EP3562934A1 - Procédé de transport de tissu cultivéin vivo - Google Patents

Procédé de transport de tissu cultivéin vivo

Info

Publication number
EP3562934A1
EP3562934A1 EP17840580.9A EP17840580A EP3562934A1 EP 3562934 A1 EP3562934 A1 EP 3562934A1 EP 17840580 A EP17840580 A EP 17840580A EP 3562934 A1 EP3562934 A1 EP 3562934A1
Authority
EP
European Patent Office
Prior art keywords
tissue
substrate
gas permeable
cells
scaffold
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17840580.9A
Other languages
German (de)
English (en)
Inventor
Michelle Barbara LOCKE
Peter Roderic DUNBAR
Vaughan John Feisst
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Auckland Uniservices Ltd
Original Assignee
Auckland Uniservices Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Auckland Uniservices Ltd filed Critical Auckland Uniservices Ltd
Publication of EP3562934A1 publication Critical patent/EP3562934A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/24—Gas permeable parts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/28—Constructional details, e.g. recesses, hinges disposable or single use
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/50—Means for positioning or orientating the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/54—Constructional details, e.g. recesses, hinges hand portable
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2535/00—Supports or coatings for cell culture characterised by topography

Definitions

  • the present disclosure relates to the improved transportation of a tissue, for example, tissue comprising epithelial cells, such as full thickness skin, produced using a device of the disclosure or by a method the disclosure, the combination of a device of the present disclosure and tissue packaged for transportation, and the use of such tissue for treating tissue damage.
  • a tissue for example, tissue comprising epithelial cells, such as full thickness skin
  • engineered tissues comprising epithelial cells, such as human skin may be used for autologous grafts for patients with burns or chronic wounds or the development and testing of pharmaceuticals, cosmetics or other topical products.
  • the cells comprising epithelial cells, such as keratinocytes, and dermal cells, such as fibroblasts are a cultured on a spun or woven substrate.
  • This substrate allows at least some of the cells (such as fibroblasts) to migrate through it and allows other cells (such as the keratinocytes) to adhere on a planar surface of the substrate.
  • the upper surface of the substrate and the epidermal cells thereon are not in direct contact with the gas permeable membrane, for example the gas permeable membrane is in the base of the device and may be in contact with a metal plate (such as a steel plate, in particular a perforated steel plate) or similar supporting the substrate.
  • a metal plate such as a steel plate, in particular a perforated steel plate
  • the substrate orientation is changed, for example by turning or rotating it through about 180 degrees, to put the epidermal cells on the "upper surface” of the substrate in direct contact with a gas permeable layer. This makes what was the upper surface of the substrate the lower (under) surface of the substrate. This rotation of the substrate also puts the cells which differentiate into an epidermis layer in direct contact with the gas permeable layer/membrane.
  • This rotation or turning may be achieved by housing the substrate on a platform that rotates relative to the main culture pot.
  • the substrate may be held on a frame which is manually removed and reinserted in an inverted orientation.
  • the culture pot can be provided with a gas permeable membrane in the base and also in the lid, which then allows the whole pot to be inverted. After inverting the pot (i.e. turning it upside down) such that it now stands on its lid then the substrate would be arranged to simply sink (or be moved) to the new bottom (which was formerly the lid] of the culture pot thereby putting the cells for differentiation into an epidermis in direct contact with a gas permeable membrane or layer.
  • the disclosure WO2016/209089 provides for the first time "synthetic" tissue which approximates very well human skin with a differentiated dermis and epidermis.
  • the synthetic skin tissue prepared is supported by the substrate which provides some structural integrity.
  • This substrate is designed to disintegrate over time and the material used starts to weaken and disappear, even as the skin tissue grows in vitro.
  • the skin tissue product is relatively delicate and difficult to handle repeatedly. It is desirable to only move the tissue only once, for example to suture it onto a patient Reducing the need for manipulating the skin tissue is important because this reduces the risk of contaminating the skin tissue product and by extension the risk of infection in the patient and the risk of the skin graft being rejected.
  • the present inventors have established that the optimised way to transport the tissue is to provide it, for example to the operating surgeon, in part or all of the device in which the tissue was cultured, thereby allowing the engineered skin tissue to remain untouched/undisturbed.
  • a method of transportation for a structured synthetic tissue cultured in vitro comprising transporting the said tissue in part or all of the manufacturing device, such that the tissue is essentially untouched before it is employed for its final purpose, for example in a procedure where it is grafted onto a patient.
  • a method of transportation for a structured synthetic tissue cultured in vitro comprising transporting the said tissue in part or all of the manufacturing device, such that the tissue is essentially untouched/undisturbed before it is employed in the procedure where it is grafted onto a patient
  • the synthetic tissue is skin, for example differentiated skin tissue, in particular comprising a dermis and an epidermis.
  • a method of transportation according to paragraph 1 or 2 where part of the manufacturing device is transported for example comprising a sub-unit of the manufacturing device, such as a frame, tray, or compartment containing the tissue.
  • tissue in the manufacturing device or part thereof are in a controlled environment.
  • the controlled environment is a temperature in the range 4 to 40 ° C, for example 37.5 ° C or 37 ° C.
  • the manufacturing device comprises: a container comprising a first endwall (bottom/base], and at least one sidewall,
  • a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells,
  • At least part of one of the base, sidewall and top comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; and wherein part of all of the device can be moved (for example can be slid and/or inverted/rotated] from (a] a first mode in which the substrate is not in direct contact with said gas permeable material, to (b] a second mode in which the substrate is in direct contact with said gas permeable material.
  • the base of the device comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange.
  • top of the device comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange.
  • top of the device is adapted to engage with the container to define a chamber (for culturing the cells].
  • gas permeable material is polydimethylsiloxane.
  • the device further comprises a 3-D substrate which presents culturing surfaces on opposite planar sides.
  • the scaffold comprises a first frame defining an interior perimeter and an exterior perimeter, said first frame comprising a substantially planar upper surface
  • a second frame defining an interior perimeter and an exterior perimeter, said second frame comprising a substantially planar upper surface
  • first frame and the second frame detachably engage around at least a part of their perimeters to define an interface to receive and hold the substrate
  • the lower surface of the scaffold comprises at least one section spanning the exterior perimeter of the scaffold and the adjacent interior perimeter, said section having a lower surface which is raised towards the upper surface of the scaffold, wherein said section defines a void when the scaffold is placed on a flat surface.
  • the section defines a recess (for example 554] provided to allow for easy removal of air bubbles when the scaffold is in submerged culture.
  • the scaffold comprises one or more transverse members spanning the interior perimeter to provide support for the substrate.
  • the substrate is a biocompatible biodegradable material, such as a biocompatible membrane.
  • a method according to any one of paragraphs 1 to 45 which comprises culturing epidermal and dermal cells with media to provide differentiated synthetic skin product comprising a dermis and an epidermis.
  • epidermal cells are keratinocytes.
  • a method according to paragraph 50, wherein the first period is in the range 12 to 60 hours, such as 24 to 48 hours, in particular 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 hours.
  • a method of engineering a structure comprising epithelial tissue comprising the steps of
  • a manufacturing device comprising:
  • a container comprising a first endwall (bottom], and at least one sidewall,
  • a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to reside upon,
  • part of all of the device can be inverted/rotated from (a] a first mode in which the substrate is not in direct contact with said gas permeable material, to (b] a second mode in which the substrate is in direct contact with said gas permeable material,
  • said device comprises a synthetic skin tissue, grown therein, and
  • said device is sealed for transportation, for example using screws or sealing clips.
  • the device containing the tissue culture is not opened before it is transported.
  • the device containing the tissue culture is opened for visual inspection of the tissue only before transportation.
  • the device containing the tissue culture is not sampled or quality tested before it is transported.
  • the tissue culture is moved or touched and is not in any way disturbed before transportation.
  • the devices containing the tissue is in exterior packaging to protect it during transportation, for example cardboard packaging, plastic packaging, a wooden box or the like.
  • the disclosed method allows the tissue to be transported directly to the patient using the device in which the tissue was cultured. This eliminates i] the need for a separate device for transporting the tissue, and ii] the need to handle the tissue in order to transfer it from the culture device to the transport device, which would require aseptic conditions.
  • the presently disclosed method greatly minimises handling of the synthetic tissue, which reduces the risk of contamination and consequently the risk of the patient getting an infection from a contaminated synthetic tissue.
  • the method also provides the tissue is pristine condition, which maximises the likely success of, for example skin grafts.
  • the synthetic tissue is skin, for example differentiated skin tissue, in particular comprising a dermis and an epidermis, such as a synthetic differentiated skin product disclosed herein or as disclosed in WO2016/209089.
  • part of the manufacturing device is transported, for example a sub-unit of the manufacturing device, for example a frame, tray, or compartment/vessel containing the tissue.
  • a sub-unit of the manufacturing device for example a frame, tray, or compartment/vessel containing the tissue.
  • the advantage of this is that only a part of the device rather than the entire device needs to be transported. This makes transporting the synthetic tissue easier and can potentially reduce transport costs.
  • the manufacturing device is designed to have a releasable container, which houses the tissue.
  • the container may be released by, for example unclipping, unscrewing, or sliding the container from a frame or housing.
  • the sub-unit of the device may for example be sterilely sealed, such as hermetically sealed before transportation.
  • the whole manufacturing device is transported.
  • the advantage of this approach is that the use of clean room clean room facilities, in particular the highest level of clean room facilities may be minimised or avoided.
  • culture media from the patient-tissue-product may be sampled by extracting fluid through a port in the device or unit, in particular a port that allows aseptic extraction of sample (such a septum or the like].
  • the container holding the tissue may be filled with a buffer, for example CO2 or an aqueous buffer (in particular one disclosed herein]. After filling the container with buffer then any ports on the container may then be sealed.
  • a buffer for example CO2 or an aqueous buffer (in particular one disclosed herein].
  • the device of sub-unit employed in the present disclosure comprises a window, which allows the tissue to inspected without opening the device.
  • the manufacturing device or part thereof is sealed, for example hermetically sealed or vacuum packed before transportation.
  • the presently disclosed device or unit can be sealed prior to transportation without the need to place the device within a separate container.
  • the device or unit is secured by one or more fixings, for example selected from the group comprising clips, screws, bolts, straps and combinations of two or more of the same.
  • the device or unit is sealed by incorporating it into a further container or packaging, for example a thermally insulated contained, a polymer film or bag, or any other suitable arrangement.
  • the manufacturing device or part thereof is disposable. This enhances the convenience of the device since the medical personnel, such as a surgeon can simply discard the device after the synthetic tissue has been recovered from the device.
  • the manufacturing device or part thereof is reusable for tissue culture.
  • the benefit of this is that reusing the device or part thereof helps to cut costs and reduce use of raw materials.
  • the device or part thereof may for example be made from more durable and/or heat resistant materials, which for example can withstand high temperatures needed to sterilise the device or part thereof before it can be reused.
  • the manufacturing device or part thereof contains cell culture media, for example as described herein.
  • the tissue in the manufacturing device or part thereof is in a controlled environment.
  • a controlled environment This may, for example be a refrigerated truck or an incubator.
  • the skilled person will be aware of other suitable controlled environments.
  • the controlled environment is a temperature in the range 4 to 40°, for example, 37 to 38°C, such as 37.5°C.
  • the synthetic tissue may be stored at a low temperature in order to extend the durability of the synthetic tissue or stored at close to body temperature so that the synthetic tissue is ready for application to the patient immediately upon arrival at the patient's location.
  • the controlled environment is a pH within a physiological range, for example pH 6.5 to 8, such as pH 7 to 7.5, in particular pH 7.35 to 7.45, especially pH 7.4.
  • the controlled environment comprises a specific concentration of CO2, such as about 5%.
  • the controlled environment is provided by an aqueous buffer system, for example "one" of the so-called 20 good buffers (i.e. MES, BIS-TRIS, ADA, ACES, BIS-TRIS propane, PIPES, MOPSO, BES, TES, HEPES, DIPSO, MOBS, TAPSO, acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, tricine, TRIZMA, glycinamide, glycyl-glycine, HEPBS, bicine, TAPS, AMPB, CHES, AMP, AMPSO, CAPSO, CAPS, CABS and combinations thereof).
  • 20 good buffers i.e. MES, BIS-TRIS, ADA, ACES, BIS-TRIS propane, PIPES, MOPSO, BES, TES, HEPES, DIPSO, MOBS, TAPSO, acetamidoglycine, TAPSO
  • the buffer is HEPES buffer (4-(2-hydroxyethyl]-l-piperazineethanesulfonic acid], for example ultrapure HEPES.
  • the buffer is at a concentration in the range ImM to 1M, for example 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425,
  • CO2 is omitted from the controlled environment
  • the manufacturing device comprises:
  • a container comprising a first endwall (bottom], and at least one sidewall, a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to deposit on,
  • part of all of the device can be inverted or rotated from (a] a first mode in which the first uppermost face of substrate is not in direct contact with said gas permeable material or said entity adapted to engage therewith, to (b] a second mode in which the first face of the substrate is in direct contact with said gas permeable material.
