Cell culture medium

Abstract

The present invention relates to a growth medium for culturing cells (e.g., epithelial cells, in particular, keratinocytes) that does not contain cholera toxin, the use of the medium for growing cells (e.g., epithelial cells, such as keratinocytes) and a kit comprising the medium. A method for generating full-thickness human skin, full human epidermis or full human dermis is also provided, the method involving culturing keratinocytes and feeder cells in the cell culture medium.

Description

[0001] The present invention relates to a growth medium for culturing cells, such as epithelial cells, in particular keratinocytes, the use of the medium for growing cells, such as epithelial cells, such as keratinocytes, and a kit comprising the medium. Background of the Invention

[0003] Engineered tissues containing epidermis (e.g., grown in vitro) have a wide range of uses. Such tissues are used in skin grafts for patients with burns or chronic wounds, or in the development and testing of pharmaceuticals, cosmetics, and other topical products. Differentiation of keratinocytes to form the epidermis requires co-culture with fibroblast feeder cells.

[0004] In the production of skin grafts, keratinocytes are often grown with Green's medium and irradiated xenogeneic mouse embryonic feeder cells (MEFs). Non-irradiated mouse feeder cells outgrow the keratinocytes and overwhelm the culture. Therefore, feeder cells are irradiated to stop or slow their replication.

[0005] Green medium is a complex mixture of the following components:

[0006] Dulbecco's Modified Eagle Medium (DMEM) contains calcium chloride, ferric nitrate, magnesium sulfate, potassium chloride, sodium bicarbonate, sodium chloride, sodium dihydrogen phosphate (anhydrous), L-arginine·HCl, L-cysteine·HCl, glycine, L-histidine·HCl·H 2 O, L-isoleucine, L-leucine, L-lysine·HCl, L-methionine, L-tryptophan, L-tyrosine·2Na·H 2 O, L-valine, choline chloride, folic acid, inositol, niacinamide, D-pantothenic acid, pyridoxal·HCl, pyridoxine, riboflavin, thiamine·HCl, D-glucose, phenol red·sodium, pyruvic acid;

[0007] Ham's F12 medium contains calcium chloride, copper sulfate·5H 2 O, ferrous sulfate·7H 2 O, magnesium chloride, potassium chloride, sodium bicarbonate, sodium chloride, disodium hydrogen phosphate (anhydrous), zinc sulfate·7H 2 O, L-alanine, L-arginine·HCl, L-asparagine·H 2 O, L-aspartic acid, L-cysteine·HCl·H 2O, L-isoleucine, L-leucine, L-lysine·HCl, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tryptophan, L-tyrosine·2Na·H 2 O, L-valine, D-biotin, choline chloride, folic acid, inositol, niacinamide, D-pantothenic acid, pyridoxine·HCl, riboflavin, thiamine·HCl, vitamin B 12 , D-glucose, hypoxanthine, linoleic acid, phenol red·Na, lipoic acid and thymidine; fetal bovine serum; cholera toxin; recombinant human insulin; hydrocortisone; L'glutamine, EGF, apo-transferrin, adenine and 3,3,5,-tri-iodinated thyronine and two or three antibiotics.

[0008] Regulatory approval of GMP manufacturing processes typically requires some validation and scientific support for the use of supplemental components (e.g., media) unless the material is classified as a “grass agent” (agents generally regarded as safe).

[0009] Green medium has never been optimized for the growth of epithelial cells such as keratinocytes. The inventors have studied the components of Green medium to determine which components have the least effect (and are therefore redundant) or may even have an adverse effect on the growth of keratinocytes. However, due to the complexity of the medium, it is difficult to determine which components are essential and which are unnecessary.

[0010] Okada et al. J. Inves. Dermatol. 1982 Jul 79(1) 42-7 disclose that cholera toxin accelerates the growth of keratinocytes. Obviously, the total processing time to produce a skin product is an important factor in processing efficiency and viability, so to date, cholera toxin has been considered an important aspect of the culture medium.

[0011] However, the presence of cholera toxin in the culture medium is a potential problem because it is a potent immunomodulator and has mucosal and systemic adjuvant activity. Adjuvant activity is desirable in vaccines because it increases the immune response to the antigens in the vaccine. However, increased immune responses are undesirable in skin grafts because these responses increase the risk of graft rejection.

[0012] Additionally, in some cases, cholera toxin is grown using bovine brain tissue, which carries the risk of contamination with prion proteins.

[0013] The use of xenogeneic feeder cells means that the skin samples grown in vitro are not fully human. Residual non-human components / proteins in the engineered tissue can cause intolerance to the transplant and generate immune responses leading to transplant rejection. Exposure of therapeutic products to xenogeneic cells can also lead to the transmission of infectious agents from non-human hosts.

[0014] Currently, no one in the art has produced a whole human skin product, for example comprising whole human epidermis, and particularly a fully differentiated full-thickness whole human skin product, particularly without the use of cholera toxin in the culture medium.

[0015] Having a cell culture medium that allows epithelial cells (e.g., keratinocytes) to be cultured with non-irradiated human fibroblasts is useful, for example, in the preparation of engineered skin products comprising a full human epidermis (e.g., full-thickness whole human skin). Therefore, there is a need for an improved culture medium for growing keratinocytes, particularly a simplified culture medium that is robust and reproducible and suitable for GMP manufacturing. SUMMARY OF THE INVENTION

[0017] The invention is summarized in the following paragraphs:

[0018] 1. A cell culture medium comprising or consisting of the following components: DMEM, Ham's F12, serum, one or more antibiotics and / or antifungal agents, keratinocyte growth factor (KGF) and / or epidermal growth factor, and optionally a keratinocyte growth promoter factor, wherein the factor is not cholera toxin.

[0019] 2. The cell culture medium according to paragraph 1, wherein the DMEM is high glucose.

[0020] 3. The cell culture medium according to paragraph 1 or 2, wherein the serum is mammalian fetal bovine serum.

[0021] 4. The cell culture medium according to any of paragraphs 1 to 3, wherein the serum is present in a concentration of 5 to 15%, such as 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15%, in particular 10%.

[0022] 5. The cell culture medium according to any of paragraphs 1 to 4, wherein at least one antibiotic (eg, 1, 2 or 3 antibiotics) is a component.

[0023] 6. The cell culture medium according to paragraph 5, wherein the antibiotic is independently selected from penicillin, streptomycin, ampicillin, amoxicillin, carbenicillin, cefotaxime, gentamicin, kanamycin, neomycin, polymyxin, blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, zeocin, and a combination of two or more thereof.

[0024] 7. The cell culture medium according to paragraph 5 or 6, wherein the antibiotic is gentamicin.

[0025] 8. The cell culture medium according to any of paragraphs 5 to 7, wherein the antibiotic is penicillin.

[0026] 9. The cell culture medium according to any of paragraphs 5 to 8, wherein the antibiotic is streptomycin.

[0027] 10. The cell culture medium according to any of paragraphs 1 to 9, wherein an antifungal agent is among the components.

[0028] 11. The cell culture medium according to paragraph 10, wherein the antifungal agent is selected from amphotericin B, nystatin, actinomycin D, fosmidomycin, mycophenolic acid, and a combination of two or more thereof.

[0029] 12. The cell culture medium according to paragraph 11, wherein the antifungal agent is amphotericin B, for example at a concentration of 0.500 to 0.750 μg / ml, for example 0.625 μg / ml.

[0030] 13. The cell culture medium according to any of paragraphs 1 to 12, wherein the ratio of DMEM:Ham's F12 is 2.5 to 3.5:1.5 to 0.5, such as 3:1.

[0031] 14. The cell culture medium according to any of paragraphs 1 to 13, wherein the concentration of KGF is in the range of 10 ng / ml to 30 ng / ml, such as 20 ng / ml.

[0032] 15. The cell culture medium according to any of paragraphs 1 to 14, wherein the keratinocyte growth promoting factor is a ROCK inhibitor, such as a small molecule ROCK inhibitor.

[0033] 16. The cell culture medium according to paragraph 15, wherein the ROCK inhibitor is selected from the group consisting of SB772077B, Y-27632, fasudil, Ripasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminofurazan, DE-104, H-1152, ROKα inhibitor, XD-4000, HMN-1152, 4-(1-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, isocyanate H-7, H-89, HA-1004, HA-1077, H-8, H-9, KN-62, GSK269962, quinazoline, and combinations of two or more thereof.

[0034] 17. The cell culture medium according to paragraph 16, wherein the ROCK inhibitor is SB772077B, Y-27632 or a combination of both, in particular SB772077B.

[0035] 18. The cell culture medium according to any of paragraphs 15 to 17, wherein the concentration of the ROCK inhibitor is in the range of 0.1 to 100 μM, for example, 0.2 to 50 μM or 0.3 to 25 μM or 0.1 to 10 μM or 0.1 to 0.95 μM, for example 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 or 0.95 μM, in particular 0.4 μM.

[0036] 19. An in vitro method for producing human full-thickness skin, full human epidermis or full human dermis (i.e. comprising full human epidermis), comprising a first culturing step, wherein human keratinocytes and feeder cells are cultured in a cell culture medium according to any of paragraphs 1 to 18.

[0037] 20. The in vitro method according to paragraph 19, wherein the feeder cells are fibroblasts.

[0038] 21. An in vitro method according to paragraph 20, wherein the fibroblasts are entirely human.

[0039] 22. The in vitro method according to any of paragraphs 19 to 21, wherein the human keratinocytes are cultured in the absence of xenogeneic feeder cells.

[0040] 23. The in vitro method according to any of paragraphs 19 to 22, wherein the human fibroblasts are not irradiated.

[0041] 24. The in vitro method according to any of paragraphs 19 to 23, wherein the human fibroblasts are sex matched to the keratinocytes (and / or the patient).

[0042] 25. The in vitro method according to any of paragraphs 19 to 24, wherein the human fibroblasts are HLA matched to the keratinocytes (and / or the patient).

[0043] 26. The in vitro method according to any one of paragraphs 19 to 25, wherein the human fibroblasts and keratinocytes are from the same donor.

[0044] 27. The in vitro method according to any of paragraphs 19 to 26, wherein the human fibroblasts are allogeneic to the human patient.

[0045] 28. The in vitro method according to any one of paragraphs 19 to 27, wherein the human keratinocytes are allogeneic to the human patient.

[0046] 29. The in vitro method according to any one of paragraphs 19 to 23 or 25, wherein the human fibroblasts are autologous to the human patient.

[0047] 30. The in vitro method according to any one of paragraphs 13 to 27 or 29, wherein the human keratinocytes are autologous to the human patient.

[0048] 31. An in vitro method according to any of paragraphs 19 to 30, wherein the method comprises a second culturing step having a first culturing phase, wherein the keratinocytes are not in contact with the gas permeable membrane (interface) and are not at the liquid interface.

[0049] 32. An in vitro method according to paragraph 31, wherein the keratinocytes in the second step are cultured for a second period in contact with a gas-permeable membrane (interface), for example, wherein the keratinocytes are in contact with a gas-permeable membrane (subsequently cultured without contact with the gas-permeable layer).

[0050] 33. The in vitro method according to any of paragraphs 19 to 32, wherein the keratinocytes are deposited on a substrate, such as a matrix, such as a rotating matrix or a mesh.

[0051] 34. An in vitro method according to paragraph 33, wherein the substrate (eg, matrix) comprises a biocompatible, biodegradable polymer.

[0052] 35. An in vitro method according to claim 33 or 34, wherein the substrate is prepared by electrospinning.

[0053] 36. The in vitro method according to paragraph 30, wherein the electrospun fibers resulting from said electrospinning have a diameter of about 0.3 μm to about 5 μm or a diameter of 2 to 5 μm, such as 1, 1.5, 2, 2.5, 3, 3.5, 4 or 4.5 μm.

[0054] 37. The in vitro method according to paragraph 35 or 36, wherein the substrate is prepared from fibers spun from a polymer 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, mixed PLA / PCL, gelatin-PVA, PCT / collagen, sodium alginate / PEO, chitosan / PEO, chitosan / PEO, Polysaccharide / PVA, gelatin / elastin / PLGA, silk / PEO, fibroin / chitosan, PDO / elastin, PHBV / collagen, hyaluronic acid / gelatin, collagen / chondroitin sulfate, collagen / chitosan, PDLA / HA, PLLA / HA, gelatin / HA, gelatin / silicone, PLLA / MWNTs / HA, PLGA / HA, dioxanone linear homopolymers (such as 100 dioxanone linear homopolymers) and combinations of two or more thereof.

[0055] 38. The in vitro method according to paragraph 37, wherein the polymer is poly(lactic-co-glycolic acid) (PLGA).

[0056] 39. The in vitro method according to paragraphs 33 to 38, wherein the concentration of the biocompatible, biodegradable polymer is 10 to 40% w / v, such as 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.

[0057] 40. The in vitro method according to any of paragraphs 33 to 39, wherein the thickness of the substrate is 10 to 100 μm.

[0058] 41. The in vitro method according to any of paragraphs 33 to 40, wherein the substrate is three-dimensional and substantially planar, having two faces.

[0059] 42. The in vitro method according to any of paragraphs 41, wherein at least one face of the substrate is micropatterned, undulated and / or dimpled.

[0060] 43. The in vitro method according to any of paragraphs 33 to 42, wherein the substrate is coated with a protein, polypeptide or peptide to assist in the adhesion of keratinocytes to the substrate and / or to allow the cells to stratify.

