WO1997026023A1 - Pansements pour blessure et appareil associe - Google Patents

Pansements pour blessure et appareil associe Download PDF

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Publication number
WO1997026023A1
WO1997026023A1 PCT/EP1997/000136 EP9700136W WO9726023A1 WO 1997026023 A1 WO1997026023 A1 WO 1997026023A1 EP 9700136 W EP9700136 W EP 9700136W WO 9726023 A1 WO9726023 A1 WO 9726023A1
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Prior art keywords
growth
growth chamber
medium
wound dressing
cells
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PCT/EP1997/000136
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English (en)
Inventor
Jiri E. Prenosil
Peter E. Villeneuve
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Eth, Eidgenössische Technische Hochschule Zürich
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Priority to AU14418/97A priority Critical patent/AU1441897A/en
Publication of WO1997026023A1 publication Critical patent/WO1997026023A1/fr

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    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • 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
    • 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
    • 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
    • C12M23/22—Transparent or translucent 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • 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
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • 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
    • 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

Definitions

  • the invention concerns a wound dressing in the form of a polymeric film of which one side is covered with cultured autologous skin cells, the preparation and the use thereof for covering and healing skin defects, an apparatus and growth chambers for the production of such a wound dressing.
  • Skin defects such as deep second and third degree burn wounds and chronic leg ulcers, are painful, heal slowly, are susceptible to contamination, and if not treated properly may not heal or form hypertrophic scars.
  • Such skin defects are normally grafted with expanded autologous split-thickness skin. Keratinocytes migrate out from the meshed autografts and close the wound in 7-14 days.
  • This effective treatment method has been utilized for many years. The method is relatively straight forward, poses little health risk to the patient and is very fast. However, this method has two disadvantages: firstly, a donor site proportional to the size of the treated wound is produced (now two scars instead of one are present) , and secondly, the treated wound generates a webbed or "fishnet" scar.
  • the grafted wound is mechanically stable and functional, however, the resulting scar is not cosmetically pleasant.
  • tissue culture is a well established technique used in basic research fields such as intracellular activity, intra ⁇ cellular flux or environmental interaction. Tissue culture has also been adopted into many routine applications in medi ⁇ cine and industry. Chromosomal analysis of cells derived from the womb by amniocentesis can reveal genetic disorders in the unborn child, viral infections may be assayed qualitatively and quantitatively on monolayers of appropriate host cells and the toxic effects of pharmaceutical compounds and poten ⁇ tial environmental pollutants can be measured in colony- forming and other in vi tro assays Freshney (1994) .
  • CEA cultured epidermal autografts
  • CEA technology has helped many patients to survive life threatening wounds, however, there are variable opinions with respect to CEA effectiveness in the literature. Critics of cultured epidermis technology cite the following problems: (1) low take rate, (2) low mechanical stability, (3) lengthy production time, (4) high costs, (5) possibility of contractions in third degree burns, (6) problems in removal of the graft without destroying the newly forming epithelium and without hurting the patient, and (7) good timing which is an important logistical problem. Most of the discussion has focused on the low “CEA take rate” (50-70%) . The CEA will "take” and heal the wound or it will not "take” and require additional grafting.
  • Deep second and third degree wound beds no longer contain papillary fibroblasts, melanocytes, keratinocytes, Merkel cells, and Langerhans cells. Each cell type has very specific interdependent homeostatic and wound healing functions. This is clearly demonstrated in a self healing wound where resident skin cell populations have been compromised but not completely destroyed. A protective scab forms under which the remaining skin cells work together as a unit to form a new epithelium. This is wh large deep wounds where the resident skin cell populations have been completely destroyed require autografting. Therefore, culture treatment methods of large- deep wounds should involve culturing as many skin cell types as possible not just keratinocytes. Culturing of melanocytes, dermal fibroblasts, and kerati ⁇ nocytes has become routine, however, an efficient culturing method for the treatment of non-self healing wounds has not been developed.
  • LaserskinTM is a 50 micron thick hyaluronic acid ester mem- brane designed for the treatment of burns and skin ulcers. Allogenic or autologous keratinocytes are seeded on the bio ⁇ degradable membrane before it is transplanted. The process was disclosed by Delia Valle et al., USP 4,851,521, and con ⁇ cerns vehicles containing a total or partial ester of hyalu- ronic acid in form of surgical articles, including foils, as vehicles for active pharmaceutical substances for topical use.
  • the hyaluronic acid esters are biodegradable and compa ⁇ tible with the organ to be treated.
  • hyaluronic acid esters may contain medicaments or keratinocytes and have been suggested as wound dressings (Andreassi et al., 1991).
  • the hyaluronic acid foil has the following disadvan ⁇ tages which limit its utility: 1) keratinocytes grow rela ⁇ tively slowly, 2) the foil is not autoclavable, 3) the foil is extremely expensive relative to other graftable mate- rials, 4) its effectiveness has not been clearly demon ⁇ strated, 5) health regulatory commissions will approve an inert material before they approve biodegradable materials.
  • Lach ann et al., EP 121981 describe an immobilised cell com- posite comprising a high surface area monolithic ceramic support having a multiplicity of mutually parallel channels in which animal tissue cells are grown.
  • the device and method cannot be used as a wound dressing.
  • Bernard et al., EP 285474 suggest skin equivalents composed of a dermis equivalent covered with an epidermis equivalent.
