AU614747B2 - Cell culture of anchorage dependent cells, materials and products - Google Patents

Description

PCT WORLD INTELLECT PR ER" yRGANT AOTI' \PCT OI n n a R I TE A OR Y INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 C12N 5/00, A61L 27/00 A3 (11) International Publication Number: WO 88/ 08448 (43) International Publication Date: 3 November 1988 (03.11.88) (21) International Application Numb (22) International Filing Date: (31) Priority Application Numbers: er: PCT/EP88/00362 21 April 1988 (21,04.88) 8709499 8711044 22 April 1987 (22.04.87) 11 May 1987(11.05.87)

GB

(32) Priority Dates: (33) Priority Country: (81) Designated States: AT (European patent), AU, BB, BE (European patent), BG, BR, CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (European patent), HU, IT (European patent), JP, KP, KR, LK, LU (European patent), MC, MG, MW, NL (European patent), NO, RO, SD, SE (European patent), SU, US.

Published With it,ernational search report Before the expiration of the time limit for amending Ie claims and to be republished in the event of the receipt of" amendments.

(88) Date of publication of the international search report: 1st December 1988 (01.12,88) (71X72) Applicant and Inventor: BAY, Michael [DK/CH]; CH-3961 Mollens (CH).

(74) Agent: JONES, Helen, Marjorie, Meredith: Gill Jennings Every, 53/54 Chancery Lane, London WC2A IHN (GB), Title; CELL CULTURE PROCESSES, MATERIALS AND PRODUCTS (57) Abstract A new process for culturing anchorage dependent mammalian cells comprises growing the cells in the presence of an aqueous medium on the surface of a she of synthetic polymer gel that is swollen by the aqueous medium, The cells are for instance fibroblasts and/or epithelial cells, The polymer gel usually comprises a copolymer of hydroxyethylacrylate and hydroxyethyl methacrylate. The medium can contain growth factors, for instance epidermal and/or fibroblast growth factor, The product can be used as a wound dressing, especially for use as a skin replacement for instance in burn treatment, i i 1 3 1

SPCT

16,852/88 WORLD INTELLECTUAL PROPERTY ORGANIZATION I~ternational Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) Internationai Patent Classification 4 (11) International Publication Number: WO 88/ 08448 C12N 5/00, A61L 27/00 A2 (43) International Publication Date: 3 November '83S (03.11.88) (21) International Application Number: PCT/E'P88/00362 (81) Designated States: AT (European patent), AU, BB, BE (European patent), BG, BR, CH (European patent), (22) International Filing Date: 21 April 1988 (21.04.88) DE (European patent), DK, FI, FR (European patent), GB (European patent), HU, IT (European patent), JP, KP, KR, LK, LU (European patent), MC, (31) Priority Application Numbers: 8709499 MG, MW, NL (European patent), NO, RO, SD, SE 8711044 (European patent), SU, US.

(32) Priority Dates: 22 April 1987 (22.04,87) 11 May 1987 (11.05.97) Published Witiout international search report and to be republished (33) Priority Country: GB upon receipt of that report (71)(72) Applicant and Inventor: BAY, Michael [DK/CH]; CH-3961 Mollens (CH).

(74) Agent: JONES, Helen, Marjorie, Meredith; Gill Jln- O. J.P. 5 JAN 1989 nings Every, 53/54 Chancery Lane, London WC2A IH N (CB),

AUSTRALIAN

2 DEC ?8 PA'tiN(i OrCFICE (54) Title: CELL CULTURE PROCESSES, MATERIALS AND PRODUCTS (57) Abstract A new process for culturing anchorage dependent mammsiian cells comprises growing the cells in the presence of an aqueous medium on the surface of a sheet of synthetic polymer gel that is swollen by the aqueous medium. The cells are for instance fibroblasts and/or epithelial cells. The polymer gel usually comprises a copolymer of hydroxyethylacrylate and S'ydroxyethyl methacrylate. The medium can contain growth factors, for instance epidermal and/or fibroblast growth factor. The product can be used as a wound dressing, especially for use as a skin replacement for instance in burn treatment.

i- I; L_ I; WC 88/08448 PCT/EP88/00362 1 I CELL CULTURE PROCESSES, MATERIALS AND PRODUCTS The present invention relates to priocesses for culturing mammalian cells, particularly epithelial cells, on a substrate and to products of the process, in particular to the use of such products as wound dressings.

Most normal mammalian cells tha' are derived from solid organs are anchorage-dependent; that is, they are substantially incapable of proliferating in suspended liquid culture but can be made to proliferate on the surface of a substrate which is in contact with growth medium containing the cells.

