GB2397824A

GB2397824A - Three dimensional cell culture

Info

Publication number: GB2397824A
Application number: GB0302258A
Authority: GB
Inventors: Ian Philip Middleton; Christopher Louis Ives
Original assignee: HiMedica Ltd
Current assignee: HiMedica Ltd
Priority date: 2003-01-31
Filing date: 2003-01-31
Publication date: 2004-08-04
Also published as: WO2004067733A1; GB0302258D0

Abstract

A cell growth scaffolding arranged in use, to mount one or more cells, comprising at least one fibre, wherein at least a portion of the fibre is mounted in a fluid perfusable support means. The invention further provides methods of growing cells and methods of manufacturing cell growth scaffolding.

Description

1 2397824

IMPROVEMENTS IN AND RELATING TO CELL GROWTH

Field of the Invention

This invention relates to cell growth scaffolding and methods of manufacturing cell growth scaffolding. The invention further relates to methods of growing cells, particularly to methods of growing cells outside (in vitro and ex viva) and inside (in vivo) the body of an organism.

Background to the Invention

Cell growth media are known to enable growth and maintenance of mammalian, and other cells in vitro, and to encourage differentiation of cells to, and maintenance of, a defined phenotype.

The practice of taking tissue from an animal or plant and breaking it down into its smallest components, the cells, and then maintaining them as living units outside the animal plant body has been carried on for over 100 years, and is commonly called cell culture. The problems associated with known techniques of cell culture are maintenance of cell viability and of the cell phenotype.

As the technique has developed, its limitations have been accepted for some model systems and various model culture systems standardized, and these have been used to develop further the science behind the practice.

It is still generally acknowledged, however, that cells grown using current methods and current cell growth media and materials immediately or progressively change and are different to their "in vivo,, cell equivalent and thus much of the potential for cell culture in testing has not been realised. The difference in cells cultured in known cell growth media outside the body, and their in vivo equivalents is thought to be due to a change in the cell phenotypic expression, i.e. a change in the RNA profile, the genes that are active within its full genotypic potential.

Use of current cell culture techniques has identified that lo the cell phenotype is altered by environmental forces, and it is now recognized that the phenotype expressed by a cell is the net result of those forces.

There are four key elements of the cell environment through which forces are exerted on the cell, namely the liquid medium surrounding the cell, the substrate on which the cell is grown, the fluid mechanical forces around the cell, and the cell membrane receptor-specific interactions.

The liquid medium is the cell culture medium, which also contains the specific membrane receptor binding molecules, growth factors, cytokines, nutrients etc. and much of the initial development work in cell culture was focused on this aspect of cell growth. I The substrates on which cells are currently grown in known I cell growth media generally comprise glass surfaces, polystyrene surfaces and the like. Such cell growth surfaces suffer problems in that they generally consist of only a single functionality on the surface, with which to adhere cells, they are nonspecific, and the surfaces are smooth and flat, non-porous and two dimensional.

Typically, a cell growth surface such as polystyrene is contacted with one or more cells, to adhere the cells to the surface, and the surface bathed in cell culture liquid. Cells grown in this manner can generally only grow to a thickness of approximately 2-3mm before becoming non- viable.

US patent 5,266,476 highlights the limitations of lo polystyrene surfaces for cell growth as an example of current two dimensional cell culture substrates. The proposed solution in US 5,266,476 is the construction of a polyester fibre matrix comprising various coatings on the fibre to enhance attachment of cells to the fibre. As a fibre, it has some three dimensional profile, but cell growth from the fibres would again be generally limited in thickness.

It would be advantageous for the substrate in cell culture to mimic that of the normal biological environment, but this would involve not only differing chemistry, but also of special organization of support structures. There are currently no culture systems available that offer biomimetic fibres with a special organization.

The third key factor in viability of cell cultures is fluid mechanical forces. Until very recently no allowance has been made for fluid mechanical forces in a life of the cell in cell culture. Most culture is typically carried out in an essentially stationary environment where there may be intermittent exposure to high levels of fluid shear, at time of changing the growth liquid bathing the cells for example. Where perfusion is used it is used as a means of refreshment of culture media to remove waste and supplement nutrients, and cells are considered to be at risk of shear damage if exposed directly to flow so this is generally avoided. In most cases, the cell culture environment contains a non- compliant substrate which will only transmit shear stress component and not shear strain. It has become clear in recent years that cells will alter shape, up regulate and down regulate secretary activity etc and generally modify gene lo expression in relation to fluid mechanical forces. No culture systems today offer perfusion as a means of cell stimulation for routine culture.

It would be advantageous to provide a cell growth medium that can control the drivers that have now been identified important in determining, and maintaining cell phenotype.

Such drivers include growth factors, cell contacts, surface functionality, surface geometry/profile, surface energy, surface porosity/permeability, surface compliance/deformability, shear stress, shear strain and/or oxygen partial pressure. It would be advantageous to provide a cell culture medium which would allow cell growth to provide tissues of much larger thickness than can be achieved in current cell culture media. It would further be advantageous to provide a cell growth media in which a pluripotent stem cell, and undifferentiated cells, could be adhered to the media and be driven down a specific differentiation pathway or conversely a differentiated cell could be adhered to the media and maintain both viability and original phenotype.

It would therefore be advantageous to provide a cell growth median which is three dimensional in nature, and enables cell growth within the three dimensional structure. It would furthermore be advantageous to provide a three dimensional cell growth structure throughout which cell growth factors and nutrients can perfuse in a all-encompassing or optionally selective manner, in order to enable continual viability and maintenance of phenotype of cells and tissues growing within the three dimensional matrix.

lo It is therefore an aim of preferred embodiments of the invention to overcome or mitigate at least one problem of the prior art, whether directly expressed hereinabove or not.

