CA2226747A1

CA2226747A1 - Cell culture products

Info

Publication number: CA2226747A1
Application number: CA002226747A
Authority: CA
Inventors: Patrick Lewis Blott; Mark Christopher Richardson; Jane Bridget Matthews; George Robert Drewery
Original assignee: Individual
Current assignee: Smith and Nephew PLC
Priority date: 1995-08-12
Filing date: 1996-08-12
Publication date: 1997-02-27
Also published as: WO1997006835A1; JP2000500032A; EP1019104A1; AU6746296A; ZA966803B; GB9516556D0

Abstract

A wound dressing comprises a carrier comprising a hydrophilic polymer layer and a layer of cultured mammalian cells anchored to the wound-facing surface of the carrier. A method of making such a dressing is also disclosed.

Description

PCl'tGB96/U1959 CF.-~ CUTTUR~PRODUCTS

T~is invention relates to the c~lturing of mara~ n anchorage dependent cells onto a con~orma~le carrier. ~ore particularly ~he invention relates to the format~on of wound dressings suita~le for treating wounds such as burns, skin graft donor sites and ulcers e.g. venous and decu~itus ulcers and to systems for use in the preparation of such dressings.

~ Am~l i an cells that are ;n~r~h1e of proliferating in suspended liquid culture but can be ~ade to proliferate on the surface of a carrier are said to be anchorage-dependent.

Epithelial cells, such as keratinocytes, are anchorage dependent. Such c~lls cultured in the presence of a carrier which is non-inhibitory and non-cytotoxic will multiply in stratified colonies and eventually produce a confluent layer. Cell cultures of this t~?e are used to investigate skin growth and have been used as skin grafts. Various terhn;cal papers have been published which describe in vitro t~chniques for growing skin cells and their subse~uent use in the treatment of full-thic~ness wounds. For example E. Bell et al (J Invest Derm 81; 2s~lOs 1983); E. Bell et al (Science 211; lOS2-1054 1981); D. Asselineau and M.
Pruneiras (Br J Derm 1984 III, Supplement 27, 219-22~) and J.F, Bur~e et al (Ann Surg 94; 413-4Z8 1981).

T~le ability of cells to anchor to a particular carrier is dependent on the properties of the carrier, the cult:urin~ conditions e.g. temperature, and the components of the culture medium. Culturing is usually carried out in hard plastic flasks made from a material which is substantially inert to the growth medium and is non-cytotoxic to the cells. Polystyrene is a commonly used material for culture flasks.

one of the problems in using hard plastic flasks for the culture of epithelial cells for use as skin grafts is that the sheet of cells normally has to reach confluence before they can be harvested. The time taken to reach confluence may be long. Furthermore the layer of cells is not very strong mech~ni cally and can easily be damaged when the unsupported cells are dislodged e.g. by using the enzyme dispase and handled unsupported.

International Patent Application No. W089/03228 discloses a synthetic surgical dressing coated with collagen having human epidermal cells attached thereto.
The method of producing the composite involves incubating the synthetic surgical dressing in a solution of collagen and evaporating the water to give a collagen coating on the surface of the synthetic dressing. The evaporation step is prefera~ly conducted under sterile conditions to impede bacterial cont~min~tion.

It is known that the sheet of cells can be supported after it has been dislodged from the surface in order to facilitate handling and transfer to the wound surface. However this techni~ue does not overcome the problems associated with hard surface cultures or the risks associated with dislodgement of the cells.

It is an object of the present invention, to overcome the above problems, by growing cells on a readily manageable, conformable carrier comprising a hydrophilic, non-inhibitory to cell growth and non-cytotoxic polymer. The polymex of the present ~ i~vention enables the transfer of a cell layer to the site of treatment before it has reached confluence.

According to the present invention a wound dressing comprises a conformable carrier havinq a wound-facing surface to which a layer of cultured mammalian cells is anchored, tLle carrier comprising a synthetic polymer layer which has a water uptake of at least 16w/w % and is non-cytotoxic and non-inhibitory to cell growth.