Epithelial cells are anchorage dependent. Cells cultured in the presence of a surface will attach to the surface and will multiply in stratified colonies which eventually become confluent. At that stage the culture enters a steady state in which dividing cells proliferate in the basal layer and the upper cells are shed from the surface, i.e. differentiation takes place and the culture behaves to some extent like intact skin. Cell cultures of this type are useful for investigating skin growth, differentiation of skin cells and how it can be controlled as well as providing sheets which can he used to graft onto skin as a permanent covrerage of wounds.

Holbrook and Fennings Invest. Dermatd. 81; 115-245, 1983) review the vitro growth of epidermal cells.

The ability of anchorage dependent cells to proliferate depends on the substrate surface, as well as the components of the liquid growth redium and the culturing conditions. The properties of the surface which allow adhesion and proliferation of the cells are as yet incompletely understood. Usually culturing is carried out in solid vessels, for instance petri dishes or flasks, II WO 88/08448 PCT/EP88/00362 made of glass or, more usually hard transparent plastics material such as polystyrene, and mammalian cells are in general adequately adherent to such surfaces to enable proliferation to occur.

In many instances it is desired to culture cells and then remove some or all of the cells, sometimes without disrupting the layer that has grown, to another location.

This may be required when samples are required to be investigated for their properties each by being submitted to different tests or to microscopys for instance in many biopsies. Layers of epithelial cells or fibroblasts may be required for use as a skin graft for use in burn treatment or other wound healing.

Studies have been carried out to investigate t-ne properties of surfaces required for cell adhesion, usually as a means for determining the factors necessary for cell surface attraction and cell adhesion. For instance in Applied Biochemistry and Biotechnology 8 115-126 (1983) Yoshii et al describe the cell culture of glial cells, pituitany tumour cells and a liver cell-in-cell line. The cells were cultured in flasks which had been coated with polymer by casting a solution of various types of acrylate ester monomers to form a coating on the inner surface of the flasks and initiating polymerisation. The polymers were swellable to a small amount, the maximum water content of a gel swollen in pure water being 25% by weight. As the polymer is coated onto the culture vessel surface adherent cells cannot easily be removed from the vessel.

Other techniques of culturing anchorage-dependent cells involve coating of the hard culture surfaces with molecules found in the extra-cellular matrix of the cells in their normal surroundings, for example using various collagens fibronectin or laminin. In J. Cellular Physiology 83, 379-379 (1973) Macieira-Coelho cultivate WO 88/08448 PCT/EP88/00362 3 fibroblasts in vessels having a coating of polymeric bovine serum albumin covered with charged materials such as poly(amino acids) histones and heparin. Although these may facilitate the growth of the cells, the methods still have the disadvantages mentioned above of growth on hard surfaces.

In Experimental Cell Re -arch 143, p. 15-25 (1983) Faris et al describe the cultivation of endothelial cells and fibroblasts is carried out on the surface of sheets of poly(hydroxyethyl meth-acrylate) (HEMA) in culture medium. The polymer is optionally prepared in the presence of protein, for instance collagen or elastin.

HEMA homopolymer has a limited swellability, being capable of absorbing 0.9% saline to give a gel havine a maximum of 38% by weight saline content.

In Cell (1977) 11, pp 405 to 416, "Terminal Differentation of Cultured Human Epidermal Cells" by Green, keratinocytes from the foreskin of new born humans were cultured to form stratified confluent colonies. The culture is used in an investigation of the differentiation. In Experimental Cell Research (1980) 125 pp 141 to 152 "Fine Structure of Sub Cultivated Stratified Squamous Epithelium" by Jepsen et al rat lingual epithelium is cultivated and the primary explants are sub-cultivated on plastic culture flask surfaces to produce long lived cultures. In the Journal of Investigative Dermatology 1987, 68:314-319 Arenholt-Bindslev et al describe experiments in which human oral keratinocytes are cultured and sub-cultured successfully in hard plastics flasks.

Often, to facilitate the growth and a differentiation of epithelial cells, feeder cells are used. For example, in Green (1977) discussed above, lethally irradiated 3T3 cells are used as feeder cells.

In Cell (1975) 6 pp 331 to 34 "Serial cultivation of :1 1 WO 88/08448 PCT/EP88/00362 4 strains of human epidermal keratinocytes: the formation of keratinising colonies from single cells", Rheinwald et al describe the use of fibroblast cells as feeder cells.

There have been various disclosures of the use of cultivated epithelial cells as skin grafts. In the Journal of Trauma (1986) 26 pp 955 to 962 Madden et al describe the use of cultured allogeneic epidermis (i.e.

derived from a person other than the patient) as a graft on second and third degree burn wounds. Although a consequence of thermal injury is immunosuppression which prevents early rejection of the allograft, these grafts are normally rejected unless drugs are used as immunosuppressents in the long term. Furthermore the use of tissue 'from other humans involves serious risks of infection, especially viral infection such as by HIV, which it is highly desirable to avoid.

