed herein is skin tissue prepared in vitro, in particular by the method of the present disclosure.

Full-thickness skin product, as employed herein refers skin tissue prepare in vitro comprising a dermis and epidermis, in particular prepared by a method of the present disclosure.

"ROCK inhibitor" as used herein refers to any compound or protein [such as an antibody or binding fragmentthereof] which has a function in reducing or blocking the activity of Rho-associated protein kinase (ROCK).

Examples of ROCK inhibitors include but are not limited to: SB 772077B, 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

The skilled person would be aware of other ROCK inhibitors.

"Trypsin" (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 used to digest both epidermis and dermis layers of skin samples. "Collagenase" as used herein 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-macroglobuhn, 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.

In one embodiment, Type 4 collagenase is employed in the methods of the present disclosure.

"Feeder cells" as used herein refers to a population of cells, typically connective tissue cells that are used to nourish cultured tissue cells, in particular 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 method of the present disclosure the feeder cells are generally not irradiated.

In one embodiment, the human fibroblast cells function as feeder cells to support the growth of the human keratinocytes. In one embodiment, the human fibroblast cells are not irradiated. This means that the human fibroblast cells are not inactivated and are able to continue growing in tandem with the human keratinocytes in the culture. This is possible because the presence of the ROCK inhibitor enables the keratinocytes to grow at a "faster rate" than the fibroblast cells and thus avoid being outgrown by the fibroblasts.

In one embodiment, the human fibroblast cells are matched to the human keratinocytes. For example, the fibroblasts may be sex matched and/or HLA matched to the keratinocytes.

In another embodiment, the human fibroblasts and human keratinocytes may both be derived from the same donor. Alternatively, the human keratinocytes and/or human fibroblast cells to be cultured are autologous, that is derived from the patient for whom the fully human epidermis is intended.

In one embodiment, the sowing of the skin cells on the matrix takes place in the presence of a physiological solution.

The term "physiological solution" as used herein refers to a solution that is similar or identical to one or more physiological conditions or that can change the physiological state of a certain physiological environment. The term "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).

In one embodiment, 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 ma be 1:1, 2:1, 3:1, 4:1, 5:1. In one embodiment, the ratio is 3:1.

Green's media as employed herein 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.

"Foetal bovine serum" or FBS is the most widely used animal serum supplement for the culture of eukaryotic cells. This is due to the very low level of antibodies and presence of growth factors which makes FBS suitable for many different cell culture applications. The presently claimed culture medium contains foetal bovine serum but the skilled person would be aware that other types of serum can also be used, for example bovine serum albumin (BSA), human platelet lysates and iron-supplemented bovine calf serum (ICS). 10% serum is typically used but other concentrations may also be used depending on the type of serum, 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%. The use of serum in the culture medium does not introduce non -human material into the final skin product.

"Penicillin" as used herein refers to a group of β-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.

In one embodiment the media does not comprise antibiotic capable of illicit an allergic reaction in a patient Thus, in some embodiments antibiotics such as penicillin and streptomycin are avoided and are replaced an antibiotic such as gentamicin.

In one embodiment, 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].

In one embodiment, 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.

In one embodiment, 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.

"Keratinocyte growth factor" or KGF, is a growth factor present in the epithelialization-phase of wound healing. KGF is encoded in humans by the FGF7 gene. KGF is a small signaling molecule that binds to fibroblast growth factor receptor 2b (FGFR2b). There are 23 known FGFs, and 4 FGF receptors. KGF is known to be a potent epithelial cell-specific growth factor, whose mitogenic activity is predominantly exhibited in keratinocytes but not in fibroblasts or endothelial cells.

In one embodiment, the culture medium is DMEM (Dulbecco's Modified Eagle Medium], M199, Ham's F12 Medium, or a combination thereof. However, any other cell culture medium which allows the cultivation of fibroblasts may also be used.

In one embodiment, Green's medium is employed.

In one embodiment, the cell culture medium consists of DMEM High glucose. Ham's F12, foetal bovine serum, penicillin, streptomycin, amphotericin B and keratinocyte growth factor (KGF].