  • the dermis equivalent is a film formed from type I collagen gel containing fibroblasts.
  • Brysk, USP 5,015,584 describes a synthetic surgical dressing consisting of a support of polyethylene/hydro-colloid type. Attached is a collagen layer.
  • non-differen ⁇ tiated epidermis cells grow separately in collagen coated culture vessels (Petri dishes) until they are substantially confluent. The cells do not grow directly on the polyethylene gamma irradiated plastic.
  • Banes WO 90/00595
  • a floating cell culture device comprising a membrane for growing a cell culture which is attached to means for imparting flotation.
  • the membrane e.g. a polyorganosiloxane foil
  • the membrane has pores and a layer of mouse 3T3 cells which are overlayed in a liquid cell culture medium containing cells, e.g. human keratinocytes.
  • a conformable wound dressing is described by Barlow et al.. Wo 91/13638.
  • the dressing comprises a layer of cultured mam ⁇ malian cells anchored to one surface of a hydrophobic, non- inhibiting to cell growth and non-cytotoxic film of a synthetic polymer.
  • the system for the manufacture of the wound dressing is a hardplastik flask of polystyrene.
  • the substrate needs a corona discharge treatment before the cells anchor thereon.
  • Eisenberg, WO 91/16010 describes a composite living skin equivalent comprising an epidermal layer of cultured kera- tinocyte cells on a layer of non-porous collagen, and a dermal layer of cultured fibroblast cells in a porous cross ⁇ linked sponge. The aim is to prepare test kits for measuring the effect of a substance on the skin equivalent. No wound dressing is suggested.
  • Rosdy FR 2665175, describes the preparation of epidermal equivalents to be used for test purposes.
  • the substrate for cell growing may be cellulose acetate, a polycarbonate, nylon, silicone or collagen, elastin, agarose and the like.
  • Tinois et al. WO 92/06179, suggest a wound dressing compri ⁇ sing a collagen substrate to which an epidermic epithelium with differentiated cells is attached.
  • WO 92/10217 describes wound dressings comprising a sterile cell-growth supporting cross-linked collagen sub ⁇ strate carrying epithelial cells.
  • Katinger et al., EP 155237 describe a method and a device for the cultivation of human, animal, plant and hybrid cells, and micro-organisms in a bioreactor having at least three adjacent chambers, whereby at least one first chamber is a cell culture chamber/ a second and third chamber is a medi ⁇ cine and a product chamber.
  • the chambers are separated by membranes, the cells are immobilised on net-like tissues which may consist of net-like fluorohydrocarbons.
  • the cell grow around the net texture.
  • Nees, DE 3317550 describes the use of a Petri dish compri ⁇ sing a porous or semipermeable substrate mounted to a holding and spanning device which is put on a plate on the bottom of the Petri dish.
  • Anderson, USP 5,073,395 describes an apparatus for isolating phage or viral vectors comprising a film carrying a gelled culture medium, a lawn of cells growing thereon, the cells being susceptible to infection by phage or viral vectors, recovery means for selectively removing a sample from the cell lawn, a drive means to move the film.
  • Cremones EP 307048, provides a cell culture flask comprising a gas permeable membrane inside of the flask and dividing the inside into a first and a second chamber, retaining means for removably securing the membrane and forming a liquid-tight seal between the first and second chamber, a gas inlet for supplying gas to the first chamber and a media inlet for supplying a cell growth medium into the second chamber.
  • Gabriels, EP 396 138 describes a method of producing cell tissues in vitro, comprising contacting first a porous sub ⁇ strate suitable for growing cells with a grow factor specific for growth of the cells in vitro, subsequently seeding the cells on to the porous the growth factor containing substrate and maintaining the seeded substrate under conditions suit ⁇ able for cell growth, to produce a confluent monolayer or multilayer tissue.
  • Butz et al., WO 92/07063, describe a tissue or cell growth device for placement within a well having side wells and a bottom, comprising a hanger supported with a portion of the bottom of the well, and a retention element, including a side well and a cell growth surface, which is detachably secured to the hanger.
  • Prenosil PCT/CH/00241
  • a computer controlled membrane reactor which consists of at least one cell growing chamber through which a stream of gas is flowing and a medium chamber through which a nourishing medium is flowing.
  • the cells are growing on a semipermeable membrane which divides the cell growing and the medium chamber.
  • the callus cell growth can be moni- tored continuously by a video camera.
  • the cell containing membrane can be moved " automatically into a transportation box attached to the system. The transfer of the cell structure to the surgeon can be achieved by said transportation box.
  • Collagen as a substrate has the advantage that it is biodegradable. However, the quality is not always constant and it cannot be sterilized easily, e.g. by UV-irradiation or heat. It is a polypeptide and as such easily decomposes under such condi ⁇ tions. If flasks are suggested as growing chambers it is difficult to remove the wound dressing from the flask. The handling, except for the reactor suggested by Prenosil, is nowhere automated, costly and time consuming. For one reason or another none of the devices described found their way into practical use.
  • Such a wound dressing is supplied by the present invention.
  • the apparatus for producing such wound dressings is another objective of the invention.
  • the hitherto used means for cultu-ring skin cells, such as Petri dishes or culture flask, are unsuitable for producing the envisioned wound dressing. Detailed Description of the Wound Dressing and the Production thereof.
  • Culturing skin cells on a graftable polymeric film provides a solution to the above mentioned problems.
  • primary keratinocyte, dermal fibroblast and melanocyte cul ⁇ tures are established and cultured for 7 days in standard tissue culture dishes before they are trypsinized, pooled, and transferred to a common culture dish containing a polymeric film or the specially designed bioreactor apparatus of the present invention containing a polymeric film.
  • the polymeric film "seeded" with the in vi tro cultured skin cells is placed directly on the wound 2-5 days post inoculation.
  • a new epithelium is formed as the individual skin cells detach from the film and grow together in the wound bed.
  • the seeded wound heals under the polymeric film which functions as an artificial scab.