  • the device can be configured between the first mode wherein the substrate (and cells thereupon] are not in contact with a gas permeable layer and a second mode wherein the substrate (and cells thereupon] are in contact with a gas permeable material.
  • the ability to switch between the two modes is important for the proper stratification of skin tissue.
  • a container comprising a first endwall (bottom], and at least one sidewall,
  • a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to deposit on,
  • At least a part of at least one of the first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • the device is configurable between (a] a first mode in which the substrate is not disposed in gaseous communication with a gas permeable material, and (b] a second mode in which the substrate is disposed in gaseous communication with a gas permeable material.
  • the device comprises
  • a container comprising a first endwall (bottom], and at least one sidewall,
  • a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to reside upon,
  • first endwall (bottom], the at least one sidewall, and the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • the substrate in a first mode the substrate is not in contact with a gas permeable material, and in a second mode the substrate is in contact with a gas permeable material.
  • the scaffold is moveable from a first position to a second position, wherein the second position disposes the substrate at a gas permeable material.
  • the substrate when present at the first position in use, the substrate is submerged, for example in cell media.
  • the scaffold and/or substrate in the first mode is separated from a gas permeable material by a removable, gas impermeable surface.
  • the removable gas impermeable surface is removed by dissolution.
  • the removable gas impermeable surface is physically removed.
  • the device comprises
  • a container comprising a first endwall (bottom] and at least one sidewall, wherein at least a part of the bottom comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • a detachable second endwall (top] adapted to engage with the container to define a chamber, wherein at least a part of the second endwall (top] or at least a part of the at least one sidewall comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • a scaffold adapted to receive a substrate for cells to reside upon, wherein the scaffold engages with the at least one sidewall to (a] allow substantially linear movement of the scaffold at least partway between the first endwall (bottom] of the chamber and the second endwall (top], and restrict rotation or inversion of the scaffold about an axis perpendicular to the at least one sidewall, or (b] allow rotational movement of the scaffold about an axis perpendicular to the at least one sidewall.
  • inversion of the device allows substantially linear movement of the scaffold at least partway between the first endwall (bottom] of the chamber and the second endwall (top] to dispose the substrate at the gas permeable material present in the second endwall (top].
  • rotation of the device allows substantially rotational movement of the scaffold about an axis perpendicular to the at least one sidewall to dispose the substrate at the gas permeable material present in the at least one sidewall.
  • the device comprises
  • a container comprising a bottom and at least one sidewall, wherein at least a part of the bottom comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • a detachable top adapted to engage with the container to define a chamber, wherein at least a part of the top comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • a scaffold adapted to receive a substrate for cells to reside upon, wherein the scaffold engages with the at least one sidewall to (a] allow substantially linear movement of the scaffold at least partway between the bottom of the chamber and the top, and (b] restrict rotation or inversion of the scaffold about an axis perpendicular to the at least one sidewall.
  • the chamber when in use and a gas permeable material is present in both the bottom and the top, the chamber is liquidly sealable from but in gaseous communication with the environment
  • the scaffold comprises:
  • a frame defining an interior perimeter and an exterior perimeter, said frame comprising a substantially planar upper surface
  • the scaffold is configured to bring substantially all of the substrate or the cells or tissues present on the substrate into contact with a gas permeable interface when the scaffold is placed in a culture device comprising at least one gas permeable interface.
  • the substrate is held in a substantially planar arrangement on the planar upper surface of the frame.
  • the substrate is held between the first frame and the second frame by friction fit engagement of the first frame to the second frame.
  • the friction fit engagement is such that it rigidly clamps the substrate maintain the substrate in a substantially planar arrangement across the interior perimeter of the scaffold.
  • the frames engage to prevent shrinking, stretching or deformation of the substrate, for example when the substrate is contacted with culture media.
  • the substrate is held between the first frame and the second frame at least in part by one or more protrusions extending between the first frame and the second frame.
  • one or more of said one or more protrusions pierce the substrate at a point within the interface formed between the first frame and the second frame on engagement
  • the lower surface of the scaffold comprises at least one section spanning the exterior perimeter of the scaffold and the adjacent interior perimeter, said section having a lower surface which is raised towards the upper surface of the scaffold, wherein said section defines a void when the scaffold is placed on a flat surface.
  • the section defines a recess 554 (shown in Figure 15] provided to allow for easy removal of air bubbles when the scaffold is in submerged culture.
  • the scaffold comprises a substrate for cells to deposit on.
  • substrate is a slice with a planar upper face and approximately parallel planar lower face and a perimeter wall joining said faces, wherein the depth dimension of the perimeter wall is smaller than the dimension of each side of the planar upper and lower face thereby providing a "long, flat" 3-D structure to the substrate.
  • the scaffold comprises one or more transverse members spanning the interior perimeter to provide support for the substrate. In one example, the scaffold comprises two or more transverse members. In one example, the scaffold comprises a lattice of transverse members spanning the interior perimeter to provide support for the substrate, for example as depicted in Figure 11.
  • the gas permeable material is a gas permeable membrane.
  • Gas permeable membrane and gas permeable layer are employed interchangeably herein unless the context indicates otherwise.
  • the gas permeable material is polydimethylsiloxane.
  • the scaffold is substantially planar. In one embodiment the scaffold is oriented substantially parallel with the bottom, or the top, or both the bottom and the top of the device. In one embodiment, the scaffold engages with the at least one sidewall to allow substantially linear movement of the scaffold between the bottom of the container and the top.
  • the scaffold has one or more lugs that engage with one or more complementary grooves in the at least one sidewall to allow translational movement of the scaffold relative to the at least one sidewall (up and/or down the container] but to restrict rotational movement of the scaffold relative to the at least one sidewall.
  • the scaffold receives the substrate to present culturing surfaces on opposite planar sides of the substrate. That is to say the scaffold is a frame, which allows the upper and lower planar faces of the substrate to be exposed to the culture media.
  • the substrate is a biocompatible material, such as a biocompatible membrane. In one embodiment the substrate is a biodegradable membrane. In various embodiments, the substrate is a co-polymer.
  • the substrate is gas permeable. In an alternative embodiment the substrate is gas-impermeable.
  • the substrate is or comprises a biodegradeable polymer selected from the group comprising: PLGA, PLA, PCL, PHBV, PDO, PGA, PLCL, PLLA-DLA, PEUU, cellulose- acetate, PEG-b-PLA, EVOH, PVA, PEO, PVP, blended PLA/PCL, gelatin-PVA, PCT/collagen, sodium aliginate/PEO, chitosan/PEO, chitosan/PVA, gelatin/elastin/PLGA, silk/PEO, silk fibroin/chitosan, PDO/elastin, PHBV/collagen, hyaluronic acid/gelatin, collagen/chondroitin sulfate, collagen/chitosan, PDLA/HA, PLLA/HA, gelatin/HA, gelatin/siloxane, PLLA/MWNTs/HA, PLGA/HA, 100 dioxanone linear homopolymer and combinations of
  • the substrate is or comprises polyfjactic co-glycolic acid] (PLGA].
  • PLGA polyfjactic co-glycolic acid
  • the substrate is or comprises electrospun PLGA.
  • the substrate comprises dioxanone linear homopolymer, for example a combination of PLGA and 100 dioxanone linear homopolymer.
  • At least one planar face of the substrate comprises is treated to improve cell adhesion, cell migration, or tissue stratification.
  • the substrate comprises one or more molecules to aid cell adhesion and/or migration and/or stratification.
  • the substrate comprises one or more proteins, such as one or more basement membrane proteins, a collagen, a fibronectin, a laminin, or a lectin, one or more carbohydrates, such as one or more saccharides, or any combination of two or more thereof.
  • the substrate comprises collagen I, fibronectin, collagen IV, collagen VII, laminin 5, one or more adhesion peptides derived from one of the aforementioned proteins, or a combination of any two or more thereof.
  • the substrate comprises one or more molecules to selectively bind or attract, or promote or enable adhesion of, a specific cell type to the substrate.
  • the substrate comprises one or more molecules to selectively bind epithelial cells, for example, keratinocytes.
  • the substrate comprises one or more molecules to selectively bind fibroblasts.
  • the substrate comprises one or more cell adhesion molecules such as one or more immunoglobulin superfamily cell adhesion molecules (IgSF CAMs], one or more integrins, one or more cadherins or one or more selectins.
  • the substrate comprises one or more epithelial cadherins (E-cadherins], one or more placental cadherins (P-cadherins], one or more neural cadherins (N-cadherins], one or more retinal cadherins (R-cadherins], one or more brain cadherins (B-cadherins or T-cadherins], or one or more muscle cadherins (M-cadherins], E- selectin, L-selectin, P-selectin, alpha 1 integrin (ITGA1], alpha 2 integrin (ITGA2], alpha 2b integrin (ITGA2b], alpha 3 integrin (IT
  • the substrate comprises one or more molecules to aid epithelial stratification, for example, epidermal stratification.
  • the substrate comprises one or more other agents to aid graft viability, maintenance, or longevity.
  • the one or more other agents can be incorporated into the substrate at manufacture, during culturing, or at any point prior to or during surgical implantation of the cells or tissue present on the substrate.
  • Exemplary agents include one or more antibiotics (such as, for example, colloidal silver or colloidal gold, or one or more chemical antibiotics], one or more immunomodulators (such as one or more anti-inflammatory agents], one or more promoters of healing or vascularisation (such as, for example, VEGF], and the like.
  • At least a part of the scaffold comprises a gas permeable material and/or is adapted to engage with a gas permeable substrate and is perforated to allow gaseous exchange across the gas permeable substrate.
  • the device when the device is in a first mode (for example, wherein the scaffold is substantially horizontal] the scaffold and substrate are disposed within the chamber and the substrate is submerged.
  • the "upper" planar face of the substrate is disposed at a gas permeable material.
  • the scaffold and substrate define the bottom of the chamber to comprise a gas permeable interface.
  • the device in use is configured so that the chamber holds a volume of culture media sufficient to submerge the substrate when the device is in a first mode (for example, when the scaffold is substantially horizontal].
  • the device in use is configured in a second mode wherein the substrate is at or adjacent a gas permeable material.
  • the substrate comprises a gas permeable interface when the device is in a second mode (e.g., an inverted or rotated orientation].
  • the container or detachable top comprises at least one fluidly-sealable access port
  • the at least one sidewall comprises at least one fluidly-sealable access port
  • the device is sterilisable, including, for example, by autoclaving or gamma irradiation.
  • step b] comprises a container comprising a first endwall (bottom], at least one sidewall, and a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to reside upon, wherein at least a part of the first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange, unless indicated otherwise.
  • the tissue is cultured by: a] providing a suspension comprising cells to be cultured and an amount of tissue culture medium sufficient to support cell growth;
  • the substrate in particular the "upper planar face" of the substrate] is optionally disposed in gaseous communication with a gas permeable material
  • the device in one embodiment in step b] is in a first mode in which the substrate (in particular the lower planar face of the substrate] is disposed in gaseous communication with a gas permeable material. In one embodiment in step d] the device is in a second mode in which the substrate (in particular the "upper planar face"] is disposed in gaseous communication with a gas permeable material, for example the upper planar face is on the gas permeable material.
  • the tissue is cultured by: step a] defined above;
  • step e] comprises incubating the cell culture device for a time sufficient to allow one or more of cell differentiation or cell proliferation.
  • the device is a device of the present disclosure.
  • the cell suspension is introduced via an access port present in the chamber, for example, an access port present in the container.
  • the cell suspension is introduced to the container prior to attaching the top.
  • the cells to be cultured are anchorage-dependent cells.
  • the cells comprise epithelial cells. In one embodiment, the cells comprise keratinocytes. In one embodiment, the cells comprise fibroblasts. In one embodiment, the cells comprise keratinocytes and fibroblasts. In one embodiment the cell suspension comprises a population of cells obtained from a tissue digest In one embodiment, the method comprises the following additional steps between steps d] and e] f] introducing a second suspension comprising cells to be cultured, for example via the access port, and
  • the method comprises one or more repeats of steps f] to h).
  • the second suspension comprises one or more cell types that differ from those present in the first suspension.
  • the first suspension comprises fibroblasts
  • the second suspension comprises keratinocytes.
  • the substrate is biocompatible. In one embodiment, the substrate is biodegradable.
  • the synthetic tissue is epithelial tissue, wherein the tissue is cultured comprising the steps of: step a] above wherein the cells are epithelial cells;
  • the method comprises one or more of the following steps following step b]: f] removing the tissue culture medium from the device,
  • the epithelial cells are keratinocytes.
  • the suspension further comprises a second population of cells, for example, fibroblasts.