In one embodiment, the ratio of DMEM High glucose:Ham's F12 is 3:1. In one embodiment, the foetal bovine serum has a concentration of 10%. In one embodiment, the amphotericin B has a concentration of 0.625μg/ml. In one embodiment, the KGF has a concentration of 20ng/ml. In one embodiment, the cell culture medium consists of: DMEM High glucose:Ham's F12 (3:1), 10% foetal bovine serum, penicillin, streptomycin, 0.625μg/ml amphotericin B and 20ng/ml keratinocyte growth factor (KGF]. The present inventors call this medium "Kelch's medium".

One of ordinary skill in the art will be aware of other defined media that may be used in accordance with the present invention. In one embodiment, a mixture of one or more defined media is employed.

In one embodiment, 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.

The first culture step in the present disclosure is to increase the number keratinocytes

(which requires the presence of fibroblast feeder cells). The first culture step may be performed in a tissue culture flasks.

Separately isolated fibroblasts are added to the in the second culture step where the keratinocytes and fibroblasts stratify and differentiate to form a dermis and an epidermis.

Antibody as employed herein refers to a full-length antibody, a binding fragment thereof, or an antibody molecule comprising any one of the same. Examples of 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.

Substrates/Matrices of the present disclosure

The term "matrix", "cell matrix", "cellular matrix", "substrate" or "cell substrate" as used interchangeably herein and refers to any physical structure including but not limited to, a solid or semi-solid structure, such as a meshwork of fibres with pores suitable for providing:

• mechanical or other support for the adherence and proliferation of cells or tissue, and

• allowing migration of the one cell types during the culturing process,

for example for ex vivo skin tissue culture.

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. Thus, 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 or CO2, in particular cell culture media). In one embodiment the distance of the relevant cells 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. In one embodiment the outer of cells for differentiation rests on the gas permeable layer/membrane. Generally, the matrix will be three dimensional, with a first 2D face and second 2D face (with a significant surface area) on which cells may deposited and a depth between the two faces giving the 3rd dimension (corresponding to a cross-section of the final skin -somewhere in the region of a 100 μπι as discussed above).

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.

In one embodiment, the matrices comprise only synthetic materials. In another embodiment the matrix comprises a mixture of synthetic and natural materials.

In one embodiment, 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.

As used here, "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 or injurious to that individual, nor does it induce immunological rejection of the material in the mammal, i.e the material is generally well tolerated in the body.

The term "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. In certain embodiments, the product is metabolized or excreted without permanent damage to the subject. An example of a biodegradable material is self-dissolving sutures.

In one embodiment, the matrix is completely resorbable by the body of a subject.

In one embodiment, a bioabsorbable matrix of the present disclosure may exist for days, weeks or months when placed in the context of a biological environment, for example in vivo. For example, 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, for example in vivo.

In one embodiment, the matrix layer is resorbed by the body of said subject at about a same rate as growth of tissue cells underlying said membrane matrix layer in said area. In certain embodiments, the cells are epithelial cells. In certain embodiments, 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. In one embodiment, the matrix incorporates one or more synthetic polymers in its construction. The matrix may be made from heteropolymers, monopolymers, or combinations thereof. Examples of 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 (1,3- dioxan-2-one), delta-valerolactone, beta-butyrolactone, epsilon-decalactone, 2,5-diketomorpholine pivalolactone, alpha, alpha-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl- l,4-dioxane-2,5-dione, 3,3-diethyl-l,4-dioxan-2,5-dione, gamma-butyrolactone, l,4-dioxepan-2- one, l,5-dioxepan-2-one, 6,6-dimethyl-dioxepan-2-one, 6,8-dioxabicycloctane-7-one and combinations thereof.

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 about95:5 to about 85:15) elastomeric copolymers of p-dioxanone (1,4- dioxan-2-one) and lactide including L-lactide, D-lactide and lactic acid (for example having a mole ratio of p-dioxanone to lactide of from about 40:60 to about 60:40) elastomeric copolymers of epsilon-caprolactone and p-dioxanone (for example having a mole ratio of epsilon-caprolactone to p-dioxanone of from about from 30:70 to about 70:30) elastomeric copolymers of p-dioxanone and trimethylene carbonate (preferably having a mole ratio of p-dioxanone to trimethylene carbonate of from about 30:70 to about 70:30), elastomeric copolymers of trimethylene carbonate and glycolide (for example having a mole ratio of trimethylene carbonate to glycolide of from about 30:70 to about 70:30), elastomeric copolymer of trimethylene carbonate and lactide including L- lactide, D-lactide, blends thereof or lactic acid copolymers (for example having a mole ratio of trimethylene carbonate to lactide of from about 30:70 to about 70:30) and blends thereof. Examples of suitable bioabsorbable elastomers are described in US4,045,418; US4,057,537 and US5,468,253 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, for example an inherent viscosity of from about 1.2 dL/g to about 2 dL/g and most preferably 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). Other materials suitable for use as a matrix of the present disclosure include, but are not limited to, polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof.

