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EP1019104A1 - Produits de culture cellulaire - Google Patents

Produits de culture cellulaire

Info

Publication number
EP1019104A1
EP1019104A1 EP96927756A EP96927756A EP1019104A1 EP 1019104 A1 EP1019104 A1 EP 1019104A1 EP 96927756 A EP96927756 A EP 96927756A EP 96927756 A EP96927756 A EP 96927756A EP 1019104 A1 EP1019104 A1 EP 1019104A1
Authority
EP
European Patent Office
Prior art keywords
carrier
layer
wound dressing
cells
wound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96927756A
Other languages
German (de)
English (en)
Inventor
Mark Christopher Richardson
Patrick Lewis Blott
Jane Bridget Matthews
George Robert Drewery
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Smith and Nephew PLC
Original Assignee
Smith and Nephew PLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Smith and Nephew PLC filed Critical Smith and Nephew PLC
Publication of EP1019104A1 publication Critical patent/EP1019104A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/08—Flask, bottle or test tube
    • 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
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • This invention relates to the culturing of mammalian anchorage dependent cells onto a conformable carrier. More particularly the invention relates to the formation of wound dressings suitable for treating wounds such as burns, skin graft donor sites and ulcers e.g. venous and decubitus ulcers and to systems for use in the preparation of such dressings.
  • Mammalian cells that are incapable of proliferating in suspended liquid culture but can be made to proliferate on the surface of a carrier are said to be anchorage-dependent.
  • Epithelial cells such as keratinocytes, are anchorage dependent. Such cells 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 type are used to investigate skin growth and have been used as skin grafts.
  • Various technical papers have been published which describe in vitro techniques for growing skin cells and their subsequent use in the treatment of full-thickness wounds. For example E. Bell et al (J Invest Derm 81; 2s-l ⁇ s 1983); E. Bell et al (Science 211; 1052-1054 1981) ; D. Asselineau and M. Pruneiras (Br J Derm 1984 III, Supplement 27, 219-222) and J.F. Burke et al (Ann Surg 94; 413-428 1981).
  • the ability of cells to anchor to a particular carrier is dependent on the properties of the carrier, the culturing 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.
  • WO89/03228 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 preferably conducted under sterile conditions to impede bacterial contamination.
  • 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.
  • this technique does not overcome the problems associated with hard surface cultures or the risks associated with dislodgement of the cells.
  • the polymer of the present invention enables the transfer of a cell layer to the site of treatment before it has reached confluence.
  • a wound dressing comprises 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 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 non-cytotoxic and having a water uptake of at least 16 w/w %, to the area to be treated.
  • kits comprising a conformable 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 require a surface on to which they can bind before they are able to proliferate.
  • the synthetic polymer layer is a synthetic polymer film.
  • the carrier comprises a single layer of polymer.
  • 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.
  • the cell layer may be anchored to this polymer layer.
  • the cell layer may be anchored to the polymer layer having a water uptake of at least 16%.
  • the carrier 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.
  • the wound dressing comprises a conformable carrier the carrier being a laminate of a polymer layer having a water uptake of at least 16 w/w % and a hydrophobic polymer layer, the polymers being non- inhibitory to cell growth and non-cytotoxic.
  • hydrophobic polymer we mean that the polymer has a water uptake less than 16 w/w %.
  • Percentage water uptake of 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 percentage water uptake w/w%. The following equation may be used to calculate the percent water uptake (w/w %) .
  • w/w% water uptake (mass of sample + water) - mass of sample) 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.
  • the carrier is transparent thus allowing the cells on the surface to be examined by a microscopy.
  • the cells 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 the cytoxicity of the carrier will not exceed 30%. More aptly the cytotoxicity of the carrier will not exceed 20%. Preferably the cytotoxicity of the carrier will not exceed 15%.
  • the carrier may be a laminate 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. PEBAX (Trade Mark), polyacrylamides and polyethylene oxide.
  • polyetherpolyester also known as a thermoplastic ether ester elastomer or a copolyester elastomer
  • polyetherpolyamide e.g. PEBAX (Trade Mark)
  • polyacrylamides e.g. ethylene oxide
  • polyethylene oxide e.g. ethylene oxide
  • the carrier may comprise a hydrogel, a hydrocolloid or other suitable polymer.
  • suitable hydrogels include polyhydroxyethylmethacrylic acid (poly HEMA) , cross-linked polyvinylacrylic acid (PVA) , polyacrylic acid cross-linked with triallylsucrose (carbopol) and polyvinylpyrrolidone.
  • suitable hydrocolloid materials include carboxymethylcellulose 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 supported 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 layer comprise the carrier.
  • the carrier comprises a hydrophobic polymer layer it may desirably be apertured to enable it to handle any exudate from the site of application particularly in the case of highly extruding wounds.
  • a continous polymer layer may be adapted to be perforated after the cell layer has reached the desired degree of confluency.
  • films are the biaxially 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 exuding 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 minimum 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 laminate.
  • the carrier is a laminate of two polymer films or nets in order that it should have the required flexibility and conformability, it should suitably have a thickness not exceeding 0.075mm. More suitably it should not have a thickness exceeding 0.05mm, and favourably not exceeding 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, 0.015mm or 0.020mm.
  • 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 dimensions.
  • Meshing is conventionally carried out before grafting of a skin graft, to increase the surface area of the graft.
  • 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.
  • the carrier is permeable 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 impervious to liquid water should have an upright moisture vapour transmission rate (MVTR) of at least 300gm —2 24 hrs—1.
  • MVTR moisture vapour transmission rate
  • 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-1.