  • the device in one embodiment in step b] the device is in a first mode in which the substrate (in particular the "lower planar face" of the substrate] optionally is disposed in gaseous communication with a gas permeable material; and in step d] the device is in a second mode in which the epithelial cells (in particular adhered to the "upper planar surface" of the substrate] are disposed in gaseous communication with a gas permeable material.
  • the epithelial cells are in direct contact with a gas permeable material.
  • the synthetic tissue is stratified epidermal tissue or full thickness skin tissue, wherein the tissue is cultured comprising the steps of: al] providing a suspension comprising keratinocytes and fibroblasts and an amount of tissue culture medium sufficient to support cell growth;
  • the cell culture device adapting the cell culture device to a second mode in which the substrate (in particular the "upper planar face”] is and/or the cells are disposed in gaseous communication with the gas permeable material, and
  • the synthetic tissue is stratified epithelial tissue, wherein the tissue is cultured comprising the steps of:
  • a2 providing a first suspension comprising cells in an amount of tissue culture medium sufficient to support cell growth
  • the device further comprises an injection port in the chamber, wherein the device is in a first mode in which the substrate is submerged in the suspension and optionally is in gaseous communication with a gas permeable material,
  • the synthetic tissue is full thickness skin tissue or stratified epidermal tissue, wherein the tissue is cultured comprising the steps of:
  • a3 providing a suspension comprising fibroblasts and an amount of tissue culture medium sufficient to support cell growth
  • the device further comprises an injection port in the chamber, wherein the device is in a first mode in which the substrate is submerged in the suspension and optionally is in gaseous communication with a gas permeable material,
  • the adaption to a second mode in step (d] exposes the surface of the substrate opposite that to which the fibroblasts are anchored (i.e. the so-called "upper planar face"] to the gas permeable material.
  • the method comprises: step a3] defined above;
  • the device furthrer comprises an injection port in the chamber, and a scaffold adapted to receive a substrate for cells to reside upon, wherein the device is in a first mode in which the substrate is submerged in the suspension,
  • the method comprises: step a3] defined above;
  • the device further comprises an injection port in the chamber, wherein the device is in a first mode in which the substrate is submerged in the suspension
  • the substrate is impermeable to the one or more cells.
  • the substrate is permeable to one or more cells.
  • the cell culture device is incubated for a time sufficient to allow the migration of at least some fibroblasts into or through the substrate.
  • the cell culture device is incubated for a time sufficient to allow for proliferation of at least some of the fibroblasts.
  • the cell culture device is incubated for a time sufficient for the fibroblasts to produce a thickened dermis. In another embodiment the cell culture device is incubated for a time sufficient for extracellular matrix deposition to occur.
  • the cell culture device is incubated for a period of at least one week, for example, at least two weeks, or at least three weeks. In certain embodiments, the cell culture device is incubated for a period of at least about one month, for example, at least about two months.
  • the cells are cultured for a first period until at least some of the epidermal cells adhere to a planar culture surface (in particular the "upper planar face"] of the substrate (in particular a pre-treated surface].
  • the first period is in the range 12 to 60 hours, such as 24 to 48 hours.
  • the orientation of the planar culture surface (in particular the "upper planar face"] of the substrate to which epidermal cells have adhered is changed to be in gaseous communication, in particular direct contact, with the gas permeable material.
  • the cells are cultured for a second period until the cells forms one or more confluent layer of cells.
  • the epidermal cells are confluent, for example on the planar culture surface (in particular the so-called “upper planar surface”] of the substrate.
  • the method further comprises one or more of the following additional steps following step b] :
  • the dermal cells are confluent at least partly within the substrate.
  • the epidermal cells are differentiated into an epidermis.
  • the second culture period in the context of the present disclosure may be any culture period following the first culture period.
  • the synthetic skin product provided is approximately the same thickness as natural human skin, for example 0.5mm to 4mm, such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4mm.
  • the synthetic tissue is stratified epithelial tissue, wherein the tissue is cultured comprising the steps of:
  • a providing a suspension comprising epithelial cells in an amount of tissue culture medium sufficient to support cell growth
  • the device comprises a container comprising a first endwall (bottom], at least one side wall, and a detachable second endwall (top] adapted to engage with the container to define a chamber, wherein the chamber comprises a scaffold adapted to receive a substrate for cells to reside upon, wherein at least a part of the first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; and
  • the device is in a first mode in which the substrate is submerged in the suspension and is not in gaseous communication with a gas permeable material
  • the synthetic tissue is stratified epidermal tissue, wherein the tissue is cultured comprising the steps of:
  • the device comprises a container comprising a first endwall (bottom], at least one sidewall, and a detachable second endwall (top] adapted to engage with the container to define a chamber, wherein the chamber comprises a scaffold adapted to receive a substrate for cells to reside upon, wherein at least a part of the first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange; and
  • the device is in a first mode in which the substrate is submerged in the suspension and is not in gaseous communication with a gas permeable material
  • the substrate is impermeable to the one or more cells.
  • the substrate is permeable to one or more cells.
  • one or more layers of fibroblasts are deposited within the three dimensional matrix of the substrate.
  • the synthetic tissue is stratified epithelial tissue, wherein the tissue is cultured comprising the steps of:
  • the device is in a first mode in which the substrate, adhered epithelial cells and/or tissue comprising epithelial cells is submerged in the media and is in gaseous communication with a gas permeable material,
  • the adhered epithelial cells are keratinocytes and the stratified epithelial tissue is stratified epidermal tissue.
  • the synthetic tissue is full thickness skin tissue, wherein the tissue is cultured comprising the steps of:
  • first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • the device is in a first mode in which the substrate, adhered cells and/or tissue is submerged in the media and is in gaseous communication with a gas permeable material, c] incubating the cell culture device containing the adhered cells or tissue for a time sufficient for epidermal stratification and/or generation of full thickness skin to occur.
  • the adhered cells or tissue comprises one or more populations of cells, in particular at least two populations of cells.
  • the adhered cells for example, keratinocytes
  • the adhered cells or tissue comprises fibroblasts.
  • the adhered cells or tissue comprises keratinocytes.
  • the adhered cells or tissue comprises fibroblasts and keratinocytes.
  • the keratinocytes are undifferentiated, for example at the point of addition to the cell culture.
  • the substrate sits in a scaffold disposed within the chamber/contained defined within the device.
  • the chamber comprises a scaffold adapted to receive the substrate.
  • the scaffold receives the substrate prior to introduction of the scaffold to the chamber.
  • the tissue is cultured comprising the steps of: a] providing a chamber having at least one gas permeable surface, wherein the chamber comprises a scaffold adapted to receive a substantially planar biocompatible gas permeable substrate, the scaffold capable of substantially linear movement from a first position to a second position at or adjacent and in gaseous communication with the gas permeable surface,
  • the scaffold in the second position defining a liquid impermeable volume within the chamber and wherein the substrate (in particular the "upper planar surface” of the substrate] is in gaseous communication with (for example disposed on] the gas permeable surface, and
  • the method is a method of culturing epithelial cells, epidermal cells, or both epithelial and epidermal cells.
  • the first suspension comprises fibroblasts and keratinocytes.
  • the tissue is cultured comprising the steps of:
  • a providing a chamber having at least two gas permeable surfaces, wherein the chamber comprises a scaffold adapted to receive a substantially planar biocompatible gas permeable substrate, the scaffold capable of substantially linear movement from a first position at or adjacent and in gaseous communication with the first gas permeable surface to a second position at or adjacent and in gaseous communication with the second gas permeable surface,
  • the method is a method of culturing epithelial cells, epidermal cells, or both epithelial and epidermal cells.
  • the first cell suspension comprises fibroblasts. In one embodiment, the second cell suspension comprises keratinocytes.
  • the formation of one or more confluent layers of cells is the formation of one or more stratified epidermal tissues.
  • the formation of one or more confluent layers of cells is the formation of full thickness skin, for example 0.5 to 4mm, such as 0.5, 1, 1.5, 2, 2.5, 3, 3.5 or 4mm.
  • the movement of the scaffold is substantially linear movement (for example up and down the chamber or container] from the first position at or adjacent and in gaseous communication with the first gas permeable surface to the second position at or adjacent and in gaseous communication with the second gas permeable surface.
  • said linear movement is without significant rotation or inversion of the scaffold about an axis perpendicular to the linear movement
  • the synthetic tissue is stratified epidermal tissue, wherein the tissue is cultured comprising:
  • a] providing a substrate wherein adhered keratinocytes are disposed over at least a part of a surface of the substrate (in particular the "upper planar face" of the substrate];
  • the method comprises providing a substrate wherein adhered fibroblasts are disposed over at least a part of a surface of the substrate, for example within the three dimensional matrix of the substrate.
  • adhered keratinocytes and adhered fibroblasts are disposed over at least a part of one surface of the substrate, for example the "the upper planar" surface of the substrate.
  • adhered keratinocytes are disposed over at least a part of one surface of the substrate and adhered fibroblasts are disposed over at least a part of an opposing surface.
  • the method is a method of producing tissue comprising stratified epidermis and dermis. In one embodiment the method is a method of producing full thickness skin.
  • the full thickness skin is fully human.
  • the biocompatible substrate is a 3-D matrix or membrane and the scaffold is adapted to hold the membrane under tension.
  • the substrate is or comprises one or more of the following: acellular de-epithelialised dermis (alloderm]; dermis; collagen including collagen gel and tissues comprising collagen; tissue or cells of an epidermal or epithelial lineage, including tissues or cells from umbilical cord, placenta, mucosa, the digestive tract; fibronectin/fibrin; platelet rich plasma; Matrigel; components of and tissues comprising extracellular matrix including extracellular matrix secreted by cells such as fibroblasts; hyaluronic acid; electrospun biocompatible materials including PLGA; biocompatible polymers or combinations of biocompatible polymers, particularly those capable of being electrospun, including polyacrylic acid, poly L Lysine, collagen, gelatin, nylon, and polyesters; gelatine; peptide hydrogels; polyglactin scaffolds; dermagraft; elastin;
  • cells and tissues recited above may be from any animal source, including human, equine, porcine, ovine, murine, canine, feline and bovine.
  • a substrate comprising one or more biologically derived products will generally provide appropriate stimulation for epidermal stratification to occur in certain embodiments of the disclosure without further modification, while substrates comprising one or more synthetic materials will typically require modification, such as a coating, to provide stimulus for epidermal stratification.
  • the substrate is electrospun.
  • the methods of the disclosure are amenable to the culturing of any animal cells.
  • Particularly contemplated are mammalian cells, including human.
  • the method of the present disclosure employs human fibroblasts, for example unirradiated human fibroblasts.
  • the method of the present disclosure employs human keratinocytes.
  • the method of the present disclosure does not employ non-human fibroblasts, for example does not employ irradiated murine fibroblast (for example as feeder cells].
  • the cells cultured are autologous to the patient, which will receive the skin graft of the synthetic tissue.
  • the methods of the disclosure can be used to prepare tissues including epithelium, for example a single cell-thick layer of keratinocytes, stratified epithelium comprising at least two of the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum, and full thickness skin comprising dermal and epidermal layers.
  • epithelium for example a single cell-thick layer of keratinocytes
  • stratified epithelium comprising at least two of the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum, and full thickness skin comprising dermal and epidermal layers.
  • the gas permeable material is selected for optimal gaseous exchange and/or cellular growth, proliferation, and/or stratification.
  • the gas permeable material is a gas permeable membrane having a thickness of 50 to 250 ⁇ , such as 75, 80, 85, 90, 95, 100, 105, 110, 120 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 ⁇ .
  • the device is of a volume sufficient to contain growth medium sufficient for atleast 10 days, for example at least 14 days, at least 15 days, at least 16 days or at least 17 days or longer.
  • the disclosure provides cells, for example, epithelial cells, keratinocytes, fibroblasts or keratinocytes and fibroblasts, prepared by a method or in a culture device as herein described.
  • the keratinocytes are differentiated keratinocytes.
  • the cells are adhered to a substrate according to the present disclosure.
  • the disclosure provides tissue, for example, epithelial tissue, stratified epithelial tissue, epidermis, stratified epidermis, stratified epidermis and dermis, or full thickness skin, prepared by a method or in a culture device as herein described.
  • tissue comprising a dermal layer comprising fibroblasts, a stratified epidermal layer comprising keratinocytes and a biocompatible substrate, prepared by a method or in a culture device as herein described.
  • a differentiated skin tissue product comprising a dermis and an epidermis obtainable from a method disclosed herein.
  • the biocompatible substrate is a biodegradable substrate. In one example, the biodegradeable substrate is present between the dermal and epidermal layers.
  • the tissue comprises a dermal layer comprising fibroblasts that are at least partially embedded in, or attached to, the substrate, and an epidermal layer.
  • the tissue comprises a substrate, a dermal layer attached to one surface of the substrate and an epidermal layer attached to the opposing surface of the substrate.
  • the tissue comprises an epidermal layer, a substrate attached on one surface of the epidermal layer and a dermal layer attached to the opposing surface of the epidermal layer.