It is therefore preferred to use autologous cultured epithelium. In the New England Journal of Medicine (1984, 16th August) pp 448 to 451, Gallico et al describe the use of cultured sheets of autologous epithelial cells as skin grafts. In the Lancet (1986) 1 pp 1123 to 1124, Cuono et al describe the use of an allograft of whole skin as a first stage of the grafting process onto a burn wound and the subsequent replacement of the epidermal layer of the allograft by cultivated of autogeneic epidermis on the dermal allograft. This method of grafting was successful since the dermis is found to be less immunoreactive than the epidermis, but the risks of viral infection are still severe.

One of the problems with the use of hard plastics or glass surfaces as the substrate for epithelial cell cultures is that the culture medium is supplied only from above the surface so that the basal cells have poor access to the medium. This leads to a slow growth rate since it is the. basal cells which are capable of WO 88/08448 PCT/EP88/00362 dividing. A further disadvantage where the cell sheets are to be used as sk.n grafts is that the sheet, which is not very mechanically strong, must be dislodged from the surface and handled unsupported, which can be inconvenient and can damage the sheet.

It is known to support the sheet after it has been dislodged from the surface in order to facilitate handling thereafter. For example in Gallico et al (op cit) the sheets are clipped to gauze, and in Cuono et al (op cit) a backing of "N-Terface" was placed on top of the culture. Although these techniques overcome some of the difficulties with handling the sheAts, the methods still have the disadvantages associate-l with growth on hard surfaces as well as still requiring that the sheets be dislodged from the surface. Dislodgment from the hard surface can be facilitated by applying an enzyme such as dispase as disclosed in US4304866.

Epithelial cells have also been grown on collagen gels and the collagen with the layer of epithelial cells has been used directly as a skin replacement. For example in Proc. Natl. Acad. Sci. USA '1979) 76 pp 1274 to 1276 El et al describe the use of a collagen qel as a substrate for epidermal cells. Human fibroblasts are seeded into the collagen lattices to be used as feeder cells. In the Journal of Investigative Dermatology (1983) 81 pp 2S to 10S, Bell et al describe the reconstitution of living skin comprising a dermal equivalent made up of fibroblasts in a collagen matrix and an epidermal equivalent developed from keratinocytes "plated" onto the collagen. The skin equivalents have been successfully grafted onto rats.

Some studies have shown that the growth of epithelial cells at the air/liquid interface, to mimic the in vivo conditions, can in some cases lea? to improved stratification (differentiation) when the cells L .1 1- WO 88/08448 PCT/EP88/00362 6 are grown on collagen gel in comparison to cells which are totally immersed in the liquid medium duri7g culturing.

Although the epithelial cells grow well on the collagen gel, there are several problems with the use of collagen. It is an expensive material, especially when it is supplied in its isolated form and even then is time consuming to make up as it is supplied as a powder which must be dissolved and gelled. The isolation and creation of the gel from the natural source in the laboratory is time consuming. Furthermore collagen is not well defined since it is a natural substance so that its behaviour from batch to batch may differ, making procedures difficult to standardise. This is particularly undesirable from a clinical point of view when the collagen is to be used as part of a skin graft, Collagen is furthermore difficult to work with in the laboratory, one of the problems being that the size of the gel and thus its water content depends upon the presence of other cells particularly fibroblasts, which are used as feeder cells. The size of the gel can vary in use which is clearly disadvantageous when the gel is to be used as part of a skin replacement. When the water content is reduced to below a certain level the gel becomes opaque which makes it less easy to observe the rate of wound healing below a graft comprising the gel. A further problem is that being a protein collagen cannot be sterilised by steam so that during storage it can only be kept free of micro-organisms by the use of antibiotics or by other methods of sterilisation. Being a natural substance collagen gel is in any case a good substrate for the growth of undesixable micro-organisms and its use on wounds can then encourage infection. A yet further disadvantage is that when the gel with the epithelial cell culture is used as a skin replacement, a further WO 88/08448 PCT/EP88/00362 7 barrier is required directly on top of the epithelial cell culture.

According to the invention there is provided a new process in which anchorage dependent mammalian cells are cultured in the presence of an aqueous culture medium on the surface of a removable substrate which is a gel comprising a water-insoluble, water-swellable hydrophilic synthetic polymer swollen with the medium, and the process is characterised in that the polymer has a swellability such that it can absorb at least its own dry weight of 0.9% by weight saline.

The process is of particular use in the culturing of human cells, preferably epithelial cells and fibroblasts, although the use of the polymer substate is also advantageous for the culture of a wide range of mammalian anchorage dependent cells.

In the new process in which the cells on the surface may be exposed to the gaseous medium above the aqueous medium during a part at least of the culturing process.

The cells may be exposed for a period early or late in the cu turing process, or throughout the process.