The present invention also provides a system for producing a wound dressing comprising a conformable carrier having a wound-facing surface to which a layer of cultured mammalian cells is anchored, the carrier being a synthetic polymer which is non-inhibitory to cell growth and non-cytotoxic and having a water uptake of at least 16 w/w %, together with a means for maintaining a aqueous culture medium containing said cells in contact with the wound facing surface of the carrier, the carrier being maintained in a submerged position in the culture medium.

The present invention further provides a method of wound treatment comprising the step of applying a wound dressing comprising a conformable carrier having a wound-facing surface to which a layer of cultured mammalian cells is anchored, the carrier being a synthetic polymer layer which is non-inhibitory to cell growth and llon-cytotoxic and having a water uptake of at least 16 w/w %, to the area to be treated.

In further embodiment of the present invention there is provided a kit comprising a conformable SUBSTITUTE SHEET (RIJI E 26) carrier having a wound facing surface, the carrier being a synthetic ploymer layer, which is non-inhibitory to cell growth and non-cytotoxic and having a water uptake of at least 16 w/w %, a container comprising a nutrient solution or growing mammalian cells and a vessel for containing the carrier and the nutrient solution.

The kit may additionally comprise a sample of mammalian cells.

The mammalian cells employed in the present invention are anchorage-dependent i.e. they re~uire a surface on to which they can bind before they are able to proliferate.

By the term 'conformable' we mean that the dressing will conform to changes in contours of the body portion to which the dressing is applied.

Preferably the synthetic polymer layer is a synthetic polymer film.

In one form, the carrier comprises a single layer of polymer. In another form the carrier comprises a laminate comprising two synthetic polymer layers, at least one polymer of said at least two layers being non-inhibitory to cell growth, non-cytotoxic and having a water uptake of at least 16 w/w%. The use of a laminate may be beneficial if the hydrophilic polymer swells or otherwise becomes fragile or difficult to handle after exposure to culture medium.

Where the wound dressing comprises a polymer layer having a water uptake of less then 16 w/w %, the cell layer may be anchored to this polymer layer.

SUBSTITUTE SHEET tRULE 2~) Alternatively the cell layer may be anchored to the polymer layer having a water uptake of at least 16~.
- In order for the carrier to have desirable surface properties which allows the anchorage of the cells, it may be surface treated. A suitable form of surface treatment is corona-discharge treatment.
Corona discharge treatment increases the surface energy of a material and may provide improved conditions for cell anchorage. The effect of corona-discharge treatment can be assessed by measuring the contact angle of water on the treated material. A
method for measuring contact angle will be described hereinafter. Aptly the contact angle should be reduced by at least 10~, favourably by at least 15 and preferably by at least 20~. Other suitable treatments which can be applied to the carrier include glow discharge or plasma treatment, chemical etching and flame treatment.

Thus in one embodiment of the present invention the wound dressing comprises a conformable carrier the carrier being a laminate o~ a polymer layer having a water uptake of at least 16 wjw % and a hydrophobic polymer layer, the polymers being non-inhibitory to cell growth and non-cytotoxic.

By hydrophobic polymer we mean that the polymer has a water uptake less than 16 w/w ~.

Percentage water uptake o~ the polymer layer is assessed by the following method. A sample of the polymer layer is weighed in its dry state. This is then immersed in excess distilled water and left for a period of 24 hours at 20~C. The hydrated sample is then removed, excess water is removed from the surface of the sample and the sample re-weighed. The percentage increase in weight obtained, is the SUB~lTUrE SHEET (RULE 26) percentage water uptake w/w%. The following e~uation may be used to calculate the percent water uptake (w/w % ) .

w/w% water uptake = (mass of sample + water) - mass of sam~le)x 100 mass of sample The percentage water uptake w/w % of the carrier may be assessed by the above described method by taking a sample of the carrier rather than a sample of the polymer layer.

Preferably the carrier is transparent thus allowing the cells on the surface to be ~A ined by a mi-croscopy. However it is not essential that the cells be viewed during the period of cell culture and therefore non-transparent or opaque carriers may also be used in the dressing of the present invention.