Summary of the Invention

According to a first aspect of the invention there is provided a cell growth scaffolding arranged, in use, to mount one or more cells, comprising at least one fibre, wherein at least a portion of the fibre is mounted in a fluid perfusable support means.

The fibre may be a hollow fibre. The hollow fibre may comprise one or more apertures in the fibre wall. In use one or more cells may be mounted inside the hollow fibre and/or on the outside of the hollow fibre.

The fibre may be a substantially solid fibre. In use, one or more cells may be mounted on the outside of the fibre.

The fibre may be a porous fibre, in which one or more cells may be mounted in one or more pores and/or on the surface of the fibre.

Preferably there are a plurality of fibres.

The fibres may be separate fibres, and may be oriented in any spatial orientation with respect to each other, such as parallel, transverse, perpendicular or any combination thereof. In one embodiment the cell growth scaffolding comprises a plurality of fibres oriented spaced apart and parallel with each other. In a preferred embodiment the lo cell growth media comprises a plurality of fibres, a first portion of which are oriented spaced apart and parallel with each other, and a second portion of which are oriented transverse to the first portion, and preferably spaced apart and parallel with each other.

At least some of the fibres are preferably connected.

Advantageously at least some of the fibres are connected to form a three dimensional network of interconnected i fibres. The three dimensional network may comprise a scaffold structure or framework of connected fibres.

The fibres may be connected by knitting, weaving, embroidery or the like, for example, or by non-weaving techniques, such as needling of fibres to form a non-woven felt, or electrodeposition of fibres to form a matted or flocked surface network, for example or by any combination of these techniques.

The density of fibres within the three dimensional network will vary according to the application, cell type and cell numbers to be cultured, as will the distance between cells. It is envisaged that in certain embodiments fibre density per unit cubed, and/or the distances between fibres, will be equal throughout the cell growth scaffolding. In alternative embodiments it is envisaged that within the growth scaffolding different regions will contain different numbers of fibres or portions of fibres such that fibre density and spatial distance between fibres will vary for each or some of the regions.

Suitable materials for use as fibres in the cell growth media of the invention may comprise any material to which lo cells are capable of anchoring to and which do not substantially damage anchored cells.

Suitable materials include synthetic material. Suitable synthetic materials include polyethylene terephthalates, polyesters, polyamides, polyolefins and copolymers thereof, polyvinyl chloride and copolymers thereof (for example vinyl chloride with acrylonitrile, vinyl acetate, polyvinylidene halide, polyether sulphone or vinylidene chloride) polyacrylics, polyacrylates, super absorbent fibres based on acrylic acid or itaconic acid, polyurethanes, polyimides, polylactides, polyglycollides and co-polymers, mixtures and salts of any of the aforesaid polymers.

However, it is preferred that the fibres used in the cell growth scaffolding of the invention comprise, at least in part, a biopolymer, such as a saccharine biopolymer, a protein or a polypeptide (whether natural or synthetic),

for example.

Particularly useful as a fibre material are saccharine polymers or oligomers. Examples of suitable polysaccharides include polymers and copolymers of: alginic acid and salts thereof; polymers of cellulose and salts thereof; polymers of carboxymethylcellulose and salts thereof; polymers of carrageenan and salts thereof; polymers of hyaluronic acid and salts thereof; polymers of heparin and salts thereof.

The fibres may comprise more than one material, and may for example comprise a composite fibre of two or more materials. For example a composite fibre may comprise lo both a biopolymer component and a synthetic plastics component. The composite fibre may be fabricated by any suitable technique such as by knitting, weaving or embroidering different materials into the composite fibre, for example. The composite fibre may comprise a plurality of thinner fibres of two or more different materials, combined to form the thicker composite fibre.

The exact makeup of the fibres will depend on which type of cell or cells are intended to be grown on the cell growth scaffolding. Thus the choice of fibre will depend on one or more of the following characteristics: nonspecific cell adhesion; specific cell adhesion; strength; durability; biodegradation; ability to provide nutrients for growing cells; porosity; fluid permeability; growth factor and ligand binding ability.

As an example a network of chitin, chitosin and/or hyaluronate fibres provides good non-specific cell adhesion through ionic groups (both positively and negatively charged) on the fibre surface, and further provides good strength, durability and flexibility.

The or each fibre may comprise surface groups able to react with or bind specific cell types or specific surface groups on cells.

The or each fibre may comprise cell adhesion molecules bound or connected to the surface of the fibre, arranged in use to adhere to a specific cell type or types.

Suitable proteins for use as cell adhesion molecules include integrin, fibrin, fibrinogen and laminin, for example. Examples of cell adhesion agents include peptides or protein molecules, which may specifically react or bind to a ubiquitous or specific protein on a cell surface, such as a receptor, membrane protein, ion channel, enzyme or the like, for example.

Preferably at least one fibre is constructed entirely from one or more biopolymers. Preferably the or each biopolymer is independently produced by a fibre spinning technique, which may be for example, wet spinning, centrifungal spinning, or electrostatic spinning.

The exact spinning technique used will depend on the desired properties of the resultant fibre.

One or more fibres may comprise one or more cell growth factors, cytokines, cell nutrients, proteins, peptides or other cell growth promotion agents, mounted within and/or on the fibre. The fibres may be fabricated by spinning the fibre material in the presence of one or more growth factors, cytokines, cell nutrients, proteins, peptide or other cell growth promotion agents, for example, such that the resultant fibre comprises the agent or agents entrapped, entrained or bonded to the fibre.

Suitably the cell growth scaffolding comprises at least one load bearing fibre and at least one fibre arranged in use to mount one or more cells. The load bearing fibre is preferably a synthetic plastics fibre, such as polyester, for example. Suitably the load bearing fibre does not comprise any growth factors, cytokines, cell nutrients, peptide, proteins or cell growth promotion agents mounted within and/or on the load bearing fibre.