[0061] 44. An in vitro method according to paragraph 43, wherein the protein is selected from extracellular matrix proteins (such as collagen, laminin and other extracellular matrix proteins) or peptides thereof, such as synthetic peptides.

[0062] 45. The in vitro method according to paragraph 44, wherein the extracellular matrix protein is selected from collagen IV, collagen I, laminin and fibronectin or a combination thereof, such as collagen IV.

[0063] 46. ​​An in vitro method according to any of paragraphs 33 to 45, wherein the substrate is located in a culture device comprising a gas permeable membrane, for example so that one side can be oriented to contact the gas permeable layer or the one side can be oriented not to contact the gas permeable layer (moved out).

[0064] 47. An in vitro method according to paragraph 46, wherein the substrate is movable (eg, by rotation or sliding) between a position in which one face is in contact with the gas permeable layer and a position in which the one face is not in contact with (moved out of) the gas permeable layer.

[0065] 48. The in vitro method according to any of paragraphs 19 to 47, further comprising a pre-treatment step of digesting the sample (eg, a skin sample) comprising human keratinocytes and human fibroblast feeder cells by protease digestion.

[0066] 49. The in vitro method according to paragraph 48, wherein the protease is selected from the group consisting of dispase, trypsin, and combinations thereof.

[0067] 50. An in vitro method according to paragraph 49, wherein a dispase enzyme is used (eg, to digest the cuticle).

[0068] 51. The in vitro method according to any one of paragraphs 48 to 50, wherein trypsin is used (eg, to digest the dermis).

[0069] 52. The in vitro method according to any of paragraphs 48 to 51, wherein the digestion is performed in the presence of collagenase.

[0070] 53. An in vitro method according to any one of paragraphs 48 to 52, wherein the digestion is performed without separating the dermis and the epidermis (referred to herein as whole cell digestion).

[0071] 54. A complete skin product comprising human epidermis cultured by the method of any of paragraphs 19 to 53.

[0072] 55. A whole human skin product according to paragraph 54 comprising differentiated dermis and epidermis.

[0073] 56. A whole human skin product according to paragraph 54 or 55 for use in therapy.

[0074] 57. The whole human epidermis according to paragraph 56, wherein the treatment is for a condition or disease selected from the group consisting of: tissue damage, such as cuts, lacerations, abrasions (such as peeling), shear injuries, bites (including animal bites, such as dog bites and insect bites); skin regeneration (such as nerves and organelles); wound healing, such as promoting / enhancing wound healing, including erosions, ulcers (such as diabetic ulcers), wounds caused by leprosy, wounds of dystrophic epidermolysis, wounds of suppurative hidradenitis, wounds of mucous membrane pemphigoid, wounds of pemphigoid, wounds of pemphigus vulgaris, wounds of gangrene pyoderma, wounds of herpes zoster; burn healing, including radiation burns, sunburns, chemical burns (such as acid burns and alkali burns), thermal burns; skin regeneration and repair (such as atrophy) or after tissue excision, for example, the excision site is: cancerous skin cells (including melanoma, basal cell carcinoma, squamous cell carcinoma (such as Bowen's disease), extra-mammary Paget's disease (extra-mammary Paget's disease), breast cancer, Darrier disease, cysts, warts (including plantar warts), necrotizing fasciitis (e.g., methicillin-resistant Staphylococcus aureus), necrotic tissue (e.g., gangrene), tissue exhibiting Hailey-Hailey disease, tissue exhibiting blisters associated with pemphigus vulgaris; epidermolysis bullosa; improving skin quality (e.g., treating ichthyosis) or appearance; preventing or repairing skin disorders, such as abscesses, dermatitis (including contact dermatitis), atopic dermatitis, acne, actinic keratosis, rosacea, carbuncle, eczema, psoriasis, cellulitis, keratosis pilaris, melasma, impetigo, or fissures; reduction or disappearance of scar tissue (e.g., treating keloids); breast skin regeneration (after surgery); cosmetic applications, such as anti-aging; dermal regeneration for wrinkles and other skin imperfections; promoting the growth of hair follicles, nerves and other organelles; healing without scarring, or re-healing to reduce scarring. In one embodiment, the condition or disease is selected from: tissue injury; skin regeneration with nerves and organelles; wound healing, such as promoting / enhancing wound healing, including ulcers, such as diabetic ulcers; burn healing; skin regeneration and repair; bullous epidermolysis; improving skin quality or appearance; preventing or remedying skin diseases; reduction or disappearance of scar tissue; breast skin regeneration (post-surgery); cosmetic applications, such as anti-aging; dermal regeneration for wrinkles and other skin defects; promoting hair follicle growth, nerve and other organelle regeneration; healing without scarring, or healing again to reduce scarring.

[0075] 58. A method of treatment comprising suturing a whole human epidermis according to paragraph 54 or 55 to a patient in need thereof, for example for treating a condition or disease selected from the following: tissue damage, such as cuts, lacerations, abrasions (e.g., peeling), shear injuries, bites (including animal bites, such as dog bites and insect bites); skin regeneration (e.g., nerves and organelles); wound healing, such as promoting / enhancing wound healing, including erosions, ulcers (e.g., diabetic ulcers), wounds caused by leprosy, wounds caused by dystrophic epidermolysis , wounds of hidradenitis suppurativa, wounds of mucous membrane pemphigoid, wounds of pemphigoid, wounds of pemphigus vulgaris, wounds of pyoderma gangrenosum, wounds of herpes zoster; healing of burns, including irradiation burns, sunburn, chemical burns (such as acid burns and alkali burns), thermal burns; skin regeneration and repair (such as atrophy) or after tissue excision, for example, the excision site is: cancerous skin cells (including melanoma, basal cell carcinoma, squamous cell carcinoma (such as Bowen's disease), extra-mammary Paget's disease ( Paget's disease), breast cancer, Darrier's disease, cysts, warts (including plantar warts), necrotizing fasciitis (e.g., methicillin-resistant Staphylococcus aureus), necrotic tissue (e.g., gangrene), tissue exhibiting Hailey-Hailey disease, tissue exhibiting blisters associated with pemphigus vulgaris; epidermolysis bullosa; improving skin quality (e.g., treating ichthyosis) or appearance; preventing or repairing skin disorders, such as abscesses, dermatitis (including contact dermatitis), atopic dermatitis, acne, actinic keratosis, rosacea, carbuncle, eczema, psoriasis, cellulitis, keratosis pilaris, melasma, impetigo, or fissures; reduction or disappearance of scar tissue (e.g., treating keloids); breast skin regeneration (post-surgery); cosmetic applications, such as anti-aging; dermal regeneration for wrinkles and other skin imperfections; promoting the growth of hair follicles, nerve and other organelles; healing without scarring, or re-healing to reduce scarring.

[0076] 59. Use of whole human epidermis according to paragraph 54 or 55 in the manufacture of a medicament for a disease or condition selected from the group consisting of: tissue injury, such as cuts, lacerations, abrasions (such as peeling), shear injuries, bites (including animal bites, such as dog bites and insect bites); skin regeneration (such as nerves and organelles); wound healing, such as promoting / enhancing wound healing, including erosions, ulcers (such as diabetic ulcers), wounds caused by leprosy, wounds with dystrophic epidermolysis, wounds of suppurative hidradenitis, Mucous membrane pemphigoid wounds, pemphigoid wounds, pemphigus vulgaris wounds, pyoderma gangrenosum wounds, herpes zoster wounds; healing of burns, including radiation burns, sunburn, chemical burns (such as acid burns and alkali burns), thermal burns; skin regeneration and repair (such as atrophy) or after tissue excision, for example, the excision site is: cancerous skin cells (including melanoma, basal cell carcinoma, squamous cell carcinoma (such as Bowen's disease), extra-mammary Paget's disease ( disease), breast cancer, Darrier disease, cysts, warts (including plantar warts), necrotizing fasciitis (e.g., methicillin-resistant Staphylococcus aureus), necrotic tissue (e.g., gangrene), tissue exhibiting Hailey-Hailey disease, tissue exhibiting blisters associated with pemphigus vulgaris; epidermolysis bullosa; improving skin quality (e.g., treating ichthyosis) or appearance; preventing or repairing skin disorders, such as abscesses, dermatitis (including contact dermatitis), atopic dermatitis, acne, actinic keratosis, rosacea, carbuncle, eczema, psoriasis, cellulitis, keratosis pilaris, melasma, impetigo, or fissures; reduction or disappearance of scar tissue (e.g., treating keloids); breast skin regeneration (post-surgery); cosmetic applications, such as anti-aging; dermal regeneration for wrinkles and other skin imperfections; promoting the growth of hair follicles, nerve and other organelles; healing without scarring, or re-healing to reduce scarring.

[0077] 60. A fully human skin product according to paragraph 54 or 55 for use in testing a topical formulation.

[0078] 61. A kit comprising the cell culture medium of any of paragraphs 1 to 18.

[0079] 62. The kit according to paragraph 61, further comprising a ROCK inhibitor in a container separate from the medium.

[0080] The inventors determined that epidermal growth factor (EGF) and cholera toxin present in Green medium are key components in Green medium. It is known that cholera toxin and xenogeneic mouse feeder cells are closely linked to the mechanism of promoting keratinocyte growth. Therefore, the inventors hypothesized that cholera toxin does not directly act on keratinocytes to promote growth, but stimulates xenogeneic mouse feeder cells to produce keratinocyte growth factor (KGF), thereby indirectly assisting the growth of keratinocytes. Therefore, the inventors chose to replace the need for cholera toxin by adding the end product of cholera toxin stimulation, namely KGF, directly to the culture medium.

[0081] Thus, in one aspect, a cell culture medium is provided, consisting of DMEM high glucose, Ham's F12, fetal bovine serum, one or more antibiotics and / or antifungal agents, and keratinocyte growth factor (KGF).

[0082] In one embodiment, the cell culture medium consists of: DMEM high glucose: Ham's F12 (3:1), 10% fetal bovine serum, penicillin, streptomycin, 0.625 μg / ml amphotericin B and 20 ng / ml KGF, and the inventors refer to this culture medium as "Kelch medium". Advantageously, the disclosed cell culture medium does not contain cholera toxin. Surprisingly, the inventors found that KGF can successfully replace cholera toxin, and when it is included in a basal medium lacking cholera toxin, it can provide keratinocyte growth kinetics similar to Green medium. In addition, the cell culture medium is a basic medium suitable for supporting the growth of keratinocytes (e.g., human keratinocytes), which can eliminate all unnecessary components that are usually present in Green medium. In addition, the culture medium of the present invention produces keratinocyte growth similar to Green medium with mouse embryonic feeder cells (MEFs) in the presence of MEFs.

[0083] However, to use non-irradiated fibroblast feeder cells, the keratinocytes require the presence of "additional" growth accelerating factors. Therefore, keratinocyte growth promoting factors, such as ROCK inhibitors, are generally required. Such factors can be added as a component of the culture medium or added directly to the culture as a component. Therefore, in one embodiment, the medium is suitable for use with a ROCK inhibitor.

[0084] In one embodiment, the cell culture medium consists of: DMEM high glucose: Ham's F12 (3:1), 10% fetal bovine serum, penicillin, streptomycin, 0.625 μg / ml amphotericin B and 20 ng / ml KGF and a keratinocyte growth promoting factor (such as a ROCK inhibitor, particularly an inhibitor disclosed in the present invention), wherein the factor is not cholera toxin.

[0085] Therefore, the cell culture medium disclosed at present can replace Green medium without using MEF, thereby eliminating the need for cholera toxin and MEF. In addition, because the cell culture medium is a basic medium, it is more convenient and easy to prepare, and has a lower cost.

[0086] In one embodiment, the keratinocyte growth promoting factor is a ROCK inhibitor, such as a small molecule ROCK inhibitor. Advantageously, the inventors have determined that including a ROCK inhibitor in cell culture results in an increase in the growth rate of keratinocytes compared to the absence of the ROCK inhibitor.

[0087] In one embodiment, the ROCK inhibitor is selected from the group consisting of: SB772077B, Y-27632, fasudil, Ripasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminofurazan, DE-104, H-1152, ROKα inhibitor, XD-4000, HMN-1152, 4-(1-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.

[0088] In one embodiment, the ROCK inhibitor is Y-27632, SB772077B or a combination of the two, in particular SB772077B. The inventors have determined that both ROCK inhibitors are particularly suitable for enhancing the growth rate of keratinocytes. However, the advantage of SB772077B is that it has a higher binding potency and specificity than Y27632, which means that it can be used at a lower concentration than Y27632 to achieve the same effect ( for 10μm).

[0089] Likewise, the addition of a ROCK inhibitor to the presently claimed cell culture medium significantly increases the growth rate of keratinocytes, thereby enabling the keratinocytes to grow so that they are not overwhelmed by non-irradiated fibroblasts (particularly non-irradiated human fibroblasts) when they are grown together in the same culture.

[0090] This is advantageous because in order to grow human keratinocytes from a skin sample, traditionally the epidermis must first be separated from the dermis, followed by digestion to isolate cells from both layers of skin for in vitro growth in cell culture. The reason for this is that fibroblasts present in the dermis generally have a tendency to outnumber keratinocytes when cultured, resulting in a culture of fibroblasts rather than the desired keratinocytes. The ability to grow human keratinocytes alongside non-irradiated fibroblasts from the same human skin sample greatly reduces the complexity of sample preparation by eliminating the need to separate the dermis from the epidermis at the beginning of sample processing. It also allows for the preparation of a completely human product.