  • the dermal layer, the epidermal layer or the dermal layer and the epidermal layer are embedded in, or attached to, the substrate.
  • the disclosure relates to the use of tissue, for example epidermis, stratified epidermis, stratified epidermis and dermis, or full thickness skin, prepared in a method as herein described in the treatment of tissue damage in a subject in need thereof.
  • tissue for example epidermis, stratified epidermis, stratified epidermis and dermis, or full thickness skin, prepared in a method as herein described in the treatment of tissue damage in a subject in need thereof.
  • the treatment is of a wound, a burn, a scar including a surgical scar, an ulcer, damage caused by necrotizing fasciitis or the like.
  • Wound as employed herein also includes anything that may require a skin graft, for example skin cancer surgery, mole removal, a cut or stab wound, a wound caused by a shearing force, a graze, an abrasion, a chemical burn, psoriasis, skin infections, bed sores or similar.
  • the disclosure provides a method of treating tissue damage in a patient in need thereof, the method comprising the steps of
  • Transportation may be any suitable means including delivery by hand, transportation by road, rail, aeroplane, or a combination of two or more of the same.
  • the application of tissue to the subject will be by surgery.
  • recovery under sterile conditions is during or immediately prior to surgery, for example in the surgical suite.
  • tissue to the subject will be at or adjacent the site of tissue damage.
  • the tissue damage is selected from a wound, a burn, a scar, a surgical site, an ulcer, damage caused by necrotizing fasciitis and the like.
  • the tissue damage is a wound, for example as disclosed herein.
  • the epithelial tissue is applied to a region of the digestive tract, integumentary system, reproductive tract, respiratory tract, sensory system, or urinary tract
  • the epithelial tissue is applied to, or forms, the submandibular gland, attached gingiva, dorsum of tongue, hard palate, oesophagus, stomach, large intestine, small intestine, rectum, anus, gallbladder, thyroid follicle, skin, sweat gland duct, mesothelium of body cavities, ovary, fallopian tube, uterus, cervix, vagina, labia majora, tubuli recti, rete testis, ductuli efferentes, epididymis, vas deferens, ejaculatory duct, bulbourethral gland, seminal vesicle, oropharynx, larynx, larynx, vocal cord, trachea, respiratory bronchiole, cornea, nose,
  • the disclosure provides a method of engineering a structure comprising epithelial tissue the method comprising the steps of
  • the disclosure provides a method of engineering a structure comprising epithelial tissue the method comprising the steps of
  • tissue such as epithelial tissue, stratified epithelial tissue, epidermis, or stratified epidermis, has been grown, for example in a method as herein described,
  • the structure comprises a structure of the digestive tract, integumentary system, reproductive tract, respiratory tract, sensory system, or urinary tract.
  • the structure comprises a duct of the submandibular gland, attached gingiva, dorsum of tongue, hard palate, oesophagus, stomach, large intestine, small intestine, rectum, anus, gallbladder, thyroid follicle, skin, sweat gland duct, mesothelium of body cavities, ovary, fallopian tube, uterus, cervix, vagina, labia majora, tubuli recti, rete testis, ductuli efferentes, epididymis, vas deferens, ejaculatory duct, bulbourethral gland, seminal vesicle, oropharynx, larynx, larynx, vocal cord, trachea, respiratory bronchiole, cornea, nose, proximal convoluted tubule
  • the disclosure provides a method of testing the toxicity of a compound or composition to a tissue, the method comprising the steps of:
  • tissue such as epithelial tissue, stratified epithelial tissue, epidermis, stratified epidermis, stratified epidermis and dermis, or full thickness skin, has been grown, for example in a method as herein described,
  • the disclosure provides a method of testing the tissue permeability, for example, the transdermal permeability, of a compound or composition, the method comprising the steps of a] transporting a synthetic tissue using a method as herein described, b] applying the compound or composition to the tissue, and
  • the disclosure provides a method of testing the efficacy of a compound or a cosmetic, therapeutic or nutraceutical composition for effecting a change in tissue, the method comprising the steps of:
  • the method further comprises the step of recovering the tissue from the device following step a). In an alternative embodiment the method comprises applying the compound or composition to tissue in situ within the device.
  • a manufacturing device comprising:
  • a container comprising a first endwall (bottom], and at least one sidewall,
  • a detachable second endwall (top] adapted to engage with the container to define a chamber, and a scaffold adapted to receive a substrate for cells to reside upon,
  • first endwall (bottom]; the at least one sidewall, and/or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • part of all of the device can be inverted/rotated from (a] a first mode in which the substrate is not in direct contact with said gas permeable material, to (b] a second mode in which the substrate is in direct contact with said gas permeable material,
  • said device comprises a synthetic skin tissue, grown therein, and
  • said device is sealed for transportation, for example using screws or sealing clips.
  • the present disclosure relates to a device and method for transporting tissue.
  • the device comprises a container comprising a first endwall and at least one sidewall, and a detachable second endwall wherein at least a part of two or more walls comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange.
  • the present disclosure further relates to tissue prepared by a method or in a culture device described herein, or to the use of such tissue for the treatment of tissue damage in a patient in need thereof.
  • the device and methods described herein provides for the culture of cells and tissues, for example, epithelial cells, stratified epithelial cells, keratinocytes, fibroblasts and/or epithelial tissue such as skin, under conditions that mimic an air-liquid interface.
  • the conditions provide a high oxygen environment that promotes epithelial cell differentiation and/or stratification, and/or the growth of multicellular layers or multilayer tissues.
  • the device and methods of the disclosure dispose growing cells and tissues in gaseous communication with, for example, at, a gas permeable interface.
  • Embodiments of the methods and device of the disclosure have numerous advantages, including but not limited to: practical and efficient engineering of cells and tissues, reduced cost of manufacture of cultured tissues and cells, reduced user handling during culture of cultured cells and tissues, reduced contamination of cultured tissues and cells, and/or reduced cell or tissue loss and/or increased yield during cell or tissue culture.
  • Tissue refers to a group of similar or associated cells that work to together in a way suitable to perform a function, in particular when incorporated into an organism.
  • tissue include skin, connective tissue, muscle and the like.
  • Synthetic tissue as employed herein refers to tissue grown ex vivo.
  • Structured synthetic tissue as employed herein refers to tissue with distinct/differentiated components, for example a dermis and an epidermis.
  • Dermis as employed herein refers to the inner of skin cells beneath the epidermis. In the body the dermis is located between the epidermis and the subcutaneous tissue.
  • Epidermis is the outer layer of cells in skin tissue, which provides a barrier to infection and regulates the amount of water released by the body.
  • Transporting refers to moving or sending the tissue to a different location, for example where the journey takes between 5 mins and 48 hours, such as 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 hours.
  • Untouched or undisturbed as employed herein refers to the absence of handling the tissue (other than in the device or part thereof). The tissue is not lifted or moved, in particular out of the device or in some instances is not moved even for testing, before it is employed for its final purpose (such as a skin graft].
  • Substrate in the context of the present disclosure is a three dimensional matrix, in particular woven, fibre and/or spun matrix and on and in which the cells adhere and grow.
  • the substrate acts a template around which the cells gather, adhere and/or grow.
  • Slice refers to a three dimensional shape, for example which is suitable template for a section of skin to grow on, for example about 100 microns in depth and with a two planar substantially faces.
  • Planar face refers to an approximately flat two dimensional surface, it does not need to be perfectly flat but simply to support the growth or cells, for example that will differentiate into an epidermis.
  • the upper planar face is micropatterned to provide textures that will ultimately increase the resistance to shear forces of the differentiated epidermis (i.e. to reduce it sliding over the dermis and instead retain the epidermis in place].
  • “Upper planar face” as employed herein is not necessarily a reference to an orientation of the substrate but instead refers to one of the two planar faces on which the keratinocytes deposit These cells ultimately differentiate into the epidermis and become the upper (outermost] layer of skin.
  • the upper planar face will generally be pre-coated to encourage the keratinocytes to adhere to it.
  • the upper planar surface at the start of the culture will generally be remote from the gas permeable layer. For example, where the scaffold holding the substrate is horizontal and parallel with a gas permeable layer in the base of the device the upper planar surface will be upper most, i.e. directed to the lid.
  • the substrate will be rotated or moved to put the upper planar face in contact with the gas permeable layer. This will generally put the upper planar face on the underside of the substrate, and thus direct the upper planar to the face base.
  • the orientation of the upper planar face changes through the process.
  • the designating upper planar face applies to the face regardless of its orientation.
  • Lower planar face as employed herein refers to the corresponding parallel face to the upper planar face. Lower is not necessarily a reference to the orientation of the substrate face.
  • First culture period refers to the period of culture when the keratinocytes adhere to the upper planar face.
  • One or more cells types may be added to the culture one or more times during the first culture period.
  • Second culture period refers to the period of culture wherein the keratinocytes adhered to the upper planar face are put into contact with the gas permeable layer.
  • One or more cells types may be added to the culture one or more times during the second culture period.
  • tissue culture medium sufficient to support cell growth includes where the requisite cells are provided in at least some media and further media (as required] to be provided separately or alternatively for the requisite cells to be provided in the (final] amount to media required to support the cell growth.
  • gas-liquid interface or "air-liquid interface” as used in this specification means a surface at which proliferating or differentiating cells or tissues are in contact with air or gas and a liquid simultaneously.
  • An air-liquid interface is required for the growth of some cells and tissues.
  • an air-liquid interface is required for the growth of full thickness skin, whereby the dermal layer of the growing skin is in contact with liquid cell culture media and the epidermal layer is in contact with air in order to induce epidermal stratification.
  • the high oxygen culture conditions at or adjacent an air-liquid interface or gas-liquid interface promote cell growth, differentiation or stratification, and/or the formation of multicellular layers or multilayer tissues.
  • This interface is usually the uppermost surface of a liquid in a container, i.e. where the liquid meets the atmosphere.
  • air-liquid interface does not normally include the interface at gas permeable material.
  • gas permeable interface means a surface located between a gaseous environment and, for example a closed environment that allows gas exchange to occur but is liquidly sealed. Alternatively a gas permeable interface is simple at the interface between a liquid and a gas permeable material.
  • the one or more gas permeable interfaces present in the device described herein provide an interface that promotes cell or tissue proliferation, differentiation and/or stratification in a similar manner to an air-liquid or gas-liquid interface.
  • the one or more gas permeable interfaces present in the device described herein provide an interface that promotes epithelial cell, for example, keratinocyte proliferation and/or differentiation and/or epithelial or epidermal stratification.
  • Gas permeable material, gas permeable membrane and gas permeable layer are used interchangeable unless the context indicates otherwise.
  • gas permeable material as used in this specification means a material through which gas exchange may occur. Gas permeable membranes are particularly contemplated for use in the device described herein.
  • in gaseous communication means that the scaffold, substrate, cells or tissue are in sufficient proximity to the gas permeable material to allow gas exchange between the environment and the substrate or cells (for example, a substrate or cells disposed on a scaffold] to occur in order to deliver, for example, increased oxygen to the substrate, cells or tissue.
  • the scaffold, substrate or cells is in direct contact with the gas permeable material. In other embodiments the scaffold, substrate or cells is not in direct contact with the gas permeable material.
  • the scaffold, substrate or cells is maintained at a distance of less than about 0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9 or about 10 mm, and useful ranges may be selected from between any of these values, for example, from about 0 to about 10 mm, about 0 to about 5 mm, about 0 to about 2 mm, about 0 to about 1 mm, about 0 to about 0.5 mm, about 0 to about 1 mm, about 0.1 to about 10 mm, about 0.1 to about 5 mm, about 0.5 to about 5 mm, or from about 2 to about 5 mm.
  • subject is intended to refer to an animal, preferably a mammal, more preferably a human.
  • Mammalian subjects include cats, dogs and horses.
  • Other mammalian subjects include an agricultural animal, including a horse, a pig, a sheep, a goat, a cow, a deer, or a fowl, or a laboratory animal, including a monkey, a rat, a mouse, a rabbit or a guinea pig.
  • treat and its derivatives should be interpreted in their broadest possible context. The term should not be taken to imply that a subject is treated until total recovery. Accordingly, “treat” broadly includes maintaining a subject's disease progression, symptoms or burn or wound healing at a substantially static level, increasing a subject's rate of recovery, amelioration and/or prevention of the onset of the symptoms or severity of a particular condition, burn, wound or other injury, or extending a patient's quality of life. The term “treat” also broadly includes the maintenance of good health for sensitive individuals and building stamina for disease, infection or infestation prevention.
  • the device of the disclosure provides for the culture or engineering of cells and/or tissues that require or benefit from exposure to an air-liquid interface to stimulate or promote their growth, differentiation or stratification.
  • the device of the present disclosure provides for the culture of cells or tissues submerged in a liquid and in gaseous communication with a gas permeable surface.