Preferably substantially the whole of the or each fibre is mounted within the fluid perfusable support means.

When a network of fibres is present, preferably the support means surrounds substantially the entire network and substantially fills the spaces between each fibre in the network.

The support means is such that one or more cells can grow, proliferate and/or differentiate within the support means.

The fluid perfusable support means is preferably a solid or gel (including hydrogel), but in certain embodiments may be a viscous liquid. By viscous liquid we mean a liquid having a centipoise value of at least 8,000 centipoise (cP) at ambient temperature, preferably at least 9,000 cP and more preferably at least 10,000 cP.

The viscous liquid is preferably such that the or each fibre mounted in the liquid is substantially immovably supported, when the cell growth media is still.

The support means may comprise one or more channels, conduits and/or cavities formed in the support means and which may extend partially or entirely therethrough.

Suitably the support means comprises a connection means, arranged in use to operably cooperate with a connection means, of a second cell growth scaffolding of the invention. The support means may comprise at least one ridge on a surface and/or at least one channel on a lo surface, which in use form the connection means, operably cooperable with a corresponding channel and/or ridge on another support means.

The fluid perfusable support means is most preferably a gel, especially a hydrogel, and may be in the dehydrated state and thus be a solid, or in the hydrated state.

Preferably the hydrogel comprises polymerized hydrophilic monomers, and may comprise a homopolymer, heteropolymer, co-polymer, or a combination thereof.

The gel, hydrogel, or solid may comprise a synthetic polymer or a natural polymer.

Suitable natural polymers include alginates, agaroses, agars, gelatines, starches, pectins, polysaccharide hydrogels, celluloses, chitosans, collagen, proteins, polypetides, and the like, for example.

Synthetic polymers suitable as hydrogels include but are not limited to, polymerized monomers of acrylamides, acrylates, pyrrolidones, acrylic acids, and derivatives and mixtures thereof; for example.

Suitable acrylamide monomers include, acrylamide per se, methacrylamide, diacetone acrylamide, N-hydroxy propylmethacrylamide, N,N,-dimethyacrylamide, N- (trishydroxymethyl) acrylamide, N-(trishydroxymethyl) methacrylamide, acrylamidopropylsulphonic acid, and salts thereof, 3-acrylamidopropyl ammonium halides, 3methacrylamidopropopyl ammonium halides, and any mixture thereof, for example.

Suitable pyrrolidones monomers include N-vinylpyrrolidone,

for example.

Suitable acrylates monomers include, for example, 2 hydroxyethylacrylate, 2-hydroxethylmethacrylate, 2- hydroxypropylacrylate, 3-hydroxpropylmethacrylate, polyethylene glycol monomethyacrylate monomers, preferably having molecular weights of the polyethylene glycol chain from 200 to 10,000, and derivatives and mixtures thereof.

Suitable acrylic acid monomers include, for example, acrylic acid, methacrylic acid, acrylic acid-(3- sulphopropyl ester) and derivates and mixtures thereof.

Particularly preferred as hydrogels are copolymers comprising an acrylamide and one or more further polymers preferably selected from an acrylate, a pyrrolidone and an acrylic acid, or derivates thereof. Suitably the copolymer comprises up to 40 molar% acrylamide.

The hydrogel may be prepared by polymerizing the monomers in the presence of a cross-linking agent and/or a free radical initiator. Suitably the cross-linking agent is a di-ethylenicaly substituted cross-linking agent. Examples of suitable di-ethylenicaly substituted cross-linking agents include di-ethylenicaly substituted dimethacrylates, acrylates, pentaerythritol tetraacrylate, methylene bis-acrylamide, ethylene bis acrylamide and dihydroxyethylenebisacrylamide.

Suitably the cross-linking agent is added to the monomer composition at a concentration of at least 0.05% by weight lo of the monomer composition, preferably at least 0.1%, and preferably no more than 20% by weight, more preferably no more than 10%.

Suitably the free radical initiator is added to the monomer composition to a concentration of at least 0.01% by weight of the monomer composition, preferably at least 0.05%, more preferably at least 0.01%, and suitably no more than 10% by weight of the monomer composition, preferably no more than 5%, and more preferably no more than 1%.

Polymerisation of the monomer composition is preferably carried out after substantially removing oxygen from the composition such as by passing a gas such as helium or nitrogen through the composition, or by ultrasonification or freeze-drying for example.

When the monomer composition comprises a cross-linking agent polymerization may be effected by heating at an elevated temperature, preferably between 50 C and 65 C.

Heating may be effected for a desired period of time, preferably at least 1 hour, more preferably at least 6 hours, still more preferably at least 12 hours. Heating is preferably effected for no more than 72 hours, more preferably no more than 60 hours and still more preferably no more than 48 hours.

After the initial heating the resultant polymer may be subjected to post curing at an elevated temperature, preferably between 60 C and 80 C for a desired period of time, preferably between 1 hour and 5 hours, more preferably around 3 hours.

When the monomer composition comprises a free radial initiator preferably polymerization of the monomers may be effected by irradiation with long wavelength ultraviolet radiation, for example. If polymerization is effected by irradiation with ultraviolet radiation preferably a free radical initiator is added to the monomers before or during irradiation. Suitable free-radical initiators include benzoyl peroxide, cumene hydroperoxide, dicumylperoxide, azobis(isobutyronitrate), azobis(2 methylpropionamide) dihydrochloride, diisopropylazodo carboxylate, Nchloro succinimide, N-bromosuccinimide, ammonium persulphate, potassium persulphate and any combination thereof, for example.