The hydrophilic polymer may be a hydrogel or a hydrocolloid. Hydrogels are preferred as they are transparent.

The carrier should not be inhibitory to cell growth. A measure of such inhibition can be expressed as a percentage cell growth reduction as measured against cells allowed to grow in the absence of a test carrier as hereinafter described. Aptly the carrier should not result in more than 50%
reduction in cell growth. More aptly it should not result in more than a 40% reduction in cell growth.
Favourably it should not result in more than a 30%
reduction in cell growth and preferably not result in more than a 20% reduction in cell growth.
The carrier should be non-cytotoxic. Cytotoxicity can be measured against a non-cytotoxic control material by a method as hereinafter described. Aptly SUBSmUTE S~ EET tRULE 26) W O 97/0683~ PCT/GB96/01959 the cytoxicity of the carrier will not ~ e~ 30%. More aptly the cytotoxicity of the carrier will not ~c~
20%. Preferably the cytotoxicity of the carrier will not exceed 15%.

Th,e carrier may be a ~ in~te of at least two films or at least two sheets or a laminate comprising a film and a sheet.

Suitable synthetic polymers include hydrophilic polyurethane, polyetherpolyester (also known as a thermoplastic ether ester elastomer or a copolyester elastomer) for e.g. HYTREL (Trade Mark), polyetherpolyamide, e.g. PEB~X (Trade Mark), polyacrylamides and polyethylene oxide. Thus an apt laminate may comprise a polyurethane film coated with a material which allows attach~ent of A~horage dependent mamm~ n cells such as e.g. ethylene vinyl acetate.

The carrier may comprise a hydrogel, a hydrocolloid or other suitable polymer. Examples of suitable hydrogels include polyhydroxyethylmethacrylic acid (po:Ly ~EMA), cross-linked polyvinylacrylic acid (PVA), polyacrylic acid cross-linke~ with triallylsucrose (carbopol) and polyvinylpyrrolidone.
Examples of suitable hydrocolloid materials include car~oxymethylcellulose eg sodium carboxymethylcellulose. The carboxyurethylcellulose may be cross-linked or non cross-linked. The hydrocolloid may further comprise a polyisobutylene which serves to hold the hydrocolloid together. The hydrogel may be in the form of a film or a sheet. The sheet may be self-supporting. Alternatively the sheet may be suppor~ed by for example a suitable support layer e.g.
a polymeric film or polymer net. Where the sheet is supported by a support layer the sheet and support W O ~7/06835 PCT/GB96/01959 layer comprise the carrier.

Where the carrier comprises a hydrophobic polymer layer it may desirably be apertured to enable it to handle any exudate from the site of application c particularly in the case of highly extruding wounds.
Thus a continous polymer layer may be adapted to be perforated after the cell layer has reached the desired degree of confluency. Examples of such films, are the bi~ lly oriented films disclosed in for example in EP-0141592, GB-1055963 and GB-914489.

Aperturing the carrier as above described ensures that there is no build up of exudate in e.g.
highly P~ll~ i ng wounds, since exudate can readily escape through the apertures and therefore does not build up under the dressings.

Perforation of the carrier may be carried out either before or after attachment of the cell layer using any suitable method such as hot pin perforation or slitting. Perforation after cell growth should be carried out with ~;ni~ll~ cell disruption and loss.
Perforation should be such as to provide an adequate open area for escape of wound exudate. The minimum open area will depend on the moisture vapour permeability of the film or the lAmi~te.

Where the carrier is a 1 A i nAte of two polymer films or nets in order that it should have the required flexibility and con~ormability, it should suitably have a thickness not excPP~lng 0.075mm. More suitably it should not have a thickness PxcPp~; ng 0.05mm, and favourably not PXcPp~ing 0.04mm. More favourably the film will have a thickness between 0.005 and 0~03mm and preferably between 0.01 and 0.025mm for example, ~ =

WO 97/06835 PCT/GB96tO1959 0.015mm or 0.020mm. Where the carrier comprises a - sheet and polymeric film or net it may have a thickness of the range of from 0.075mm up to about 5mm, the polymeric film or net having the above defined cions.