[0091] Additionally, since non-irradiated human fibroblasts could not previously be cultured with human keratinocytes, irradiated xenogeneic feeder cells, such as mouse feeder cells, are often included in the culture to provide the extracellular secretions required for keratinocyte growth. When producing keratinocytes for skin transplantation in the clinic, the need to rely on feeder cells from a different organism is far from ideal due to the risk of immunogenicity and transmission of infectious agents.

[0092] The presently disclosed culture medium is able to significantly enhance the growth of keratinocytes, so that non-irradiated human fibroblasts can be used as feeder cells for keratinocytes instead of using irradiated xenogeneic feeder cells, such as MEF, because the fibroblasts no longer outgrow the keratinocytes. Therefore, the methods disclosed herein produce less immunogenic and infectious products than prior art methods.

[0093] In one embodiment, the method further comprises a pretreatment step of digesting a sample (e.g., a skin sample) comprising human keratinocytes and human fibroblast feeder cells in the presence of trypsin and optionally collagenase. Advantageously, the inventors have found that digestion of skin samples with trypsin alone or with trypsin and collagenase results in significantly higher cell yields compared to the commonly used prior art dispase-collagenase sequential digestion method.

[0094] The pre-treatment steps used in the present invention are only used to emphasize that the steps are related to sample preparation and are not intended to limit the order of the steps performed in the present invention. Therefore, any reasonable order can be provided to perform the steps of the method.

[0095] In one embodiment, keratinocytes and fibroblasts are isolated from the same skin sample in a single digestion reaction. Advantageously, the disclosed method allows for the isolation of keratinocytes from the epidermis and the isolation of fibroblasts from the dermis using a single digestion reaction. This eliminates the need to digest the epidermis and dermis in two separate reactions, thereby saving time and simplifying the process.

[0096] In one embodiment, the sample is digested in the presence of trypsin and collagenase. The advantage of digesting in the presence of collagenase is that collagenase helps digest the dermis and basement membrane, thereby releasing more cells from the skin tissue. This can result in a higher cell yield compared to using trypsin alone. In addition, the inventors have found that keratinocytes and / or fibroblasts isolated by digesting the skin in trypsin and collagenase can proliferate faster when grown in culture than keratinocytes and / or fibroblasts isolated from dispase-digested epidermis and collagenase-digested dermis.

[0097] In one embodiment, the collagenase is collagenase type I. Advantageously, collagenase type I has collagenase, tyrosine, clostripain and trypsin activities, making it well suited for digesting skin samples containing a range of different cell types.

[0098] In one embodiment, the skin sample is obtained from a human, particularly a patient, from any suitable location, such as the shoulder, arm, thigh, calf, breast, abdomen, foreskin and / or buttocks.

[0099] In one embodiment, human keratinocytes are not cultured in the presence of xenogeneic feeder cells. This has the advantages discussed above. In one embodiment, human fibroblasts are not irradiated. The advantage of this is that the fibroblasts remain active and are ultimately able to grow and differentiate into the dermis. Another advantage is that the process of growing keratinocytes is greatly simplified by not needing to irradiate the cells or by ensuring through a quality control system that the irradiation process does not allow any cells to escape irradiation and proliferate.

[0100] In one embodiment, human fibroblasts are autologous to human patients. In one embodiment, human keratinocytes are autologous to human patients. Advantageously, when a whole human epidermis is produced from the patient's own skin cells, this can greatly reduce the patient's immunogenicity risk. In addition, the skin graft product prepared by the method of the present invention can be produced in as short as 3 weeks, which makes it possible to produce a graft for an individual patient. Autologous and allogeneic (also referred to as heterologous) are relevant in the case of applying a skin graft to a specific patient, and may be relevant in the case of obtaining an original sample. It is not a parameter that affects the individual steps of the method itself.

[0101] In one embodiment, keratinocytes are cultured for a period of time without contacting a gas permeable membrane (interface). In one embodiment, keratinocytes are cultured for a period of time in contact with a gas permeable membrane (interface). The latter helps cell differentiation. In one embodiment, the culture period in which keratinocytes are in contact with a gas permeable membrane is followed by a culture period in which they are not in contact with a gas permeable layer.

[0102] In one embodiment, the distance of the relevant cells to the gas permeable layer (e.g. where the cells are in contact with the gas permeable layer) is 2 cm or less, such as 1 cm or less, in particular 0.5, 0.4, 0.3, 0.2, 0.1 or 0.05 cm.

[0103] In one embodiment, the substrate is coated with extracellular matrix proteins or peptides thereof, such as synthetic peptides (e.g., collagen, laminin, and other extracellular matrix proteins). Advantageously, the presence of the coating generates a second cell signal (the first signal causing the skin tissue to grow at the air-liquid or air-permeable interface), which enhances the proper stratification of the skin tissue.

[0104] As discussed above in one embodiment, the substrate is located in a culture device comprising a gas permeable membrane, for example, the substrate can be moved to a position where the cells deposited thereon are in contact with the gas permeable layer, or can be moved to a position where the cells are not in contact with the gas permeable layer. Suitable devices are disclosed in WO2016 / 209089, which is incorporated herein by reference.

[0105] In one embodiment, a culture device comprises: a container comprising a first end wall (bottom) and at least one side wall, a removable second end wall (top) adapted to engage with the container to define a chamber, and a support adapted to accommodate a substrate for cell residence, wherein at least a portion of at least one of the first end wall (bottom), the side wall, or the second end wall (top) comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gas exchange; and wherein the device is configurable between the following modes: (a) a first mode, wherein the substrate is not in gas communication with the gas permeable material; and (b) a second mode, wherein the substrate is moved so as to be in gas communication with the gas permeable material.

[0106] Advantageously, the ability to move the substrate between the two modes allows cells to be submerged in culture medium during the initial stages of growth and then readily exposed to the atmosphere (eg, contact with a gas permeable membrane) to properly differentiate into full-thickness skin.

[0107] In one embodiment, the bracket is engaged with at least one side wall to (a) allow the bracket to move at least partially in a substantially linear manner between a first end wall (bottom) and a second end wall (top) of the chamber and limit rotation or inversion of the bracket about an axis perpendicular to at least one side wall, or (b) allow rotational movement of the bracket about an axis perpendicular to at least one side wall.

[0108] In one embodiment, the scaffold comprises a frame defining an inner perimeter and an outer perimeter, the frame comprising: a substantially planar upper surface; a substrate for cell residence, which is retained on the inner perimeter of the frame in a substantially planar arrangement, wherein when the scaffold is placed in a culture device comprising at least one gas permeable interface, the scaffold is configured to bring substantially all of the substrate or cells or tissues present on the substrate into contact with the gas permeable interface.

[0109] In one aspect, human full-thickness skin cultured by the methods of the present invention is provided, for example including both epidermis and dermis.

[0110] In one aspect, there is provided intact human epidermis cultured by the methods described herein.

[0111] In one aspect, there is provided human full-thickness skin, full human epidermis or full human dermis cultured by the method of the present invention for use in treatment, in particular, a full human skin product comprising epidermis is provided.

[0112] In one embodiment, the treatment is for a condition or disease selected from the group consisting of: tissue damage; skin regeneration of nerves and organelles; wound healing, such as promoting / enhancing wound healing, including ulcers, such as diabetic ulcers; burn healing; skin regeneration and repair; epidermolysis bullosa; improving skin quality or appearance; preventing or repairing skin diseases; reduction or disappearance of scar tissue; breast skin regeneration (post-surgery); cosmetic applications, such as anti-aging; dermal regeneration for wrinkles and other skin defects; promoting hair follicle growth, nerve and other organelle regeneration; healing without scarring, or healing again to reduce scarring.

[0113] In one aspect, there is provided use of human full-thickness skin, whole human epidermis or complete human dermis cultured by the method of the present invention in the preparation of a medicament for a condition or disease such as that disclosed above in the present invention.

[0114] In one aspect, a method of treatment is provided, which comprises suturing human full-thickness skin, whole human epidermis or whole human dermis cultured by the method described in the present invention to a patient in need thereof.

[0115] In one embodiment, the treatment is of a condition or disease, such as disclosed elsewhere herein. DETAILED DESCRIPTION OF THE INVENTION

[0117] "DMEM" or "Dulbecco's Modified Eagle's Medium" is a modified form of the basal medium Eagle that contains four times the concentration of amino acids and vitamins, as well as other ingredients. The specific ingredients are listed in the Background of the Invention section of this invention. DMEM typically contains about 1000 mg / L of glucose. "DMEM High Glucose" refers to a form of DMEM that contains 4500 mg / L of glucose instead of the usual 1000 mg / L.

[0118] "Ham's F12" is a medium designed for the growth of low-density, serum-free Chinese hamster ovary (CHO) cells. Ham's F12 is based on Ham's F10 medium, but has increased concentrations of choline, inositol, putrescine, and other amino acids. The specific ingredients are listed in the background of the invention section of the present invention.

[0119] The ratio of DMEM:Ham's F12 in the cell culture medium can be 1: 1, 2: 1, 3: 1, 4: 1, 5: 1. In one embodiment, the ratio is 3:1.

[0120] The Green medium used in the present invention is DMEM:Ham's F12 (Life Technologies 31765-035) in a ratio of 3:1. Fetal calf serum (FCS) is usually used as serum, although NCS and serum replacement products are also suitable, and Hepes buffer, for example, is used as buffer. The pH value of the solution of cell culture medium, buffer and serum is usually in the range of 6.0 to 8.0, such as in the range of 6.5 to 7.5, more particularly in the range of 7.0.

[0121] "Fetal bovine serum" or FBS is the most widely used animal serum supplement for eukaryotic cell culture. This is due to its very low antibody levels and the presence of growth factors, which makes FBS suitable for many different cell culture applications. The culture medium currently claimed contains fetal bovine serum, but the skilled person will appreciate that other types of serum may also be used, such as bovine serum albumin (BSA), human platelet lysate, and iron-supplemented calf serum (ICS). Typically 10% serum is used, but other concentrations may be used, such as 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%, depending on the type of serum.

[0122] As used herein, "penicillin" refers to a group of beta-lactam antibiotics, including penicillin G, penicillin V, procaine penicillin, and benzathine penicillin. Penicillin works by inhibiting the formation of peptidoglycan crosslinks in the bacterial cell wall. This weakens the cell wall of the dividing bacteria, and eventually the cell wall ruptures due to osmotic pressure and the bacteria die. Gram-positive bacteria have thicker cell walls containing high levels of peptidoglycan, while Gram-negative bacteria are characterized by thinner cell walls with lower peptidoglycan content. Therefore, penicillin is most effective against Gram-positive bacteria.

[0123] "Streptomycin" is an antibiotic originally purified from Streptomyces griseus. It works by binding to the 30S subunit of bacterial ribosomes, resulting in the inhibition of protein synthesis and the death of susceptible bacteria. Streptomycin is able to cross the outer cell wall of gram-negative organisms by passive diffusion through water channels. In contrast, the thicker cell wall of gram-positive bacteria inhibits the transport of streptomycin. Therefore, streptomycin works better on gram-negative bacteria.

[0124] In one embodiment, the cell culture medium contains both penicillin and streptomycin to help protect cells grown in culture from both Gram-positive and Gram-negative bacteria.

[0125] "Gentamicin" is an antibiotic that contains three different closely related aminoglycoside sulfates, gentamicin C1 Gentamicin is a complex of C1a, C2 and C1a obtained from Micromonospora rubrum and related species. Gentamicin is a broad-spectrum antibiotic commonly used for serious infections with the following microorganisms: Pseudomonas aeruginosa, Proteus species (indole-positive and indole-negative), Escherichia coli, Klebsiella-Enterobacter-Serratia species, Citrobacter species, and Staphylococcus species (coagulase-positive and coagulase-negative).

[0126] In one embodiment, the cell culture medium comprises gentamicin.

[0127] In one embodiment, the culture medium does not contain one or more antibiotics that have been associated with allergic reactions, such as penicillin and / or streptomycin.

[0128] Amphotericin B is an antifungal drug used for serious fungal infections and leishmaniasis. It works by binding to ergosterol, a component of the fungal cell membrane, forming small pores that allow monovalent ions (such as K) to pass through. + , Na + , H + and Cl - ) leaks rapidly, leading to fungal cell death.

[0129] In one embodiment, the cell culture medium comprises an antibiotic targeting Gram-positive bacteria (eg, penicillin), an antibiotic targeting Gram-negative bacteria (eg, streptomycin), and an antifungal drug.

[0130] "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 the 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 primarily manifested in keratinocytes and not in fibroblasts or endothelial cells.

[0131] As used in the present invention, "ROCK inhibitor" refers to any compound or protein having the function of reducing or blocking the activity of Rho-associated protein kinase (ROCK), such as a small molecule ROCK inhibitor or an antibody.

[0132] Examples of ROCK inhibitors include, but are not limited to, SB 772077B, Y-27632, fasudil, Ripasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminofurazan, DE-104, H-1152, ROKα inhibitor, XD-4000, HMN-1152, 4-(1-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.

[0133]

[0134]

[0135]

[0136] Other ROCK inhibitors are also known to the skilled person.

[0137] The terms "epithelial cells" and "epithelium" refer to cells covering the internal and external body surfaces (skin, mucus and serous fluid), including glands and other structures derived therefrom, such as corneal, esophageal, laryngeal, epidermal, hair follicle and urothelial cells.

[0138] In one embodiment, the cells employed are skin cells, such as human skin cells, such as cells forming the epidermis and dermis, such as fibroblasts and keratinocytes.