  • a first embodiment of the device is shown in Figure 1.
  • Device 100 comprises a container 110 formed by first endwall 111 and at least one sidewall 112, and a detachable second endwall 120.
  • Container 110 is configured to sealingly engage with second endwall 120 to form a liquid seal. When engaged, container 110 and second endwall 120 define a chamber 130.
  • a flange 114 extends laterally from container 110 at or towards end 113.
  • flange 114 defines a groove 115 configured to retain an O-ring (not shown], for example, a rubber O-ring. The O-ring forms a liquid seal when the container engages with second endwall 120.
  • second endwall 120 is fastened to container 110.
  • second endwall 120 is fastened to container 110 using nuts and bolts.
  • second endwall 120 is configured to engage with container 110 by friction fit, a threading arrangement or one or more clamps.
  • a sidewall 112 or second endwall 120 comprises at least one fluidly-sealable access port 117.
  • the device further comprises a bung, such as a bung, or other member that engages with port 117 to form a liquid seal.
  • the device comprises a rubber bung (not shown] that engages with port 117 to form a liquid seal, wherein a needle may be inserted through the rubber bung to inject cells or culture medium into the chamber. When the needle is removed, the liquid seal is restored.
  • container 110 and/or second endwall 120 are formed substantially of polycarbonate. Suitable materials for use in forming container 110 and/or second endwall 120 are materials that are sterilisable, for example, materials that are autoclavable or that may be irradiated. At least a part of two or more of second endwall 120, endwall 111 and the at least one sidewall 112 comprise or is configured to engage with a gas permeable material. In an exemplary embodiment at least a part of first endwall 111 and second endwall 120 comprise or are configured to engage with a gas permeable material.
  • gas permeable material is located on the interior surface of the endwall or sidewall. In one embodiment the gas permeable material is attached using an adhesive.
  • the part of second endwall 120, first endwall 111 and/or sidewalls 112 that comprises or engages with the gas permeable material is perforated to allow gaseous exchange as shown in Figure 1.
  • the part of the second endwall, first endwall and/or sidewall that comprises or engages with the gas permeable material comprises a plurality of perforations 140.
  • perforations 140 are about 3 mm in diameter.
  • the perforations extend to substantially the same area as the gas permeable material, thereby forming a gas permeable interface on the interior surface of each of endwalls 120 and 111.
  • a flange extends from first endwall 111 and/or second endwall 120, the flange configured such that when device 100 is placed on an external surface, the exterior face of first endwall 111 and/or second endwall 120 does not contactthe external surface.
  • the presence of one or more flanges in the device may enhance gas exchange between the exterior environment and the chamber of the device through perforations 140 and the gas permeable material.
  • chamber 130 when device 100 is in use and a gas permeable material is present and extended to entirely cover perforations 140, chamber 130 is liquidly sealable from but in gaseous communication with the environment
  • the gas permeable material is a gas permeable membrane.
  • the gas permeable material has a thickness of less than about 50, 75, 100, 120, 125, 140, 145, 150, 155, 160, 170, 175, 200, 225, 250, 300, 350, 400, 450 or less than about 500 ⁇ , and useful ranges may be selected from between any of these values, for example, from about 50 to about 500 ⁇ , from about 100 to about 200 ⁇ , or from about 125 to about 175 ⁇ .
  • the gas permeable material is polydimethylsiloxane, silicone, fluoroethylenepolypropylene, polyolefin, or ethylene vinyl acetate copolymer.
  • Device 100 further comprises a scaffold 150 located in chamber 130 that is adapted to receive a substrate for cells to reside upon.
  • scaffold 150 is detachable from the device.
  • scaffold 150 is oriented substantially parallel with first endwall 111 and/or second endwall 120.
  • scaffold 150 is substantially planar and may be in the form of a frame adapted to retain a substrate.
  • the scaffold comprises a clamp or clip that retains the substrate in or on the scaffold under tension.
  • the substrate is directly bound or applied to scaffold 150.
  • the device comprises a plurality of scaffolds wherein each scaffold comprises a discrete substrate. In one embodiment each scaffold comprises a different substrate. In one embodiment each scaffold comprises the same substrate.
  • the scaffold comprises a plurality of separated substrates.
  • the separated substrates are the same substrate. In another embodiment the separated substrates are different substrates.
  • a member comprising the substrate is fitted to scaffold 150, for example by adhesive, clamping or friction fit
  • Scaffold 150 is configured to engage with one or more sidewalls 112 to allow substantially linear movement of scaffold 150 between a first position at or towards first endwall 111 and a second position at or towards second endwall 120, or vice versa, when, in use, device 100 is inverted.
  • the substrate In either the first position or the second position, or both the first position and the second position, the substrate is in gaseous communication with the gas permeable material. In one embodiment the substrate is in contact with the gas permeable material.
  • scaffold 150 is located in a first position at or towards first endwall 111.
  • scaffold 150 moves linearly by force of gravity to a second position at or towards second endwall 120.
  • Scaffold 150 is configured to engage with the one or more sidewalls 112 such that rotation or inversion of the scaffold about an axis perpendicular to at least one sidewall 112 is restricted.
  • scaffold 150 has one or more lugs that engage with one or more complementary grooves in the at least one sidewall 112 to allow translational movement of scaffold 150 but to restrict rotational movement.
  • the at least one sidewall 112 has one or more lugs that engage with one or more complementary grooves in scaffold 150.
  • scaffold 150 is formed from stainless steel. It will be appreciated that other suitable materials include materials of sufficient density that when chamber 130 is filled with a volume of liquid and the device is inverted, scaffold 150 moves through the liquid from the first position to the second position. Suitable materials to form the scaffold include materials that are biocompatible and resistant to oxidation or degradation caused by tissue culture media.
  • scaffold 150 comprises one or more apertures to allow movement of liquid in chamber 130 and to release trapped air when device 100 is inverted.
  • container 110 is cylindrical as shown in Figure 2.
  • firstendwall 111, second endwall 120, flange 114 and/or scaffold 150 may also be circular in shape.
  • a second embodiment of the device is shown in Figure 3 comprising an alternative system to guide translational movement but restrict rotational movement of the scaffold.
  • Device 200 comprises a scaffold 250 operated by a double arm lever to achieve substantially linear movement between first and second positions.
  • scaffold 250 is in the form of a frame fixedly attached, or integral with, pins 251 extending from opposing sides of scaffold 250 towards sidewalls 212.
  • Hinged levers 253 moveably engage with pins 251 and extend in opposing orientations to fixedly engage with pins 254 that extend through sidewalls 212.
  • device 200 comprises one or more bungs 255 that sealably engage with the apertures in sidewalls 212 housing pins 254.
  • Device 200 further comprises one or more opposing handles 256 located on the exterior of opposing sidewalls 212 and fixedly engaged with pins 254. In embodiments comprising more than one handle, conveniently the handles 256 extend in opposing directions.
  • Translational, linear movement of scaffold 250 from a first position shown in part A of Figure 3 is achieved by rotating one or both handles 256 about an axis perpendicular to sidewalls 212 to drive hinged levers 253 to open and raise scaffold 250 to the second position shown in part B of Figure 3.
  • FIG. 4 A third embodiment of the device is shown in Figure 4 comprising a further alternative system to guide translational movement but restrict rotational movement of the scaffold.
  • scaffold 350 moveably engages with lever arms 353 extending from opposing sides of scaffold 350 towards sidewalls 312.
  • Lever arms 353 engage with scaffold 350 at or towards opposing corners of scaffold 350.
  • Lever arms 353 extend in opposing orientations to fixedly engage with pins 354 that extend through sidewalls 312.
  • Device 300 comprises one or more opposing handles 356 located on the exterior of opposing sidewalls 312 and fixedly engaged with pins 354. In embodiments comprising more than one handle, conveniently the handles 356 extend in opposing directions.
  • Translational, linear movement of scaffold 350 from a first position to a second position is achieved by rotating one or both handles 356 about an axis perpendicular to sidewalls 312 to force lever arms 352 to open and raise or lower scaffold 350.
  • a fourth embodiment of the device is shown in Figure 5.
  • Device 400 comprises a container 410 formed by first endwall 411 and sidewalls 412, and a second endwall 420.
  • Container 410 is configured to sealingly engage with second endwall 420 to form a liquid seal and define a chamber 430.
  • Device 400 may comprise a flange 414 and sealing arrangement as described above for device 100.
  • At least one sidewall 412 comprises at least one fluidly-sealable access port.
  • the device further comprises a bung 418 or other member that engages with the port to form a liquid seal.
  • At least a part of second endwall 420 comprises or is configured to engage with a gas permeable material.
  • two or more separated regions of second endwall 420 comprise or are configured to engage with a gas permeable material as shown in Figure 4.
  • the one or more regions of second endwall 420 that comprise or engage with the gas permeable material are perforated to allow gaseous exchange as described for device 100.
  • the one or more regions of the second endwall that comprise or engage with the gas permeable material comprises a plurality of perforations 440.
  • chamber 430 is liquidly sealable from but in gaseous communication with the environment
  • Device 400 further comprises a scaffold 450 located in chamber 430 that is adapted to receive a substrate for cells to reside upon.
  • the substrate may be applied or fitted to scaffold 450 as described above for device 400.
  • scaffold 450 is detachable. In another embodiment scaffold 450 is fixed to device 400.
  • Scaffold 450 is configured to engage with one or both sidewalls 412 to allow substantially rotational movement of scaffold 450 about an axis perpendicular to one or both sidewalls 412.
  • scaffold 450 engages with a pin 451 that extends across the width of chamber 430 between sidewalls 412.
  • scaffold 450 is fixedly attached to pin 451.
  • Pin 451 rotates freely about an axis perpendicular to one or both sidewalls 412.
  • the device comprises a handle 452 located on the exterior of container 410 and fixedly attached to an end of pin 451.
  • the device comprises a second handle extending from the opposing end of pin 451.
  • Scaffold 450 moves between a first position shown in part A of Figure 6 and a second position shown in part B of Figure 6. In the first position a side of scaffold 450 contacts a first region of endwall 420.
  • the substrate is in gaseous communication with or directly contacts the gas permeable material in the first position.
  • a user moves scaffold 450 by rotating handle 452 about pin 451 to move scaffold 450 to the second position so that the opposing planar surface of scaffold 450 contacts a second region of endwall 420.
  • the substrate is in gaseous communication with, or contacts, the gas permeable material in the second position.
  • device 400 comprises a partition (not shown] extending from the interior surface of first endwall 411 towards second endwall 420 and extending between sidewalls 412 to bisect chamber 430 and form two sub chambers.
  • the partition is configured so as to not obstruct rotational movement of scaffold 450.
  • pin 451 may be housed within the partition.
  • Culture media may be added to one or both subchambers. Addition of culture media to one subchamber provides an adequate volume of media for the growth and differentiation of cells or tissue located in the subchamber, while providing a large gaseous volume of the chamber, thus maximising gas exchange at the interface between the media and the gas in the chamber.
  • one or more of the sidewalls of the container are deformable.
  • manual manipulation of the deformable sidewalls is used to effect or allow translational movement of the scaffold at least partway between the first and second endwalls of the container.
  • the device comprises a removable or dissolvable cover at least partially covering the gas permeable material.
  • the cover may be removed by a user to allow media, cells, tissue or the substrate to come into contact with the gas permeable material.
  • the removable cover comprises a dissolvable material. The cover is dissolved when liquid is added to the chamber of the device.
  • a fifth embodiment of the device is shown in Figure 7.
  • Device 500 comprises a body 510 formed by sidewalls 512, back wall 513, a front wall (not shown], and opposing detachable endwalls 520 and 521.
  • body 510 is configured to sealingly engage with endwalls 520 and 521 to form a liquid seal.
  • the liquid seal is formed when endwalls 520 and 521 are fastened to body 510 using screws.
  • Other suitable fastening means that may be used will be apparent to those skilled in the art, including those discussed above.
  • body 510 and endwalls 520 and 521 When engaged, body 510 and endwalls 520 and 521 define a chamber.
  • any wall of body 510 comprises at least one fluidly-sealable access port 517.
  • a bung or other member that engages with access port 517 is used to form a liquid seal as described above.
  • Endwalls 520 and 521 comprise a plurality of perforations 540 as shown in Figure 7 to allow gaseous exchange between the chamber and the environment. At least a part of endwalls 520 and 521 is configured to engage with a gas permeable material (not shown] that extends to entirely cover perforations 540 such that, when device 500 is in use, the chamber is liquidly sealed from, but in gaseous communication with, the environment A gas permeable interface (GPI] is thereby formed on the interior surface of each of endwalls 520 and 521.
  • a gas permeable material not shown
  • the GPIs provide for gas diffusion into media within the device and promote cell or tissue proliferation, differentiation and/or stratification when cells seeded on the substrate are in direct contact with a GPI.
  • Device 500 comprises a scaffold 550 located in the chamber that is adapted to receive a substrate 551.