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], copolyfether-esters], polyalkylenes oxalates, polyamides, tyrosine derived polycarbonates, poly(iminocarbonates], polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amine groups, poly(anhydrides], polyphosphazenes, and blends thereof.

In one embodiment, 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 polyglycohc 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. By adjusting the ratio of the polymer blend the final rate of degradation of the matrix can be controlled.

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 polyglycohc acid alone, as well as combinations of PGA and other natural and/or synthetic biocompatible materials, are within the scope of the present disclosure.

In one embodiment, the matrix comprises poly(lactic-co-glycolic acid] (PLGA], such as PLGA microfiber or nanofibres.

In another embodiment, the matrix comprises dioxanone linear homopolymer, such as 100 dioxanone linear homopolymer (e.g. Dioxaprene 100M].

In one embodiment, 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 μιη to 5 μιη, for example 1 μιη, 1.5 μιη, 2 μπι, 2.5 μηι, 3 μιη, 3.5 μιη, 4 μπι, 4.5 μπι or 5 μιη, in particular in the range 1 to 3 μιη or 2 to 5 μπι.

The term "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]. 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.

In one embodiment, a matrix of the present disclosure is formed using electrospinning. The term "electrosplnning" 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). To perform the electrosplnning process using these materials, an electrosplnning liquid (i.e. a melt or solution of the desired materials that will be used to form the fibers) is generally first loaded into a syringe and is then fed at a specific rate set by a syringe pump.

As the liquid is fed by the syringe pump with a sufficiently high voltage, 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. Further, as the jet is stretched and the solvent is evaporated, 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. In the context of the present disclosure, due to the high ratio of surface area to volume and the one-dimensional morphology, 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).

PLCL DCM PLLA-DLA Chloroform

PEUU HFIP Cellulose acetate Acetic acid/water

PEG-b-PLA Chloroform PVA Water

Collagen/chondroitin 70% propan-2-

TFE/water EVOH

sulfate ol/water

PEO Water PVP Ethanol/water

Blended

PLA/PCL Chloroform Gelatin/PVA Formic acid

PCL/collagen HFIP Sodium aliginate/PEO Water

Chitosan/PEO Acetic acid/DMSO Chitosan/PVA Acetic acid

Gelatin/elastin/PLGA HFIP Silk/PEO Water

Silk fibroin/chitosan Formic acid PDO/elastin HFIP

PHBV/collagen HFIP Hyaluronic acid/gelatin DMF/water

Collagen/chitosan HFIP/TFA

Composites

PDLA/HA Chloroform PCL/CaC03 Chloroform /methanol

PCL/CaC03 DCM/DMF PCL/HA DCM/DMF

PLLA/HA Chloroform Gelatin/HA HFIP

PCL/collagen/HA HFIP Collagen/HA HFIP

Acetic acid/ethyl

Gelatin/siloxane PLLA/MWNTs/HA 1,4-dioxane/DCM

acetate/water

PLGA/HA DCM/water

In one embodiment, 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, for example made by electrospinning using the materials listed in Tables 1 and 2, may be used to form the fibres to improve their biocompatibility as well as their mechanical, physical, and chemical properties.

Once the desired microfiber or nanofiber matrices have been produced, in one embodiment two or more fibre matrices of the present disclosure are layered together. By layering multiple fibre matrices, advantages of each fibre matrix can be combined.. For example, 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. In this example, 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 is able to employ various combinations of two or more different matrices in order to achieve desired properties.

In one embodiment, the matrix of the present disclosure may be treated via a single procedure or a combination of procedures to reduce the number of microorganisms capable of growing in the matrix, for example under conditions at which the matrix is stored and/or distributed. The matrix may be sterilised, for example by irradiated and then stored under sterile or aseptic conditions.