  • the wound forms a cosmetically pleasant skin that is similar to skin of self healing wounds.
  • the polymeric film dries up and detaches from the wound as the wound heals.
  • Skin cells are attachment dependent, therefore, the graftable material must provide a desirable surface for the attachment and growth of attachment dependent cells.
  • Tissue culture plastic, treated polystyrene is commonly used to grow attachment dependent cell lines in vi tro and is considered superior to other non-coated surfaces. Therefore, the skin cells should grow on the graftable material at least as well as they do on tissue culture plastic.
  • the graftable material is transparent to allow the non-invasive inspection of the wound.
  • the pre-sent graftable material adheres tightly to the wound, because the material is extremely flexible. Rigid materials are unsuitable because they tend to pull away from the wound.
  • the present graftable material according to the invention poses no health risk to the patient and it does not inhibit the healing process, as it is bio-inert and bio- compatible.
  • the graftable material maintains gas flux characteristics that closely approximate that of skin, as it is permeable to both carbon dioxide and oxygen gas. It provides an instant protective barrier against air-born contaminants, as it has no pores or holes that exceed 0.22 microns in diameter.
  • the graftable material can be physically removed from the wound in such a manner that the healing process is not disturbed.
  • Polymeric films e.g. silicone, vinyl copolymers, polyurethane, polycaprolactone, polyvinyl chloride, polytetrafluoroethylene are routinely used as wound dressings.
  • a Teflon FEP flourocarbon film preferably used according to the present invention meets all the objectives. It demon ⁇ strates the following excellent culturing and wound dressing characteristics: The film 1) is autoclavable, 2) is trans ⁇ parent, 3) promotes cell attachment and subsequent growth as well as tissue culture plastic, 4) is flexible, 5) is perme ⁇ able to carbon dioxide and oxygen, 6) adheres tightly to the wound 7) prevents water lose, 8) protects the wound from air borne contamination, and 9) detaches from the wound without harming the newly formed epithelium. 10) Any additional layers of dressing required above the FEP film may be applied and/or changed without disturbing the healing process.
  • the FEP film performs both as a growth substrate and a trans ⁇ parent wound dressing.
  • the above mentioned objectives associated with current CEA technology are either entirely or partially achieved when skin cells are grown on FEP hydrocarbon film.
  • treatment time is reduced from more than 3 weeks to 2 weeks, production costs are greatly reduced, take rate is increased, and mechanical stability is increased.
  • the cellular components responsible for pigmentation and dermal matrix remodeling have been placed onto the present film and into the wound.
  • a computer controlled bioreactor has been built and tested to further streamline the process. Process automation will re ⁇ cute manual labor, largely eliminate human error, and increase product uniformity.
  • the bioreactor is composed of growth chamber(s) that are con ⁇ nected to auxiliary equipment which automatical-ly changes the medium and maintaines optimal culture conditions.
  • An automatic bioreactor with 12 growth chambers is capable of producing a 3500 cm 2 transplantable sub-confluent sheet of autologous keratinocytes or a combination of keratinocytes, dermal fibroblasts, and melanocytes in as little as 8-14 days after obtaining the biopsy.
  • the automated bioreactor is designed for the production of large wound dressings for the treatment of large burns.
  • Similar stand alone growth chambers are constructed for placement in a standard incubator where the medium is changed manually. They are designed for the production of small wound dressings for the treatment of small wounds.
  • the present invention provides in a first aspect a wound dressing comprising a polymeric film of which one side is covered with cultured skin cells.
  • the wound dressing may comprise a polymeric film selected from the group consisting of a polyhydrocarbon, e.g. polypro ⁇ pylene, a polyalcohol, e.g. polyvinylalcohol, a polyester, e.g. polysulfone, a polyamide, e.g. nylon, and particularly a fluorinated polyhydrocarbon, e.g. Teflon 1 ".
  • a polyhydrocarbon e.g. polypro ⁇ pylene
  • a polyalcohol e.g. polyvinylalcohol
  • a polyester e.g. polysulfone
  • a polyamide e.g. nylon
  • fluorinated polyhydrocarbon e.g. Teflon 1 ".
  • Said polymeric film has at least one side suitable for culturing attachment dependent cells.
  • it is gas permeable, has a thickness of 10-200 microns, preferably 12.5-75 microns, is optically transparent, does not fluoresce, has a UV-transmittance >200 n , is inert against most chemicals, is not toxic, and does not inhibit cell- growth.
  • the Teflon 1 " film is selected from the group consi ⁇ sting of a type C-FEP 50, 100, 200, 300 and type C-20-FEP 100, 200.
  • the skin cells attached to the one side of the film are in particular human skin cells, such as either only human kera ⁇ tinocytes, or preferably a combination of keratinocytes, dermal fibroblasts, and melanocytes. If a combination of these three types of cells is desired the ratios are about those occuring in the human skin.
  • Skin cells derived from the patient to be treated are named autologous skin cells.
  • the culturable surface of the wound dressing is either fully, e.g. 95 to 100%, or only partially, e.g. 5 to 95%, covered with a layer of in vi tro grown skin cells.
  • the invention concerns a method for the preparation of a wound dressing described above, comprising the inoculation of skin cells from primary cultures obtained by known methods on the polymeric film placed in a growth chamber, culturing the inoculated cells under standard condi ⁇ tions until the desired surface coverage is obtained, repla ⁇ cing the growth medium with a suitable serum-free isotonic solution, and removing the film covered by the cultured skin cells from the growth chamber in a form for placing on the wound.
  • Primary keratinocyte cultures are established according to the method described by Green et al. (1979) .
  • Primary mela- nocyte cultures are established according to the method described by Freshney (1994) .
  • Primary dermal fibroblast cul ⁇ tures are established according to the method described by Harper and Grove (1979) .
  • the three cell types are cultured in standard polystyrene tissue culture dishes for 5-7 days. Secondary cultures and wound dressings on a small scale are produced in standard " vessels, e. g. a Petri dish, such as the