[0139] Other exemplary epithelial tissues include: olfactory epithelium, which is a pseudostratified epithelium located in the olfactory region of the nasal cavity and contains receptors for the sense of smell; glandular epithelium, which refers to epithelium composed of secretory cells; squamous epithelium, which refers to epithelium composed of flat, plate-like cells.

[0140] The term "tissue" is used to refer to an aggregation of similar specialized cells united in the performance of a specific function. Tissue is intended to cover all types of biological tissues, both hard and soft. A "tissue" is a collection or aggregation of specialized cells embedded in their natural matrix, where the natural matrix is ​​produced by specific living cells. The term may also refer to an ex vivo aggregation of similar specialized cells that has been expanded in vitro, such as in an artificial organ.

[0141] As used herein, the term "skin tissue" or "skin" refers to any tissue, including the epidermis, dermis, and may also include basement membrane tissue, such as full-thickness skin.

[0142] The method according to the invention enables the production of full-thickness human skin consisting of two tissue-specific layers, dermis equivalent and epidermis equivalent. Said skin tissue substantially corresponds histologically and functionally to native skin.

[0143] As used herein, "epidermis" refers to the outer layer of the two layers that make up the skin, the inner layer being the "dermis".

[0144] As used herein, "fully human" refers to tissues that do not contain and are not prepared with any non-human cells, such as xenogeneic feeder cells, such as mouse embryonic fibroblasts. The cells and tissues according to the present invention do not contain any non-human components because they are completely from humans. However, the culture medium used to culture the cells may contain, for example, fetal bovine serum. This serum does not make the cells and tissues of the present invention non-human.

[0145] As used herein, "fibroblasts" are understood to be naturally occurring fibroblasts or precursor cells thereof, such as adipose-derived stromal cells, more particularly, fibroblasts present in the dermis, genetically modified fibroblasts or fibroblasts resulting from spontaneous mutation or their precursors.

[0146] As used in the present invention, "keratinocytes" are understood to be epidermal cells, genetically modified keratinocytes, or keratinocytes produced by spontaneous mutations or their precursors that form cornified plate epithelium, and the keratinocytes can be of animal or human origin. Alternatively, for normal skin keratinocytes, mucosal keratinocytes or intestinal epithelial cells can be applied to matrix. These are, for example, pre-cultured cells, and in one embodiment, are keratinocytes in the first or second cell passage, although cells from higher passages can also be used.

[0147] Fibroblasts and keratinocytes are obtained and cultured by methods known to those skilled in the art, which methods may be adapted to the desired properties of the skin tissue to be produced.

[0148] In one embodiment, other cell types and / or other cells of other tissue types, such as 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, especially sebaceous gland cells or sebaceous gland tissue or sebaceous gland explants, sweat gland cells or sweat gland tissue or sweat gland explants, hair follicle cells or hair follicle explants; and cells from tumors or metastases of other organs can be cultured with human keratinocytes. The cells mentioned can be of human and animal origin, but unless otherwise mentioned, they will be human, so as to produce a full human epidermis. Stem cells, tissue-specific stem cells, embryonic and / or adult stem cells from various sources can also be integrated into the skin model.

[0149] "Trypsin" (EC number 3.4.21.4) used in the present invention is a serine protease from the PA clan superfamily, present in the digestive system, for example in the pancreas of many vertebrates, where trypsin hydrolyzes proteins. Trypsin cleaves peptide chains primarily at the carboxyl side of lysine or arginine amino acids. If acidic residues are on either side of the cleavage site, the rate of hydrolysis is slower; if proline residues are on the carboxyl side of the cleavage site, no cleavage occurs. As used in the presently disclosed method, trypsin is capable of digesting the epidermis and dermis of a skin sample.

[0150] As used herein, "collagenase" refers to a group of enzymes that decompose natural collagen that holds animal tissues together. Collagenase is made from a variety of different microorganisms and many different animal cells. Crude collagenase preparations contain several isoforms of two different collagenases, namely sulfhydryl proteases, clostripain, trypsin and aminopeptidase. This combination of collagenase hydrolysis and proteolytic activity effectively decomposes the intercellular matrix, which is an important part of tissue dissociation. One component of the complex is a hydrolase that preferably degrades the helical region in natural collagen at the Y-Gly bond in the Pro-Y-Gly-Pro sequence, where Y is most commonly a neutral amino acid. This cleavage produces a product that is easy to further peptidase digestion. Crude collagenase is inhibited by metal chelators (such as cysteine, EDTA or o-phenanthroline), but not by DFP. It is also inhibited by large plasma glycoprotein α2-macroglobulin. Ca 2+ Required for enzyme activity. Four main types of collagenase are commonly used, depending on the requirements:

[0151] Crude collagenase type 1 has an original balance of collagenase, tyrosine, clostripain and trypsin activities.

[0152] Type 2 contains higher relative levels of protease activity, especially clostripain.

[0153] Type 3 contains the lowest levels of secondary proteases.

[0154] Type 4 is designed to have particularly low trypsin activity to limit damage to membrane proteins and receptors.

[0155] In one embodiment, type 1 collagenase is used in the methods of the invention.

[0156] As used herein, "feeder cells" refer to a cell population, typically connective tissue cells, that are used to nourish cultured tissue cells, particularly human keratinocytes as described herein. Feeder cells provide metabolites and other nutrients to the cells they support. Feeder cells typically do not grow or divide, and are typically inactivated by irradiation, such as gamma irradiation. However, in the present invention, feeder cells are not irradiated.

[0157] In one embodiment, human fibroblasts act as feeder cells to support the growth of human keratinocytes. In one embodiment, human fibroblasts are not irradiated. This means that the human fibroblasts are not inactivated and can continue to grow with the human keratinocytes in culture. This is possible because the presence of a ROCK inhibitor can allow keratinocytes to grow at a rate that is not exceeded by the growth of fibroblasts.

[0158] In one embodiment, the human fibroblasts are matched to human keratinocytes. For example, the fibroblasts can be sex matched and / or HLA matched to the keratinocytes.

[0159] In another embodiment, both human fibroblasts and human keratinocytes can be derived from the same donor. Alternatively, the human keratinocytes and / or human fibroblasts to be cultured are autologous, derived from a patient in whom a whole human epidermis is intended to be used. As described above, autologous / allogeneic is technically irrelevant to the execution of the actual process steps, but is relevant to the administration to the patient.

[0160] In one embodiment, seeding of the skin cells on the matrix is ​​performed in the presence of a physiological solution.

[0161] As used in the present invention, the term "physiological solution" refers to a solution that is similar or identical to one or more physiological conditions or that can change the physiological state of some physiological environment. As used in the present invention, the term "physiological solution" also refers to a solution that can support the growth of cells (including but not limited to mammals, vertebrates and / or other cells).

[0162] In one embodiment, the physiological solution comprises a defined culture medium in which the concentration of each culture medium component is known and / or controlled. A defined culture medium typically contains all nutrients required to support cell growth, including but not limited to salts, amino acids, vitamins, lipids, trace elements, and energy sources (e.g., carbohydrates). Non-limiting examples of defined culture media include DMEM, Basal Media Eagle (BME), culture medium 199; F-12 (Ham) nutrient mixture; F-IO (Ham) nutrient mixture; minimal basal medium (MEM), culture medium E (Williams' Media E), and RPMI 1640.

[0163] In one embodiment, the physiological solution is a cell culture medium disclosed herein.

[0164] In one embodiment, the culture medium may contain other factors, such as hormones, growth factors, adhesion proteins, antibiotics, selection factors, enzymes and enzyme inhibitors, etc. For example, growth factors can help enhance the proliferation of seeded cells.

[0165] The antibody used in the present invention refers to a full-length antibody, a binding fragment thereof or an antibody molecule comprising any of them. 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, bivalent, trivalent or tetravalent 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).

[0166] The peptides used in the present invention are sequences of 2 to 50 amino acids.

[0167] As used herein, synthetic peptides refer to peptides prepared by synthetic chemistry techniques (as opposed to recombinantly expressed peptides).

[0168] As used herein, substantially planar refers to having a surface that substantially (a major portion of which) lies in one plane.

[0169] Substrate / matrix of the present invention

[0170] Unless the context indicates otherwise, the terms "matrix", "cell matrix", "cell matrix", "substrate" or "cell substrate" are used interchangeably in the present invention. It refers to any physical structure, including but not limited to solid or semi-solid structures, such as fibrous mesh structures with holes, which can provide:

[0171] Mechanical or other support for cell or tissue adhesion and proliferation, and

[0172] Allow at least one cell type to migrate during culture.

[0173] For example, it can be used for in vitro skin tissue culture.

[0174] As used herein, contact with a gas permeable layer / membrane means that the relevant cells are on or near the membrane / layer so that 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 between them would be separated by, for example, culture medium, buffer or CO). 2 In one embodiment, the distance between the cells of interest and the gas permeable layer is 2 cm or less, such as 1 cm or less, in particular 0.5, 0.4, 0.3, 0.2, 0.1 or 0.05 cm. In one embodiment, the outer portion of the cells for differentiation rests on the gas permeable layer / membrane.

[0175] Typically, the matrix will be three-dimensional, with a first 2D face on which cells can be deposited and a second 2D face (with a larger surface area), and the depth between the two faces giving the third dimension (corresponding to a cross-section of the final skin - as mentioned above, approximately in the range of 100μm).

[0176] The matrix of the present invention can be constructed of natural or synthetic materials. The matrix can be a specific shape or form to affect or define a three-dimensional shape or form assumed by a group of proliferating cells. Such shapes or forms include, but are not limited to, films (e.g., forms in which two dimensions are significantly greater than the third dimension), ribbons, ropes, sheets, flat disks, cylinders, spheres, three-dimensional amorphous shapes, etc.

[0177] In one embodiment, the matrix comprises only synthetic materials. In another embodiment, the matrix comprises a mixture of synthetic and natural materials.

[0178] In one embodiment, the synthetic materials used to prepare the matrices of the invention are biocompatible and biodegradable (eg, subject to enzymatic and hydrolytic degradation), such as biodegradable polymers.

[0179] As used herein, "biocompatible" refers to any material that does not cause adverse reactions in a mammal when implanted in the mammal. A biocompatible material is not toxic or harmful to an individual when introduced into the individual, nor does it induce immune rejection of the material in a mammal.

[0180] As used herein, the term "biodegradable" or "bioabsorbable" is intended to describe a material that exists in a biological environment for a limited time and degrades under physiological conditions to form a product that can be metabolized or excreted without harming the subject. In some embodiments, the product is metabolized or excreted without permanent damage to the subject.

[0181] In one embodiment, the matrix is ​​completely absorbable by the subject's body.

[0182] In one embodiment, when placed in a biological environment, the bioabsorbable matrix of the present invention can exist for several days, weeks or months. For example, when placed in a biological environment, the bioabsorbable matrix can 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 longer.

[0183] In one embodiment, the matrix layer is absorbed by the body of the experimenter at a rate approximately the same as the growth of the tissue cells below the membrane matrix layer in the region. In some embodiments, the cell is an epithelial cell. In some embodiments, the matrix layer is substantially completely absorbed by the body within approximately 3 to 12 months after applying the skin graft. In some embodiments, the matrix is ​​substantially completely absorbed within approximately 3 months.

[0184] Biodegradable materials (eg, polymers) are hydrolytically degradable and may require cellular and / or enzymatic action for complete degradation, such as hydrolysis, oxidation, enzymatic processes, phagocytosis, or other processes, including combinations of the foregoing.

[0185] Biodegradable polymers are known to those of ordinary skill in the art and include, but are not limited to, synthetic polymers, natural polymers, mixtures of synthetic and natural polymers, inorganic materials, and the like.

[0186] In one embodiment, the matrix incorporates one or more synthetic polymers in its construction. The matrix can be made of heteropolymers, monomers or combinations thereof. Examples of polymers suitable for making cell matrices include, but are not limited to, aliphatic polyesters, copoly (ether esters), polyalkylene oxalates, polyamides, poly (imino carbonates), polyorthoesters, polyoxaesters (polyoxaesters), polyamidoesters, amine-containing polyoxaesters, poly (anhydrides), polyphosphazenes, biomolecules and mixtures thereof. Suitable aliphatic polyesters include homopolymers, copolymers (random, block, segmented, tapered blocks, grafted, triblocks, etc.) with linear, branched or star structures. Suitable monomers for preparing aliphatic homopolymers and copolymers can be selected from, but not limited to, the group consisting of: lactic acid, lactide (including L-, D-, meso and D, L mixtures), glycolic acid, glycolide, ε-caprolactone, p-dioxanone (1,4-dioxane-2-one), trimethylene carbonate (1,4-dioxane-2-one), δ-valerolactone, β-butyrolactone, ε-decanolactone, 2,5-diketolide 1,4-dioxane-2,5-dione, 1,5-dioxane-2,6-dimethyldioxane-2-one, 6,8-dioxabicyclooctane-7-one and combinations thereof. 1,4-dioxane-2,5-dione, 1,5-dioxane-2,6-dimethyldioxane-2-one, 6,8-dioxabicyclooctane-7-one and combinations thereof. In one embodiment, the synthetic polymer is selected from PLGA, PLA, PCL, PHBV, PDO, PGA, PLCL, PLLA-DLA, PEUU, cellulose acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP, mixed PLA / PCL, PDLA / HA, PLLA / HA, PLLA / MWNT / HA, PLGA / HA, 100 dioxanone linear homopolymer and a combination of two or more thereof.