In one embodiment, the matrix is sterilised using gamma radiation. In another embodiment, the matrix is sterilised using ethylene oxide (EtO). In another embodiment, the matrix is sterilised using Revox which utilises percetic acid.

In one embodiment, 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 product that is packaged in its final shipping container. As the beam is scanned through the product, 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. The skilled addressee is aware of other possible methods for sterilising the matrices of the present disclosure.

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 product.

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.

In one embodiment, 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 .

In one embodiment, 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. Particular seeding densities a specific application may require, individual layers of the matrix to be seeded at different 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.

Spreading involves the use of an instrument such as a spatula to spread the inoculum across the spongiform matrix. 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. First, it must not subject the cells in solution to shearing forces or pressures that would damage or kill substantial numbers of cells. Second, it should not require that the cellular suspension be mixed with a propellant fluid that is toxic or detrimental to cells or wound beds. A variety of 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.

According to another embodiment of the invention, 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.

Typically, the dimensions of the matrix should be substantially planar and of a thickness that gives seeded cells sufficient access to a nutrient medium. 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. Accordingly, in one embodiment, the matrix has a thickness of 100 μπι or less, for example 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 μιη. By keeping the matrix 100 μιη 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. Alternatively, the matrices may be pre-joined, and the selected population(s) of cells introduced at a selected location. Seeding is distinct from the spontaneous infiltration and migration of cells into the matrix from the culture or a wound site when the matrix is placed at the wound site.

In one embodiment the matrices are seeded on at least one surface before the respective cell- seeded surfaces are collocated (for example opposed to each other) to form a layered arrangement.

Various 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.

In one embodiment, cell factors are coated/attached to the matrix of the present disclosure. As used herein, the term "cell factors" refers to substances that are synthesized by living cells (for example stem cells) and which produce a beneficial effect in the body (for example, mammalian or human body). Cell factors include, but are in no way limited to, growth factors, regulatory factors (such as cytokines or chemokines), 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.

Some non-limiting examples of 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 (for example. 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-β, bone morphogenetic proteins (BMPs), Hedgehog molecules, Wnt-related molecules, and combinations thereof.

In one embodiment, a growth factor such as EGF (Epidermal Growth Factor), IGF-I (Insulin- like Growth Factor), a member of Fibroblast Growth Factor family (FGF), Keratinocyte Growth Factor (KGF), PDGF (Platelet-derived Growth Factor AA, AB, BB), TGF-β (Transforming Growth Factor family - βΐ, β2, β3), 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.

In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PDGF. The skilled addressee would be 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.

In one embodiment, an extracellular matrix protein, such as, fibronectin, laminin, and/or collagen, is further attached to or coated on the matrix. Thus, in one embodiment, 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 at an air liquid interface, causes proper stratification of skin cells grown using the matrix.

In one embodiment, 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.

In another embodiment, a therapeutic agent is further attached to the matrix. The term "therapeutic agent" as used herein 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. Examples of therapeutic agents that may be used include, without limitation, proteins, peptides, drugs, cytokines, extracellular matrix molecules, and/or growth factors. One of skill in the art will be aware of other suitable and/or advantageous therapeutic agents that may be used in accordance with the present disclosure.

In one embodiment, the therapeutic agent is an an ti -inflammatory agent or an antibiotic.

Examples of 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.

Various antibiotics can also be employed in accordance with the presently-disclosed subject matter. 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 telithromycin; cephalosporins, such as cefadroxil, cefazolin, cephalexin, cephalothin, cephapirin, cephradine, cefaclor, cefamandole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, loracarbef, cefdinir, cefditoren, cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, or cefepime; monobactams, such as aztreonam; nitroimidazoles, such as metronidazole; oxazolidinones, such as linezolid; penicillins, such as amoxicillin, amoxicillin/clavulanate, ampicillin, ampicillin/sulbactam, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, piperacillin/tazobactam, ticarcillin, or ticarcillin/clavulanate; streptogramins, such as quinupristin/dalfopristin; sulfonamide/folate antagonists, such as sulfamethoxazole/trimethoprim; tetracyclines, such as demeclocycline, doxycycline, minocycline, or tetracycline; azole antifungals, such as clotrimazole, fluconazole, itraconazole, ketoconazole, miconazole, or voriconazole; polyene antifungals, such as amphotericin B or nystatin; echinocandin antifungals, such as caspofungin or micafungin, or other antifungals, such as ciclopirox, flucytosine, griseofulvin, and/or terbinafine. In one embodiment, various analgesic and/or anesthetic are attached to or incorporated into the matrices of the presently disclosure. As used herein, the term "analgesic" refers to agents used to relieve pain and, in some embodiments, can be used interchangeably with the term "antiinflammatory agent" such that the term analgesics can be inclusive of the exemplary anti- inflammatory 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.