The support means may comprise one or more cell growth factors, cytokines, cell nutrients, peptides, proteins or other cell growth promotion agents, mounted within and/or on the support means. When the support means comprises a hydrogel formed by polymerizing monomers, suitably polymerization takes place in the presence of the or each cell growth factor, cytokine, cell nutrient, peptide, protein or cell growth promotion agent, in order that said agent is contained within and/or on the resultant polymer.

In one embodiment of the cell growth scaffolding of the invention comprising a hydrogel, monomers may be bulk polymerized in the dehydrated state, (to form a xerogel) and the resultant bulk dehydrated hydrogel polymer may be machined into the desired size and shape. The dehydrated hydrogel polymer may then be contacted with a hydrating solution to form the hydrated hydrogel.

lo In an alternative embodiment the hydrogel monomers may be dispersed, dissolved or mixed with a hydrating solution and polymerized therein to form the hydrated hydrogel.

The hydrating solution may be water or a simple salt solution. Preferably the hydrating solution is a cell growth medium, preferably comprising one or more cell growth factors, cytokines, nutrients, proteins and/or inorganic salts, for example.

Cell growth media suitable as hydrating solutions include Dulbecco's Modified Eagle Medium (DMEM), Ham's Growth Medium, Ames' medium, BGJb medium, Basal medium Eagle, CMRL-1066, Dulbecco's modified Eagle's medium, Fischer's medium, Glasgow minimum essential medium, Ham's F-12 Coon's modification, Iscove's modified Dulbecco's medium, L-15 medium Leibovitz, McCoy's 5A medium, Medium 199, Minimum essential medium eagle, nutrient mixture F-10 Ham, RPMI-1640 medium, Swimm's s-77 medium, Waymouth MB 752/1 medium and Williams' medium E. Monomers particularly suited for polymerization within a hydrating solution include acrylamide-based monomers, which are generally readily soluble in most aqueous-based cell growth media solutions.

Suitably the monomer solution comprises monomers in at least 1% by weight of the total weight of the solution, preferably at least 2% by weight, more preferably at least 5% by weight and most preferably at least 10% by weight.

Suitably the monomer solution comprises monomers in an amount of no more than 50% by weight of the total weight lo of the solution, preferably no more than 40% by weight, more preferably no more than 30% by weight and most preferably no more than 20% by weight.

Suitably the monomer solution comprises a cross-linking agent, preferably a di-ethylenically substituted cross- linking agent, in an amount of at least 0.05% by weight of the total weight of the solution, preferably at least 0.1% by weight and more preferably at least 0.5% by weight.

Suitably the monomer solution comprises the cross-linking agent in an amount of no more than 20% by weight, preferably no more than 10% by weight and more preferably no more than 5% by weight of the total weight of the solution.

Alternatively or additionally to a cross-linking agent the monomer solution may comprise a free radical initiator, which is preferably substantially water-soluble. Suitably the initiator is present in an amount of at least 0.01% by weight, preferably at least 0.05% by weight, and more preferably at least 0.1% by weight of the total weight of the solution. Suitably the initiator is present in an amount of no more than 10% by weight, preferably no more than 5% by weight and more preferably no more than 1% by weight of the total weight of the monomer solution.

Thus in preferred embodiments the monomers are dissolved in a hydrating solution which further comprises a cross- linking agent and/or free radial initiator, and then the monomers polymerized by any suitable means such as curing or ultraviolet irradiation for example.

lo Suitably the monomers or monomer solution is placed in a mould of desired dimensions and polymerised therein, to effect a desired shape of the resultant polymer.

The mould may include one or more protrusions around which the monomer solution is located. The or each protrusion may comprise a member of uniform cross-section, such as a cylinder, pyramid, parallelepiped and the like for example, such as to create cavities within the polymerized hydrogel. The cavities may take the form of channels, conduits and the like for example. The protrusions may be members of irregular crosssection such that the resultant cavities within the polymerized hydrogel take the form of, for example, cavities spaced apart by conduits or channels. There may be one or more protrusions in the mould dimensioned to create a corresponding channel or conduit extending substantially entirely through the polymerized hydrogel.

In alternative embodiments the hydrogel may comprise channels, conduits and/or cavities extending therethrough, formed into the hydrogel after polymerization, by for example, insertion of a suitably shaped member or drill into the hydrogel.

The mould may be shaped to effect incorporation of at least one connection means. Such as a ridge and/or channel, for example, in a surface of the hydrogel.

Alternatively the connection means may be formed into the surface after polymerization of the monomer by stamping, boring, drilling or the like, for example.

The or each fibre may be mounted in the hydrogel before, during or after polymerization of the monomers.

Preferably the or each fibre is located in the monomers or monomer solution before polymerization, in the desired orientation, and the monomers or solution subsequently polymerized around the or each fibre.

Thus for a fibre network, the network is preferably constructed then placed in a monomer solution and the hydrogel polymerized around the fibre network.

The or each fibre may be rigidly or fixedly mounted within the support means.

If the support means comprises channels, conduits or cavities extending therein, the or each fibre may be mounted within the or each channel, conduit and/or cavity.

The fluid perfusable support means may comprise one or more water-soluble polymers, as an alternative to, or additional to a hydrogel.

One or more water-soluble polymers may be cross-linked by ionic bonds and/or by inter-polymer complexation.