[0187] Elastomeric copolymers are also particularly useful in the presently disclosed matrices. Suitable bioabsorbable biocompatible elastomers include, but are not limited to, elastomers selected from the group consisting of: elastomeric copolymers of ε-caprolactone and glycolide (a suitable molar ratio of ε-caprolactone to glycolide is about 35:65 to about 65:35, for example, 45:55 to 35:65); elastomeric copolymers of ε-caprolactone and lactide, including copolymers of L-lactide, D-lactide, mixtures thereof, or lactic acid (a molar ratio of ε-caprolactone to lactide is suitably about 35:65 to about 65:35, and for example, 45:55 to 30:70 or about 95:5 to about 85:15); elastomeric copolymers of p-dioxanone (1,4-dioxane-2-one) and lactide (including L-lactide, D-lactide and lactic acid) (a molar ratio of p-dioxanone to lactide is preferably about 1,2-dioxanone to 1,4-dioxane-2-one); of about 40:60 to about 60:40); elastomeric copolymers of ε-caprolactone and p-dioxanone (suitably the molar ratio of ε-caprolactone to p-dioxanone is about 30:70 to about 70:30); elastomeric copolymers of p-dioxanone and trimethylene carbonate (suitably the molar ratio of p-dioxanone to trimethylene carbonate is about 30:70 to about 70:30); elastomeric copolymers of trimethylene carbonate and glycolide (suitably the molar ratio of trimethylene carbonate to glycolide is about 30:70 to about 70:30); elastomeric copolymers of trimethylene carbonate and lactide, including copolymers of L-lactide, D-lactide, blends thereof or lactic acid (suitably the molar ratio of trimethylene carbonate to lactide is about 30:70 to about 70:30); and blends thereof. Examples of suitable bioabsorbable elastomers are described in US 4,045,418; US 4,057,537 and US 5,468,253, all of which are incorporated herein by reference. These elastomeric polymers have an intrinsic viscosity of about 1.2 dL / g to about 4 dL / g, preferably an intrinsic viscosity of about 1.2 dL / g to about 2 dL / g and, for example, an intrinsic viscosity of about 1.4 dL / g to about 2 dL / g measured at 25°C in a 0.1 gram per deciliter (g / dL) solution of the polymer in hexafluoroisopropanol (HFIP).

[0188] Other materials suitable for use as the matrix of the present invention include, but are not limited to, polylactic-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes and combinations thereof. Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetate, polyvinyl alcohol, polylactide, chondroitin sulfate (a proteoglycan component), polyesters, polyethylene glycol, polycarbonates, polyvinyl alcohol, polyacrylamide, polyamides, polyacrylates, polyesters, polyetheresters, polymethacrylates, polyurethanes, polycaprolactones, polyphosphazenes, polyorthoesters, polyglycolides, copolymers of lysine and lactic acid, copolymers of lysine-RGD and lactic acid, and the like, and copolymers thereof. Synthetic polymers may further include those selected from the following: aliphatic polyesters, polyamino acids, polypropylene fumarate, copolyetheresters, polyalkylene oxalates, polyamides, tyrosine-derived polycarbonates, polyiminocarbonates, polyorthoesters, polyoxyesters, polyamide esters, amine-containing polyoxyesters, polyanhydrides), polyphosphazenes and blends thereof.

[0189] In one embodiment, the matrix incorporates polylactic acid (PLA). PLA is particularly suitable for tissue engineering methods using cell matrices because it degrades in the human body to form lactic acid (a naturally occurring chemical that is easily removed from the body). The cell matrix of the present invention may also incorporate polyglycolic acid (PGA) and / or polycaprolactone (PCL) as matrix materials. PGA and PCL have similar degradation pathways as PLA, but the degradation rate of PGA in the body is faster than that of PLA, while the degradation rate of PCL is slower than that of PLA.

[0190] PGA has been widely used in tissue engineering. PGA matrix can be easily prepared into various three-dimensional structures and provides excellent support and transportation for cells (Christenson L, Mikos AG, Gibbons DF, et al.: Biomaterials for tissue engineering: summary. Tissue Eng. 3 (1): 71-73; discussion 73-76, 1997). Matrixes made of polyglycolic acid alone and made of PGA in combination with other natural and / or synthetic biocompatible materials are within the scope of the present invention.

[0191] In one embodiment, the matrix comprises poly(lactic-co-glycolic acid) (PLGA), such as PLGA microfibers or nanofibers.

[0192] In another embodiment, the matrix comprises a dioxanone linear homopolymer, such as a 100 dioxanone linear homopolymer (eg, Dioxaprene 100M).

[0193] In one embodiment, the matrix comprises a combination of PLGA and 100 dioxanone.

[0194] The term "fiber" as used herein refers to a material in the form of continuous filaments or discrete elongated forms, which generally include or consist of biodegradable polymers such as those described above. The fibers of the present invention generally have a diameter in the micrometer range, such as 0.5 μm to 5 μm, for example 1 μm, 1.5 μm, 2 μm, 2.5 μm, 3 μm, 3.5 μm, 4 μm, 4.5 μm or 5 μm, in particular, 1 to 3 μm.

[0195] The term "fibrous matrix" as used herein refers to an arrangement of fibers into a support framework, such as in the form of a sheet of fibers, which can then be used to support cells or other additional materials (see also the definition of "matrix"). Various methods are known to the skilled person that can be used to produce suitable fibers, including but not limited to interfacial polymerization and electrospinning.

[0196] In one embodiment, electrospinning is used to form the matrices of the present invention.

[0197] The term "electrospinning" generally refers to a technique that utilizes a high voltage power source, a spinneret (e.g., a hypodermic needle), and a conductive collector plate (e.g., aluminum foil or stainless steel). To perform an electrospinning process using these materials, the electrospinning solution (i.e., a melt or solution of the desired material to form the fiber) is typically first loaded into a syringe and then fed at a specific rate by a syringe pump.

[0198] When the liquid is fed with a sufficiently high voltage by a syringe pump, the repulsive force between the charges fixed on the resulting droplet surface overcomes the limitation of surface tension and causes the liquid to be ejected from the orifice. Then, the charged jet undergoes a whipping and stretching process, which subsequently results in the formation of uniform nanofibers. In addition, with the stretching of the jet and the evaporation of the solvent, the diameter of the fiber can then be continuously reduced to a desired scale, such as microns, even as small as nanometers, and under the influence of the electric field, the fiber can then be forced to advance toward a grounded collector, on which they are usually deposited as nonwoven mats. In the context of the present invention, due to the high ratio of surface area to volume and one-dimensional morphology, electrospun fibers can simulate the structure of the extracellular matrix.

[0199] Examples of materials for producing the nanofibers of the present invention are selected from those listed in Tables 1 and 2 below.

[0200] Table 1 - Exemplary materials for producing electrospun fibers (natural polymers).

[0201]

[0202]

[0203] In one embodiment, the matrix of the invention is composed of synthetic microfibers or nanofibers, for example using the materials listed in Table 2.

[0204] The selection of a particular polymer and its use in a specified amount or concentration or range thereof provides the ability to control, tailor and adjust the rate of degradation of the polymer and, therefore, the rate of degradation 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 for the growing skin tissue, but once the patient's own cells have absorbed the skin graft, the matrix will eventually degrade and be bioabsorbed by the patient's body, thereby eliminating the requirement to later recover the matrix from the patient's body.

[0205] Various polymer blends can be used, for example, by electrospinning fibers using the materials listed in Tables 1 and 2, to improve the biocompatibility of the fibers as well as their mechanical, physical, and chemical properties.

[0206] Once the desired microfiber or nanofiber matrix is ​​produced, in one embodiment, two or more fiber matrix layers of the present invention are laid together. By layering multiple fiber matrixes, the advantages of each fiber matrix can be combined, and in some cases a synergistic effect can be generated.

[0207] For example, the first matrix can include the micropores for receiving one or more related cells and / or skin tissues, and then its layer is laid on the second matrix with radially arranged fiber. In this example, the first matrix can provide the benefit of increasing damaged skin repair by providing related cells and / or skin tissues, and the second matrix can provide guidance and strengthen the benefit of cell migration to the center from the periphery of the matrix of layer laying. Two or more matrix layers can also help to strengthen the water tightness of matrix. Those skilled in the art can derive the various combinations of two or more different matrixes to realize the characteristic of expectation.

[0208] In one embodiment, the substrate of the present invention may be further treated by a single method or a combination of methods that reduces the number of microorganisms that are able to grow in the substrate under the conditions of storage and / or distribution of the substrate.

[0209] In one embodiment, gamma rays are used to sterilize the matrix. In another embodiment, ethylene oxide (EtO) is used to sterilize the matrix. In another embodiment, Revox is used, which utilizes peracetic acid to sterilize the matrix.

[0210] In one embodiment, ionizing radiation such as electron beam radiation is used to sterilize the matrix. In any currently recognized sterilization method, the processing cycle of electron beam treatment is the shortest. Under electron beam irradiation, the product will be exposed to radiation for a few seconds, and the product will be transported in and out of the radiation shielding layer and will consume a lot of processing time. The whole process time (including transportation time) is 5 to 7 minutes. Electron beam treatment involves the use of high-energy electrons, which usually have an energy of 3 million to 10 million electron volts (MeV) to irradiate disposable medical products. Electrons are produced by accelerators working in pulse and continuous beam modes. These high energy levels are needed to penetrate products such as those packaged in their final transport containers. When the beam scans through the product, the electron interacts with the material and produces secondary high-energy substances, such as electrons, ion pairs and free radicals. These secondary high-energy substances inactivate microorganisms because they destroy the DNA chain of the microorganism, thereby making the product sterile. The technician knows other possible methods for sterilizing the matrix of the present invention.

[0211] The seeding density of the cell matrix can vary, and each layer of the cell matrix can have the same or different seeding densities. The seeding density can vary depending on the specific application to which the cell matrix is ​​applied. The seeding density can also vary depending on the cell type used to make the cell matrix.

[0212] The number and concentration of cells seeded into or onto the matrix can be varied by varying the concentration of the cells in the suspension, or by varying the amount of the suspension dispersed over a given area or volume of the matrix.

[0213] 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 .

[0214] 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 .

[0215] The inoculation density of each layer of the matrix will depend on the use of the matrix. Although a person skilled in the art will understand the specific inoculation density that a specific application will require, the various layers of the matrix can be inoculated with a variety of inoculation densities. It will be understood by a person skilled in the art that the inoculation density of each layer of the matrix can vary depending on the use of the matrix. Spreading involves spreading the inoculum on a sponge-like matrix using a tool such as a spatula. The matrix is ​​inoculated by spraying by dipping a brush into the inoculum, extracting it, and then wiping the brush filled with the inoculum on the matrix. This method has the following disadvantages: a large number of cells may be attached to the brush and will not be applied to the grid. However, it can still be useful, especially in the case of a pattern or area of ​​a grid where the distribution of the inoculum needs to be carefully controlled. Inoculating the matrix by spraying generally involves passing the inoculum through any type of nozzle that converts the liquid into small air-suspended droplets. This embodiment is subject to two constraints. First, it must not subject the cells in the solution to shear forces or pressure, which can destroy or kill a large number of cells. Secondly, the cell suspension should not be required to be mixed with a propellant fluid that is toxic or harmful to the cells or wound bed. A variety of nozzles that are generally available meet these two constraints. Such a nozzle may be connected to a reservoir containing the epithelial stem cell inoculum in any conventional manner.

[0216] It is to use pipette, common " dropper " or other similar devices that can place a small amount of inoculum on the matrix surface of the present invention to complete by pipetting that matrix is ​​inoculated. Aqueous liquid will penetrate through porous matrix. The cell in the suspension tends to be trapped on the matrix surface, and therefore remains on the matrix surface.

[0217] According to another embodiment of the present invention, the cell inoculum can be inoculated by a hypodermic syringe equipped with a hollow needle or other catheter. The cell suspension is injected into the syringe barrel and the needle is inserted into the matrix. Pressing the plunger of the syringe can eject a certain amount of solution from the cylinder through the needle and into the scaffold.

[0218] An important advantage of utilizing an aqueous suspension of cells is that it can be used to greatly expand the matrix area where the effective inoculum is distributed. This provides two significant advantages. First, if a very limited number of intact tissues can be used for autotransplantation, various suspension methods can be used to significantly increase the area or volume of the matrix that can be inoculated with a limited number of available cells. Secondly, if it is necessary to inoculate cells on a matrix of a given area or volume, the quantity of intact tissue that needs to be harvested from the donor site can be greatly reduced. The optimal inoculation density for a specific application can be determined by those skilled in the art via routine experiments.

[0219] Generally, the dimensions of the matrix should be substantially planar and its thickness should be such that the seeded cells have adequate access to the nutrient medium. After implantation, the cell matrix must have adequate access to body fluids to remove nutrients and waste products. The thickness of the matrix can vary with changes in the porosity of the matrix. Thus, an increase in matrix porosity can allow the matrix to have a greater thickness, while a larger pore size improves access to the external medium and body fluids.

[0220] Thus, in one embodiment, the thickness of the matrix is ​​100 μm or less, such as 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 μm. By maintaining the thickness of the matrix at 100 μm or less, the seeded keratinocytes can receive nutrients and remove waste by diffusion alone, without the need for a vascular system to survive.

[0221] The matrix of inoculation layering involves introducing one or more required cell groups into selected substrate material, for example, subsequently the material is combined to form a layered matrix. Alternatively, the matrix can be connected in advance, and the selected cell group is introduced into the selected position. Inoculation is different from spontaneous infiltration and migration of cells from the wound site where the matrix is ​​placed, for example.