As used herein, the term "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.

Culture devices of the present disclosure

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.

In one embodiment, the present disclosure provides a matrix, skin tissue and method useful in the regeneration of damaged, lost and/or degenerated tissue. For example, 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", "Regenerate", "Regenerative" as used herein refer 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.

As used herein, the term "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, burns and skin removed to excise cancerous lesions or infected/necrotic tissue, and ulceration (such as chronic ulceration)) 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).

As used herein, the term "partial thickness wound" refers to wounds that encompass Grades I-III; e.g., burn wounds, pressure sores, venous stasis ulcers, and diabetic ulcers. As used herein, the term "deep wound" is used to describe to both Grade III and Grade IV wounds. In one embodiment, there is provided is 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.

As used herein, a "graft" refers to a cell, tissue or organ that is implanted into an individual, typically to replace, correct or otherwise overcome a defect Thus, a "skin graft" is 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. Autologous and allogenic is only an issue at the point of treating the patient, for the purpose of culturing the cells the methods are usually the same.

In one embodiment the tissue is prepared using cells that are autologous to the subject. For example, in various embodiments the tissue is prepared using fibroblasts, keratinocytes, or fibroblasts and keratinocytes, for example that are autologous to the subject. In an alternative embodiment the tissue is prepared using cells that are heterologous to the subject. In a further embodiment 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.

It will be appreciated that cells including autologous to the subject and heterologous cells may be isolated using any method known in the art. For example, 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.

In one embodiment the tissue is an autograft, for example, a skin autograft. In various embodiments the tissue is an epidermal autograft, a split thickness skin autograft or a full thickness skin autograft. In another embodiment the tissue is an allogeneic graft.

It will be appreciated that the application of tissue prepared using cells autologous to the patient, such as an autograft, is highly desirable to reduce or prevent immune rejection of the tissue and to reduce the requirement for ongoing immunotherapy or another ancillary treatments.

In one embodiment the tissue further comprises the matrix. In another embodiment the tissue is separated from the matrix before application to the patient.

Generally, the application of tissue to the patient will be by surgery. In one embodiment, recovery under sterile conditions is during or immediately prior to surgery, for example in the surgical suite.

Generally, the application of tissue to the patient will be at, on or adjacent to the site of tissue damage. In various embodiments the tissues is applied to at least partially cover the site of tissue damage or to completely cover the site of tissue damage.

In one embodiment the 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. In one embodiment the graft is secured, for example sutured in place.

In one embodiment the graft is covered by a dressing, for example to keep it clean and moist after it is secured on the wound.

Thus, 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.

Other non-medical uses

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.

Morphological differences between animal and human skin means thatthe excised animal skin or live animals for the testing of products is not optimal. Furthermore, there is considerable ethical concern about the use of live animals or animal skins for testing cosmetic products, including bans on such testing in some countries. For these reasons, there is a strong desire to identify alternatives to animal models for the testing of such products.

Inconsistent and highly variable results have been observed when human cadaver skin is used for product testing.

Accordingly, 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.

In various embodiments 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.

More particularly, 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. These 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 or the metabolism or gene expression.

In various embodiments 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. Using processes for quantifying cell damage, more particularly using a vital dye, such as a tetrazolium derivative, it is possible, for example, to detect cytotoxic effects on skin cells. The testing of substances or agents using the skin tissue may comprise both histological processes and also immunological and/or molecular-biological processes.

A "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.

In another embodiment, 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. For example, 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.

In one embodiment, 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. In addition, 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.

In various embodiments 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.

In various embodiments the compound is a pharmaceutical compound, a cosmetic compound or a nutraceutical compound.