Mech;ng is conventionally carried out before grafting of a skin graft, to increase the surface area of the graft. In the same way, where the wound dressing of the present invention consists of polymeric films it may be conveyed through a conventional mesh -grafting device.

Films suitable for serving as the carrier may be flat or contoured. The contours may be produced for example by embossing. Suitably contoured films may also have apertures. Such films are described in W090/00398.

Aptly the carrier is per~eable to moisture vapour, oxygen and carbon dioxide. In this way a dressing when in place on the wound will provide moist conditions allowing for the cells to remain viable while the wound heals. The carrier, whilst desirably being impe~vious to liquid water should have an upright moisture vapour tr~nC~;csion rate (MVTR) of at least 300c~m 2 24 hrs 1. Aptly the carrier should have an upright MVTR of less than 2000 gm 2 24 hrs 1. The carrier should have an inverted MVTR of at least 2500 gm 2 24 hrs 1. Suitably the inverted MVTR should not exceed 25000 gm 2 24 hrs l. A method for determining the upright: and inverted MVTR of a substrate is given as follows:

The moisture vapour transmission rate (MVTR) may be measured by the Payne Cup method. This method uses a cup 1.5cm deep which has a flanged top. The inner diameter of the flange provides an area of locm2 of test material through which moisture vapour may pass.
In this method lOml of distilled water is added to the cup and a sample of the test material, large enough to completely cover the flange, is clamped over the cup.
When the test material has an adhesive surface it is clamped with the adhesive surface facing into the cup.
The complete assembly is then weighed and placed in a fan assisted electric oven where the temperature and relative humidity are maintained at 37~C and 10%
respectively. The relative humidity within the oven is maintained at 10% by placing lkg of anhydrous 3-8 mesh calcium chloride on the floor of the oven. After a suitable period of time, for example 17 hours, the cup is removed from the oven and allowed to cool for 20 minutes to reach room temperature. Ater reweighing the mass of water lost by vapour transmission is calculated. The moisture vapour permeability (MVP) is expressed in units of gm 2 24 hrs 1 at 37OC, 100% to 10% relative humidity difference. This is the MVP when the test material is in contact with moisture vapour.
To calculate the MVTR, the reading is corrected for st~n~Ard thickness of test material i.e. a thickness of 1 thou. The MVP when the material is in contact with water may be measured using the same apparatus and making the necessary adjustments for thickness of test material, by simply placing the Payne cup in an inverted position in the oven so that liquid water (and not moisture vapour) is in contact with the test material.

In a modification of the invention the carrier may be formed from knitted, woven or non-woven synthetic polymers to form a tight web of small mesh size. After cell culture the web may be stretched to form a web having a larger mesh size without loss of cells.

It is preferred to use autologous cultivated epithelial cells since these have little or no immunological rejection problems when applied to the host (patient). However it may also be possible to use non-autologous cells e.g. to produce an allograft or xenograft~ Preferably the cells are keratinocytes. we have found that it is desira~le not to allow the cell layer to reach confluence before transferring the wound dressing onto the wound site, thus enabling a suitable wound dre~ising to be produced within a few hours. It will be understood that as the cell layer is sub-confluent it will comprise a monolayer of cells.

The carrier may be sterilised by any suitable known meth,ods of sterilisation. Suitable forms of sterilisation include ethylene oxide (allowing the required time for de-gassing) gamma-irradiation or steam sterilisation. The carrier should be washed after sterilisation, to remove any low molecular weight contaminants, for example where the carrier is a synthetic polymer to remove any unpolymerised monomer as such monomers can be cytotoxic. The washing process may comprise several sequential washes using sterile de-ionised water in sequential steps.

Alternatively the carrier may be aseptically produced.

In the system of the invention the carrier on which the cells are grown shou]d preferably be easily removable from the other parts of the system, namely the means ~or maintaining the aqueous culture medium containing said cells in contact with the wound facing surface of the film. For example, where the carrier W O 97/06835 PCT/GB96/ol959 forms a wall of the vessel cont~i ninq the culture medium, it should be readily removable from the other parts of the vessel. The carrier may form a part of all of the container in which the cell culture is grown.
The other parts of the container or culture vessel may be formed from suitable materials conventionally used for the manufacture of tissue culture vessels. High impact polystyrene is preferred.