Throughout the culturing process the synthetic polymer gel is swollen with aqueous medium, so that nutrients are supplied to the cells via the gel substrate. Any exposure of the cells to gaseous medium during the process is achieved by maintaining the surface on which the cells are growing in contact with the surrounding air or other gaseous medium while at the same time supplying aqueous medium continuously to the gel.

For instance the gel can be partially immersed in the aqueous medium, e.g. by supporting it at the surface using solid supports or by allowing the gel to float on the surface of the aqueous medium.

This process may be useful as a research tool to investigate the growth and differentiation of epithelial WO 88/08448 PCr/EP88/00362 8 cells in vitro by mimicking the in vivo conditions in which the surface layer of skin is exposed to the atmosphere, nutrients being supplied from underneath the skin.

Apart from the use of the special substrate the cell culture is conventional containing the usual nutrients and components for controlling the rate of growth. Thus for epithelial cells the culture medium may contain substances known to facilitate the growth of epithelial cells. These may be growth factors, for example epidermal growth factor (EGF) usually combined with substances derived from feeder cells, for example normal human fibroblasts, 3T3 cells or glial or other brain cells.

in a 15referred process feeder cells are grown on the surface of the container for the culture medium and, when they have established their growth the epithelial cells are introduced, A swollen synthetic polymer gel may be introduced onto the established feeder cell layer. This may allow the growth of epithelial cells in the presence of, but physically separated from, feeder-cells.

Alternatively feeder cells may be grown on a surface of swollen synthetic polymer gel and, after the growth has been established, the epithelial cells are introduced, Sometimes epithelial cells are grown on the same surface as feeder cells but, as indicated, different cells t-ypes can be grown on different surfaces, When the feeder cells are fibroblasts these can be treated to Prevent them multiplying as described by Green in US4016036.

A Furthermore the cells can be grown b the procedure described in FP2589165 where the culturing comprises two steps between which the cells are f4ro?en# the second step being carried out in accordance with the present invention The culture medium may contain any of the conventional components well known to those skilled in T WO 98/0848 PCT/EP88/00362 9 the art, for instances as described in any of the above-mentioned publications. Thus the culture medium may contain other growth-affecting substances, serum, nutrients, enzymes, sequestrants, buffers and/or antibiotics or other conventional substances. The substrate may have proteing or other natural substances incorporated into it or cOated onto it, to affect the growth of the cells, for instance protein such as elastin or collagen. Such components can, as disclosed by Faris et al (op cit), allow selection of particular types of cells for growth.

The polymer gel used in the process is preferably wholly synthetic. It is generally in the form of a swollen sheet, for example 0.1 to 5mm thick preferably 0.5 to 2mm thick.

In order for the polymer to be sufficiently permeable to nutrients and water it should have a swellability such that it can absorb at least its own weight of 0.9% by weight saline, that is, it swells in 0.9% saline to give a gel which comprises at least 50% by weight saline. Usually the polymer can absorb 0.9 9 saline to give a gel which comprises more than 60% saline preferably 70 to 95% saline. The polymer from which the substate is formed is often such that 't will absorb up to 100 times its dry weight of water. Since the aqueous culture will contain dissolved substances, for example any of those mentioned above, the swellability of the polymer in that medium is likely to be lower than in pure water the swellability usually being about the same as in 0.9% saline. Preferably the polymer is capable of absorbing the aqueous mediun .n an amount in the range 1 to 50 times of its own dry weight, pteferably 1.5, mor preferably 2, or 3 to 20 times its own weight, for example about 10 times ivs weight in the aqueous medium.

The swellability of the polymer is as high as possible in wo 88/08448 rCTr/EP88/00362 order to improve the permeability of the polymer to oxygen and to substances reqruired for epithelial cell growth. Preferably the gas permeability of the polymers substrate is such that it has a PK value of at least preferably in the range 20 to 35. Gels with high swellability also allow quicker equilibration of the liquid phase within the gel and the liquid medium when the gel is first introduced into the medium and this is advantageous for cell growth.