Solutions of water soluble polymers and co-polymers containing acidic groups (or their monovalent salts) may be mixed with divalent or trivalent cations, or mixtures thereof, to form an tonically cross-linked gel or alternatively mixed with water soluble polymers or co- polymers bearing positively charged groups to yield an inter-polymer complex. Examples of water soluble polymers and co-polymers containing acidic groups (or their lo monovalent salts) that may be used are polyacrylic acid, polymethacrylic acid, alginic acid, carrageenan, hyaluronic acid, heparin, chondroitin sulphate, polystyrene sulphonicid- co-malic acid co-polymers, polyacrylic acid -(3-sulphopropyl ester), polymethacrylic acid -(3-sulphopropyl ester) and polyacrylamido propyl sulphonic acid. Typical examples of divalent and trivalent ions that may be used to produce ionic cross- links are Ca2+, Ba2+, Cu2+, Zn2+, Sr2+ and A13+. Examples of water soluble polymers or co-polymers bearing positively charged groups to yield an inter-polymer complex are chitosan, polyethyleneimine, polydimethylaminoethyl I acrylate, polydimethylaminoethyl methacrylate, poly(3 acrylamido propyl ammonium chloride) and poly(3 methacrylamido propyl ammonium chloride). Additionally some pairs of water soluble polymers are known to form gel complexes when mixed via strong interchain hydrogen bonds. Examples of such pairs of polymers are polyacrylic acid and polyethylene oxide and polyacrylic acid and polyvinyl pyrrolidone. It is of course possible to form a hydrogel containing mixtures of polymers containing acidic groups with divalent cations, polymers bearing positive charges and inter-polymer complexes. The fluid perfusable support means may comprise a combination of one or

more hydrogels and one more tonically cross-linked water-soluble polymers or polymer complexes, to yield an interconnected polymer network.

This is most conveniently achieve by first degassing both a hydrophilic monomer composition to be polymerized in cell growth media, and the or each water soluble polymer, before mixing, transferring to a mould containing the 3- dimensional matrix of fibres and effecting polymerization lo as outlined above. The resulting material offers a good compromise between the excellent transport properties of an tonically cross-linked polymer(s) and the increased strength of a covalently bonded hydrogel system.

The fluid perfusable support means may itself be used to set or stabilise the spatial organization of the or each fibre mounted in the support means, which may be achieved by selective hardening of the support means, in the case of gels and viscous liquids, or through rigidly mounting the or each fibre in a solid or gel support means.

If cell growth factors, cytokines, cell nutrients, proteins and the like are present on a fibre, or within or on the fluid perfusable support means, they may be present in a defined or selective concentration gradient within the cell growth scaffolding, such as to drive cell growth migration towards or away from defined or desired areas of the scaffolding.

The or each fibre, and/or the fluid perfusable support means may comprise different compliance characteristics at different portions thereof, so as to encourage, in use, cell growth or drive cell differentiation at different rates in different regions of the cell growth scaffolding.

The cell growth scaffolding, or part thereof, may be s degradable, preferably biodegradable. The rate of degradation or biodegradation of the cell growth scaffolding, or part thereof may be tailored by selection of particular fibres and/or fluid perfusable support means having desired degradation characteristics. Degradation lo in vitro may be due to oxidation, reduction or degradation by growing cells, for example. Degradation in vivo or ex vivo may be as above, or by the organism's immune system,

for example.

The materials used for the fibres and support means of the cell growth scaffolding may be different for in vitro and in vivo/ex vivo use, for the same type of growing cells, due to the need to consider immunogenicity and antigenicity of the cell growth scaffolding in viva and ex viva.

In preferred embodiments of the invention the or each fibre, and/or the fluid perfusable support means comprise(s) substantially all of the cell growth factors, nutrients, cytokines, proteins, peptides, and the like, mounted in or on the fibre(s) and/or support means, and which are needed for efficient cell growth and viability, and thus any external cell growth media added to the scaffolding may need only be a saline or salt solution and incorporate no, or little, cell growth material.

If the cell growth scaffolding includes one or more cell growth materials such as growth factors, nutrients, cytokines etc. then preferably the cell growth scaffolding effects both a support and nutrient delivery system, stabilizing the or each growing cell and enabling direct presentation of any growth material to the or each cell's luminal or abluminal surface. Thus high concentrations of cell growth media liquid or solutions would not be needed; as is currently necessary in known techniques; as the entrained cell growth material in the cell growth scaffolding of the invention would be directly and evenly lo accessible to a growing cell or cells, throughout cell growth and differentiation. As stated hereinabove, concentration gradients may be set-up within the cell growth scaffolding to enable selective cell growth and differentiation.

According to a second aspect of the present invention there is provided a method of growing cells, the method comprising the steps of: (a) providing at least one fibre and a fluid perfusable support means; (b) mounting at least a portion of the or each fibre in the fluid perfusable support means to provide a cell growth scaffolding; and (c) optionally contacting the cell growth scaffolding with a solution of one or more cell growth factors and/or nutrients; and wherein the method comprises seeding at least one cell to the or each fibre and/or the support means.

The or each cell may be seeded at any point after step (a) in the method but is preferably performed between steps (a) and (b). In alternative embodiments cells may be seeded after steps (b) and/or (c).

The or each fibre and the fluid perfusable support means are preferably as described for the first aspect of the invention.

The or each cell growth factor and/or nutrient are preferably as described for the first aspect of the lo invention.

The cell growth scaffolding is preferably as described for the first aspect of the invention.

According to a third aspect of the present invention there is provided a method of manufacturing a cell growth scaffolding of the first aspect of the invention, comprising mounting at least one fibre in a fluid perfusable support means or precursor thereof.

The fluid perfusable support means or precursor may be monomers or a monomer solution as hereinabove described.

The method may comprise mounting the cell growth scaffolding in a support frame or support apparatus. The support frame or apparatus may comprise one or more cell growth solution conduits, cell growth waste conduits, oxygenation units, detoxification units, fluid pumps, and/or thermostats.

According to a fourth aspect of the invention there is provided a method of growing cells in vivo or ex viva, the method comprising the steps of: (a) mounting at least one fibre in a fluid perfusable support means; and (b) inserting the resultant cell growth scaffolding into a living tissue or organism.

Preferably the fibres, support means and cell growth scaffolding are as described for the first aspect of the invention.

