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.

Background of the Invention

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.

By combining basic tissue culture techniques and engineering principles it is possible to produce artificial auto skin equivalents and to treat large and small skin defects there¬ with without split-thickness donor skin.

In vi tro cultivation of animal cells was first introduced at the beginning of this century (Harrison, 1907; Carrel, 1912) . Today, 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) .

The pioneering work of Green et al. (1979) in the field of keratinocyte culture has lead to the application of cultured

epidermal autografts (CEA) for the treatment of burns. It has become common practice to biopsy a burn patient' s skin, propagate the three-dimensional epidermis composed of keratinocytes in culture 10,000 fold over a three week period. The cultured epidermis is placed directly on the wound where it attaches to the body and closes the wound in 10-15 days. In principle, this technique is very similar to the traditional autograft method were the keratinocytes from donor skin grow out of the meshed skin to close the wound. 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. The combination of low take rate, 20 day production time and $15,000 per square foot means only severely burned patients that do not have enough donor skin to cover their wounds receive cultured epidermal autografts. Only 240 out of 1,600 catastrophic burn patients have benefited from this technology between 1988 and 1992, which reflects a low acceptance level among burn surgeons. It is presently more efficient from a clinical view point to autograft large wounds. Logical process improvements will include increasing take rate, increasing mechanical stability, reducing production time and costs, and solving the contraction problem. As mentioned above the known culture treatment technique involves transplanting an entire ultilayered epidermis composed primarily of cultured keratinocytes. However, it is well known that besides keratinocytes other cell types are involved in the healing process. 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.

State of the Art Concerning Wound Dressings

An efficient method for the application of cultured-undiffe- rentiated-subconfluent-autologous skin cells for the treat- ment of chronic skin wounds so far has not been established. Previous attempts were either ineffective, costly, or time consuming. None of the known methods is in routine use to¬ day. Epicel™ is a wound graft, originally according to Green et al. CEA technology, is commercially available through Genzyme Tissue Repair Cambridge, MA.

Laserskin™, 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. Such hyaluronic acid esters may contain medicaments or keratinocytes and have been suggested as wound dressings (Andreassi et al., 1991). However, 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.

Early methods of growing cells were not yet directed to wound dressings but had first to solve the problems of anchoring and growing. Later also wound dressings have been proposed. Such methods are discussed in the following.

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. Within 4 - 6 hrs 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, describes 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, 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.

Barlow, WO 92/10217, describes wound dressings comprising a sterile cell-growth supporting cross-linked collagen sub¬ strate carrying epithelial cells.

State of the Art Concerning Bioreactors

Several processes and devices have become known especially designed for growing cells in vi tro .

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. These nets cannot be used as wound dressings because the net cannot be removed from the wound without tearing off cells. The apparatus is not suit¬ able for recovery of the cells or the membrane with the cells.

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, describes a method for producing a cell structure in 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.

All these prior art methods have certain drawbacks. 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.

There is still a great need for a wound dressing containing the patients own skin cells which is rapidly produced in large amounts at an acceptable price.

Objectives of the Invention

It is an objective of the present invention to overcome the drawbacks of the hitherto used methods for healing non-self- healing wounds and to provide an apparatus for the efficient cost-neutral production of a wound dressing that comprises the patients own skin cells, which is easily producedin a timely manner at a relatively low price, which in addition for the medical personnel are easy to handle, simple to apply to the wound and which do not interfere with the newly developing epithelium. In addition the final scar should be cosmetically acceptable. It is an objective of the invention to provide a wound dressing whith the above mentioned advantages and furthermore which:

- is sterilizable before the skin cells are cultured thereon and which can be kept sterile until they are applied to the patient,

- provides a suitable surface for the growth of skin cells,

- is transparent, so that wound inspection is possible,

- is resistant to water transport so that water loss from evaporation is minimized, - does not absorb liquid so that it does not adhere to the wound,

- is only permeable to gases so that it reduces the risk of air born bacterial contamination,

- is strong enough for handling and transport, - is easily transportable,

- can be cut to the desired size,

- can be easily applied to the wound,

- protects the wound from the environment,

- promotes healing,

- once applied does not require further manipulation,

- need not be replaced until the wound is closed,

- is easily detachable from the wound without disturbing the healing process, - circumvents anaesthesia and surgical techniques, and

- provides cosmetically acceptable scars.