  • Petriperm® of Heraeus containing a film according to the invention, in particular a FEP hydrocarbon film defined above.
  • the method for the preparation of a secondary culture comprises trypsinization of the primary culture, eventually pooling the various cell type cultures, and inoculating the polymeric film in the culture chamber with the primary culture.
  • the growth medium is maintained at pH 7.2 by applying an appropriate stream of air and carbon dioxide.
  • the temperature is maintained at 37°C.
  • the skin cells attach to the surface of the film and start to form colonies.
  • the inoculation medium is changed after 48 hours and replaced by growth medium.
  • the cells are cultivated for 2 to 10 days.
  • the wound dressing film with the attached cell lawn is washed. It may be cut out with a sterile scalpel as a whole just before applying to the wound, or if desired in form of stripes, or may be perforated before or after the cell growth in order to allow drainage of the wound.
  • the wound dressing with the attached autologous skin cells can be prepared for use either 1-2 hours before the operation in the laboratory or just before use in the operating room. If the final wound dressing is prepared in the laboratory the cells are washed twiced with DMEM. The film is cut out manually with a sterile scalpel and transferred to a sterile dish containing a transport medium, e. g. Eagle's MEM Hanks' salts with NaHC0 3 4 mM, HEPES 10 mM. The dish with the seeded film is placed in an insulated box and transferred to the operating room. The film, if desired, may be perforated with a scalpel if the wound is draining excessively, or placed directly on the wound with the cell side down. If the final wound dressing is prepared in the operating room the dishes and the growth chambers with the seeded film are washed twice automatically with transport medium, placed in an insulated box and transferred to the operating room directly, where it is processed as described above.
  • a transport medium e. g.
  • the invention concerns in another aspect the use of the wound dressings obtainable according to the present invention for protecting and healing wounds.
  • the present wound dressings are particular useful for protecting and healing large wounds, such as occur in burnings, or only slowly healing wounds, such as in diabetic patients. They can be further used in cosmetic surgery, where scars are particular important to be avoided.
  • a sterile second bandage may be used to tighten the foil to the wound surface and to protect the perforations against infections.
  • Another object of the invention is an apparatus for the automated production of a wound dressing according to the invention and culturing of attachment dependent cells, comprising one or more growth chambers containing a polymeric film which has at least one side suitable for culturing attachment dependent cells, means for supplying inoculation medium, means for keeping sterile the means for supplying the inoculation medium, means for supplying cell growth medium, means for removing and accepting waste medium, means for transportation of cell growth medium and waste medium connec ⁇ ted to said growth chamber(s), means for pumping the media to the growth chamber(s) and for emptying the growth chamber(s), means for tilting the growth chamber(s), means for supporting the growth chambers, means for heating and for keeping the temperature constant, means for covering the growth cham ⁇ ber(s), means for controlling the flow of the inoculum to the growth chambers, means for controlling the flow of the growth medium to the growth chambers, means for controlling the flow of the waste medium out of the growth chamber(s), means for controlling air mass flow, means for
  • the stack of growth chambers is built in such way that the polycarbonate cover plate- of each growth chamber forms concurrently the lower polycarbonate plate of the growth chamber directly above of it.
  • the gasket O-rings are on both sides of such plate (Fig. 8) .
  • the stack of the growth chambers is held watertight by severl, preferably six, screw rods attached to two strong plates, metal base of the stack and metal top of the stack. The apparatus and its components will be more easily understood by inspecting the FIGS. 1 to 9.
  • FIG. 1 shows the entire bioreactor apparatus and support equipment.
  • FIG. 2 shows a cross section of an example A of a growth chamber.
  • FIG. 3 shows a cross section of an example B of a growth chamber.
  • FIG. 4 shows a cross section of an example C of a growth chamber.
  • FIG. 5 shows cross section of an example D of a growth chamber.
  • FIG. 6 shows two examples of a holding frame
  • FIG. 7 shows a three dimensional expanded view of the growth chamber A.
  • Fig. 8 shows a cross secttion of a growth chamber E from the stack.
  • Fig 9 shows schematically a complete stack of six growth chambers with the bae and the top holding frames.
  • peristaltic bi-directional pump 7a pump direction fill
  • growth chamber body e.g. polycarbonate
  • liquid medium inlet/outlet
  • Means for supplying inoculation medium is for example a glass or plastic vessel (1) of sufficient size containing the necessary amount of inocculation medium and the primary cul ⁇ ture of the cells. It is placed in the sterile bench and is connected via tubings (6a) with the computer actuated pneu ⁇ matic valve (16) .
  • Means for keeping sterile the means for supplying the inocu ⁇ lation medium and the medium itself is for example a sterile bench or hood (2) .
  • Means for supplying cell growth medium is for example a glass or plastic vessel, e. g. the medium reservoir (3), of suffi ⁇ cient size containing the necessary amount of growth medium. It is connected via tubings ( 6a) with the computer actuated valve (14) and can be thermostated to at least 37°C or slightly above.
  • Means for removing and accepting waste medium is for example a glass or plastic vessel, e. g. the waste reservoir (4), of sufficient size for collecting the produced waste medium. It is connected via tubings (6a) with the computer actuated valve (15) .
  • Means for transportation of liquid incubation medium, cell growth medium and waste medium are for example sterilizable tubes made of glass or metal or are preferably silicone tu ⁇ bings (6a) of sufficient diameter to allow unihibited trans ⁇ portation of the fluids from the vessels to the growth cham- ber(s), or from the growth chamber(s) to the waste reservoir.
  • Means for pumping the liquid media to the growth chamber(s) and for emptying the growth chamber(s) are pumps, e. g. a bi ⁇ directional peristaltic pump (7) to which more than one tubing (6a) may be attached and which allows to pump the liquids in either direction (7a) or (7b) .