In an alternative embodiment the carrier may be laid down within a flask of an appropriate design adapted to allow the removal of the substrate. Where the carrier is an integral part of the culture flask, it may be remova~ly sealed to the other parts of the flask for example by heat sealing or by means of an ahdesive. Preferably the carrier will form a flat surface and will aptly form the base of the flask.

Where the carrier is to be contained within the flask, the flask will be provided with a closeable opening having dimensions sufficient to enable the carrier to be readily removed without disruption of the cells anchored thereto.

Where apertured carrier are used to form an integral part of the culture flask these may be overlaid by e.g. a continuous film to keep the flask water tight and to maintain sterility. If the carrier is laid down within the flask it may be ret~;ne~ by, for example, a pre-sterilised stainless steel ring or, alternatively, by coating the carrier on one side with a layer of non-cytotoxic adhesi~e which is capable of maintaining tack in the presence of tissue culture medium.

Nutrients, growth factors or medicaments such as -antibiotics or antiflammatories may be incorporated into the a~ueous medium in which the cell culture is grown. The nature and weight and/or volume of such additional ingredients are co~ventionally well known.
J

The wound dressing of this invention may be used for a variety of wounds. The dressing of the invention is particlllarly suitable for treating partial-thickne~
wounds that is those where only the epidermis and possibly part of the dermis is lost. Such woun~s include for example skin graft donor sites, first or possibly second degree burns, shallow leg ulcers or pressure sores. The dressing is also suitable for treating ~ull thickness wounds eg. venous ulcers.
Continuous polymeric film carriers aptly act as barriers t:o bacteria whilst being sufficiently permea~le to moisture vapour, oxygen and carbon dioxide to allow wound healing to occur at a desira~le rate. If the substrate is perforated, a secondary dressing could be appliecl to maintain the desired degrees of moisture vapour, oxygen and car~on dioxide permeabilities. A
suitable material is a polyurethane film dressing such as OPSITE (Trade Mark) which may be placed over the wound to create the same conditions. The dressing can suitably be left in place on the wound for a suitable period to allow the wound to become from 30-90%
re-epithelialised, (healed) depen~;nq on the nature of the particular wound and the condition of the patient.
At this time the dressing can be removed and replaced with a conventional wound dressing.

In 1975 Green et al proposed the use of transformed cell line 3T3 cells derived from the mouse as a feeder layer system in order to expand skin cultures. 3T3 cells synthesize factors which are essential for the growth of keratinocytes which are seeded at very low densitites. Furthermore 3T3 cells inhibit unwanted fibroblast growth. Skin cell preparations cont~;ni ng 3T3 cells first have to be g-irradiated (typically about 6000 rads) to inhibit cell division yet not kill the cells. Such irradiated cells survive for several days and during that time will synthesize and supply materials for the host keratinocyte cells. Eventually the 3T3 cells are expelled from the skin cell layer. ~owever inevitably some mouse cells remain and thus will be grafted with the host keratinocytes.

In the dressings of the present invention the feeder cells such as 3T3 may be seeded into a medium in contact with the reverse side of the carrier. These cells will then synthesize materials for the host cells. Since the feeder cells do not come into contact with the host cells there is no need to irradiate them.
When the carrier is removed from the culture flask, the reverse side may be washed to free floating feeder cells.

Where a feeder cell layer is employed with an apertured carrier, the apertures should be large enough to allow free ~ch~nge of the culture medium, but not large enough to allow cells to pass through. Aptly the aperture size should not be larger than 5~ across its largest dimensions. Suitably the aperture size will be from 0.5 to 2~.