Thus the cell growth scaffolding of the invention may be implanted into a tissue and/or organism enabling cell migration from the tissue or organism into the scaffolding for subsequent growth.

One or more cells may be seeded onto at least one fibre before insertion into the support means. Alternatively or additionally one or more cells may be seeded into the support means. Thus cells may be introduced to the cell growth scaffolding before insertion into the tissue and/or organism for subsequent growth and/or migration into the tissue or organism. This can be useful in the repair of damaged tissues.

The use of a support means containing one or more fibres allows for enhanced three-dimensional growth of cells in the cell growth scaffolding of the invention. The combination of a fluid perfusable support means and one or more anchoring fibres increases the compliance of the cell growth scaffolding to enhance the phenotypic drivers of the cell growth and expression such as cell contact, cell growth media surface functionalities, and geometries, surface energy, surface porosity/permeability, surface compliance/deformability, shear stress, shear strain and available oxygen to growth cells; as compared to known cell growth media.

The cell growth scaffolding of present invention is not only suitable for in vitro growth and culture of cells, but may also be used for in viva and ex vivo growth and culture of cells. The cell growth scaffolding of the present invention is suitable for implantation in existing lo tissues or organisms for in vivo and ex vivo culture of cells and tissues.

The cell growth scaffolding of the invention is also useful for handling and culturing stem cells, which can be difficult or impossible to culture effectively in known growth media.

According to a fifth aspect of the invention there is provided a cell growth media comprising a fluid perfusable solid cell support means, wherein the support means comprises at least one cell growth agent fixedly mounted in or on the support means.

Preferably the fluid perfusable solid cell support means comprises a solid support means as described hereinabove for the cell growth scaffolding of the first aspect of the I invention, and is more preferably a gel (including hydrogel).

Preferably the or each cell growth agent is selected from a growth factor, a cytokine, a cell nutrient, a protein, a peptide, and the like, for example.

The fluid perfusable solid cell support means may have one or more cells mounted in or on the support means.

The support means may be mounted in a support frame or I support apparatus. The support frame and support apparatus may be as described for the third aspect of the invention.

The cell growth media of the fifth aspect of the invention lo may be used in viva, ex vivo or in vitro, as described for the third aspect of the invention.

Brief Description of the Drawings

In order to provide a better understanding of the present invention and to exemplify how embodiments of the same may be put into effect, the various aspects of the invention will now be described with reference to the accompanying: drawings in which: Figure 1 illustrates a part plan view, part perspective view of a cell growth scaffolding of the present invention; Figure 2 illustrates a side elevation of the cell growth scaffolding of Figure 1; I Figure 3A illustrates a portion of a fibre of the cell growth scaffolding of Figures 1 and 2; Figure 3B illustrates a portion of a fibre of the cell growth scaffolding of Figures 1 and 2; Figure 3C illustrates a portion of a second fibre type of the cell growth scaffolding shown in Figures 1 and 2; Figure 4 illustrates an asymmetric view of a portion of the cell growth scaffolding of Figures 1 and 2 in more detail; Figure 5 illustrates an asymmetric view of a portion of the a second embodiment of the cell growth scaffolding of lo the invention; Figure 6 illustrates a cell growth apparatus of the invention; and Figure 7 illustrates a second embodiment of the cell growth apparatus of the invention in block diagram format.

Description of the Preferred Embodiments

We refer firstly to Figures 1 and 2.

A preferred embodiment of a cell growth scaffolding 2 of the invention comprises a three dimensional network of fibres 6 mounted and encapsulated in a fluid perfusable support means in the form of a hydrogel disc 4. The hydrogel disc 4 includes, running therethrough, a perfusion channel 12, which extends entirely through the hydrogel disc 4. Radiating from the perfusion channel 12 on the upper surface of the hydrogel disc 4, are a plurality of connection means in the form of connection ridges 8, machined into the upper surface of the hydrogel disc 4. The ridges 8 extend from the perfusion channel 12 to the peripheral edge of the hydrogel disc 4. Also present on the lower surface of the hydrogel disc 4, extending from the perfusion channel 12 to the peripheral edge of the disc, are a plurality of radially extending connection means in the form of channels 10, formed into the hydrogel disc 4. Thus, in use, a number of cell growth scaffolding 2 may be stacked on top of each other, with the ridges 8 cooperating with the channels 10 of adjacent discs 4.

The cell growth scaffolding 2 was constructed as follows.

A mould (not shown) was fabricated in the shape of the hydrogel disc 4, which also included protrusions corresponding with the ridges 8 and channels 10 of the disc 4. The three dimensional fibre network 6 was constructed by wet spinning polyalginate fibres, and knitting or weaving the fibres into the desired three dimensional network. The three dimensional fibre network 6 was then placed in the mould. A monomer solution of acrylamide was prepared, and ethylenically substituted pentaerythritol tetraacrylate cross linking agent added to the liquid acrylamide monomer composition. The liquid monomer composition was then added to a cell growth media solution, Dubbs Modified Eagle solution. The resultant solution was poured into the mould, such that it filled the mould around the fibre network 6. The monomer solution was then polymerized by curing (60 C for 24 hours) and the resultant hydrogel disc 4 containing the three dimensional fibre network 6 removed from the mould.

The perfusion channel 12 was then drilled through the centre of the hydrogel disc 4 using a suitable machine tool.

We refer now to Figure 3A to 3C. In order to effect cell culture in the cell growth scaffolding 2 of Figures 1 and 2, desired cells may be seeded into the cell growth scaffolding 2 in a variety of ways. Cells may be adhered to the three dimensional fibre network 6 before placing the network 6 in the mould, and therefore the cells will be seeded on the network 6 when the monomer solution is polymerised to form the hydrogel disc 4. In this lo situation, differing polymerization strategies may need to be incorporated in order not to damage or kill off the cells due to excessive heat or ultraviolet radiation on polymerizing the monomer solution.