  • Means for tilting the growth chamber(s) comprise for example a lever, an axis for rotation and a electrical motor or a manually operated handle.
  • Means for supporting the growth chamber(s) is for example a table (9) made of a stable material, e. g. wood, metal or strong plastic.
  • Means for heating and for keeping the temperature around the growth chamber(s) constant are for example a hot-plate or a metal block, e. g. of aluminum (10), to which an electrical heater (11) is attached, and a ther ostate for keeping the temperature within the growth chamber(s) at 37°C.
  • Means for covering the growth chamber(s) is for example a five-sided box (12) made of a transparent plastic or plexiglas over which is placed a removable five-sided non- tranparent isolating box (13), e. g. made of styrofoam.
  • Means for controlling the flow of the inoculum to the growth chamber(s) is a valve, for example a pneumatic computer actuated valve (16) .
  • Means for controlling the flow of the growth medium to the growth chamber(s) is a valve, for example a pneumatic com ⁇ puter actuated valve (14) .
  • Means for controlling the flow of the waste medium out of the growth chamber(s) is a valve, for example a pneumatic com- puter actuated valve (15) .
  • Means for controlling air mass and carbon dioxide mass flow are for example air mass (18) and carbon dioxide mass (17) flow controllers allowing the regulation of the desired pro- portions of both gases.
  • Means for storing air and carbon dioxide are for example pressure cylinders of sufficient size containing compressed clean air and carbon dioxide, respectively.
  • Means for humidifying air and carbon dioxide is for example a bubble column allowing to add the desired amount of water to the gases.
  • Means for filtering air and carbon dioxide are for example one or more sterile filters.
  • Means for transporting the gases from the storing means to the growth chamber(s), are for example sterilizable tubes made of glas or metal or are preferably silicone tubings (6b) of sufficient diameter to allow unihibited transportation of the fluids from the vessels to the growth chamber(s), or from the growth chamber(s) to the waste reservoir.
  • Means for controlling the bioreactor functions in real time is for example a "computer, such as a Macintosh or PC containing the necessary software, e. g. LabviewTM in conjunction with National Instruments analog in-put/out-put board to control the bioreactor operations.
  • the inventions concerns in particular an apparatus for the envisioned use, comprising a medium reservoir, one or more growth chambers each containing a polymeric film of which at least one side is suitable for culturing of attachment- dependent cells, pumps for pumping the cell growth medium to the growth chambers and for emptying the growth chambers, a tilting mechanism for tilting the growth chambers, at least the growth medium reservoir and the growth chambers being thermostated to 37°C, computer actuated valves controlling the flow of liquids, devices for controlling the air and carbon dioxide mass flow, and a computer controlling the functions in real time.
  • the invention concerns in particular an apparatus for the envisioned use as shown in FIG. 1, comprising an inoculation vessel (1), placed in a sterile hood (2), a medium reservoir
  • a waste reservoir (4) one or more growth chambers (5), silicone tubings (6) connecting the reservoirs and the growth chambers, a multihead-bidirectional-peri-staltic pump (7), a tilting mechanism (8) for tilting the growth chambers, a table (9), an aluminum block (10), an electrical heater (11), a plexiglas five-sided box (12) , a styrofoam five-sided box (13), a pneumatic waste valve (15), a pneumatic medium valve (14), a pneumatic inoculation valve (16), a carbon dioxide mass flow controller (17), an air mass flow controller (18), air supply (19), carbon dioxide supply (20), humidifier (21), and an air and carbon dioxide filter (22) .
  • the invention concerns further a growth chamber, e. g. of a length of about 20 to 60 cm, a breath of about 6 to 15 cm, and a height of about 1 to 3 cm, in particular of about 40 to 10 to 2 cm, for the production of a wound dressing and culturing of attachment dependent cells according to FIG. 2, 6 and 7, which is used alone or preferably in the above described apparatus, comprising a polycarbonate growth chamber body (23), liquid medium inlet/outlet (24), air/carbon dioxide mixture inlet/outlet (25) , a holding frame
  • a particular growth chamber is such, wherein the holding frame (26) keeps the upper and lower parts attached to each other by attaching means, e.g. screws, as is possible through the holes in the holding frame (26) shown in FIG. 7.
  • a particular growth chamber is such, wherein the holding frame (26) is made from one piece as shown in FIG. 6 left hand.
  • a particular growth chamber is such, wherein the holding frame (26) keeps the upper and lower parts attached to each other by keys sliding in slots of the polycarbonate growth chamber body (23), (32), (34) or (36) as shown in FIG. 6 right hand side.
  • a particular growth chamber is such, whereein its upper part (28) is concurrently the lower part (28) of the adjacent growth chamber above, and its lower part (28) is concurrently the upper part (28) of the adjacent growth chamber bellow.
  • the invention covers further a growth chamber for the produc ⁇ tion of a wound dressing and culturing of attachment depen ⁇ dent cells according to FIG. 3, comprising a polycarbonate growth chamber body (32), a detachable cover lid made of transparent material (37), a lower transparent plate (28), covered with the polymeric film according to claim 1 (29) , being attached and kept tight by the seal (27) , using a locking mechanism, e.g. bars of stainless steel (30) .
  • the invention covers further a growth chamber for the pro ⁇ duction of a wound dressing and culturing of attachment dependent cells accoring to FIG. 4, comprising a transparent polycarbonate growth chamber body (34), a liquid medium inlet/outlet (24), an air/carbon dioxide mixture inlet/outlet (25), a holding frame (26), a seal (27), a transparent plate
  • the invention covers further a growth chamber for the produc ⁇ tion of a wound dressing and culturing of attachment depen ⁇ dent cells according to FIG. 5, comprising a polycarbonate growth chamber body (36), a detachable cover lid (37) made of transparent material sitting snugly on the upper seal (27), the bottom of the growth chamber body (36) covered with the polymeric film according to claim 1 (29) .