In various embodiments the compound for testing is applied to tissue alone or in an admixture with pharmaceutically or cosmetically acceptable carriers, excipients or diluents.

In various embodiments 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. The term "gas permeable material" or "gas permeable membrane" as used herein means a material or membrane through which gas exchange may occur.

"Comprising" in the context of the present specification is intended to mean "including". Where technically appropriate, embodiments of the invention may be combined.

Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.

Technical references such as patents and applications are incorporated herein by reference. Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

The background of the present specification contains technical information relevant to the disclosure herein and may be used as the basis for an amendment.

The present application claims priority from GB1715928.6 filed 30 September 2017. This priority document is incorporated by reference and may be used as the basis for corrections.

The invention will now be described with reference to the following examples, which are merely illustrative and should not in any way be construed as limiting the scope of the present invention.

BRIEF SUMMARY OF THE FIGURES

Figure 1 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 2 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.

Figure 3 shows a comparison of keratinocyte growth rates with or without ROCK inhibitor in a range of different cell culture media. Epidermal cells were grown in Greens medium with MEFs, CnT Prime, Epilife medium supplemented with S7 or EDGS. All media were tested with and without ROCK inhibitor Y27632. 550,000 epidermal cells were grown in each medium until one sample reached 80% confluence, at this point all samples were fixed and stained for rhodamine B. Sample confluency was then assigned a score between 0 and 4, 0 having no cells, and 4 having maximum confluency. N=2.

Figure 4 shows that ROCK inhibitors Y27632 and SB772077B have comparable effects on keratinocyte growth. Frozen human keratinocytes were grown in Base medium, Base medium plus KGF ("Kelch's medium" in the figure legend), or Green's medium, supplemented with either Y27632 or SB772077B. For evaluation of keratinocyte growth cells were fixed, stained with rhodamine B, and confluency was measured using the Operetta plate reader. Results are shown for 3 donors in triplicate (+SD). N = 3. These results show that when supplemented with a ROCK inhibitor, Kelch's medium (Base medium plus KGF)] drives superior keratinocyte proliferation to either Green's medium or Base medium plus ROCK inhibitor without KGF, demonstrating that KGF is a crucial component of Kelch's medium.

Figure 5 shows that Kelch's medium plus ROCK inhibitor SB772077B) provides equivalent keratinocyte growth to Green's medium plus a ROCK inhibitor,. Trypsin and collagenase digested skin cells were grown in Kelch's medium v Greens medium, both containing ROCK inhibitor SB772077B. Triplicate samples were grown for up to 30 days, and were passaged 1:9 when they reached 80-90% confluency. N=l.

Figure 6 shows the effect of serum-free Optipeak medium (InVitria) on Keratinocyte growth.

Human primary keratinocytes from cryopreserved epidermal preparations were grown in 4 different formulations of serum-free Optipeak media or Green's medium with mouse embryonic feeder cells (MEFs) for 8 days. All media were tested with and without ROCK inhibitor Y27632. For evaluation of keratinocyte cell growth cells were fixed, stained with rhodamine B, and confluency was measured using the Operetta plate reader. Results are shown as triplicates of one representative donor (+SD). N = 2. These results show that while ROCK inhibitor enhanced growth of keratinocytes in serum-free Optipeak medium, the resulting proliferation was still inferior to that resulting from culture in Green's medium in the presence of MEFs. These results also show that ROCK inhibitor can enhance growth of keratinocytes in Green's medium in the presence of MEFs.

CnT-Prime is a fully defined, animal-component-free culture medium available from CellnTec. It is suitable for isolation and expansion of epithelial cells from skin, cornea, gingiva, mammary and bladder tissue. It was developed using human tissue, but may also be used with other species (e.g. mouse).

Supplement S7 is available from ThermoFisher. It is a sterile, concentrated (100X), ionically balanced solution intended for use as one component in a complete culture environment for human epidermal keratinocytes. Supplement S7 is chemically defined and animal origin-free. Each 5 ml bottle of Supplement S7 is the correct amount of supplement for a 500 ml bottle of EpiLife® basal medium.