  • scaffold 550 is configured to engage with one or more side walls 512 to allow substantially linear translational movement of scaffold 550 between a first position (A] at or towards endwall 520 and a second position (B] at or towards endwall 521. In use, scaffold 550 moves between the first and second positions when device 500 is inverted.
  • the dimensions of the device 500 and scaffold 550 are such that the scaffold is greater in one dimension (width or length] than the device is deep. These dimensions ensure smooth linear transitional movement of scaffold 550 between the first and second positions while restricting rotational movement to avoid buckling or jamming of scaffold 550 in the device.
  • scaffold 500 when scaffold 500 is in the first position shown in Figure 8A, the upper surface of substrate 551 is in contact with the GPI on the interior surface of endwall 520.
  • cells or tissues that require contact with a GPI in order to proliferate, differentiate or stratify, such as keratinocytes, should be seeded on the upper surface of substrate 551.
  • differentiate or stratify such as keratinocytes
  • a part of endwall 521 is raised to form a platform that engages the gas permeable material.
  • the platform is configured such that, in use, the lower surface of substrate 551 contacts the GPI at endwall 521 when scaffold 550 is in the second position.
  • device 500 can be manipulated in use such that the upper surface of substrate 551 contacts a GPI in the first position and the lower surface contacts a GPI in the second position.
  • cells added to the chamber adhere to the substrate 551.
  • Cells may adhere to the upper surface of substrate 551, the lower surface of substrate 551 or both the upper and lower surfaces of substrate 551.
  • scaffold 550 is in the form of a frame as shown in Figure 9.
  • Scaffold 550 is configured to retain substrate 551 under tension.
  • substrate 551 covers substantially all of the top edge of scaffold 550.
  • the scaffold comprises a lower member 552 and an upper member 553 as shown in Figure 10.
  • lower member 552 forms a grid as shown in Figure 11 that supports the substrate and allows liquid and air to pass through the scaffold when the device is inverted in use.
  • lower member 552 and upper member 553 are configured to retain a substrate on the scaffold.
  • a substantially planar substrate 551 is placed across the top surface of lower member 552.
  • Lower member 552 is shaped around its perimeter to engage upper member 553 to form a friction fit that retains substrate 551 under tension.
  • lower member 552 is shaped around its perimeter to engage upper member 553 to form a friction fit that retains substrate 551 under tension.
  • scaffold 550 comprises one or more clips or clamping members to fixedly engage lower member 552 and upper member 553.
  • lower member 552 comprises one or more protrusions 5521 on its top surface. In use, then upper member
  • scaffold 550 comprises one or more apertures 554 as shown in Figure 15 that allow air trapped under the substrate to pass through the scaffold.
  • apertures 554 are located at two or more corners of scaffold 550.
  • scaffold 555 comprises one or more recesses 555 located at a position along the length of a side of the scaffold frame as shown in Figures 16 and 17.
  • a tool may be inserted in recess 555 to lever apart lower member 552 and upper member 553 to release the substrate from the scaffold.
  • Culture or engineering of some cells and tissues benefits from or requires an oxygen-rich environment This can be achieved by using culturing techniques that dispose the growing cells or tissue at an air-liquid interface. These techniques typically involve positioning the cells or tissue at the surface of the liquid tissue culture media in proximity to the gas present in the chamber of the tissue culture vessel. Such techniques are very difficult to implement precisely, particularly in order to grow tissues having a large area. Maintenance of these cultures over many weeks inevitably results in sub-optimal conditions for cell growth, due to the practical difficulty of maintaining a consistent depth of medium over all parts of the tissue. Multiple changes of the culture media is typically required which risks contamination of the cells or tissue.
  • the disclosure provides an efficient and precise method of culturing or engineering cells or tissues that require exposure to an air liquid interface, or that benefit from culture in a high oxygen environment
  • the methods and device described herein may be used to culture or engineer complex tissues from cells.
  • the methods and device provide an environment that promotes proliferation and differentiation of cells to form complex tissue structures.
  • the method is a method of culturing epithelial cells, for example, epidermal cells such as keratinocytes.
  • the method is a method of stimulating or maintaining epithelial cell proliferation or differentiation.
  • the method is a method of culturing one or more confluent layers of cells on a substrate, wherein said one or more confluent layers are disposed over at least part of the substrate.
  • the method is a method of culturing cells in multicellular layers.
  • the method is a method of culturing tissue.
  • the method is a method of culturing tissue comprising epithelial tissue (epithelium], stratified epithelial tissue, epidermal tissue (epidermis], stratified epidermal tissue, or stratified epidermal tissue and dermal tissue (dermis].
  • the method is a method of culturing epithelium. In various embodiments the method is a method of culturing a single cell-thick layer of keratinocytes, or stratified epithelium comprising at least two of the stratum basale, stratum spinosum, stratum granulosum, stratum lucidum and stratum corneum.
  • the method is a method of culturing skin or skin tissue.
  • the method is a method of culturing skin comprising dermal and epidermal layers or full thickness skin.
  • cells, tissue, epithelium or skin are derived from humans or non-human animals.
  • the cells, tissue, epithelium or skin are mammalian.
  • the cells, tissue, epithelium or skin are human, monkey, rabbit, equine, porcine, ovine, murine, canine, feline, bovine, caprine, or avian.
  • the epithelial tissue comprises simple epithelium that is only one cell thick, stratified epithelium that is two or more layers thick, columnar epithelium, squamous epithelium, cuboidal epithelium, transitional epithelium or pseudostratified epithelium.
  • the epithelium is keratinised or non-keratinised.
  • the epithelium is ciliated.
  • the epithelium comprises microvilli.
  • the epithelium is simple columnar epithelium, stratified squamous epithelium, simple cuboidal epithelium or pseudostratified columnar epithelium.
  • the epithelium is alveolar epithelium, endothelium, mesothelium, germinal epithelium, respiratory epithelium, corneal epithelium, olfactory epithelium, or urothelium.
  • the disclosure provides a method of culturing cells, the method comprising a] providing a suspension comprising cells to be cultured in an amount of tissue culture medium sufficient to support cell growth;
  • iii. for cell proliferation to occur iv. to form one or more confluent layers of cells, for example, one or more confluent layers of cells disposed over at least part of the substrate,
  • stratified tissue for example, stratified epidermal tissue
  • vii. to allow migration of at least some cells, for example, fibroblasts, into or through the substrate, or
  • the suspension comprises a homogenous cell population. In one embodiment the suspension comprises a heterogenous cell population.
  • the suspension comprises cells obtained from a tissue digest or partially purified tissue digest
  • the suspension comprises cells obtained from a skin digest
  • the cells are anchorage-dependent cells or adherent cells.
  • the cells comprise keratinocytes, fibroblasts or keratinocytes and fibroblasts.
  • the cells comprise pigment-producing cells, vascular cells, pluripotent stem cells or immune cells.
  • the cells comprise melanocytes, endothelial cells, smooth muscle cells, monocytes, macrophages, T lymphocytes, platelets, mast cells, adipose cells, or mesenchymal cells.
  • tissue culture medium is Green's medium.
  • tissue culture medium comprises salts, growth factors, hormones and/or antibiotics. It will be appreciated that different tissue culture media may be appropriate for the growth of other cell or tissue types and/or different media additives may be used.
  • the device is a device of the disclosure.
  • the cell suspension is introduced via an access port present in the chamber, for example, an access port present in the container.
  • the cell suspension is introduced to the container prior to attaching the second endwall (top].
  • the substrate is biocompatible. In one embodiment, the substrate is biodegradable. In one embodiment, the substrate is impermeable to the cells. In another embodiment, the substrate is permeable to the cells. In one embodiment the substrate is gas permeable. In another embodiment the substrate is gas impermeable.
  • the substrate is or comprises one or more of acellular de-epithelialised dermis (alloderm]; dermis; collagen including collagen gel and tissues comprising collagen; tissue or cells of an epidermal or epithelial lineage, including tissues or cells from umbilical cord, placenta, mucosa, the digestive tract; fibronectin/fibrin; platelet rich plasma; Matrigel; components of and tissues comprising extracellular matrix including extracellular matrix secreted by cells such as fibroblasts; hyaluronic acid; electrospun biocompatible materials including PLGA, Dioxanone Linear Homopolymer (for example Dioxaprene 100M] or a combination thereof; biocompatible polymers or combinations of biocompatible polymers, particularly those capable of being electrospun, including polyacrylic acid, poly L Lysine, collagen, gelatin, nylon, and polyesters; gelatine; peptide hydrogels; polyglactin scaffolds; dermagraft; elastin;
  • the substrate is or comprises a biodegradeable polymer described, for example on page 11 herein.
  • the substrate is or comprises poly(lactic co-glycolic acid] (PLGA].
  • PLGA poly(lactic co-glycolic acid]
  • substrate is or comprises electrospun PLGA.
  • the substrate is or comprises a combination of PLGA and Dioxanone Linear Homopolymer, such as 100 Dioxanone Linear Homopolymer.
  • the substrate comprises one or more molecules to aid cell adhesion or migration, differentiation, proliferation and/or stratification.
  • the substrate comprises one or more proteins, such as one or more basement membrane proteins, a collagen, a fibronectin, a laminin, or a lectin, one or more carbohydrates, such as one or more saccharides, or any combination of two or more thereof.
  • gas permeable materials for use in the method of the disclosure are discussed above.
  • the gas permeable material is selected for optimal gas exchange, cellular growth, cell or tissue proliferation, tissue stratification and/or biocompatibility with the substrate, cells or tissue.
  • the substrate is disposed at a gas permeable material in the first mode. In one embodiment the substrate is disposed at a gas permeable material in the first mode and the second mode.
  • the method comprises the following additional steps between steps d] and e] f] introducing a second suspension comprising cells to be cultured, for example via the access port, and
  • incubation conditions commonly used in the art for cell or tissue culture may be used.
  • the device is incubated at a temperature of about 37 Q C in a humidified atmosphere comprising 5% carbon dioxide.
  • Full thickness skin comprising a dermal layer (dermis] and an epidermal layer (epidermis] may be formed using the methods described herein. Fibroblasts are added to the device and cultured to form the dermis.
  • Keratinocytes added to the device and cultured differentiate and proliferate to form a stratified epidermis. The applicant has observed that keratinocytes adhered to the substrate must directly contact the gas permeable interface for optimal epidermal stratification to occur.
  • fibroblasts and keratinocytes are introduced to the device simultaneously.
  • fibroblasts and keratinocytes are introduced to the device sequentially.
  • the method comprises introducing a first suspension comprising fibroblasts, incubating the device for a time sufficient for the fibroblasts to adhere or to proliferate, or to form a dermis of a required thickness, then introducing a second suspension comprising keratinocytes. This embodiment may allow for the development of a thicker dermis.
  • fibroblasts are cultured first and a combination of fibroblasts and keratinocytes are introduced together at a later stage. In one embodiment the fibroblasts and keratinocytes are seeded on one surface of the substrate. In another embodiment fibroblasts are seeded on one surface of the substrate and keratinocytes are seeded on the opposing surface of the substrate.
  • the engineered tissue comprises the substrate.
  • fibroblasts may migrate into the substrate to form the dermal layer of full thickness skin.
  • a suspension comprising a heterogenous population of keratinocytes and fibroblasts.
  • the cells are provided in a volume of suitable tissue culture medium, for example, Green's medium, sufficient to support growth of the cells for a period of at least about 14 days.
  • a device comprising a scaffold comprising electrospun PLGA coated with collagen IV.
  • the device is provided in an orientation such that a first wall comprising a gas permeable material and having perforations is located at the top to form a gas permeable interface.
  • the device optionally comprises a gas permeable material located on the opposing second (bottom] wall, which is also perforated.
  • the scaffold is inserted into the device such that the substrate side faces the bottom wall.
  • the scaffold is inserted in the device so that the top surface of substrate 551 faces the first (top] wall.
  • the cell suspension is added to the device and the device is sealed.
  • the device is incubated for a period of about 48 hours to allow cell adherence to at least part of the surface of the substrate to occur.
  • the device is inverted so that the scaffold moves to the opposing end (first wall] of the device.
  • the adhered cells are now in contact with the gas permeable interface at the top wall of the device.
  • the device is incubated for a period of about 14 days to induce production of a stratified epithelium. No further manipulation of the device is required.
  • the scaffold After 14 days the scaffold comprises full thickness skin.
  • the skin may be removed from the scaffold using a scalpel or other tool.
  • fibroblasts and keratinocytes are added separately, and the device is inverted twice.
  • the device as described above for the first exemplary embodiment is used.
  • the device is provided in a first mode such that the substrate is in contact with the gas permeable material.
  • a first cell suspension comprising a homogeneous population of fibroblasts is added to the device.
  • the device is sealed and incubated for 48 hours.
  • the device After 48 hours the device is inverted so that the scaffold moves to the opposing end of the device.
  • a second cell population comprising a homogenous population of keratinocytes is added, for example, using an access port on a sidewall of the device.