The polymer is preferably formed from ethylenically unsaturated monomer, preferably comprising acrylic monomer. The monomer most preferably comprises a hydroxy alkyl (meth) acrylate, for example selected from those in which the alkyl group has 2 to 4 carbon atoms. Examples of such monomers are hydroyethyl acrylate (EA), hydroxyethyl methacrylate (HEMA) hydroxypropyl methacrylate, hydroxypropyl acrylate and hydroxytrimethylene acrylate. The monomer preferably comprises HEA and/or HEMA. Such hydroxy alkyl (moth) 2Q acrylates and generally co-polymerised with one or more co-monomers which may be hydrophilic or hydrophobic and which are selected to impart specific chemical or physical properties to the resulting co-polymer, Examples of co-monomers are alkyl (meth) acrylates, alkoxy alkyl (meth) acrylates, polyalkylene glycol (meth) acrylatos, (meth) acrylic acid, (meth) acrylamide, styrane, N-vinyl lactam, e.g. N-vinyl pyrrolidone. Thcorporation of hydrophilic monomer, e.g. (meth) acrylic acid, in general increases the swellability of the polymer, The preferred polymers are formed from a maior amount, i.e. 50 to 100%, of hydroy alkyl (moth) acryn te, with a minor amount, i.e. 50 to 0% of a co-monomer. Tho most preferred mixture comprises 50 to 100%, preferably 75 to 99%, more preferably 90 to 98% WO 88109448 W088/8448PCT/EP88/00362 12.

H-EMA and/or H-EA with minor a. of co-monomer, preferably a hydrophilic co-monomer.

The polymer is preferably cross-linked, cross-linking rendering it insoluble, controlling the swellability and giving it physical strength, Cross-linking is preferably covalent and is generally achieved by incorporating into the polymerisation mixture of di- or poly-functional ethyleniically unsaturated compounds in appropriate amounts, generally less than 2$ by weight of the monomer mixture for example in amounts in the range .001 to 2%1 preferably .05 to generally in the range .05 to Examples of cross-linkingj agents o.,1 this type are well known in the art and may includ~e di- or tri-esters of (meth) acrylic acid, for mnpl1e alkylene di" (moth) acrylates, generally in which the alkylene has from 2 to 4 carbon atoms, or di- or poly-alkylene glycol (meth) acrylates, g~enerally in which the alkylene groups have 2 to 4 carbon ,,toms, and also alkylone bis (meth) aorylamidest usually mnethylene bis (moth) acrylamide, The poll;Merization mav be carrte out Without any diluent or may Lja carried out in the presence wa3ter or other squitabl.e diluqnt by 'qP7'S Polymerisation of aquouts solutions of monwlm_'7 is 4esc; iboO in VS297657G. Polymerioation in thu subotantial aLsFence of l~q*,i6 4iluents is described in tlqS332O949.

Polymerisation in the presnce 04 non-acruootaz Oilu~nts is described in GR-A-2097305 which descr~ibe-- the pOl~Ymerisation in the pre;3ence 04. an ester formed from boric acid and a compound. containing I or' more hyrox~yl giroups, In tP-A-0182655 polymerisation is carrtted otut in the presence Of a range of wator-displacable solvenrtal including ester roaction products of carboxylic ac"6s o4r anhydrides andI polyols, often di-functionial arbo. ytio n'acida, an(I polyqls themselves and, mixturesi Any CL" the WO 88/08448 PCT/EP88/00362 12 methods described in those references may be used to form the polymer used in the present invention. Polymerisation may be carried out in the presence of protein or other natural compounds. Proteins may be, for instance, collagen or elastin.

The polymerisation is initiated by any known means, for example by us, g thermal initiators, redox initiators and/or maybe initiated using irradiation, optionally including an irradiation-sensitive catalyst. The 1V irradiation may comprise u-v -,rradiation, electron beam irradiation or irradiation from a radioactive source.

Curing agens sui'7able for use with these forms of irradiation are woll known in the art.

Sometimes it may aid adhesion of the cells to render the surface of the gel rough or porous and this may be achieved by including suitable diluents in the polymerisation mixture.

Although the polymer may b, formed into the desired shape after polymerisation, it is generally polymerised to the desired shape, i.e. as a sheet. The polymerisation mixture is therefore polymerised wthin a mould or, usually, cast onto a flat surface, optionally covered with a protective "heet substance and then polymerised.

The layer of mixture rMay be in the range 0,1 to 2mm, prefe.ably 0.2 and 1mm, more preferably about thick.

Following completion of the polymerisation the polymer must be washed to rid it of any low molecular weight contaminants, which may comprise for insta :ce excess unpolymerised monomer. Since the polymer is to be used inr direct contact with cells, it is most important for excess monomor to be washed out of it. Washing can be carried out by any of the known techniques which are suitable tor this. The washing process may comprise several sequential washes using Iemineralised water or WO 88/08448 PCT/EP88/00362 13 using water of different conductivities in sequential steps. One suitable process comprises soaking the polymer in successively more conductive water.

The polymer may be provided in the form of a package and containing polymer as defined above containing also components of the cell culture medium, the contents of the package being suitable for use in the new process.

The package may contain dried polymer containing any of the substances, preferably, however, it comprises polymer in the form of a gel swollen with an aqueous liquid containing any of the components. Thus it may comprise serumi, growth-effecting substances, nutrients, enzymes, sequestrants, buffers and/or antibiotics.