Of course if in alternative embodiments the fluid perfusable support means may be a preformed gel, a solid or a viscous liquid and the or each fibre may have one or more cells seeded to the fibre(s) before directly inserting the fibre(s) into the support means.

An alternative method of introducing cells into the hydrogel disc 4 is to insert a fibre such as a porous fibre 14 or hollow fibre 20 shown in Figures 3A and 3C, into the perfusion channel 12 and/or through the hydrogel itself in a desired position. The fibre 14 shown in Figure 3A is a solid but porous fibre of any suitable polymer, especially a biopolymer such as chitosan, alginate etc. Cells 16 are adhered to the fibre 14 and within the pores of the fibre 14 until they become fully adhered and flattened as shown in cells 18 of Figure 3A.

As shown in Figure 3B, eventually a plurality of cells will be adhered to the solid fibre 14. The fibre 14 may then be inserted into the perfusion channel 12 of the hydrogel disc 4, and suitable growth factor solution containing nutrients and growth factors drawn through the channel 12 as and when desired. Alternatively the fibre 14 may be inserted through the soft hydrogel disc 4 in any desired position to extend therethrough.

We refer now to Figure 3C. In an alternative embodiment to the solid porous fibre 14 shown in Figure 3A and 3B, a hollow fibre 20 may be incorporated into the hydrogel disc 4. The hollow fibre 20 enable cells to be adhered on the outside of the surface as shown in cells 18 of Figure 3C, and also on the inside of the hollow fibre 20, as shown in cells 22 of Figure 3C. The hollow fibre 20 may be inserted into the perfusion channel 12 such that a bore still remains through the centre of the perfusion channel 12. Alternatively the hollow fibre 20 may be inserted into the hydrogel disc 4 itself as described above.

We refer now to Figure 4 which illustrates a close up perspective view of a portion of the hydrogel disc 4 of a second preferred embodiment of the cell growth scaffolding 2 of the invention, in which a hollow channel 24 has been cast through part of the hydrogel disc 4, and a cell- containing porous fibre 26 has been inserted into the hollow channel 24. As shown in Figure 4, the hollow channel 24 extends through the hydrogel disc 4, and the cell containing fibre 26 extends entirely through the hollow channel 24. The hydrogel disc 4 is bathed in cell growth solution such as Dubbs Modified Eagle solution, and kept at optimum conditions for cell growth. As time passes, cells on the fibre 26 will begin to multiply and form cell growth which can pass through the pores or apertures of the porous hollow channel 24 and into the hydrogel disc 4. The cell growth becomes cell growth capillaries 28, which extend through the hydrogel disc 4, and adhere to fibres in the three dimensional fibre network 6 within the hydrogel disc 4, as shown in Figure 4. Further growth of the cell capillaries 28 cause cells to adhere to further fibres in the three dimensional network 6, such that they are adequately supported for continued growth, and in this way tissue growth may be maintained over and above conventional depth and lo thicknesses achieved in conventional cell cultures without the cells becoming non- viable or losing their phenotype.

Thus a capillary network of cells is developed which is surrounded by a fluid perfusable support, mitigating or removing the dependency on local diffusion for nutrient supply and gaseous exchange which limit the growth of cells in known cell growth media.

In respect of cell growth between fibres in the support means, cells may secrete their own extra-cellular matrix as they grow, which they can adhere to and migrate in relation to; in the latter situation in an amorphous support means, with no fibres, cells will usually clump together showing little migration, but contrastingly will migrate if there is a fibrous component to adhere to.

Thus cell orientation, position and numbers in the support means can be influenced by the presence, orientation and density/spacing of the fibres as well as the composition of the support means (e.g. its water content, nutrients, growth factors, compliance eta).

Cells may also be seeded into the support means as well as or alternatively to anchoring cells to one or more fibres in the media. Thus in alternative embodiments of the invention cells may be seeded only onto and/or into the support means or both on one or more fibres and in/on the support means. Delivery of cells into the support means may be by addition of the cells in the monomer solution (in the case of a polymerized support means) prior to polymerisation/gelation, by injection into specific sites of the support means, or by loading the cells in a carrier, such as a fibre and sewing the carrier into a specific place in the support means.

The cells can be directed in their growth by positioning of fibres strategically within the support means and/or be driven along concentration gradients set up within the support means and/or fibres.

We turn now to Figure 5, which illustrates a perspective view of a portion of a third preferred embodiment of cell growth scaffolding 2 of the invention. In this embodiment, the hydrogel disc 4 includes a number of different channels 30, 32, 34 running therethrough. A nutrient canal 30 is inserted through the hydrogel disc 4 and connected to a nutrient reservoir (not shown) which may contain growth factors and nutrients and the like, for example. The nutrient canal 30 is fluid perfusable such that nutrients flowing into the nutrient canal 30 may perfuse into the hydrogel disc 4 and throughout the fibre network 6. A cell coated solid fibre 32 is also present extending through the hydrogel disc 4 and from which cell growth capillaries 36 have extended into the three dimensional fibre network 6. A hollow fibre 34 in which cells are adhered to the inside surface of the hollow channel 34 has also been inserted into the hydrogel disc 4. Again cell growth capillaries 36 have extended from the hollow fibre 34 into the hydrogel disc 4 and begun to adhere onto the three dimensional fibre network 6. As can be seen in Figure 5, extended cell capillary growth 38 has begun along one or more of the fibres of the fibre network 6.

In alternative embodiments of the hydrogel disc 4 shown in Figure 5, the nutrient canal 30 may simply be a bore machined into the hydrogel disc 4 by a suitable machine lo tool to create a hollow channel through the disc 4. The hydrogel disc 4 may comprise many bores drilled therethrough into which cell-adhered fibres, whether porous or hollow may be inserted, or which may be used as nutrient canals or inlets/outlets for fresh nutrient solution and waste nutrient solution.