The dressings of the present invention may be prepared by suitably qualified personnel at the location where they are to be applied to the host. Thus suitably the dressing may be prepared according to the above disclosed method by a cell-culturist in a hospital cell-culture laboratory. Alternatively the WO 97/06835 PCT/GB96~1959 dressing may be prepared at a location remote from the location ~.g. hospital where it is to be applied. In the latter case, the dressing ~ay be transported under suitable conditions e.g. under a precisely controlled t,--,~~ature. Thus the dressin~ may be cryopreserved ie. maintained at - 190~C and thereafter brought back to room temperature just prior to use. Alternatively the dressing may be transported in a ready-to-use state. Thus the dressing may be tra~sported in a suitable ;ncllh~tor set at the desire~ temperature.

Contact angles are measured by the Wilhelmy Plate dynamic contact angle mea~u~ nt system using a Cahn DCA-322 Dynamic Contact Angle Analyser. Both advancing and rPC~; ng contact angles may be measured. The test li~uid used may be water (HPLC grade). An immersion/withdrawal speed of 150 micron/sec may be used, Samples of films may be prepared by sticking the film onto a glass coverslip approx. 24mm by 30mm or by dip coatinq a slide from solution. T~e testing may be carried out: at 23 deg. C/50% RH.

A suitable method for measuring cell growth reduction is the method disclosed in W0 91/13638.

The method of measuring percentage cellular cytotoxicity of the substrates is as disclosed in W0 91/13638.

Embo~imPnts of the dressing systems of the invention will be illustrated by reference to the accompanying drawings. Referring to Figure 1, one wall the base, of a culture flask 1 comprises a laminate of an aperture~ film 2 and a continuous film 3. The edges of the ~aminate are removably bonded to the other wall portions 5 of the flask 3. Culture media 4 and donor cells can be introduced into flask 3 through neck 6 and sealed therein by stopper 7.

After the system has been incu~ated under suitable cell culture conditions, the laminate 2, 3 may be removed by peeling from flask 1.

Figure 2 illustrates a structure2, 3 formed by casting a first film 2 over a second film 3, film 3 having a plurality of raised portions 8. The resulting film 2, has a plurality of thin 9 and thirk~ne~ 10 areas. On separation of film 2 from film 3, the thin areas 9 ru~L~Le to form apertures 12 as shown in Figure 3. The resulting apertured from which may subsequently be l~ in~ted to a polymer layer to form a laminate, suitable for use as a carrier in the dressing of the present invention.

Referring to Figure 3, the culture flask 1 is divided into compartments 21, 31 by a carrier comprisiny a hydrophilic polymer layer 14 and a perforated hydrophobic film 2.
Culture medium 4 is introduced into the flask and occupies both compartments 21, 31. Skin cells are seeded into romp~rtment 21 through neck 6 whilst feeder cells such as 3T3 cells are seeded into compartment 31 through access port 11.

Nutrients, growth factors or medicaments such as anitbiotics or antiinflammatories may be incorporated into the aqueous medium in which the cell culture is grown. The nature of weight and/or volume of such additional ingredients are conventionally well known.

After the layer of skin cells has reached the re~uired degree of confluence, the carrier is detached CA 02226747 l998-02-ll W O 9710683~ PCT/GB96tO1959 from the wall portion 5 of the flask and removed from the access port 13.
The cell layer is preferably sub-confluent.
Alternatively it may be confluent.
The invention may be illustrated by using any of the polymers given in the following table.

Product Polymer Water Uptake w/w %
Aquasorb 'pnlyviny_py~~o_idone) 4~0 ConCeel Ulcus ca~boxymethv_cellulose) ~o Elastogel po~yacry~.am_~e; 2 o ~,e._perm po_yacry_am ee/agar) 1 n C-ranu~lex E cmc) :PU ~ po~yurethane) n :PU ~ po_yurethane) ~u-ge_ pvp) Cutinova Hydro ~polyurethane) SUBSTITUTE SHEET (RULE 26) Exam~les Human epithelial cells (keratinocytes) were grown on films and laminates comprising a polymer having a water uptake of greater than 16%. The materials and method used are detailed below.

The cells used were from the SCaBER cell line, supplied by American Type Culture Collection of Maryland USA.