  • the device is incubated for a period sufficient to achieve keratinocyte adherence, for example, about 48 hours.
  • the device is inverted a second time so that the scaffold moves to the opposing end of the device, and the keratinocytes are in contact with a gas permeable material.
  • the device is incubated for a period of about 14 days to induce production of a stratified epithelium.
  • Prior art methods of engineering full thickness skin require the use of an air-liquid interface (ALI].
  • ALI air-liquid interface
  • the disadvantage of such methods is that close monitoring of medium levels is required to maintain the growing tissue at the optimum ALI to achieve growth and epidermal stratification.
  • the device and methods of the disclosure provide for the growth, differentiation and/or engineering of cells or tissues using a gas permeable interface (GPI] that is not dependent on culture level and volume in the device, which eliminates the need for careful monitoring during culture.
  • GPI gas permeable interface
  • a further method of the disclosure provides a method for culturing stratified epidermal tissue, or full thickness skin tissue, comprising the steps of:
  • first endwall (bottom], the at least one sidewall, or the second endwall (top] comprises a gas permeable material or is adapted to engage with a gas permeable material and is perforated to allow gaseous exchange;
  • the device is in a first mode in which the substrate, adhered cells or tissue is submerged in the suspension and is in gaseous communication with a gas permeable material, c] incubating the cell culture device containing the adhered cells or tissue for a time sufficient for epidermal stratification, or for the generation of full thickness skin, to occur.
  • the adhered cells or tissue may be prepared by culturing keratinocytes, or fibroblasts and keratinocytes, on a de-epidermised dermis (DED] in a culture vessel. Once the cells have adhered, the cells disposed on the substrate are removed from the culture vessel and transferred to the device.
  • DED de-epidermised dermis
  • the device and methods of the disclosure are suitable for the culture and/or engineering of a range of epithelium or tissues comprising epithelial cells or tissue.
  • the device and methods of the disclosure are particularly suitable for the culture of multicellular layers or multilayer tissues, which typically requires an oxygen-rich environment
  • the methods and device of the disclosure are suitable for the engineering of tissues or structures comprising simple epithelium, including simple squamous epithelium, simple cuboidal epithelium and simple columnar epithelium, stratified epithelium, including stratified squamous epithelium, stratified cuboidal epithelium and stratified columnar epithelium.
  • Simple epithelium comprises a single layer of epithelial cells.
  • Stratified epithelium comprises two or more layers of epithelial cells.
  • the stratified epithelium may be keratinised or non-keratinised, or transitional epithelium.
  • the methods and device of the disclosure are suitable for preparing epithelium including the outer layer of the skin (the epidermis], the cornea, the inner lining of structures of the gastrointestinal tract including the mouth, oesophagus, and rectum, the lining of structures of the urinary or reproductive tract such as the vagina or ureter, the lining mucosa of the lungs, or the epithelium forming the walls of the pericardium, pleurae or peritoneum.
  • the device and methods of the disclosure are suitable for the culture of fibroblasts, keratinocytes and the production of stratified epidermis and/or full thickness skin.
  • Full thickness skin comprises epidermal and dermal layers.
  • the epidermis is a stratified epithelium comprising keratinocytes.
  • the dermis is a layer beneath the epidermis comprising fibroblasts and matrix components including collagen.
  • a challenging aspect of engineering full thickness skin in vitro is the production of stratified epidermal tissue.
  • Stratification of the epidermis is crucial for skin function, and is required for the formation of a stratum corneum, the tissue layer that provides the barrier function of skin.
  • the device and method of the disclosure provide for the engineering of full thickness skin comprising a dermis and stratified epidermal tissue.
  • the disclosure provides for growth of full thickness skin on a suitable substrate while providing a gas permeable interface in contact with or in close proximity to the growing epidermis allowing gas exchange required for the differentiation of keratinocytes and stratification of the epidermis. It is not necessary to transfer the growing tissue to another culture vessel at any time, and few media changes or user interventions are required.
  • the device and method of the disclosure further provide for full thickness skin comprising additional cells and tissues to improve the function of full thickness skin produced by methods of the disclosure after transplantation.
  • tissue comprising differentiated or stratified epithelium produced using a method or device of the disclosure is further manipulated to form a structure.
  • any structure comprising an epithelial surface or lining may be formed.
  • tissue may be wrapped or folded to form a tubular structure, such as a vessel, urethra or oesophagus.
  • Such methods are known in the art, for example, the methods described in Green et al, 2010. Tissue Engineering Vol 16, No 3, pages 1052-1064; Bhargava et al, 2004. BJU International, Vol 93: pages 807-811; and Bhargava et al, 2008. European Urology, Vol 53: pages 1263-1271], which are hereby incorporated by reference.
  • Structures comprising tissue produced using the methods or device of the disclosure may be suitable for transplantation into a subject in need thereof.
  • epithelial tissue produced by a method or using an device of the disclosure is recovered from the device and formed into the structure in situ during a surgical transplantation procedure.
  • tissue produced by a method or using an device of the disclosure is formed into a urethra in situ during a urethroplasty procedure.
  • tissues produced using an device of the disclosure according to the methods described herein may be transported in the device from the laboratory to the operating theatre.
  • the disclosure provides for the use of tissue, such as epithelium, epidermis, stratified epithelium, stratified epidermis and dermis, split thickness skin or full thickness skin, prepared using a method described herein for the treatment of tissue damage in subject in need thereof.
  • tissue such as epithelium, epidermis, stratified epithelium, stratified epidermis and dermis, split thickness skin or full thickness skin, prepared using a method described herein for the treatment of tissue damage in subject in need thereof.
  • the disclosure further relates to a method of treating tissue damage in a subject in need thereof comprising the steps of
  • tissue such as epithelium, stratified epithelium, epidermis, stratified epidermis, stratified epidermis and dermis, split thickness skin or full thickness skin, has been grown, for example in a method as herein described,
  • the tissue damage is a wound, a chronic wound, a surgical wound, damage caused by an animal bite, an ulcer, a non-healing wound, a scar, a surgical scar, a scald or a burn, to replace the cancerous skin tissue excised by surgery (such as sarcoma, melanoma or the like], to replace skin tissue excised to try contain infection with necrotising fasciitis.
  • the burn is a first degree burn, a second degree burn, a third degree burn, a deep dermal burn or a full thickness burn.
  • the tissue damage is epithelium located on a mucosal surface.
  • the epithelium is located on or in skin, the lungs, the gastrointestinal tract (for example, the oesophagus or mouth], reproductive tract, or the urinary tract (for example, the urethra].
  • the tissue is prepared using cells that are autologous to the subject.
  • the tissue is prepared using fibroblasts, keratinocytes, or fibroblasts and keratinocytes that are autologous to the subject.
  • the tissue is prepared using cells that are heterologous to the subject.
  • the tissue is prepared using cells that are autologous to the subject and cells that are heterologous to the subject
  • cells autologous to the subject may be isolated using any method known in the art.
  • autologous cells may be isolated from a skin sample or skin biopsy taken from the subject by digesting the sample tissue and separating fibroblasts and/or keratinocytes from the digested tissue.
  • the tissue is an autograft, for example, a skin autograft.
  • the tissue is an epidermal autograft, a split thickness skin autograft or a full thickness skin autograft.
  • the tissue is an allogeneic graft
  • tissue prepared using cells autologous to the patient is highly desirable to reduce or prevent immune rejection of the tissue and to reduce the requirement for ongoing immunotherapy or another ancillary treatments.
  • tissue comprises the tissue further comprises the substrate. In another embodiment the tissue is separated from the substrate before application to the patient
  • the application of tissue to the patient will be by surgery. In one embodiment, recovery under sterile conditions is during or immediately prior to surgery, for example in the surgical suite. Generally, the application of tissue to the patient will be at or adjacent the site of tissue damage. In various embodiments the tissues is applied to at least partially cover the site of tissue damage or to completely cover the site of tissue damage.
  • tissue is applied to temporarily cover the site of tissue damage. In an alternative embodiment the tissue is applied to permanently cover the site of tissue damage. In vitro testing
  • the efficacy and safely of topically applied pharmaceutical, nutraceutical or cosmetic products are typically tested using animal skin or live animals, human cadaver skin or synthetic human skin models.
  • Cells or tissues prepared using the device or methods described herein are useful for in vitro testing of pharmaceuticals, nutraceuticals or cosmetic products.
  • cells or tissue prepared using the device or methods described herein are used to test transdermal penetration of a compound, to test the permeation of a compound across the epidermis, dermis or basement membrane, to test the efficacy of an active ingredient for treating or preventing a condition, for example, a skin condition, or to test the toxicity of a compound.
  • the cells or tissue are used to determine if a compound of interest is a skin irritant, for example, to determine if a compound of interest induces a skin rash, inflammation, or contact dermatitis.
  • the cells comprise fibroblasts, keratinocytes or immune cells, or a combination of any two or more thereof. In one embodiment the cells comprise fibroblasts and keratinocytes. In various embodiments the tissue is selected from the group comprising epidermis, stratified epidermis and dermis, stratified epidermis and dermis, split thickness skin or full thickness skin.
  • the compound is a pharmaceutical compound, a cosmetic compound or a nutraceutical compound.
  • the compound for testing is applied to tissue alone or in an admixture with pharmaceutically or cosmetically acceptable carriers, excipients or diluents.
  • the compound for testing is applied topically to the tissue in the form of a sterile cream, gel, pour-on or spot-on formulation, suspension, lotion, ointment, dusting powder, a drench, spray, drug- incorporated dressing, shampoo, collar or skin patch.
  • Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.
  • Figure 1 is two exploded perspective views of a first embodiment of the device
  • Figure 2 is an exploded perspective view of the device shown in Figure 1;
  • Figure 3 is a perspective view of a second embodiment of the device showing movement of the scaffold between A] a first position and B] a second position;
  • Figure 4 is a perspective view of a third embodiment of the device showing views from A] a first perspective and B] a second perspective;
  • Figure 5 is an exploded perspective view of a fourth embodiment of the device.
  • Figure 6 is a perspective view of the device shown in Figure 5 showing movement of the scaffold between A] a first position and B] a second position;
  • Figure 7 shows an upper front perspective view of a fifth embodiment of the device with a cross section taken through the device in the plane of back wall 513;
  • Figure 8 shows a front view of the embodiment shown in Figure 7 showing the scaffold in A] a first position and B] a second position;
  • Figure 9 shows a top perspective view of the scaffold of the embodiment of Figure 7 showing the scaffold engaging a substrate
  • Figure 10 shows a left view of the embodiment of Figure 9
  • Figure 11 shows a bottom perspective view of the embodiment of Figure 9 without a substrate
  • Figure 12 shows A] a left exploded view of a cross section of the embodiment of Figure 10 taken through plane A-A (no substrate]; and (B] a left view of a cross section through plane A-A showing the scaffold engaging a substrate;
  • Figure 13 shows a top perspective view of an alternative embodiment of the lower member of the scaffold of Figure 9;
  • Figure 14 shows A] a cross section of the lower scaffold member of the embodiment shown in
  • FIG. 13 taken through plane B-B; and (B] the same view showing the scaffold engaging a substrate anchored by an upper scaffold member;
  • Figure 15 shows a view highlighting the trapped air release mechanism of the embodiment of
  • Figure 16 shows a perspective view of the underside of the scaffold of Figure 9.
  • Figure 17 shows a right view of the scaffold of Figure 9.
  • Sterilised substrate (Electrospun PLGA or any other dermal substitute compatible with skin cell growth] is attached to a stainless-steel scaffold.
  • the substrate is optionally coated with collagen IV (Collagen IV Sigma-Aldrich C5533, used at lOug/cm 2 ] for 2 hours then washed three times with phosphate buffered saline (PBS].
  • collagen IV Collagen IV Sigma-Aldrich C5533, used at lOug/cm 2 ] for 2 hours then washed three times with phosphate buffered saline (PBS].
  • the scaffold with attached substrate is placed into a gas permeable interface (GPI] device so that the collagen IV-coated side is facing towards the opening.
  • GPI gas permeable interface
  • Greens medium 250ml of Greens medium (DMEM:Hams F12 (Life Technologies 31765-035] 3 :1, 10% FCS, lOng/ml EGF (Sigma-Aldrich E9644], 0 ⁇ g/ml hydrocortisone (Sigma-Aldrich H0396], O.lnM choleratoxin (Sigma-Aldrich C8052], 180 ⁇ adenine (Sigma-Aldrich A2786], 5ug/ml insulin (Sigma-Aldrich 19278], 5 ⁇ g/ml apotransferrin (Sigma-Aldrich T2036], 2nM 3,3,5,-tri-idothyronine (Sigma-Aldrich T2752], lx Penicillin/Streptomycin, 0.625 ⁇ g/ml Amphotercin B (Sigma-Aldrich A2942]] is added to the device.