The gel may be provided in a container which is suitable for use directly as a container for the culture medium in the process of the invention. More conveniently however t e polymer is provided in a sterile sealed package of a foil, generally a metal or a plastics foil, which is easy to use in the laboratory. The sealed package iu preferably sterilised using steam sterilisation whilst subjecting the outside of the package to external pressure to prevent it bursting. The pressure may be exerted by holding the package in a mould, but is more conveniently by gas pressure, sterilisation is preferably carried out using an autoclave, preferably one in which the temperature and pressure are independently variable.

In the invention there is also provided a new product which comprises a gel substrate which comprises a water-insoluble, water-swellable synthetic hydrophilic polymer swollen with an aqueous liquid and having a laver of cultured mammalian cells on its surface. The new product is the product of a new process according to the invention, thus the c -lls are preferably fibroblasts or epithelial cells, ~i WO 88/08448 PCT/EP88/00362 14 One particularly useful application of the product is as a skin graft, for example in animals or humans. In a such an application the gel covered by a laver (generally substantially a monolayer) of epithelial or fibroblast cells is placed cell side down upon the wound, for example a burn wound. The gel acts as a barrier in particular to bacteria whilst being sufficiently oxygen permeable to allow healing to continue. For further protection and to keep the product in place and to keep the gel moist it is preferred for the dressing to be covered by further coverings. The gel will generally be left in place for several days, until the cells are grafted onto the wound and then the gel may be pealed off and replaced with further swollen gel of the same type, or with conventional wound coverings. Nutrients and growth factors, antibiotics, analgesics, anti-inflammatonics etc. may be incorporated in the aqueous liquid with which the gel is swollen, either before application to the patient or after application.

If a product carrying a layer of fibroblast cells is applied to a wound, the fihroblasts may act as feeder cells for the natural epithelial cells present at the site of the wound, stimulating these to multiply and differentiate, thereby healing the wound. The epithelial cells are stimulated to grow under the wound-contacting side of the gel, so that the gel can subsequently be removed from the wound. Because the gel is permeable to nutrients for the epithelial cells these can diffuse to the cells through the gel. Preferably the fibroblast culture is developed from the wound of the patient. This application is particularly useful since the fibroblasts are relatively easy to culture in vitro and it is not necessary to culture epithelial cells in vitro, as their growth is stimulated in vivo by. the cultured fibroblasts. This is especially advantageous in view of WO088/08448 PCT/EP88/00362 the greater difficulty in growing epithelia! cells in vitro. For particularly bad burn wounds it may be helpf"Ll to se'ed the wound with epithelial cells or the use of t cultured epithelial cells simultaneously with the use of the gel carrying the cultured fibroblasts.

A product which has a layer of cultured epithelial cell1s could alternatively be applied to a wound.

Such a product may be used to replace the sheets of cultured epithelia or skin replacements comprising collagen gel carrying epithelial cell cultures used in any of the methods described in the above mentioned prior art. If necessary the gel can be separated from the adherent cells by the use of dispase, as disclosed by Green in US4016036, either immediately after application to the wound or later.

The gel used in the present invention has numerous advantages over su~bstrates used in the prior art. It has the advantage over hard surfaces that it is permeable so that nutrients and other conwronents of the culture medium 2C2 are supplied to basal cel',s of a culture to provide a more efficient growth. It is much cheaper than coll~cjer and is well defined which is e,-,ential for clinical u-~e, It is far easier to handle physically and since it is well defined chemically, is easier to standardise and control. Being synthetic it is a).ro a worse substrate for micro-organism cell growth which is advantageous in the process as well as in the final product reducing the risk of bacterial or other infection. The swollen gal is relatively permeable to oxygen so that healing is maintained whilst acting as a barrier to bacteria and other unwanted components. The gel can be sterilised before use using steam sterilisation which is highly advantageous. Furthermore the gel is transparent so that wound healing can be observed through the gel.

16 Use of the products as aids to wound healing can avoid completing the use of foreign tissue and natural substances such as collagen, thus minimising risk of infection and immunological rejection.

Another use of the process of the invention is for conducting investigations on the cells themselves particularly where subcultivation is necessary or where a number of different tests need to be carried out on samples of the cultured cells.

The following examples illustrate the invention:

EXAMPLE

Formation of polymer gel 97.5 parts HEMA (containing 200 PPM hydroquinone monomethyl ether HQME), 1 part containing HEA (containing 200 PPM HQME) and 0.75 parts methacrylic acid with parts a cross-linking monomer comprising ethylene glyol di-methacrylate, were mixed together. 0.25 parts of a curing agent (Darocur 1173) was added to the polymerisation mixture which was then cast onto a flat surface to a thickness of 0.5mm. The mixture was cured by irradiating with U. at a wavelength of 365nm using an intensity of 200mW/cm for a period of about 60 minutes.