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. In particular, 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. However, none of the mentioned films has demonstrated utility with respect to cell culture and subsequent grafting. 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. In particular, 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¬ duce 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 ".

Said polymeric film has at least one side suitable for culturing attachment dependent cells. In particular 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. Preferably 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.

In a second aspect 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.

Secondary cultures and wound dressings on a small scale are produced in a growth chamber according to the invention of a type described further below (FIGS. 2-5) that contain a film according to the invention, in particular a FEP hydrocarbon film defined above.

Secondary cultures and wound dressings are produced on a large scale by using an apparatus according to the invention defined further below.

Methods for the preparation of the secondary cultures are in principle also known. In the proper medium, in the presence of feeder cells and under carefully monitored temperature and moisture conditions the cells will grow to confluency within about 7 to 10 days (total culture time 12 to 14 days) .

More particularly, 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.

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.

Detailed Description of the Apparatus

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 storing air, means for controlling carbon dioxide mass flow, means for storing car¬ bon dioxide, means for humidifying air and carbon dioxid, means for filtering air and carbondioxide, and means for con¬ trolling the bioreactor functions in real time.

If more than one growth chamber is intended 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.

Short description of the Figures.

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.

The numbers in the Figures and the following description in parenthesis have the following meanings:

I. inoculation vessel 2. sterile bench

3. medium reservoir

4. waste reservoir

5. growth chamber according to claim 12 6a. silicone tubing transporting liquid 6b. silicone tubing transporting gas

7. peristaltic bi-directional pump 7a. pump direction fill

7b. pump direction empty

8. tilting mechanism 9. table

10. aluminium block

II. electrical heater

12. Plexiglas 5 sided box

13. Styrofoam 5 sided box 14. pneumatic medium valve (computer actuated)

15. pneumatic waste valve (computer actuated)

16. pneumatic inoculation valve (computer actuated)

17. carbon dioxide mass flow controller

18. air mass flow controller 19. air supply

20. carbon dioxide supply

21. humidifier

22. sterile filter

23. growth chamber body (e.g.polycarbonate)

24. liquid medium inlet/outlet

25. air/carbon dioxide mixture outlet

26. aluminum holding frame

27. seal 28. glass plate

29. polymeric sheet (25 microns thick)

30. locking bar (stainless steel construction)

31. growth chamber according to claim 14

32. growth chamber body according to claim 14 33. growth chamber according to claim 15

34. growth chamber body according to claim 15

35. growth chamber according to claim 16

36. growth chamber body according to claim 16

37. detachable cover lid

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. Labview™ 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

(3), 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

(26), seals, e.g. silicone tubing (27), upper and lower transparent plates (28), the lower plate being covered with the polymeric film (29) , both plates being attached and kept tight by the lower seal (27), using a locking mechanism, e.g. bars of stainless steel (30) . 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

(28) being attached and kept tight by the seal (27), using a locking mechanism, e.g. bars of stainless steel (30), the bottom of the growth chamber body (34) being covered with the polymeric film according to claim 1 (29) .

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

PEST: penicillin/streptomycin 5000 units/ml

T3T: mouse myeloma cells TBSA: total body surface area

The following examples serve to further describe the present invention, however should not be construed as a limmitation thereof.

Example 1. Preparation of a Wound dressing with Keratinocytes

The following solutions are used: Transport medium:

- 1.0 bottle of minimum essential medium eagle (Sigma M- 0268) ,

- 1 liter of double distilled water

- 336 mg of sodium hydrogen carbonate - 10 ml of 1.0 molar HEPES (Sigma H-0887)

- adjusted to pH 7.2 with HCL.