  • Fibroblasts and keratinocytes are detached from culture dishes and counted. 300,000 keratinocytes and 100,000 fibroblast per cm 2 are added into the GPI device. The lid is placed on to seal the GPI device. The device is incubated at 37°C, 5% CO2 for 48 hours.
  • the GPI device is inverted, ensuring that the scaffold moves to the opposite end of the device and the substrate is in direct contact with the gas permeable membrane.
  • the device is incubated at 37°C, 5% C0 2 for 14 days.
  • the GPI device is opened, all liquid is discarded, and the scaffold is removed. A scalpel cut around the edges is used to release the skin from the scaffold.
  • the method will produce full thickness skin comprising a dermis and stratified epidermis suitable for grafting on to a patient.
  • This example outlines an investigation of the preparation of full thickness skin using an device and method of the disclosure.
  • Sterilised substrate is attached as described for Example 1 to stainless steel scaffold.
  • the substrate is optionally coated with collagen IV (Collagen IV Sigma-Aldrich C5533, used at lOug/cm 2 ] for 2 hours then washed three times with phosphate buffered saline (PBS].
  • collagen IV Collagen IV Sigma-Aldrich C5533, used at lOug/cm 2 ] for 2 hours then washed three times with phosphate buffered saline (PBS].
  • the scaffold with attached substrate is placed into a gas permeable interface (GPI] device so that the collagen IV coated side is facing away from the opening.
  • GPI gas permeable interface
  • 100,000 fibroblasts per cm 2 are added into the GPI device.
  • the lid is placed on the GPI device and sealed.
  • the device is incubated at 37°C, 5% CO2 for at least 48 hours.
  • the GPI device is inverted, ensuring that the scaffold moves to the opposite end of the device. 300,000 keratinocytes per cm 2 are added into the GPI device through the injection port such thatthe keratinocytes settle on the unseeded side of the substrate. The device is incubated at 37°C, 5% CO2 for 48 hours. The GPI device is inverted a second time, ensuring that the scaffold moves to the opposite end of the device and the substrate is in direct contact with the gas permeable membrane. The device is incubated at 37°C, 5% C0 2 for 14 days.
  • the GPI device is opened, all liquid is discarded, and the scaffold is removed. A scalpel cut around the edges is used to release the skin from the scaffold.
  • the method will produce full thickness skin comprising a dermis and stratified epidermis suitable for grafting on to a patient.
  • This example outlines an investigation of the preparation of a stratified epidermis using a device and method of the disclosure.
  • Sterilised substrate is attached as described for Example 1 to stainless steel scaffold.
  • the substrate is optionally coated with collagen IV (Collagen IV Sigma- Aldrich C5533, used at lOug/cm 2 ] for 2 hours then washed three times with phosphate buffered saline (PBS].
  • collagen IV Collagen IV Sigma- Aldrich C5533, used at lOug/cm 2 ] for 2 hours then washed three times with phosphate buffered saline (PBS].
  • the scaffold with attached substrate is placed into a gas permeable interface (GPI] device so that the collagen IV coated side is facing towards the opening.
  • GPI gas permeable interface
  • 300,000 keratinocytes per cm 2 are added into the GPI device such that the keratinocytes settle on the unseeded side of the substrate.
  • the lid is placed on the GPI device and sealed.
  • the device is incubated at 37°C, 5% C0 2 for 48 hours.
  • the GPI device is inverted ensuring that the scaffold moves to the opposite end of the device and the substrate is in direct contact with the gas permeable membrane.
  • the device is incubated at 37°C, 5% C0 2 for 14 days.
  • the GPI device is opened, all liquid is discarded, and the scaffold is removed.
  • a scalpel cut around the edges is used to release the stratified epidermis from the scaffold.
  • the method will produce a stratified epidermis suitable for grafting on to a patient
  • DED De-epidermised acellular dermis
  • Full thickness skin was prepared using a prior art method utilising an air-liquid interface as follows. After 48 hours the ring was removed from the DED and the DED comprising adhered fibroblasts and keratinocytes transferred onto a stainless steel rack in a tissue culture dish comprising Greens medium.
  • the rack consisted of a grid of holes, raised 7 mm off the base of the culture dish, through which medium can contact the DED. The level of medium in the culture dish was maintained such that the base of the DED, resting on the metal rack, was in contact with the medium and the top surface of the DED, upon which the keratinocytes and fibroblasts had been seeded, was exposed to air creating an air-liquid interface.
  • the cells were cultured for 14 days at the air-liquid interface with complete medium changes every two to three days.
  • the ring was removed from the DED and the DED transferred into an device comprising a gas permeable membrane.
  • the DED was placed in the device so that the adhered fibroblasts and keratinocytes were in contact with the gas permeable membrane located at the bottom of the device.
  • the cells were cultured for 14 days at the gas permeable interface.
  • tissue was harvested for analysis to assess the quality of the skin formed in contact with an air-liquid interface or with a gas permeable membrane.
  • the skin produced using a GPI was of a similar thickness and appearance to the skin produced using the ALL
  • cytokeratin 19 a marker of keratinocyte stem cells
  • cytokeratin 14 a basal keratinocyte marker
  • cytokeratin 10 a suprabasal keratinocyte marker
  • fluorescent microscopy Five ⁇ thick transverse sections of each frozen skin sample were fixed with acetone and blocked with a 0.25% casein solution.
  • Samples were washed once with TBS, then three times with rocking for five minutes each time.
  • Secondary antibodies specific for each primary antibody with Alexa 488 dye conjugated, in TBS with 1% FBS containing nuclear stain 4',6-diamidino-2-phenylindole (DAPI] covered the sample sections. Samples were incubated for 30 minutes at room temperature. Samples were washed once with TBS, then twice with rocking for 15 minutes each time. Samples were covered with Prolong Gold mounting solution and a coverslip placed on top. Images were obtained for all samples of DAPI stain and each Alexa 488 stain using a fluorescent microscope.
  • keratinocytes were present in both sample types. Changes in the shape of the nucleus of the keratinocytes in the epidermis, from round in the basal region, to flattened in the upper regions, indicated that a stratified epidermis had formed in skin grown at both the ALI and the GPI. Skin grown at an ALI or GPI demonstrated expression of cytokeratin 10, a suprabasal keratinocyte marker, in the top layer of the epidermis indicating that a stratum corneum layer had been successfully formed, which in turn indicated that the keratinocyte differentiation and epidermal stratification process had been successfully completed.
  • cytokeratin 10 a suprabasal keratinocyte marker
  • cytokeratin 14 a basal keratinocyte marker, in the layers of keratinocytes below the stratum corneum, indicating these keratinocytes were in a proliferative state, which is required for formation of a stratified epidermis.
  • Skin grown at an ALI and GPl contained keratinocytes that stained positive for cytokeratin 19, a keratinocyte stem cell marker. The presence of keratinocyte stem cells indicates that all of the keratinocyte cell types required for continued renewal of the epidermis were present.
  • a comparison of skin produced at an ALI with skin grown at a GPl indicates that the GPl may result in a greater number of keratinocyte stem cells present in the epidermis, potentially producing a better stratified epidermis.
  • This example demonstrates the preparation of skin tissue using a method and device of the disclosure.
  • Electrospun PLGA was coated with collagen IV solution (lOug/cm 2 ] for 2 hours at 37°C to form the substrate.
  • the coated PLGA was washed three times with phosphate buffered saline (PBS] before seeding fibroblasts and keratinocytes onto the coated surface.
  • 100 cm 2 substrate was used for method (1], 6 cm 2 for method (2] and 1 cm 2 for method (3].
  • Collagen-coated electrospun PLGA was clamped into the scaffold of a GPl device of the disclosure such that the coated side was flush with the top surface of the scaffold.
  • the scaffold was placed in the bottom of the GPl device such that the coated side of the electrospun PLGA faced upwards.
  • the GPl device was filled with 300 ml of Green's medium.
  • the composition of Green's medium is described in Example 1.
  • the lid (comprising a GPl] was placed on the GPl device and the GPl device was sealed.
  • the GPl device was inverted to move the scaffold to the opposing end of the device (the lid]. In this position, the adhered fibroblasts and keratinocytes were in direct contact with the GPl in the lid.
  • the cells were cultured for 14 days and required no medium changes for that period of time.
  • Method (2) Preparation of skin using a prior art GPI device
  • a stainless-steel ring with a 25 mm diameter aperture and 10mm depth was set on coated electrospun PLGA inside a 5 cm diameter culture dish and filled with Green's medium. 750,000 keratinocytes and 200,000 fibroblasts were added into the centre of the ring. The media was changed twice within 24 hours.
  • the ring was removed from the coated electrospun PLGA and the coated electrospun PLGA was transferred from the culture dish into a G-RexlO device (Wilson Wolf) with 20 mL of Green's medium, such that the adhered fibroblasts and keratinocytes were in contact with the GPI located at the bottom surface of the G-RexlO.
  • a G-RexlO device Wang Wolf
  • the cells were cultured for 14 days and required no medium changes for that period of time.
  • a stainless-steel ring with a 10 mm diameter aperture and 10mm depth was set on coated electrospun PLGA inside a six well culture plate and filled with Green's medium.
  • the ring was removed from the coated electrospun PLGA and the coated electrospun PLGA comprising adhered fibroblasts and keratinocytes was transferred onto a stainless-steel rack in a tissue culture dish comprising Green's medium.
  • the rack consisted of a grid of holes, raised 7 mm off the base of the culture dish, through which medium can contact the coated electrospun PLGA.
  • the level of medium in the culture dish was maintained such that the base of the coated electrospun PLGA, resting on the metal rack, was in contact with the medium and the top surface of the coated electrospun PLGA, upon which the keratinocytes and fibroblasts had been seeded, was exposed to air creating an air-liquid interface.
  • the cells were cultured for 14 days at the ALI with complete medium changes every two to three days.
  • Samples of each skin were stained with antibodies to pan-cytokeratin, a marker of all keratinocyte cells to assess epidermal quality, and vimentin, a marker of fibroblasts, to assess dermal quality, and examined by fluorescent microscopy.
  • Methods (1], (2] and (3] all produced full thickness skin comprising a dermal layer with a stratified epidermis on top of the dermal layer.
  • the dermal layer was the bottom layer of the skin produced, as evidenced by positive staining for the fibroblast marker Vimentin.
  • the dermal layer was a single cell thick for skin tissue produced by all three methods.
  • a stratified epidermal layer formed above the dermal layer for skin tissue produced by all three methods.
  • the epidermis comprised many layers of keratinocytes, as evidenced by positive staining for keratinocyte marker pan-cytokeratin. Stratification of the epidermis was observed in the pan- cytokeratin staining of skin samples from the layering of the cytokeratin. Keratinocytes in the basal layer were rounded, becoming flattened out in the intervening layers until a stratum corneum forms the top layer.
  • Stratification of the epidermis was also demonstrated by the morphology of the keratinocyte cell nuclei, shown by DAPI staining, in the layers of the epidermis.
  • keratinocyte cell nuclei were rounded, indicating healthy, basal keratinocytes capable of proliferation. Moving up through the epidermal layers the keratinocyte cell nuclei flatten out, indicating they have undergone the differentiation process required to achieve stratification.
  • the cell nuclei were either very thin or had disappeared completely producing a stratum corneum layer consisting of dead keratinocyte cells.
  • the device and methods of the disclosure have utility for the engineering of many cells and tissues having a wide range of therapeutic, pharmaceutical, cosmeceutical, nutraceutical and other laboratory applications, including skin grafting and testing of pharmaceutical and cosmetic products.

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Abstract

La présente invention concerne un procédé de transport d'un tissu synthétique structuré cultivé in vitro comprenant le transport dudit tissu dans une partie ou une totalité du dispositif de fabrication, de telle sorte que le tissu est essentiellement non touché/non perturbé avant qu'il ne soit utilisé dans la procédure au cours de laquelle il est greffé sur un patient.
EP17840580.9A 2016-12-28 2017-12-28 Procédé de transport de tissu cultivéin vivo Withdrawn EP3562934A1 (fr)

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GBGB1622341.4A GB201622341D0 (en) 2016-12-28 2016-12-28 Method
PCT/NZ2017/050178 WO2018124888A1 (fr) 2016-12-28 2017-12-28 Procédé de transport de tissu cultivé in vivo

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EP3562934A1 true EP3562934A1 (fr) 2019-11-06

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EP3926036A1 (fr) * 2020-06-15 2021-12-22 ESTR Biosystems GmbH Réservoir pour un processus bio-pharmaceutique

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AU2002230479A1 (en) * 2000-11-21 2002-06-03 Biocrystal Ltd. Cell culture apparatus and methods of use
US10793816B2 (en) * 2013-08-30 2020-10-06 Augusta University Research Institute, Inc. Tissue culture chip
EA201792635A1 (ru) 2015-06-25 2018-06-29 Окленд Юнисервисиз Лимитед Устройство и способ культивирования тканей

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