Following polymerisation the polymer was removed from the surface and was immersed in water having a conductants of 750 microsiemens/cm 2 for about 12-hours.

The polymer gel was then placed in water having a conductants of 1550 microsiemens/cm for a further period of about 12 hours. The polymer was then immersed in a 0.9% sodium chloride solution in redistilled water for several hours. Pieces of the gel sheet were then packed in polypropylene foil containers containing excess impregnant sodium chloride solution. The pouches were WO 88/08448 PCT/EP88/00362 17 heat sealed and were then sterilised in an autoclave at a temperature of 121 0 C for a period of 15-20 minutes.

The polymer produced by this process was capable of absorbing 0.9% saline to form a gel having 70% ry weight impregnant liquid.

Use as culture substrate Sheets of polymer hydrogel produced as described S above were incubated in sterile MEM (Mirimal Essential Medium) a 34 degrees centigrade for a period of 3 days before the experiment. The gaseous enironment consisted of 5 percent CO 2 in air.

The polymer sheets were cut into 1 sq cm pieces and placed in culture flasks (Nunclon R) and fixed to the surface of the culture flask with a small clot of sterile silicone fat to prevent floating of the polymer sheet.

Three pieces of gel were placed in each culture flask.

CELLS

a) Cultures of rat palatal epithelial (RPE) cells were established, maintained and subcultivated according to methods previously described in Jepsen et al (op. cit).

Cells in 20 passage were used in the present study, b) A transformed tumorigenic cell line derived from the RPE cell line mentioned above was maintained and subcultivated following the same procedures as for non-tumorivenic RPE cells.

c) Cultures of normal human oral epithelial cells were established, maintained and subcultivated according to methods previously described in Arenholdt-Bindslev et al (op. cit).

Immediately following the introduction of gel fragments into culture flasks, 7.5 ml of cell suspension containing a total of 5 x 10 cells was added to each flask.

i. .1 WO 88/08448 PCT/EP88/00362 18 Each cell type was introduced into two flasks. Cells were left to attach for 4 days. Cell attachment was followed in phase contrast microscope and biopsies taken out and processed for light microscopy on day 6. Photos were taken at day 6.

After 4 days all three cell types had attached to the surface of the gel fragments. During the following days cell proliferation could be observed on the gels. On day 6 the gels incubated with both tumorigenic and non-tumorigenic rat oral epithelial cells were covered by a confluent layer of epithelial cells. The human epithelial cells proliferated but more slowly.

Claims (13)

1. A process in which anchorage dependent mammalian cells are cultured in the presence of an aqueous medium on the surface of a removable substrate which is a sheet- form gel comprising a water-insoluble, water-swellable hydrophilic synthetic polymer swollen with the aqueous medium characterised in that the polymer has a swellability such that it can absorb at least its own weight of 0.9% by weight saline.

2. A process according to claim 1 characterised in that the gel has a swellability such that when swollen in 0.9% saline the gel comprises 70% to 95% by weight of saline.

3. A process according to claim 1 or 2 in which the polymer from which the gel is formed is wholly synthetic.

4. A process according to any preceding claim in which the polymer is formed from ethylenically unsaturated monomer, A process according to claim 4 wherein the ethylenically unsaturated monomer comprises an acrylic monomer.

6. A process according to any preceding claim in which the cells comprise fibroblasts and/or epithelial cells.

7. A process according to claim 6 in which the cells are epithelial cells and in which the culture medium contains one or more substances to facilitate the growth of the epithelial cells. C 91Ostt4jpe.O2l1,t8S~spe~19

8. A process according to c are derived from feeder cell

9. A process according to c2 cells comprise brain cells, Laim 7 wherein the substances Laim 7 in which the feeder fibroblasts or 3T3 cells. S* 0* S S S S. S S S. 0* 5 9 *5OS S sees S S 9SSSSS 5 0O 5@ S S. S S .55. S. S 8.S S A process according to any preceding claim in which the culture medium contains components selected from grawth-affecting substances, serum, nutrients, enzymes, sequenstrants, buffers and antibiotics.

11. A process according to any preceding claim in which the anchorage dependent cells are beneath the surface of the aqueous medium throughout the process.

12. A product comprising a sheet-form gel comprising water-insoluble, water-swellable synthetic hydrophilic polymer swollen with an aqueous liquid wherein the polymer has a swellability such that it can absorb at least its own weight of 0.9% saline by weight and having a layer of cultured anchorage dependent mammalian cells.