EDGS is also available from ThermoFisher. It is a defined, sterile, concentrated (100X) solution intended for use with EpiLife® medium to culture human epidermal keratinocytes. EDGS is not intended for use with Medium 154. EDGS contains all of the growth factors and hormones necessary for the growth of human epidermal keratinocytes. Each bottle contains the correct amount of supplement for a 500 ml bottle of EpiLife® medium. EDGS contains: · purified bovine serum albumin; · purified bovine transferrin; · hydrocortisone; · recombinant human insulin-like growth factor type-1 (rhIGF-1); · prostaglandin E2 (PGE2); · recombinant human epidermal growth factor (rhEGF). The bovine products in EDGS have been isolated from animals of North American origin. The isolation process for these components includes steps that inactivate viruses. All components of EDGS are greater than 95% pure.

EXAMPLES

Example 1 - Materials and Methods

Materials

DO - DMEM with glutamax, penicillin and streptomycin. DF10 - DMEM with 10% foetal bovine serum, glutamax, penicillin and streptomycin. Greens medium - DMEM High glucose:Hams F12 3:1, 10% foetal bovine serum, lOng/ml EGF, O.lnM choleratoxin, O^g/ml hydrocortisone, 180μΜ adenine, 5μg/ml insulin, 5μg/ml apotransferrin, 2nM 3,3,5,-tri-idothyronine, 2mM glutamine, gentamicin, 0.625μg/ml Amphotercin B. ROCK inhibitors - added to Greens medium for keratinocyte growth - Y27632 lOum, SB 772077B 400Nm. Collagen IV - Sigma catalogue # C5533. PBS - Life Technologies catalogue # 10010023. Gas permeable interface (GPI] device - 100cm 2 and 16cm 2 sizes

Preparation of skin samples

All subcutaneous fat and hypodermis was trimmed away. Next, the skin was cut into 1cm 2 pieces and weighed. Finally, the underside of the dermis was heavily scored using a scalpel.

Dispase-CoIIa enase sequential digest method ftraditional method to separate and purify epidermal cells from human skin separately from dermal cells)

The pieces of skin were placed in the wells of a 6 well plate. Next, lOmg/ml dispase with MilliQ water was prepared and the enzyme stock was filtered through a 0.2 μιη syringe filter.

600μ1 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 next day, the epidermis was separated from the dermis using a scalpel. The epidermis was placed in Greens medium and a scalpel was used to cut the epidermis into small pieces. Next, 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 ΙΟΟμιη 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 Greens medium to assess cell number and viability.

Trypsin, and trypsin and collagenase. digest methods (to generate mixed populations of epidermal and dermal cells from human skin)

The pieces of skin were placed in the wells of a 6 well plate. Next, 5mg/ml collagenase [type I] was prepared with MilliQ water and the enzyme stock was filtered through a 0.2um syringe filter in hood. 0.25% trypsin was diluted to 0.1% in DO and for trypsin and collagenase digests, 600μ1 of collagenase was added making a total volume of 6ml. 6ml of trypsin or trypsin and collagenase was added to each well, and the plate was placed in an incubator at 37°C with 5% C0 2 overnight. The next day, the skin and enzyme containing medium were transferred to 6ml of DF10 in a 10cm dish and teased with a scalpel. The skin was next broken up further by passing through a pipette repeatedly. Next, the cell mix was passed through a ΙΟΟμιη strainer.

Next, the mix was centrifuged at 1800rpm for lOmin and as much supernatant as possible was removed without disturbing cell pellet. Finally, the cell pellets were resuspended in DF10 to assess cell number and viability.

Dispase digested sample keratinocvte culture and passage

All the cells from the dispase digested 1cm 2 piece of epidermis were seeded into a T25 flask with Greens medium containing ROCK inhibitor Y27632 at ΙΟμΜ and 300,000 irradiated mouse feeder cells. The medium was changed after 24 hours. Subsequently the medium was changed every 2-3 days.

Once the culture in the flask reached 80-90% confluence, the cells were passaged into a new flask. First, all the medium was removed and the cells were washed with 1ml DPBS. Next 2ml of TrypLE™ (Thermo Fisher Scientific] was added and incubated at 37°C until all the cells detached when the flask was struck. 4ml of DF10 was added to neutralize TrypLE. The liquid was aspirated and dispensed 2-3 times to break up cell clumps and detach any remaining cells attached to the flask. Next the cells were centrifuged at 1300rpm for 5 min. All the medium was removed and the cell pellet was resuspended in 1ml of keratinocyte medium with ROCK inhibitor added. One fifth of the cells were re-seeded into a fresh flask along with 300,000 irradiated mouse feeder cells.