0.1% Trypsin/antibiotic solution:

- (ICN Biochemicals 103140, 100 grams) - 0.3 g of trypsin

- 300 ml of PBS

- 300 mg of glucose

- 0.15 ml (1%) of phenol red

- PEST, oxacillin, methicillin all 1.0 mg/ml filter and freeze back 5 ml aliquots

0.04% EDTA solution:

10 ml of 2% EDTA in 490 ml of PBS 0.05% trypsin 0.02% EDTA solution:

5 ml of 0.1% trypsin/antibiotic solution in 5 ml of 0.02% EDTA solution

Keratinocyte growth medium + EGF: - 400 ml of DMEM (Seromed T041-01)

- 100 ml of HAMS F-12 (Seromed T081-1)

- 55 ml of FCS (Hyclone A-6166-L) Lot 61662304

- 5 ml of PEST

- 5 ml of 200 mM glutamine - 1.0 ml of hydrocortisone

- 0.5 ml of cholera toxin

- 0.5 ml of EGF

- 12 ml of adenine (fresh)

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,

Petriperm®, hydrophilc side upward) . 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. Labview™, 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),

(15) , and (16) . A programmed medium change proceeds as follows:

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:

Laboratory preparation of the wound dressing: 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) .

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 6) The seeded films are removed from the dishes and placed directly on the wound (cell side down) .

Operating room preparation of the wound dressing: 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) . 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. Step 6) The FEP film is placed directly on the wound (cell side down) .

Large quantities of dressing are efficiently produced with a specially designed fully automated apparatus acording to the invention, e. g. as shown in FIG. 1 and 2.

Example 2: Preparation of a Wound Dressing with a Mixture of Keratinocytes, Melanocytes, and Dermal Fibroplasts In a similar manner as described in Example 1 is 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.

Results of two patients with keratinocyte wound dressings 1. Patient KM: Keratinocytes were obtained from a 5 cm ² biopsy taken from the leg. The wound of. this 55 ^ear old male patient had a surface area of 480 cm . A 75 cm primary culture grew to confluence in 8 days. Secondary cultures were diluted 1:50 and cultured on FEP film for 5 days at which time they were 5% confluent. At that time they were trans¬ planted. KM.'s keratinocytes were cultured for 5 days on 24 20 cm ² Petri dishes containing FEP film and in one incubator growth chamber. All the dishes and the bioreactor growth chamber were incubated in a humidified carbon dioxide controlled incubator (37C, 95% RH, 15% C02) . Six percent of the FEP film was covered with cells at the time of trans¬ planting. Thirteen days were required to obtain 3500 cm ² dressing. All the wounds treated with the keratinocyte seeded film

"closed" within 7-9 days and remained closed. The wounds were second degree. The resultant scars resembled scars of self healing wounds with respect to appearance and texture.

2. Patient HG: The wound of this 47 year old female patient had a surface area of about 60% TBSA. Two 75 cm ² primary cul¬ tures grew to confluence in 5 days. Secondary cultures were started 5 days after the start of the primary culture and grew for 6 days. HG.'s keratinocytes were cultured for 6 days on FEP film in two bioreactor growth chambers (640 cm ² ) and in 24 20 cm ² Petri dishes containing FEP film. The dishes were incubated in a humidified carbon dioxide controlled incubator (37°C, 95% relative humidity, 15% Cθ2) • 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

Andreassi, L. et al., WOUNDS 1991, 3(3), 116-126;

Harrison, R.G., Proc Soc Exp Biol Med, Vol. 4 pp. 140-143, 1907;

Carrel, A. J. Exp. Med. Vol 15, pp. 516-528, 1912;

Green, H., et al., P roc. Natl. Acad. Sci. USA Vol. 76, No. 11, pp. 5665-5668, 1979;

Freshney, I., Culture of Animal Cells third Ed., Wiley-Liss 1994; Harper, R. and Grove, G., Science, Vol. 204, pp. 526, 1979. Dulbecco, R., Virology Vol. 8 pp 396, 1959.