  • the invention covers further a method for the production of a wound dressing and for culturing of attachment dependent cells, wherein the wound dressing is prepared in an incubator using anyone of the growth chambers accordimg to FIG. 3, 4 or 5.
  • the following abbreviations are used throughout this descrip ⁇ tion:
  • EDTA ethylene dinitrolotetra-acetic acid
  • HEPES 4- (2-hydroxyethyl)-1-piperazineethane sulphonic acid
  • PBS phosphate buffered saline
  • DMEM Dulbecco's minimum essential medium
  • EGF epidermal growth factor
  • HAMS Ham's F-12 nutrient medium
  • FCS fetal calf serum
  • T3T mouse myeloma cells
  • TBSA total body surface area
  • Transport medium
  • Keratinocyte growth medium + EGF - 400 ml of DMEM (Seromed T041-01)
  • Keratinocytes may beObtained from the patients leg using for example a 5 cm 2 biopsy.
  • a primary culture of keratinocytes is obtained by growing the obtained keratinocytes for 5 to 8 days in a Petri dish accor ⁇ ding to Reinwald J.G. & Green H., Nature 265, 421-424, 1977, in an incubator. Secondary cultures are grown in the computer-controlled fully-automated skin bioreactor according to FIG. 1, 2, and 7.
  • Keratinocytes from the primary culture are trypsinized with a trypsin EDTA solution (10 ml EDTA in 490 ml PBS), resuspended in 100 ml keratinocyte medium minus EGF and transferred into a sterile 500 ml Schott bottle (1) in a sterile hood (2) .
  • the Schott bottle is connected to the growth chambers (5) with 4 mm silicone tubing (6a) .
  • the glass bottoms (28, Figure 2) of the growth chambers of the bioreactor are covered with a hydrophilic 25 micron-thick optically-clear Teflon sheet (29) (Heraeus-Biofolie 25,
  • a multihead peristaltic pump (7) is used to transfer the keratinocyte suspension into the bioreactor chambers (5) .
  • the suspension is mixed in the bioreactor by actuating the bioreactor tilting mechanism (8) three times. Over the next 48 hours the bioreactor is not disturbed.
  • the temperature is automatically maintained at 37°C by placing the bioreactor on a 50 X 60 X 5 cm aluminum plate (10) , which is maintained at 46°C with an electrical heater (11) .
  • the bioreactor and aluminum plate is covered with a 50 X 60 X 50 five sided Plexiglas box (12) and a five sided Styrofoam box (13) .
  • Medium pH is maintained at 7.2 by mixing the air stream entering the bioreactor with a pure stream of carbon dioxide gas.
  • Mass flow controllers (17 and 18) are used to adjust the relative carbon dioxide percentage to 10%.
  • the individual keratinocytes attach and begin to form colonies on the hydrophilic side of the teflon sheet (29) .
  • the medium is exchanged after the first 48 hours with keratinocyte growth medium automatically.
  • a computer software e. g. LabviewTM, is used in conjunction with a National Instruments analog in-put out-put board to control all bioreactor operations. Analog output signals are connected to TTL logic relays that are connected to a peristaltic . pump (7), and pneumatic solenoid valves (14),
  • Step 1) The spent medium is removed by actuating valve (15) (open waste valve), actuating the tilt mechanism (8) for tilting the bioreactor, and changing the direction of the peristaltic pump (7) to the empty direction (7b) . Spent medium is collected in the waste reservoir (4) .
  • Step 2) Fresh keratinocyte growth medium is added by actuating valve (14) and changing the direction of the peristaltic pump (7) to the feed direction (7a) .
  • Step 3) The medium is evenly distributed in the individual chambers by actuating the tilting mechanism (8) three times at 30 second intervals.
  • Step 4) The cells are cultivated for 2-10 days on the FEP film.
  • Step 5) The grafts are either prepared in the operating room or in the laboratory 1-2 hours before the operation:
  • Step 1) The 20 cm 2 Petri dishes and the growth chamber(s) that have been cultured in an incubator are manually washed twice with DMEM.
  • the growth chambers of the bioreactor are washed twice with DMEM automatically.
  • Step 2) The growth chambers and Petri dishes are opened manually.
  • Step 3 The FEP film is cut out of each dish and growth cham ⁇ ber manually with a sterile scalpel and transferred to a sterile 90 mm Petri dish containing 4 ml of transport medium (Eagle's MEM Hanks' salts with NaHC0 3 4 mM, HEPES 10 mM) .
  • transport medium Eagle's MEM Hanks' salts with NaHC0 3 4 mM, HEPES 10 mM
  • Step 4) The Petri dishes containing the colonized FEP film are placed in a insulated box and transferred to the operating room.
  • Step 5) The FEP film is sliced with a scalpel if the wound is draining excessively.
  • Step 1) The 20 cm 2 dishes and the growth chamber(s) that have been cultured in the incubator are manually washed twice with transport medium (Eagle's MEM Hanks' salts with NaHC0 3 4 mM, HEPES 10 mM) .
  • transport medium Eagle's MEM Hanks' salts with NaHC0 3 4 mM, HEPES 10 mM
  • the growth chambers of the bioreactor are auto ⁇ matically washed with the same medium.
  • Step 2) The Petri dishes and the growth chambers containing the seeded FEP film are placed in an insulated box and transferred to the operating room. Step 3) The growth chambers and Petri dishes are opened manually.
  • Step 4) The FEP film is cut out of each dish and growth chamber manually with a sterile scalpel.
  • Step 5) The FEP film is sliced with a scalpel if the wound is draining excessively.
  • Example 2 Preparation of a Wound Dressing with a Mixture of Keratinocytes, Melanocytes, and Dermal Fibroplasts
  • a wound dressing prepared comprising on the FEP film a lawn of keratinocytes, melanocytes and dermal fibroplasts from the patient.
  • the primary cultures for each cell type are individually prepared and mixed in the Petri dish or the growth chamber in proportions as they occur in the patient, and cultured as in Example 1.
  • the wound dressing is prepared for use either in the labora ⁇ tory or just befor the operation in the operating room.
  • the dishes were incubated in a humidified carbon dioxide controlled incubator (37°C, 95% relative humidity, 15% C ⁇ 2) •
  • the bioreactor film was 42% confluent and the dishes were 90% confluent at the time of transplantation.
  • the overall time for obtaining the wound dressing with the keratinocytes was 11 days. All small third and deep second degree wounds that were treated with the keratinocyte seeded FEP film closed per enately and the resultant scars resembeled scars of self healing wounds with respect to appearance and texture. All the large third degree wounds, e.g., the entire right arm, that were treated with the keratinocyte seeded FEP film initially closed after 10-14 days. However, these wounds were sensitive to mechanical shear and 50% percent required autografting. References