[0222] In one embodiment, the matrix is ​​seeded on at least one surface before the individual cell-seeded surfaces are opposed to each other to form a hierarchical structure.

[0223] Various additional materials and / or biomolecules can be attached to the matrix of the present invention. The term "attachment" includes but is not limited to coating, embedding or incorporating additional materials and / or biomolecules in any manner, and attachment can refer to incorporating such components into the entire matrix or only a portion thereof.

[0224] In one embodiment, cytokines are coated / attached to the matrix of the present invention. As used herein, the term "cytokine" refers to a substance that is synthesized by living cells (e.g., stem cells) and produces a beneficial effect in the body (e.g., a mammal or human body). Cytokines include, but are not limited to, growth factors, regulatory factors, hormones, enzymes, lymphokines, peptides, and combinations thereof. Cytokines can have a variety of effects, including but not limited to in vivo and / or in vitro effects on the growth, proliferation, stereotyping, and / or differentiation of cells (e.g., stem cells).

[0225] Some non-limiting examples of cytokines include, but are not limited to, cytokines (e.g., common beta chain, common gamma chain, and IL-6 cytokine family), vascular endothelial growth factors (e.g., VEGF-A, -B, -C, -D, and -E), adrenomedullin, insulin-like growth factor, epidermal growth factor EGF, fibroblast growth factor FGF, autotaxin, GDF, IGF, PDGF, growth differentiation factor 9, erythropoietin, activin, TGF-α, TGF-β, bone morphogenetic protein (BMP), Hedgehog molecule, Wnt-related molecules, and combinations thereof.

[0226] In one embodiment, growth factors such as EGF (epidermal growth factor), IGF-I (insulin-like growth factor), members of the fibroblast growth factor family (FGF), keratinocyte growth factor (KGF), PDGF (platelet-derived growth factor AA, AB, BB), TGF-β (transforming growth factor family-β1, β2, β3), CIF (chondrology inductive factor), at least one of BMP 1-14 (bone morphogenetic protein), granulocyte-macrophage colony stimulating factor (GM-CSF), or a combination thereof, which can promote tissue regeneration, can be attached or coated onto the matrix of the present invention.

[0227] In one embodiment, the growth factor is VEGF. In another embodiment, the growth factor is PDGF. Those skilled in the art are aware of various other materials and biomolecules that can be attached to or used to coat the matrix of the presently disclosed subject matter and can select them for a particular application based on the tissue to which they will be applied.

[0228] In one embodiment, extracellular matrix proteins such as fibronectin, laminin and / or collagen are further attached 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 inventors have found that these proteins help provide secondary cell signals that, in conjunction with growth at an air-liquid interface (e.g., on a gas permeable membrane), lead to proper stratification of skin cells grown using the matrix.

[0229] In one embodiment, collagen IV is used. Collagen IV has been shown to be particularly effective in producing proper epidermal stratification.

[0230] The extracellular matrix protein may be in the form of a full-length protein or a peptide thereof, such as a synthetic peptide.

[0231] In another embodiment, the 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 the methods, matrix and / or skin tissue of the present invention. Examples of therapeutic agents that can be used include, but are not limited to, proteins, peptides, drugs, cytokines, extracellular matrix molecules and / or growth factors. Those skilled in the art will appreciate other suitable and / or advantageous therapeutic agents that can be used in accordance with the present invention.

[0232] In one embodiment, the therapeutic agent is an anti-inflammatory agent or an antibiotic. Examples of anti-inflammatory agents that can be incorporated into the matrix include, but are not limited to, steroidal anti-inflammatory agents such as betamethasone, triamcinolone acetonide, 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 meclofenamic acid, mefenamic acid, piroxicam and suprofen.

[0233] Various antibiotics may also be used 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, grefloxacin, 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 cefdroxil, cefazolin, cephalexin, cephalothin, cefpirin, cephradine, cefaclor, cefmandole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, loracarbef, cefdinir, cefditoren, cefixime, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, and ceftriaxone or cefepime; monocyclic β-lactams, such as aztreonam; nitroimidazoles, such as metronidazole; oxazolidinones, such as linezolid; penicillins, such as amoxicillin, amoxicillin / clavulanic acid, ampicillin, ampicillin / sulbactam, bacillin, carbenicillin, cloxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillin V, piperacillin, piperacillin / tazobactam, ticarcillin, or ticarcillin / clavulanic acid; streptogramins, For example, 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, or terbinafine.

[0234] In one embodiment, various analgesics and / or anesthetics are attached to or incorporated into the matrix of the present invention. As used herein, the term "analgesic" refers to a drug used to relieve pain, and in some embodiments, can be used interchangeably with the term "anti-inflammatory agent" so that the term analgesic can include exemplary anti-inflammatory agents described herein. Exemplary analgesics include, but are not limited to, paracetamol and nonsteroidal anti-inflammatory agents, COX-2 inhibitors, and opiates (e.g., morphine) and morphine mimetics.

[0235] As used herein, the term "anesthetic" refers to an agent used to induce a reversible loss of sensation in a subject and thus can 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, dicaine, cocaine, lidocaine, prilocaine, bupivacaine, levobupivacaine, ropivacaine, carbocaine, and dibucaine.

[0236] Culture device of the present invention

[0237] Any cell culture device suitable for producing a whole human epidermis can be used to carry out the method of the present invention. WO2016 / 209089 describes such an apparatus, the contents of which are incorporated herein by reference. The skilled person will be aware of other alternative culture devices.

[0238] Use of the whole human epidermis of the present invention

[0239] The present invention provides the use of tissues, such as epithelium, epidermis, stratified epithelium, stratified epidermis and dermis, split-thickness skin or full-thickness skin, prepared using the methods of the present invention, for example, using the matrix of the present invention for treating tissue damage in a subject in need thereof.

[0240] The term "subject" is used herein to refer to both human and animal subjects, but is generally intended to refer to a human patient in need of treatment.

[0241] As used herein, the term "treat" includes, but is not limited to, inhibiting the progression of damage to tissue, arresting the development of damage to tissue, reducing the severity of damage to tissue, ameliorating or alleviating symptoms associated with tissue damage, and repairing, regenerating and / or causing regression of damaged tissue or regression of one or more symptoms associated with damaged tissue.

[0242] The present invention provides a method of treating tissue damage in a subject in need thereof, the method comprising:

[0243] a) providing a skin tissue, such as epithelium, stratified epithelium, epidermis, stratified epidermis, stratified epidermis and dermis, split-thickness skin or full-thickness skin, which has been grown using a method such as described herein, such as using a method for growing whole human epidermis produced using a method such as described herein,

[0244] b) restoring the tissue using a matrix under aseptic or sterile conditions, and

[0245] c) applying the tissue to a patient.

[0246] In various embodiments, the tissue damage is a wound, a chronic wound, a surgical wound, an ulcer, a non-healing wound, a scar, a surgical scar, a scald or a burn. In various embodiments, the burn is a first degree burn, a second degree burn, a third degree burn, a deep skin burn or a full thickness burn.

[0247] In various embodiments, the tissue damage is an epithelium located on a mucosal surface. In various embodiments, the epithelium is located on or in the skin, lungs, gastrointestinal tract (e.g., esophagus or oral cavity), reproductive tract, or urinary tract (e.g., urethra).

[0248] Other examples of treatment include, but are not limited to: skin regeneration of nerves and organelles; wound healing, such as promoting / enhancing wound healing, including ulcers, such as diabetic ulcers; burn healing; skin regeneration and repair; epidermolysis bullosa; improving skin quality or appearance; preventing or repairing skin diseases; reduction or disappearance of scar tissue; breast skin regeneration (post-surgery); cosmetic applications, such as anti-aging; dermal regeneration for wrinkles and other skin defects; promoting hair follicle growth, nerve and other organelle regeneration; healing without scarring, or re-healing to reduce scarring.

[0249] In one embodiment, the invention provides a matrix, skin tissue and method that can be used to regenerate damaged, lost and / or degenerated tissue. For example, the matrix, method or skin tissue of the invention can be used to initiate, increase, support, promote and / or guide the regeneration of damaged, lost and / or degenerated tissue, particularly the regeneration of damaged skin.

[0250] As used herein, "regeneration" refers to any method or property that initiates, increases, regulates, promotes, supports and / or directs the growth, regrowth, repair, function, patterning, connectivity, reinforcement, vitality, and / or natural wound healing process of fragile, damaged, lost and / or degenerated tissue. These terms may also refer to any process or property that initiates, increases, regulates, promotes, supports and / or directs the growth, reinforcement, function, vitality, resilience, effectiveness and / or health of fragile, tired and / or normal tissue.

[0251] As used herein, the term "wound" broadly refers to injuries to the skin and subcutaneous tissue in different ways (e.g., bedsores caused by prolonged bed rest and wounds caused by trauma) and with different characteristics. Wounds are generally classified into one of four grades based on the depth of the wound: Grade I: wounds limited to the epithelium; Grade II: wounds extending to the dermis; Grade III: wounds extending to the subcutaneous tissue; and Grade IV (or full-thickness wounds), i.e. wounds with exposed bones (e.g., bone pressure points, such as the greater trochanter or sacrum).

[0252] As used herein, the term "superficial wound" refers to wounds including grades I-III; for example, burns, bedsores, venous congestion ulcers, and diabetic ulcers. As used herein, the term "deep wound" is used to describe grade III and IV wounds.

[0253] In one embodiment, a skin tissue, such as epithelium, epidermis, stratified epithelium, stratified epidermis and dermis, split-thickness skin, or full-thickness skin, is provided, which is prepared using the methods described herein for facilitating skin transplantation by covering an area of ​​damaged, injured, traumatized, diseased, removed, or missing skin tissue from a subject's body.

[0254] As used in the present invention, "graft" refers to cells, tissues or organs that are usually implanted into an individual to replace, correct or otherwise overcome defects. Therefore, "skin graft" is a skin tissue that can be implanted into an individual, such as sutured to the individual. Graft can further include a matrix of the present invention, such as wherein the matrix is ​​integrated into the skin graft. Tissue or organ can be composed of cells originating from the same individual. The graft is referred to by the following interchangeable terms in the present invention: "allograft", "autologous transplant", "autologous implant" and "autologous graft". The present invention refers to a graft comprising cells from genetically different individuals of the same species by the following interchangeable terms: "allograft", "allogeneic transplant", "allogeneic implant", "allogeneic graft" and "heterologous graft". "Xenograft," "xenogeneic transplant," or "xenogeneic implant" refers to a transplant from one individual to another species.

[0255] 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 that are autologous to the subject. In alternative embodiments, the tissue is prepared using cells that are allogeneic to the subject. In another embodiment, the tissue is prepared using a combination of cells, some of which are autologous to the subject and some of which are allogeneic to the subject.

[0256] It should be understood that cells (e.g., autologous to the subject) can be isolated using any method known in the art. For example, cells (e.g., autologous cells) can be isolated from a skin sample or a skin biopsy obtained from a subject by digesting the sample tissue and isolating fibroblasts and / or keratinocytes from the digested tissue.

[0257] In one embodiment, the tissue is an autologous graft, such as a skin autologous graft. In various embodiments, the tissue is an epidermal autologous graft, a split-thickness autologous graft, or a full-thickness autologous graft. In another embodiment, the tissue is an allograft.

[0258] It will be appreciated that there is a great need for tissues prepared using a patient's own cells, such as autologous transplants, to reduce or prevent immune rejection of the tissue and reduce the need for ongoing immunotherapy or other adjunctive therapies.

[0259] In one embodiment, the tissue further comprises a matrix. In another embodiment, the tissue is separated from the matrix prior to administration to a patient.

[0260] Typically, the tissue is administered to the patient surgically. In one embodiment, the recovery under sterile or aseptic conditions is during or prior to surgery, such as in an operating room.

[0261] Typically, the tissue is administered to a patient at or near a site of tissue damage. In various embodiments, the tissue is administered to at least partially cover a site of tissue damage or to completely cover a site of tissue damage.

[0262] In one embodiment, the tissue is administered to temporarily cover a site of tissue damage. In an alternative embodiment, the tissue is administered to permanently cover a site of tissue damage.

[0263] Other non-medical uses

[0264] The efficacy and safety of topically applied drugs, nutraceuticals or cosmetics are often tested using animal skin or live animal, human cadaver skin or synthetic human skin models.

[0265] The morphological differences between animal and human skin mean that cut animal skin or live animals for product testing are suboptimal. In addition, there are considerable ethical concerns regarding the use of live animals or animal skins to test cosmetics, including bans on such testing in some countries. For these reasons, there is a strong desire to find alternatives to animal models for testing such products.

[0266] Inconsistent and highly variable results have been observed when human cadaver skin has been used for product testing.

[0267] Therefore, cells or tissues, such as skin tissues, prepared using the device or method of the present invention can be used for in vitro testing of drugs, nutritional products and / or cosmetics.

[0268] In various embodiments, cells or tissues prepared using the devices or methods described herein are used to test transdermal penetration of compounds, to test penetration of compounds across the epidermis, dermis, or basement membrane, to test the efficacy of active ingredients in treating or preventing a condition, such as a skin condition, or to test the toxicity of compounds.