13. A wound-dressing which is a product according to claim 12 and/or the product of a process according to any of claims 1 to 11. DATED this 12th day of June, 1991 MICHAEL BAY BY DAVIES COLLISON PATENT ATTORNEYS FOR THE APPLICANT(S) 910612,ejhspe.021,1685,spe20 'rN INTERNATIONAL SEAFF1CH REPORT N International Appication No PCT EP 8 8 0 03 6 2 1. CLASSIFICATION OF SUEJECT MATTER lif several classi!fication symbol& apply, indicate all)t According to International Patent Classification (IPC) or to both National Claslfication and JPC IPC 4 C 12 N 5/00; A 61 L 27/00 11, FIELDS SEARCHED Minimum Documentation SearchedI Cli i~on System I Classification Symbols 'PC 4C 12 N; A 61 L Documentation Searched other than Minimum Documentation to the Extent that such Documents are tncluded In the Fields Searched IlL. DOCUMENTS CONStDERED TO 9E RELEVANT' Category *I Citation ot Document, "1 with Indication, where appropriate, of the relevant passee i ieant to Claim No. 1 Y Chemical Abstracts! vol. 99, n~o. 13, 1-3,7,8,12 26th September 1983 (Columnbus, Ohio, US) F. Yoshaii et al.: "Cell culture on polymers prepared by radiation- AIuced polymerization of various glass-forming monomers see page 317, abstract no. 101878s, Appl. Biochem. Biotechnol. 1983, 8(2)f 115-26 (cited in the application) Y chemical Abstracts, vol. 98, no. 17, 1-3,7,8,12 April 1983 (Columbus, Ohio, us) B. Faris et al.; "Effect of protein- hydroxy'ethy. methacrylate hydrogels on cultured endothelial cells", see page 294, abstract no. 140076c, EXP. CELL RES. 1983, 143(1)0 15-25 (cited in the applicationl Y' chemical Abstracts, vol. 107, no, 18, 13781 2nd November 1987 (Columbus,. Qhioo US) *Special categories of cited dlocumenter to later document published car the Initrnational filing date document defining the general elate ot the cii which Is not or priority data and not tn cOnfliCt with the applicatiorn but Considered to be of particuiar relevances cited to understand the principle or theory undierlitng the "Fearlier document but published an or shtte the International OX" document of particular relevancet the claimned Invenion filing date at be considered novel at cannot be considered to OL" document which may throw double on priority clalm~s or Invv an Inventive mtep which Is cited to scalis 1h the publication date ofat anot document ot paritUlat relevainco,' the claimed Invention citation of other special reason lis ,specined) cannot be considered t0 Involve en Inventive 6te0 whnen Ihe I'0" document (sterring to en cad dieclosuire, Use, eshibition Or document ia combined with one or more other such docu. other mAs omenit such combination beting obvious to a person skilled O" documnt published prior to the International filing dale but In the art, laterf than the piatity date claimed document Member Of the same patent family IV. CERTIFICATION Dale ot the Actual Completion at the International Search, 19th September 1988 Da at, Mailing of thie International St~lrch Mellott 1 25. 1 88 international Searching Authority I EUROPEAN PATENT OFFICE Form PCT/II5AI (escond ehoot) (jarwary 1,M) Illi. DOCUMENT~ Internalional Apphcallom No. PCT/EP 88/00362 -2- ~s CONSIDERED TO 59 RELEVANT (CONTINUED FROM THE SECOND SHEET) -e Category At A, P; Citation~ of Docurrnolt, with indCtor, whate appropriate, of the. relevant passages Relevant to Cteimn No m. Sugarnori et al.: "Microencapsulation of mamm~alian cells in polyacrylate membranes for metabolic prostheses", see page 477, abstract no. 161613v, Prog. Artif. Organs, (Pap. Int. Soc. Artif. orga nsl World Ccngr.), 1985 (Pub. 1986) 630-4 GP, A, 2094833 (UNITED STATES OF AMERICA) 22 September 1982 FR, At 2589165 (INSTITUT NATIONAL DE LA SANTE ET DE LA RECH-ERCHE MEDICALE) April 1987 (cited in the application) Farm PCT ISA Ito (efti Sheet) (JanusV$ 10351 s ~i ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. EP 8800362 SA 21888 This annex lists the patent family members relating to the patent documents !id in (h abov.Vitottioned international search report, The members are as contined in the European Patent OTffice EDP rite on 11110/ The European Patent Office i n no way liable for these particula.s hitch arg ircelty g fr f the purpose of information, Patent document cited in search report Publication date Publication date L- i GB-A- 2094833 22-09-82 BE-A- FR-A B SE-A- DE-A, C JP-A US-A- CA-A- CH-B- JP-A- SE-B- $92479 2503183 8201554 3209127 58016693 4409331 1172961 654328 61088893 454780 01-07-82 08-10-82

14-09-82 09-12-82

31-01-83 11- 10-83 21-08-84 14-02-86 07- 05-86 30-05-88 r- FR-A- 2589165 30-04-87 None er r or more details about, this annex st Ofricial Journal of the E~uropesan P'atent Mecr So. 1%182