The above passaging process was repeated when the culture reached 80-90% confluency.

Trypsin collagenase digested sample keratinocvte culture and passage method

All the cells from trypsin collagenase digested 1cm 2 piece of skin were seeded into a T25 flask with Greens medium containing ROCK inhibitor Y27632 at 10μΜ or SB772077B at 400nM as appropriate. The medium was changed after 24 hours. Subsequently the medium was changed every 2-3 days. Once the culture in the flask reached 80-90% confluence, the cells were passaged into a new flask. First, all the medium was removed and the cells were washed with 1ml DPBS. Next 2ml of TrypLE™ (Thermo Fisher Scientific] was added and incubated at 37°C until all the cells detached when the flask was struck. 4ml of DF10 was added to neutralize TrypLE. The liquid was aspirated and dispensed 2-3 times to break up cell clumps and detach any remaining cells attached to the flask. Next the cells were centrifuged at 1300rpm for 5 min. All the medium was removed and the cell pellet was resuspended in 1ml of keratinocyte medium with ROCK inhibitor added. One fifth of the cells was re-seeded into a fresh flask

The above passaging process was repeated when the culture reached 80-90% confluency.

Skin graft culture method

Scaffold coating -

Dilute lmg/ml collagen IV stock in PBS to 50ug/ml. Coat PLGA with collagen IV solution, use 200ul of50μg/ml solution per cm 2 . Incubate at 37°C for 2 hours. Wash three times with PBS. Place scaffold into GPI devices and fill with Kelch's medium.

Skin graft growth - Seed 15-30 million keratinocytes into the 100cm 2 GPI device. Seed 5-10 million fibroblasts into the 100cm 2 GPI device. Seed 2.4-4.8 million keratinocytes into the 16cm 2 GPI device. Seed 0.8-1.6 million fibroblasts into the 16cm 2 GPI device.

Incubate at 37°C/5% C0 2 for 48 hours. Invert GPI devices such that the surface of the synthetic scaffold seeded with fibroblasts and keratinocytes is now in direct contact with a gas permeable membrane. Incubate at 37°C/5% CO2 for 7-14 days.

Results

Figure 1 shows the results of keratinocytes grown from full thickness human skin samples digested with trypsin only, or trypsin and collagenase, or from epidermis separated using dispase. These results show that in the presence of ROCK inhibitor it is possible to grow keratinocytes in the absence of mouse feeder cells when human dermal fibroblasts are present. Crucially, the presence of the ROCK inhibitor enables the human keratinocytes to outgrow the human fibroblasts, obviating the need for separation of dermal cells from keratinocytes, and the traditional need to add xenogeneic fibroblastic cells to enable keratinocyte growth.

Figure 2 shows the results of keratinocytes from trypsin collagenase digested full thickness human skin samples grown in keratinocyte growth medium containing ROCK inhibitor Y27632 or SB 772077B. Keratinocytes from dispase digested epidermis samples grown in keratinocyte growth medium with ROCK inhibitor Y27632 and irradiated xenogeneic mouse feeder cells is shown as a comparison of a culture that requires irradiated xenogeneic mouse feeder cells, where the trypsin collagenase digested samples did not require irradiated xenogeneic mouse feeder cells for keratinocyte growth. Keratinocyte growth was equivalent whether ROCK inhibitor Y27632 or SB772077B was used.

Figure 3 shows the effects on keratinocyte growth when different cell culture media are used with or without ROCK inhibitor. S7 and EDGS are used to supplement EpiLife® medium, from the Gibco range. CnT Prime was obtained from Cellntech. All commercial media were designed to be serum and feeder free. Green's medium uses both serum and mouse embryonic feeder cells. All media were used with and without ROCK inhibitor Y27632. Green's medium supplemented with ROCK inhibitor and mouse embryonic feeder cells provided the fastest keratinocyte growth. ROCK inhibitor enhanced keratinocyte growth in Green's medium, S7 medium and CnT Prime medium. Hence, the results provide strong evidence of the ability of ROCK inhibitors to enhance keratinocyte growth rates when included in cell cultures and that this effect is applicable across a range of different cell culture media.