[0269] More particularly, the skin tissue produced according to the invention is suitable for testing products, for example effectiveness, undesirable side effects, such as irritation, toxicity and inflammation or allergenic effects, or compatibility of substances. These substances can be substances that are potentially used as drugs, such as substances used as dermatological drugs, but also substances that are ingredients of cosmetics or even consumer products that come into contact with the skin (such as washing powders, etc.).

[0270] The skin tissue of the present invention can also be used, for example, to study the absorption, transfer and / or penetration of substances. It is also suitable for studying other agents (physical quantities), such as light or heat, radioactivity, sound, electromagnetic radiation, electric fields, for example, for studying phototoxicity, i.e. the destructive effect of light of different wavelengths on cell structure. The skin tissue can also be used to study wound healing, and is also suitable for studying the effects of gases, aerosols, smoke on cell structure or metabolism or gene expression.

[0271] In various embodiments, cells or tissues are used to determine whether a compound of interest is a skin irritant, for example, to determine whether a compound of interest induces a rash, inflammation, or contact dermatitis.

[0272] The effects of substances or agents on human skin can be determined, for example, by the release of substances (e.g., cytokines or mediators) by cells of human or animal skin model systems and the effects on gene expression, metabolism, proliferation, differentiation and reorganization of these cells. Using methods for quantifying cell damage, more particularly, using vital dyes such as tetrazolium derivatives, cytotoxic effects on skin cells, for example, can be detected. Testing of substances or agents using skin tissue can include histological procedures as well as immunological and / or molecular biological procedures.

[0273] As used herein, a "test agent" is any substance that is evaluated for its ability to diagnose, cure, alleviate, treat or prevent disease in a subject or is intended to change the structure or function of the subject's body. Test agents include, but are not limited to, chemical compounds, biological agents, proteins, peptides, nucleic acids, lipids, polysaccharides, supplements, signals, diagnostic agents, and immunomodulators. The test agent may further include electromagnetic and / or mechanical forces.

[0274] In another embodiment, the skin tissue produced according to the present invention can be used as a model system for studying skin diseases and for developing new treatments for skin diseases. For example, cells from patients with certain genetic or acquired skin diseases can be used to establish patient-specific skin model systems, which in turn can be used to study and evaluate the effectiveness of some therapies and / or drugs.

[0275] In one embodiment, the skin tissue may be populated with microorganisms, more particularly pathogenic microorganisms, having a population of pathogenic or parasitic microorganisms, especially including human pathogenic microorganisms.

[0276] As used herein, "microorganism" generally refers to fungi, bacteria and viruses. Microorganisms are preferably selected from fungi or pathogens and / or parasitic bacteria known to infect the skin. These include, but are not limited to, species of Candida albicans, Trichophyton mentagrophytes, Malassezia furfur and Staphylococcus aureus.

[0277] Using the correspondingly filled skin tissue, it is possible to study the processes of the microbial population of microorganisms themselves, in particular the infection processes of the microorganisms themselves, and also the reaction of the skin to this population. In addition, it is possible to study the influence of substances applied before, during or after the colonization on the population itself or the influence of the population on the skin tissue.

[0278] In various embodiments, the cells comprise fibroblasts, keratinocytes or immune cells, or any combination of two or more thereof. In one embodiment, the cells comprise fibroblasts and keratinocytes. In various embodiments, the tissue is selected from epidermis, stratified epidermis and dermis, stratified epidermis and dermis, split-thickness skin or full-thickness skin.

[0279] In various embodiments, the compound is a pharmaceutical compound, a cosmetic compound, or a nutraceutical compound.

[0280] In various embodiments, the compound for testing is administered to the tissue alone or in admixture with a pharmaceutically or cosmetically acceptable carrier, excipient, or diluent.

[0281] In various embodiments, the compounds for testing are applied topically to tissue in the form of a sterile cream, gel, pour-on or spot-on formulation, suspension, lotion, ointment, sprinkle, dip, spray, medicated dressing, shampoo, collar, or skin patch.

[0282] As used in the present invention, the term "breathable material" or "breathable membrane" refers to a material or membrane through which gas exchange can occur.

[0283] In the context of this specification, "comprising" is intended to mean "including".

[0284] The embodiments of the present invention may be combined where technically appropriate.

[0285] The present invention describes embodiments as comprising certain features / elements. The present invention also extends to separate embodiments consisting of or consisting essentially of said features / elements.

[0286] Technical references such as patents and applications are incorporated herein by reference.

[0287] Any embodiment of the invention specifically and explicitly described may form the basis for a disclaimer either alone or in combination with one or more other embodiments.

[0288] The background of the present specification contains technical information related to the disclosure of the present invention and can be used as a basis for modification.

[0289] This application claims priority to GB1715930.2 filed on September 30, 2018, which is incorporated herein by reference. The priority document may be used as a basis for correction of errors in this application.

[0290] The present invention will now be described with reference to the following examples, which are merely illustrative and should not be construed in any way as limiting the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0292] Figure 1 It shows that some components in Green medium are not essential for the growth of keratinocytes in the presence of ROCK inhibitors. Human primary keratinocytes were recovered from cryopreservation and grown in Green medium, Kelch medium ("basal medium" + KGF in the figure) or DF10 (all with the addition of ROCK inhibitor Y27632) until 80% confluence was reached. In order to determine the non-essential components in Green medium, one component was removed from the Green mixture at a time. KGF was added to identify its effect on the growth of keratinocytes in each culture medium. Keratinocytes were grown in the presence (right) or absence (left) of mouse embryonic feeder cells (MEF). To evaluate the growth of keratinocytes, the cells were fixed, stained with rhodamine B, and the confluence was measured using an Operetta plate reader. The results are shown in triplicate for a representative donor (+SD), with the growth level of Green medium + Y27632 indicated by the dotted line. N=2. These results indicate that even in the absence of MEFs, Kelch's medium is as effective as Green's medium in inducing keratinocyte proliferation when ROCK inhibitor is added. This also suggests that both cholera toxin and EGF are important components of Green's medium in generating keratinocyte proliferation.

[0293] Figure 2 Comparison of keratinocyte growth in Kelch medium and Green medium is shown. Human primary keratinocytes from frozen epidermal preparations were grown to 80% confluence in different medium supplemented with the ROCK inhibitor SB772077B. Cells were grown in regular Green medium with or without MEF (right), or in basal medium supplemented with KGF, MEF, or both (left). Kelch medium is represented in the figure by basal medium plus KGF. To assess the growth of keratinocytes, cells were fixed, stained with rhodamine B, and confluence was measured using an Operetta plate reader. The results show the mean of 3 donors (+SD) and were statistically analyzed using a two-tailed Student's t-test. N=3. These results indicate that Kelch medium produces keratinocyte proliferation at least as well as Green medium, regardless of the presence or absence of MEF feeder cells. It also indicates that KGF is a key component in Kelch medium, as the proliferation of keratinocytes in basal medium plus ROCK inhibitor is significantly lower than the proliferation of keratinocytes in basal medium plus ROCK inhibitor plus KGF. In the absence of feeder cells, the addition of KGF to Kelch's medium resulted in proliferation of keratinocytes statistically equivalent to proliferation in Green's medium in the presence of feeder cells, confirming the ability of Kelch's medium to eliminate xenogeneic feeder cells.

[0294] Figure 3 It shows that ROCK inhibitors Y27632 and SB772077B have a comparable effect on the growth of keratinocytes. Frozen human keratinocytes were grown in basal medium supplemented with Y27632 or SB772077B, basal medium plus KGF ("Kelch medium" in the legend), or Green medium. To evaluate the growth of keratinocytes, the cells were fixed, stained with rhodamine B, and the degree of confluence was measured using an Operetta plate reader. The results are shown in triplicates (+SD) from three donors. N=3. These results indicate that after adding ROCK inhibitors, Kelch medium (basal medium plus KGF) can drive the proliferation of keratinocytes, which is better than Green medium or basal medium without KGF plus ROCK inhibitors, indicating that KGF is a key component of Kelch medium.

[0295] Figure 4It was shown that Kelch medium plus ROCK inhibitor SB772077B) and Green medium plus ROCK inhibitor provide equivalent keratinocyte growth. Skin cells digested with trypsin and collagenase were grown in Kelch medium and Green medium, both containing ROCK inhibitor SB772077B. Triplicate samples were grown for up to 30 days and passaged 1:9 when they reached 80-90% confluence. N=1.

[0296] Figure 5 The effect of serum-free Optipeak medium (InVitria) on keratinocyte growth is shown. Human primary keratinocytes in cryopreserved epidermal preparations were cultured with mouse embryonic feeder cells (MEFs) in 4 different formulations of serum-free Optipeak medium or Green medium for 8 days. All culture media were tested with and without the ROCK inhibitor Y27632. To evaluate the growth of keratinocytes, the cells were fixed, stained with Rhodamine B, and the confluence was measured using an Operetta plate reader. The results are shown in triplicate for one representative donor (+SD). N=2. These results indicate that although ROCK inhibitors enhance the growth of keratinocytes in serum-free Optipeak medium, the resulting proliferation is still not as good as the proliferation produced by culturing in Green medium in the presence of MEFs. These results also indicate that ROCK inhibitors can enhance the growth of keratinocytes in Green medium in the presence of MEFs.

[0297] Figure 6 A comparison of keratinocyte growth rates between Green medium and commercially available keratinocyte growth medium is shown. Epidermal cells were grown in Green medium, CnT Prime, Epilife medium supplemented with S7 or EDGS containing MEFs. All media were tested with and without the ROCK inhibitor Y27632. 550,000 epidermal cells were grown in each medium until one sample reached 80% confluence, at which time all samples were fixed and stained with Rhodamine B. The confluence of the sample was then assigned a score between 0 and 4, with 0 representing no cells and 4 representing maximum confluence. N=2. These results indicate that ROCK inhibitors can also enhance the growth of keratinocytes in other keratinocyte growth media, but unlike Kelch medium, none of these media produced keratinocyte growth comparable to Green medium plus MEFs. Example

[0298] CnT-Prime is a fully defined animal component-free culture medium available from CellnTec. It is suitable for the isolation and expansion of epithelial cells from skin, cornea, gingival, breast and bladder tissues. It was developed using human tissue but can also be used with other species such as mice.

[0299] Supplement S7 is available from ThermoFisher. It is a sterile, concentrated (100X), ionically balanced solution intended for use as a component in the complete culture environment of human epidermal keratinocytes. Supplement S7 is chemically defined and free of animal origin. Each 5 ml bottle of Supplement S7 is available in 500 ml bottles. Correct supplementation dosage for basal medium.

[0300] EDGS is also available from ThermoFisher. It is a defined, sterile, concentrated (100X) solution designed for use with EDGS is not intended for use with Medium 154. EDGS contains all the growth factors and hormones required for the growth of human epidermal keratinocytes. Each bottle contains the correct amount of 500 ml bottle of Supplement for culture 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 are isolated from animals of North American origin. The isolation process for these components includes a step to inactivate viruses. All components of EDGS are greater than 95% pure.

[0301] Example 1 - Methods

[0302] Material

[0303] Green medium - DMEM high glucose:Hams F12 3:1, 10% fetal bovine serum, penicillin, streptomycin, 10ng / ml EGF, 0.1nM cholera toxin, 0.4μg / ml hydrocortisone, 180μM adenine, 5ug / ml insulin, 5μg / ml apo-transferrin, 2nM 3,3,5,-tri-iodinated thyronine, 2mM glutamine, 0.625μg / ml amphotericin B, 10μM Y27632

[0304] Kelch medium - DMEM high glucose: Hams F12 3:1, 10% fetal bovine serum, penicillin, streptomycin, 0.625 μg / ml amphotericin, 20 ng / ml KGF

[0305] Basal medium - DMEM high glucose: Hams F12 3:1, 10% fetal bovine serum, penicillin, streptomycin, 0.625 μg / ml amphotericin

[0306] The final concentration of Y27632 was 10 μM. The final concentration of SB772077B was 400 nM.

[0307] CnT Prime – Cellntech catalog number CNT-PR; Epilife medium – Thermofisher catalog number MEPI500CA; S7 supplement – ​​Thermofisher catalog number S0175; EDGS – Thermofisher catalog number S0125; coating matrix – Thermofisher catalog number R011K; Y27632 – Selleckchem catalog number S1049; SB772077B - Tocris catalog #4118

[0308] Tryple–Life Technologies Catalog #12604021

[0309] Comparison of Green, Cellntec, S7, and EDGE media

[0310] Pre-coat flasks with coating matrix of Epilife medium samples supplemented with S7 and EDGS. Add 1.7 ml of dilution buffer to each T25 flask. Then add 17 μl of coating matrix. Incubate the mixture at room temperature for 30 minutes. Next, remove the dilution buffer and coating matrix mixture from the flasks before adding cells and medium.

[0311] 550,000 epidermal cells and 5 ml of each required culture medium (with or without 10 μl Y27632) were added to a T25 flask. The flask was incubated at 37°C with 5% CO. 2 Incubate until one sample reaches 80% confluency.

[0312] Next, remove the medium from the flask and fix the cells with 4% formaldehyde for 30 minutes. Add 1% Rhodamine B solution and incubate the cells at room temperature for 10 minutes. Remove the Rhodamine B solution and rinse the cells thoroughly with water. Finally, remove all liquid from the flask using a pipette. Image the cells and calculate the degree of confluence.

[0313] Trypsin and collagenase digestion methods

[0314] All subcutaneous fat and subcutaneous tissue were trimmed from the skin sample. Next, the skin was cut into 1 cm 2 Finally, a deep scarification was made on the underside of the dermis using a scalpel.