We turn now to Figure 6 which illustrates a cell growth apparatus 53 of the invention. The apparatus comprises a support body 54 into which is inserted stacked layers of cell growth scaffolding 2 of the invention, preferably stacked layers of the cell growth media 2 of the embodiment shown in Figures 1 and 2. In the apparatus 53 shown in Figure 6, the support body 54 includes a perfusion inlet 56 which is arranged to cooperate with the perfusion channels 12 in the cell growth scaffolding 2 to form a continuous channel therethrough. The support body 54 also includes a plurality of perfusion outlets 58 extending through the support body 54 and cooperable with the hydrogel discs of the cell growth scaffolding 2.

Thus, in use cell growth factors, nutrients and other solutions may be perfused into the hydrogel discs by movement through the perfusion inlet 56 into the perfusion channels 12 and then through the hydrogel discs. As the cell growth factors, nutrients and solutions spread through the hydrogel discs, any waste solution may exit i the hydrogel discs through the perfusion outlets 58 to be recycled and/or disposed of.

We turn now to Figure 7. Figure 7 illustrates a block diagram of a second embodiment of a cell culture apparatus I 53 of the invention. The cell culture apparatus includes three stacked cell growth scaffolding 2 through which is lo extended a continuous perfusion channel 43. In this embodiment, running between the cell growth scaffolding 2 are perfusion channels 40 formed from hemispherical channels cut into the lower and upper surfaces of the hydrogel disc 4 of the cell growth media 2. The cell growth scaffolding 2 are mounted in a controlled environment 42 which controls temperatures, pressures eta at optimum conditions for cell growth. Connected to one side of the cell growth scaffolding 2 are nutrient conduits 44, and connected to the opposite side of the cell scaffolding media 2 are waste conduits 45, with both nutrient conduits 44 and waste conduits 45 being in fluid communication with the cell growth scaffolding 2. The nutrient inlets 44 are connected to a oxygenation unit 46, which is in turn connected downstream of a water and nutrient replenishment unit 52. The water and nutrient replenishment unit 52 is connected downstream of a pump I 50. Connected to the waste conduits 45 is a detoxification unit 48 which is connected upstream to the pump 50, thereby completing a circuit.

In use, pump 50 is operated to transport water and nutrients from the replenishment unit 52 through the i oxygenation unit 46 in order to oxygenate the water, and therefrom through the nutrient conduits 44 into the cell growth scaffolding 2. The nutrients and water are also i pumped into the nutrient channels 40 between the cell growth scaffolding 2 in order that nutrients may perfuse into all parts of the cell growth scaffolding 2.

Nutrients and water which are passed entirely through the I cell growth scaffolding 2 exit via the waste conduits 45 and are transported via the pump 50 to the detoxification lo unit 48 which removes waste materials, and the detoxified nutrients and water solution are then pumped via pump 50 back to the replenishment unit 52.

Although specific chemicals have been indicated in the examples described hereinabove, a person skilled in the art would be able to provide alternative compounds, polymers and the like to replaced those described for the fibres 6 and hydrogel 4. For example, fibres 6 may be any: suitable synthetic or natural polymer on which cells may be grown, and particularly preferred are biopolymers such as chitosan, alginates, polysaccharides and the like for example. Furthermore, the fluid perfusable solid support means of the invention may be other than a hydrogel, such as a viscous liquid or solid fluid perfusable support, such as a porous or absorbent solid. Alternatives to polyacrylamide hydrogels include polyacrylate, polyacrylic I acids, polyvinylpyrrolidones, or any other gel which is able to absorb a sufficient quantity of water and cell growth factor nutrient solution and through which the solution may perfuse easily to provide nutrients to growing cells.

Furthermore, although in vitro use of the cell growth scaffolding of the invention has been exemplified, in i other embodiments, use may be made in viva or ex vivo, for example by implantation of the scaffolding into an existing tissue or organism. The scaffolding may or may not include one or more cells seeded within it. If cells are seeded into the scaffolding before implantation the I scaffolding may effect growth and migration of the cells into the tissue or organism to which it is implanted. If lo no cells are seeded, then cells from the tissue or organism may migrate into the scaffolding and grow therein. Thus the scaffolding could be used to bridge a wound or injury to tissue and encourage or begin healing through enhanced cell growth.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, I except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, i equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiment(s). The invention extend to any novel one, or any novel combination, of the features disclosed I in this specification (including any accompanying claims, : abstract and drawings), or to any novel one, or any novel lo combination, of the steps of any method or process so disclosed. :

Claims

Claims 1. A cell growth scaffolding arranged in use, to mount one or more

cells, comprising at least one fibre, wherein at least a portion of the fibre is mounted in a fluid perfusable support means.
2. A cell growth scaffolding as claimed in Claim 1, wherein the or each fibre is a hollow fibre.
3. A cell growth scaffolding as claimed in Claim 1, : wherein the or each fibre is a porous fibre.
4. A cell growth scaffolding as claimed in any one of Claims 1 to 3, wherein there are a plurality of fibres.
5. A cell growth scaffolding as claimed in Claim 4, wherein at least some of the fibres are connected. :
6. A cell growth scaffolding as claimed in Claim 5, wherein at least some of the fibres are connected to form a three dimensional network of interconnected fibres.
7. A cell growth scaffolding as claimed in Claim 5 or 6, wherein the fibres are connected by knitting, weaving, embroidery, or by non-weaving techniques, or any; combination thereof.
8. A cell growth scaffolding as claimed in any preceding claim wherein the or each fibre is constructed from one or more synthetic materials.