The cells were labelled with a dye marker in order to facilitate their detection by optical microscopy. This was necessary because the cells were difficult to detect against the non-homogenous surfaces of some samples. The cells were labelled using a Sigma Immuno Chemicals PKH26 red fluorescent general cell linker kit supplied by Sigma Chemical Company of St Louis USA, according to the instructions suplied with the kit. The cells were then washed with serum-free culture medium before being re-suspended in fresh medium for culture on the samples.

Control experiments were performed in order to determine whether the fluorescent dye marker was taken up by the polymer substrates. The results showed that the dye was not absorbed by the polymers and was specific to the cells, so fluorescence observed on the experimental samples could be attributed to cells attached to the polymer surfaces.

The culture medium used was Earle's ~;n; ~tl~
Essential Medium (MEM) + 10% foetal calf serum made up as follows:-348 ml Earle's MEM (without L - glutamine), supplied by Gibco BRL.

o, 40 ml heat-inactivated foetal calf serum 4 ml penicillin/streptomyci.n (5000IU/ml-5000 ~g/ml), supplied by Gibco BRL.
4 ml non-essential amino acids 4 ml L-glutamine (100x) 200 mM, supplied by Gibco BRL.

The cells were seeded at a density of 106 cells per 3ml of medium.

The culture medium contai~ing cells at the above concentrati.on was placed in a petri-dish in contact with the sample film being tested and incubated at 37~C for 24 hours initially and then for further periods of 24 hours up to 3 days. The polymer substrate was then removed from the petri dish, carefully washed and then ~A~; ned for the presence of attached. cells under ultra-violet light using a NIKON inverted microscope fitted with a rhodium filter to d.etect the labelled red-fluorescing cells.

The polymer layers used were all thin films and were laid on the bottom of the petri dishes using metal rings to keep the film flat and make subsequent handling easier.

Polymer layers used for carrier were samples of hydrophilic films which are used in wound dressing constructions and so are known to be suitable for application to skin for medical purposes.

Example 1 Thermoplastic polyether-polyurethane extruded to form a film approximately 20~m thick, having a water uptake of 64% as measured by the method described SUBSmUTE SHEET (RlJLE 26) above, was used as the carrier layer. The film was laid on the base of a petri dish and culture medium containing cells was introduced onto the upper surface of the film. After incubation for 24 hours the_carrier was removed and washed. Observation under u.v. light showed that cells had attached to the film surface. The film had swollen by absorbtion of culture medium.

Exam~le 2 A film of thermoplastic polyether - polyurethane 20~m thick was laminated to a 50~m film of ethylene-vinyl acetate (EVA) to form a carrier layer. The EVA
material had a water uptake of about 0.3%. The culture medium and cells were placed on the polyether polyurethane side of the laminate. Some cells were observed to have attached after 24 hours, with increasing numbers of cells attaching after 2 and 3 days.

The sample-appeared to have remained generally flat and ridges were seen in the surface of the film, due probably to the swelling of the hydrophilic film in the culture medium.

Example 3 The laminate described in Example 2 was used as the carrier layer and the cells were grown on the EVA
side of the laminate. Cells were observed to have attached and grown on the film after 24 hours.

SUBSTITUTE SHEET tRULE 26) Example 4 The carrier used was a commercial dressing sold under the trademark "OPSITE IV3000" by Smith & NepheW
Healthcare Ltd, which comprises a backing layer of a hydrophilic polyurethane film and a layer of acrylic pressure-se!nsitive adhesive attached to the wound-facing side thereof. The water-uptake of the dressing was measured as appro~imately 68%.

Cells were observed to colonise the non-adhesive, polyurethane side of the dressing.

~y~mnle 5 The carrier was the dressing used in example 4 with the adhesive side of the dressing used as the cell-carrying layer. Cells were observed to attach and proliferate on the adhesive.

Exam~le 6 The carrier used was a laminate of two 20 micrometre thermoplastic polyether-polyurethane films as used in Example 1. As expected, cells were observed to be well disper,sed over the film area in contact with the medium and attached to the film surface. The sample had a bubbled appearance after contact with the medium but this did not appear to unduly affect the attachment of cells to the surface.