  • the moisture vapour transmission rate may be measured by the Payne Cup method. This method uses a cup 1.5cm deep which has a flanged top. The inner 2 diameter of the flange provides an area of 10cm of test material through which moisture vapour may pass.
  • test material 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 1kg of anhydrous 3-8 mesh calcium chloride on the floor of the oven.
  • 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 37 ⁇ C, 100% to
  • the reading is corrected for standard thickness of test material i.e. a thickness of
  • 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.
  • 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 .
  • autologous cultivated epithelial cells since these have little or no immunological rejection problems when applied to the host (patient) .
  • non-autologous cells e.g. to produce an allograft or xenograft.
  • the cells are keratinocytes.
  • the carrier may be sterilised by any suitable known methods of sterilisation. Suitable forms of sterilisation include ethylene oxide (allowing the required time for de-gassing) gamma-irradiation or steam 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.
  • the carrier may be aseptically produced.
  • the carrier on which the cells are grown should preferably be easily removable from the other parts of the system, namely the means for maintaining the aqueous culture medium containing said cells in contact with the wound facing surface of the film.
  • the carrier forms a wall of the vessel containing 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.
  • the carrier may be laid down within a flask of an appropriate design adapted to allow the removal of the substrate.
  • the carrier may be removably sealed to the other parts of the flask for example by heat sealing or by means of an ahdesive.
  • the carrier will form a flat surface and will aptly form the base of 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.
  • apertured carrier 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 retained by, for example, a pre-sterilised stainless steel ring or, alternatively, by coating the carrier on one side with a layer of non-cytotoxic adhesive 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 aqueous medium in which the cell culture is grown.
  • the nature and weight and/or volume of such additional ingredients are conventionally well known.
  • the wound dressing of this invention may be used for a variety of wounds.
  • the dressing of the invention is particularly suitable for treating partial-thickness wounds that is those where only the epidermis and possibly part of the dermis is lost.
  • Such wounds 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 full thickness wounds eg. venous ulcers. Continuous polymeric film carriers aptly act as barriers to bacteria whilst being sufficiently permeable to moisture vapour, oxygen and carbon dioxide to allow wound healing to occur at a desirable rate. If the substrate is perforated, a secondary dressing could be applied to maintain the desired degrees of moisture vapour, oxygen and carbon 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) depending 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.
  • 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 containing 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. However inevitably some mouse cells remain and thus will be grafted with the host keratinocytes.
  • g-irradiated typically about 6000 rads
  • 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.
  • the apertures should be large enough to allow free exchange of the culture medium, but not large enough to allow cells to pass through.
  • the aperture size should not be larger than 5 ⁇ across its largest dimensions.
  • 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.
  • the dressing may be prepared according to the above disclosed method by a cell-culturist in a hospital cell-culture laboratory.
  • the dressing may be prepared at a location remote from the location e.g. hospital where it is to be applied.
  • the dressing may be transported under suitable conditions e.g. under a precisely controlled temperature.
  • the dressing may be cryopreserved ie. maintained at - 190°C and thereafter brought back to room temperature just prior to use.
  • the dressing may be transported in a ready-to-use state.
  • the dressing may be transported in a suitable incubator set at the desired temperature.
  • Contact angles are measured by the Wilhelmy Plate dynamic contact angle measurement system using a Cahn DCA-322 Dynamic Contact Angle Analyser. Both advancing and receding contact angles may be measured.
  • the test liquid 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 coating a slide from solution. The testing may be carried out at 23 deg. C/50% RH.
  • a suitable method for measuring cell growth reduction is the method disclosed in WO 91/13638.
  • the method of measuring percentage cellular cytotoxicity of the substrates is as disclosed in WO 91/13638.
  • one wall the base, of a culture flask 1 comprises a laminate of an apertured film 2 and a continuous film 3.
  • the edges of the laminate 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.
  • 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 thickened 10 areas.
  • the thin areas 9 rupture to form apertures 12 as shown in Figure 3.
  • the resulting apertured from which may subsequently be laminated to a polymer layer to form a laminate, suitable for use as a carrier in the dressing of the present invention.
  • the culture flask 1 is divided into compartments 21, 31 by a carrier comprising 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 compartment 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.
  • the carrier is detached 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.
  • keratinocytes 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.
  • the culture medium used was Earle's Minimum Essential Medium (MEM) + 10% foetal calf serum made up as follows:-
  • the cells were seeded at a density of 10 cells per 3ml of medium.
  • the culture medium containing cells at the above concentration 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 examined for the presence of attached cells under ultra-violet light using a NIKON inverted microscope fitted with a rhodium filter to detect 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.
  • 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.
  • thermoplastic polyether - polyurethane 20 ⁇ m thick was laminated to a 50 ⁇ m film of ethylene- vinyl acetate (EVA) to form a carrier layer.
  • EVA material had a water uptake of about 0.3%.
  • the culture medium and cells were placed on the polyether
  • Example 4 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.
  • Example 4 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.
  • 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-sensitive adhesive attached to the wound- facing side thereof.
  • the water-uptake of the dressing was measured as approximately 68%.
  • 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.
  • 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 dispersed 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.