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Abstract

On décrit des pansements pour blessures, sous la forme de films polymères dont un côté est recouvert de cellules cutanées autologues cultivées in vitro, la préparation de ces pansements ainsi que leur utilisation pour couvrir et cicatriser des pertes de substance cutanée. On décrit également un appareil de production automatique de tels pansements et des chambres de culture spécialement conçues à cet effet.
PCT/EP1997/000136 1996-01-19 1997-01-14 Pansements pour blessure et appareil associe WO1997026023A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU14418/97A AU1441897A (en) 1996-01-19 1997-01-14 Wound dressing and apparatus

Applications Claiming Priority (3)

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US58893996A 1996-01-19 1996-01-19
US08/588,939 1996-01-19
GB96810624.5 1996-09-23

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WO1997026023A1 true WO1997026023A1 (fr) 1997-07-24

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013076A1 (fr) * 1996-09-24 1998-04-02 Brigham And Women's Hospital Reseau fibreux ensemence avec des cellules de culture et destines au remplacement de tissus
FR2821853A1 (fr) * 2001-03-09 2002-09-13 Natural Implant Sa Bioreacteur pour tissu cultive en couche mince et utilisations
DE10208311A1 (de) * 2002-02-27 2003-09-11 Tuhh Tech Gmbh Vorrichtung und Verfahren zur Kultivierung von Gewebezellen
DE10326747A1 (de) * 2003-06-13 2005-01-05 Gerlach, Jörg, Dr.med. Perfusionseinheit und Perfusionsstation zur Hautwundbehandlung sowie dessen Verwendung
DE102007036853A1 (de) * 2007-08-06 2009-02-19 Günther, Ralf, Dr. Vorrichtung zum Auflegen und zur Manipulation von Geweben
DE102004054125B4 (de) * 2004-11-08 2011-01-05 Minucells And Minutissue Vertriebs Gmbh Gradientenkammer zum Kultivieren und/oder Differenzieren von Zellen/Geweben
DE10128810B4 (de) * 2001-06-18 2013-11-07 Pan-Biotech Gmbh Einrichtung zur Kultivierung von Zellen, insbesondere menschlicher oder tierischer Zellen