[0315] The skin fragments were placed in the wells of a 6-well plate. Next, 5 mg / ml collagenase (type I) was prepared with MilliQ water and the enzyme stock solution was filtered through a 0.2 um syringe filter in a fume hood. 0.25% trypsin was then diluted to 0.1% in D0 and 600 μl of collagenase was added to make a total volume of 6 ml. The plate was placed at 37°C, 5% CO 2 incubator overnight.

[0316] The next day, the culture medium containing the skin and enzyme was transferred to 6 ml DF10 in a 10 cm dish and the skin was separated with a scalpel. Next, the skin was further broken up by repeated passage through a pipette. Next, the cell mixture was passed through a 100 μm filter.

[0317] Next, the mixture was centrifuged at 1800 rpm for 10 min and as much supernatant as possible was removed without disturbing the cell pellet. Finally, the cell pellet was resuspended in DF10 to assess cell number and viability.

[0318] Keratinocyte expansion

[0319] Add 4.5 ml of keratinocyte growth medium with 400 nM SB 772077B to the T25 flask.

[0320] Add 500 μl of the single-cell suspension from the trypsin / collagenase digestion process to a T25 flask. Then incubate the cells at 37 °C / 5% CO. 2 Incubate overnight.

[0321] All medium was removed and replaced with 5 ml of keratinocyte growth medium with 400 nM SB772077B.

[0322] Cell growth was monitored until the flasks reached 80-90% confluence and then cells were passaged for continued expansion.

[0323] Passage 1 Keratinocytes

[0324] Remove all culture medium from the T25 flask.

[0325] Use 1 ml of TrypLE TM Wash the cells once. Add 2 ml of Tryple and incubate the mixture at 37°C / 5% CO. 2 Incubate at 4 °C until most of the cells detach by tapping the flask vigorously.

[0326] Add 4 ml of keratinocyte growth medium with 400 nM SB772077B to the T25 flask. Next, rinse the bottom of the flask 3 times using a serological pipette. This detaches any remaining cells and breaks up the cell clumps.

[0327] Add 10 ml of keratinocyte growth medium with 400 nM SB772077B to the T75 flask. Add one third of the dissociated cell mixture to the T75 flask.

[0328] Next, incubate the cells at 37°C / 5% CO 2 Monitor cell growth until the flask reaches 80-90% confluency, then passage the cells for continued expansion.

[0329] Passaging of keratinocytes from P2 to Px

[0330] Remove all medium from the T75 flask. Next, wash the cells with 2 ml of TrypLE TM Wash once. Add 4 ml TrypLE TM Incubate cells at 37°C / 5% CO 2 Incubate at 4 °C until most of the cells detach by tapping the flask vigorously.

[0331] Next, add 8 ml of Keratinocyte Growth Medium with SB772077B to the T75 flask. Then rinse the bottom of the flask 3 times with a serological pipette. This will detach any remaining cells and break up any cell clumps.

[0332] Add 11 ml of keratinocyte growth medium with 400 nM SB772077B to the T75 flask. Then transfer one tenth of the separated cell mixture to the T75 flask.

[0333] Incubate cells at 37°C / 5% CO 2 Monitor cell growth until the flask reaches 80-90% confluency and then subculture cells for further expansion.

[0334] Example 2 - Determination of which components of Green's medium are not required for keratinocyte growth.

[0335] exist Figure 1In the study, the active components of Green medium were evaluated by excluding them one at a time from the mixed medium. Importantly, the cells were grown in the presence of a ROCK inhibitor (Y27632), which stimulates cell growth and obsoletes the effects of some of the Green medium components. The right figure shows that if keratinocytes are co-cultured with mouse embryonic feeder cells (MEFs), they grow rapidly in both mixtures. The absence of any of the individual components did not have a noticeable effect. However, if MEFs were not present (left figure), the absence of some components had a negative impact on keratinocyte growth, namely epidermal growth factor (EGF) and cholera toxin. We later found that EGF did not enhance keratinocyte growth (data not shown).

[0336] In summary, cholera toxin is one of the most important single components of Green medium. Interestingly, keratinocyte growth factor (KGF) added to basal medium (DMEM:F12 3:1, 10% fetal bovine serum, penicillin, streptomycin, amphotericin) + Y27632 stimulated growth, but failed to enhance keratinocyte growth in Green medium + Y27632 and failed to rescue growth in medium lacking cholera toxin. Overall, keratinocyte growth in basal medium + KGF + Y27632 (Kelch's medium) was similar to or even better than growth in Green medium Y27632.

[0337] Deletion of adenine alone from Green medium + Y27632 enhances keratinocyte growth.

[0338] Keratinocytes also grew much better in Green or Kelch medium compared to DF10+KGF.

[0339] Example 3 - Comparison of keratinocyte growth in Kelch's medium and Green's medium

[0340] Figure 2The growth of keratinocytes in Kelch medium and Green medium, both of which were supplemented with ROCK inhibitors, was compared. Keratinocytes grew best in the presence of MEFs compared to medium alone, and this was true for both Kelch and Green medium. In the presence of ROCK inhibitors, adding KGF to the basal medium (basal medium + KGF = Kelch medium) had a similar effect to adding MEFs to the basal medium, indicating that KGF is an effective substitute for MEFs in keratinocyte culture. In Kelch medium, the growth of keratinocytes was the same as Green medium (+ / -MEF). Keratinocytes in Kelch medium with ROCK inhibitors grew significantly better than basal medium with ROCK inhibitors in the absence of KGF, but there was no significant difference compared to Kelch medium + MEF + ROCK inhibitor or Green medium + ROCK inhibitor (+ / -MEF).

[0341] Therefore, Kelch's medium resulted in similar keratinocyte growth rates compared to Green's medium.

[0342] Example 4 - Comparison of keratinocyte growth using Y27632 and SB772077B

[0343] Figure 3 The similarity of the effects of ROCK inhibitors Y27632 and SB772077B on keratinocyte growth was shown. Therefore, SB772077B can be used as an alternative to Y27632 for keratinocyte growth.

[0344] Example 5 - Comparison of the growth of keratinocytes obtained from skin digested with trypsin collagenase in Kelch's medium and Green's medium

[0345] Skin samples digested with trypsin and collagenase were grown in Green medium supplemented with SB772077B or in Kelch medium supplemented with SB772077B. Figure 4 A graph showing keratinocyte proliferation rates is shown. Over a 30-day period, keratinocyte growth in Kelch's medium was comparable to that in Green's medium. Therefore, Kelch's medium can be substituted for Green's medium as it provides equivalent keratinocyte growth kinetics and does not require cholera toxin.

[0346] Example 6 - Determination of serum-free Opti-Peak TM Experiments on potential uses of the culture medium

[0347] Figure 5 Serum-free Optipeak media were examined for potential use in growing keratinocytes. We tested four Opti-Peak TM Media formulations. When any of these media were used alone, the cells did not show any noticeable growth. If the ROCK inhibitor Y27632 was added to the cultures, some colonies began to form. However, none of the 4 formulations was as good as Green medium, which by itself, especially in the presence of Y27632, had a significant effect on the growth of keratinocytes.

[0348] Example 7 - Comparison of Keratinocyte Growth Between Green Medium and Commercial Keratinocyte Growth Medium

[0349] Figure 6 A comparison of keratinocyte growth between Green medium and commercially available keratinocyte growth medium is shown. S7 and EDGS are used to supplement Gibco's Media. CnT Prime was obtained from Cellntech. All commercially available media are designed to be serum-free and feeder-free. Green medium uses serum and mouse embryonic feeder cells. All media are used with and without the ROCK inhibitor Y27632, which is known to have a positive effect on the growth of keratinocytes. Green medium is supplemented with ROCK inhibitor and mouse embryonic feeder cells to provide the fastest keratinocyte growth. ROCK inhibitor enhances the growth of keratinocytes in Green medium, S7 medium, and CnT Prime medium.

Claims

1. A cell culture medium without cholera toxin, comprising the following components: DMEM, Ham's F12, serum, one or more antibiotics and / or antifungal agents, keratinocyte growth factor, and ROCK inhibitor, wherein the cholera toxin-free cell culture medium does not contain cholera toxin.

2. The cholera toxin-free cell culture medium according to claim 1, wherein the DMEM is high glucose.

3. The cholera toxin-free cell culture medium according to claim 1 or 2, wherein the serum is mammalian.

4. The cholera toxin-free cell culture medium according to claim 3, wherein the serum is fetal bovine serum.

5. The cholera toxin-free cell culture medium of claim 1, wherein the serum is present at a concentration of 5 to 15%.

6. The cholera toxin-free cell culture medium according to claim 5, wherein the serum is present at a concentration of 10%.

7. The cholera toxin-free cell culture medium of claim 1, comprising at least one antibiotic.

8. The cholera toxin-free cell culture medium according to claim 7, wherein The antibiotics are independently selected from penicillin, streptomycin, ampicillin, amoxicillin, carbenicillin, cefotaxime, gentamicin, kanamycin, neomycin, polymyxin, blasticidin, geneticin, hygromycin B, mycophenolic acid, puromycin, bleomycin and combinations of two or more thereof.

9. The cholera toxin-free cell culture medium according to claim 7, wherein the antibiotic is gentamicin.

10. The cholera toxin-free cell culture medium according to claim 7, wherein the antibiotic is penicillin.

11. The cholera toxin-free cell culture medium according to claim 7, wherein: The antibiotic is streptomycin.

12. The cholera toxin-free cell culture medium according to claim 1 or 7, comprising an antifungal agent.

13. The cholera toxin-free cell culture medium according to claim 12, wherein: The antifungal agent is selected from the group consisting of amphotericin B, nystatin, actinomycin D, fosmidomycin, mycophenolic acid, and a combination of two or more thereof.

14. The cholera toxin-free cell culture medium according to claim 13, wherein The antifungal agent is amphotericin B.

15. The cholera toxin-free cell culture medium according to claim 14, wherein The antifungal agent is amphotericin B at a concentration of 0.500 to 0.750 μg / ml.

16. The cholera toxin-free cell culture medium according to claim 14, wherein: The antifungal agent was amphotericin B at a concentration of 0.625 μg / ml.

17. The cholera toxin-free cell culture medium according to claim 1, wherein: The ratio of DMEM:Ham's F12 is 2.5 to 3.5:1.5 to 0.

5.

18. The cholera toxin-free cell culture medium according to claim 17, wherein: The ratio of DMEM:Ham's F12 was 3:

1.

19. The cholera toxin-free cell culture medium according to claim 1, wherein the concentration of the keratinocyte growth factor is in the range of 10 ng / ml to 30 ng / ml.

20. The cholera toxin-free cell culture medium according to claim 19, wherein the concentration of the keratinocyte growth factor is 20 ng / ml.

21. The cholera toxin-free cell culture medium according to claim 1, wherein: The ROCK inhibitor is a small molecule ROCK inhibitor.

22. The cholera toxin-free cell culture medium according to claim 21, wherein The ROCK inhibitor is selected from: SB 772077B, Y-27632, fasudil, Ripasudil, Y39983, Wf-536, SLx-2119, azabenzimidazole-aminofurazan, DE-104, H-1152, ROKα inhibitor, XD-4000, HMN-1152, 4-(1-aminoalkyl)-N-(4-pyridyl)cyclohexane-carboxamide, rhostatin, BA-210, BA-207, BA-215, BA-285, BA-1037, Ki-23095, VAS-012, RKI-1447, GSK429286A, Y-30141, HA-100, H-7, iso H-7, H-89, HA-1004, HA-1077, H-8, H-9, KN-62, GSK269962, quinazoline, and combinations of two or more thereof.

23. The cholera toxin-free cell culture medium according to claim 21 or 22, wherein: The concentration of the ROCK inhibitor ranged from 0.1 to 100 μM.

24. The cholera toxin-free cell culture medium according to claim 23, wherein: The concentration of the ROCK inhibitor ranges from 0.2 to 50 μM, or 0.3 to 25 μM, or 0.1 to 10 μM, or 0.1 to 0.95 μM, or is present at a concentration of 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, or 0.95 μM.

25. The cholera toxin-free cell culture medium according to claim 24, wherein The ROCK inhibitor was present at a concentration of 0.4 μM.

26. The cholera toxin-free cell culture medium of claim 21, wherein the ROCK inhibitor is SB772077B, Y-27632, or a combination of both.

27. The cholera toxin-free cell culture medium of claim 26, wherein the ROCK inhibitor is SB772077B.

28. An in vitro method for producing human full-thickness skin, whole human epidermis or whole human dermis comprising a first culturing step, wherein human keratinocytes and feeder cells are cultured in a cholera toxin-free cell culture medium according to any one of claims 1 to 27.

29. The in vitro method according to claim 28, wherein Human keratinocytes were cultured without the presence of xenogeneic feeder cells.

30. The in vitro method according to claim 28 or 29, wherein The feeder cells are fibroblasts.

31. An in vitro method according to claim 30, wherein the fibroblasts are fully human.

32. The in vitro method according to claim 30, wherein Fibroblasts were not irradiated.

33. The in vitro method according to claim 28 or 29, wherein the feeder cells and keratinocytes are from the same donor.

34. The in vitro method according to claim 30, wherein the fibroblasts and keratinocytes are from the same donor.

35. A complete skin product comprising human epidermis cultured by the method of any one of claims 28 to 34.

36. The whole skin product of claim 35, comprising differentiated human dermis and human epidermis.

37. A total skin product according to claim 35 for use in therapy.

38. A total skin product according to claim 36 for use in therapy.

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