SUBSTITUTE SHEET (RULE 26)

Claims

1. A wound dressing comprising a conformable carrier having a wound facing surface to which a layer of cultured mammalian cells is anchored, the carrier comprising a synthetic polymer layer which has a water-uptake of at least 16 w/w % and is non-cytotoxic and non-inhibitory to cell growth.

2. A wound dressing as claimed in claim 1, wherein said polymer layer is a synthetic polymer film,

3. A wound dressing as claimed in claim 1, wherein said polymer layer is a knitted, woven or non-woven fabric.

4. A wound dressing as claimed in any of claims 1 - 3, wherein said carrier comprises a single layer of a synthetic polymer which has a water uptake of at least 16% w/w.

5. A wound dressing as claimed in any of claims 1 - 3, wherein said carrier comprises a laminate comprising two synthetic polymer layers, at least one polymer of said at least two layers being non-inhibitory to cell growth, non-cytotoxic and having a water uptake of at least 16 w/w%.

6. A wound dressing as claimed in claim 5 comprising a conformable carrier which is a laminate of a polymer layer having a water-uptake of at least 16% w/w and a polymer layer having a water-uptake of less than 16w/w%, the polymers being non-inhibitory to cell growth and non-cytotoxic.

7. A wound dressing as claimed in claim 6, wherein the cells are anchored to said polymer layer having a water-uptake of at least 16 w/w%.

8. A wound dressing as claimed in claim 6, wherein the cells are anchored to said polymer layer having a water-uptake of less than 16%w/w.

9. A wound dressing as claimed in any preceding claim, wherein said wound-facing surface of said carrier has been surface-treated.

10. A wound dressing as claimed in claim 9, wherein said surface treatment has reduced the contact angle of said surface by at least 10%.

11. A wound dressing as claimed in any preceding claim, wherein said polymer layer comprises a film of a hydrophilic polyurethane, polyether-polyester, polyether-polyamide, polyacrylamide or polyethylene oxide.

12. A wound dressing as claimed in any preceding claim, wherein said carrier comprises an apertured polymer layer.

13. A wound dressing as claimed in any preceding claim, wherein the cells are epithelial cells.

14. A wound dressing as claimed in any preceding claim, wherein the cell layer is a sub-confluent layer.

15. A wound dressing as claimed in any preceding claim, wherein the cell layer is from 40% - 70% confluent.

16. A wound dressing as claimed in any preceding claim, wherein the cell layer is a monolayer.

17. A system for producing a wound dressing comprising a conformable carrier having a wound facing surface to which a layer of cultured mammalian cells is anchored, the carrier comprising a synthetic polymer layer which has a water-uptake of at least 16 w/w %
and is non-cytotoxic and non-inhibitory to cell growth, together with a means for maintaining an aqueous cuture medium containing said cells in contact with the wound-facing surface of the carrier, the carrier being maintained in a submerged position in the culture medium.

18. A system as claimed in claim 17 wherein said means comprises a culture vessel and said carrier layer forms an integral part of said vessel.

19. A system as claimed in claim 17, wherein said means comprises a vessel in which said carrier layer is arranged and supported.

20. A system as claimed in any of claims 17 - 19, wherein said carrier is supported upon a further liquid-impervious layer which forms a part of said containing means.

21. A system as claimed in any of claims 17 - 20 wherein said carrier layer is supported on a further layer, said further layer having projections and said carrier layer having a plurality of thinner layers corresponding to the areas of projections of the further layer.

22. A method of treating a wound comprising the step of applying a wound dressing comprising a conformable carrier having a wound facing surface to which a layer of cultured mammalian cells is anchored, the carrier comprising a synthetic polymer layer which has a water-uptake of at least 16 w/w % and is non-cytotoxic and non-inhibitory to cell growth, to the area to be treated.

23. A kit comprising a conformable carrier having a wound facing surface, the carrier comprising a synthetic polymer layer which has a water-uptake of at least 16 w/w % and is non-cytotoxic and non-inhibitory to cell growth, a container comprising a nutrient solution for growing mammalian cells and a vessel for containing the carrier and nutrient solution.

24. A kit as claimed in claim 23 further comprising a sample of mammalian cells.