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EP0155237A2 (fr) * 1984-03-15 1985-09-18 MBR Bio Reactor AG Procédé et appareil pour la culture de cellules humaines, animales, végétales ainsi que de cellules hybrides et de micro-organismes
EP0307048A2 (fr) * 1987-09-11 1989-03-15 Joseph Guy Cremonese Flacon pour la culture de cellules muni d'une membrane
WO1989003228A1 (fr) * 1987-10-14 1989-04-20 Board Of Regents, The University Of Texas System Systeme de greffe cutanee
EP0396138A2 (fr) * 1989-05-04 1990-11-07 Millipore Corporation Procédé pour la croissance de tissu cellulaire
WO1991013638A1 (fr) * 1990-03-05 1991-09-19 Smith & Nephew Plc Produits de culture cellulaire
WO1992010217A1 (fr) * 1990-12-05 1992-06-25 Vitaphore Wound Healing Inc. Pansements et procedes de fabrication
WO1995017526A1 (fr) * 1993-12-22 1995-06-29 Prenosil, Jiri, E. Procede et dispositif de production d'une structure cellulaire sur une membrane semi-permeable

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0155237A2 (fr) * 1984-03-15 1985-09-18 MBR Bio Reactor AG Procédé et appareil pour la culture de cellules humaines, animales, végétales ainsi que de cellules hybrides et de micro-organismes
EP0307048A2 (fr) * 1987-09-11 1989-03-15 Joseph Guy Cremonese Flacon pour la culture de cellules muni d'une membrane
WO1989003228A1 (fr) * 1987-10-14 1989-04-20 Board Of Regents, The University Of Texas System Systeme de greffe cutanee
US5015584A (en) * 1987-10-14 1991-05-14 Board Of Regents, The University Of Texas System Epidermal graft system
EP0396138A2 (fr) * 1989-05-04 1990-11-07 Millipore Corporation Procédé pour la croissance de tissu cellulaire
WO1991013638A1 (fr) * 1990-03-05 1991-09-19 Smith & Nephew Plc Produits de culture cellulaire
WO1992010217A1 (fr) * 1990-12-05 1992-06-25 Vitaphore Wound Healing Inc. Pansements et procedes de fabrication
WO1995017526A1 (fr) * 1993-12-22 1995-06-29 Prenosil, Jiri, E. Procede et dispositif de production d'une structure cellulaire sur une membrane semi-permeable

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013076A1 (fr) * 1996-09-24 1998-04-02 Brigham And Women's Hospital Reseau fibreux ensemence avec des cellules de culture et destines au remplacement de tissus
CN100408670C (zh) * 2001-03-09 2008-08-06 天然植入物公司 薄层中组织培养用的生物反应器及其应用
FR2821853A1 (fr) * 2001-03-09 2002-09-13 Natural Implant Sa Bioreacteur pour tissu cultive en couche mince et utilisations
WO2002072750A1 (fr) * 2001-03-09 2002-09-19 Natural Implant Bioreacteur pour tissu cultive en couche mince et utilisations
US7163825B2 (en) 2001-03-09 2007-01-16 Henkel Kgaa Bio-reactor for tissue cultivated in form of a thin layer and uses thereof
DE10128810B4 (de) * 2001-06-18 2013-11-07 Pan-Biotech Gmbh Einrichtung zur Kultivierung von Zellen, insbesondere menschlicher oder tierischer Zellen
DE10208311A1 (de) * 2002-02-27 2003-09-11 Tuhh Tech Gmbh Vorrichtung und Verfahren zur Kultivierung von Gewebezellen
DE10208311B4 (de) * 2002-02-27 2005-01-13 Tuhh-Technologie-Gmbh Vorrichtung und Verfahren zur Kultivierung von Gewebezellen
DE10326747B4 (de) * 2003-06-13 2006-11-02 Gerlach, Jörg, Dr.med. Perfusionseinheit und Perfusionsstation zur Hautwundbehandlung sowie dessen Verwendung
DE10326747A1 (de) * 2003-06-13 2005-01-05 Gerlach, Jörg, Dr.med. Perfusionseinheit und Perfusionsstation zur Hautwundbehandlung sowie dessen Verwendung
DE102004054125B4 (de) * 2004-11-08 2011-01-05 Minucells And Minutissue Vertriebs Gmbh Gradientenkammer zum Kultivieren und/oder Differenzieren von Zellen/Geweben
DE102007036853A1 (de) * 2007-08-06 2009-02-19 Günther, Ralf, Dr. Vorrichtung zum Auflegen und zur Manipulation von Geweben
DE102007036853B4 (de) * 2007-08-06 2014-02-13 Ralf Günther Vorrichtung zum Auflegen und zur Manipulation von Geweben

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