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  • Biochemistry (AREA)
  • Sustainable Development (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Botany (AREA)
  • Transplantation (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Dermatology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Hematology (AREA)
  • Materials Engineering (AREA)
  • Clinical Laboratory Science (AREA)
  • Materials For Medical Uses (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un pansement comprenant un support constitué d'une couche polymère hydrophile et une couche de cellules de mammifères ancrées à la surface du support en contact avec la plaie. Elle concerne également un procédé de fabrication de ce pansement.
EP96927756A 1995-08-12 1996-08-12 Produits de culture cellulaire Withdrawn EP1019104A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB9516556 1995-08-12
GBGB9516556.9A GB9516556D0 (en) 1995-08-12 1995-08-12 Cell culture laminate
PCT/GB1996/001959 WO1997006835A1 (fr) 1995-08-12 1996-08-12 Produits de culture cellulaire

Publications (1)

Publication Number Publication Date
EP1019104A1 true EP1019104A1 (fr) 2000-07-19

Family

ID=10779152

Family Applications (1)

Application Number Title Priority Date Filing Date
EP96927756A Withdrawn EP1019104A1 (fr) 1995-08-12 1996-08-12 Produits de culture cellulaire

Country Status (7)

Country Link
EP (1) EP1019104A1 (fr)
JP (1) JP2000500032A (fr)
AU (1) AU6746296A (fr)
CA (1) CA2226747A1 (fr)
GB (1) GB9516556D0 (fr)
WO (1) WO1997006835A1 (fr)
ZA (1) ZA966803B (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU8224598A (en) * 1997-06-26 1999-01-19 Smith & Nephew Plc Cell culture products
US6071690A (en) * 1998-02-02 2000-06-06 Denco, Inc. Ionomeric modified poly-ether-ester plastic container for cryogenic fluids
CN100408098C (zh) * 1999-12-08 2008-08-06 范赛尔生物技术有限公司 聚丙烯酰胺凝胶在哺乳动物的机体组织中形成胶囊的应用、培养细胞的方法和治疗肿瘤和糖尿病的方法
US6673603B2 (en) 2000-09-01 2004-01-06 Modex Therapeutiques, S.A. Cell paste comprising keratinocytes and fibroblasts
US7144729B2 (en) 2000-09-01 2006-12-05 Dfb Pharmaceuticals, Inc. Methods and compositions for tissue regeneration
CN101601858B (zh) 2009-05-27 2012-09-19 上海交通大学 中期因子蛋白的用途及含该蛋白的医用装置
JP6447125B2 (ja) * 2014-12-26 2019-01-09 大日本印刷株式会社 細胞培養容器

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1201400A (fr) * 1982-04-16 1986-03-04 Joel L. Williams Surfaces chimiquement specifiques pour influencer l'activite cellulaire pendant la culture
JPH02504221A (ja) * 1987-04-22 1990-12-06 ベイ　ミカエル 細胞培養法、培養物及び培養製品
US5015584A (en) * 1987-10-14 1991-05-14 Board Of Regents, The University Of Texas System Epidermal graft system
US5122470A (en) * 1988-07-05 1992-06-16 Banes Albert J Floating cell culture device and method
GB9004911D0 (en) * 1990-03-05 1990-05-02 Smith & Nephew Cell culture products
CZ281269B6 (cs) * 1994-05-31 1996-07-17 Ústav Makromolekulární Chemie Av Čr Biologicky aktivní kryt rozsáhlých ranných ploch a způsob jeho přípravy

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9706835A1 *

Also Published As

Publication number Publication date
GB9516556D0 (en) 1995-10-11
ZA966803B (en) 1997-02-12
AU6746296A (en) 1997-03-12
JP2000500032A (ja) 2000-01-11
WO1997006835A1 (fr) 1997-02-27
CA2226747A1 (fr) 1997-02-27

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