WO2007060382A1 - Cell aggregates - Google Patents

Abstract

A method of aggregating a population of cells comprising causing one or more cells in the population to be cross-linked with one or more other cells in the population and/or one or more micro-particles added to the population of cells.

Description

CELL AGGREGATES

This invention relates to cell aggregates, and in particular to cell aggregates produced by cross-linking cells to each other and/or to micro- particles.

The function of tissues and organs in the body is determined by the co-ordinated activities of the constituent populations of cells. This coordination is dependent to a large extent on the three dimensional architectural organisation of the cells. The three dimensional structure of a tissue is determined, at least in part, by the physical interaction of cells within the structure with each other, with the extracellular matrix and with cytokines. These physical interactions evolve over time during tissue development and repair.

Within the field of tissue engineering, and more broadly regenerative medicine, cell-to-cell interactions are very important. These interactions control the three-dimensional arrangement of cells within a tissue which are integral to the tissue development and function, and thus are vital in the creation of bioartificial tissue and organs. There is therefore a need to control the three-dimensional arrangement of cells in tissue engineering. The control of cell-to-cell interactions is also important in other fields, such as in vitro assay systems. By creating tissue models for use in in vitro assays which more accurately represent the in vivo conditions, advances in the accuracy and relevance of in vitro models can be made.

According to a first aspect, the present invention provides a method of aggregating a population of cells comprising causing one or more cells in the population to be cross-linked with one or more other cells in the population and/or one or more micro-particles added to the population of cells.

Preferably the cells and/or micro-particles are covalently cross-linked.

The rapid kinetics of covalent bond formation may allow cells and/or micro-particles to be aggregated according to the method of the invention more rapidly than by normal cell-to-cell or cell-to-matrix interactions, which are typically receptor-ligand interactions. In addition, the generally non-reversible nature of the covalent bonds in the aggregate mean that the aggregate formed is more stable, and the covalent bonds formed will usually not break until they are eventually hydrolysed. The greater strength of covalent bonds, compared to, for example, ionic or Van Der Waals interactions, means the aggregates produced are, at least initially, more stable.

The rapid kinetics of covalent bond formation means that if a population of cells and/or micro-particles is administered to a patient, then according to the method of the invention cell and/or micro-particle aggregation will begin almost immediately after administration of the cells and/or micro-particles and the cells and/or micro-particles will not be able to disperse significantly from a site of administration.

A population according to the invention comprises at least two cells. More preferably, a population comprises in the order of tens of cells or more, or in the order of hundreds of cells or more, or more preferably in the order of thousands of cells or more, or more preferably still in the order of millions of cells or more,

A population of cells according to the invention may comprise cells of only one cell type, or it may comprise cells of two or more different cell types. The method of the invention may cause cells of the same type to be cross-linked to each other and/or to micro-particles, or it may cause cells of more than one cell type to be cross-linked to each other and/or to micro-particles

Aggregating is used in the context of this invention to mean the bringing together and cross-linking of cells and/or micro-particles to form an aggregate. An aggregate comprises a population of cells and/or micro-particles in which at least some of the cells and/or micro-particles are cross-linked. The aggregate may comprise one cell type or it may comprise two or more cell types, furthermore the aggregate may also comprise micro-particles. The micro-particles in an aggregate may all be the same type or they may be of more than one type.

Preferably the one or more cells which are cross-linked to one or more other cells and/or one or more micro-particles have a modified region on the cell surface which is used in the cross-linking reaction. The one or more other cells may also have a modified region on their surface. If there is a modified region on the surface of cells which are cross-linked together, then these modified regions may be used to cross-link these cells .

Alternatively, or in addition, the modified region may be used to cross-link the one or more cells to one or more micro-particles.

If more than one cell has a modified region on the cell surface the surface modifications may be the same or different. If a micro-particle forms part of the aggregate then it may also have the same group on its surface as the modification on the surface of the one or more cells.. Alternatively the one or more cells in the population may be cross-linked to one or more other cells in the population via naturally occurring groups on the cell surface. For example, naturally occurring lysines found on the cell surface could be used to cross-link cells.

Preferably the one or more cells in the population are cross-linked to one or more other cells in the population by means that would not naturally occur in the organism from which the cells are derived.

Preferably the one or more cells in the population are cross -linked to one or more other cells and/or micro-particles using a linker molecule. The linker molecule used preferably does not naturally cross-link the cells used, that is, the cross-linking reaction of the method of the invention would not normally occur in the organism from which the cells are derived. The linker used could be arranged to recognise naturally occurring groups on the surface of a cell, for example, it may recognise lysine molecules, and by bonding to these molecules may cause the cells to be cross-linked. Alternatively the linker molecule may cross-link to one or more modified regions on a cell surface .

Preferably the method of the invention is used to control the architecture in a cell aggregate.

The term architecture is used in relation to this invention to refer to the three-dimensional structure of the aggregate produced by any method of the invention which is determined by which cells are cross-linked to which other cells and/or to which micro-particles. The method of the invention may allow the orientation of cells relative to other cells and/or micro-particles to be controlled, and may also allow the distance between the cells and/or micro-particles within the aggregate to be controlled. The present invention may also allow cell type specific interactions to be designed and controlled, and accordingly the architecture of the aggregate produced can be controlled. For example, cell type interactions may be controlled to cause, or substantially cause, only homotypic or only heterotypic cell interactions, or to quantitatively vary the ratio of heterotypic to homotypic interactions, or indeed to prevent or discourage interaction between particular cell types, or particular cell types and particular micro-particles. The present invention therefore allows the production of cell aggregates with a predetermined architecture.

For example, the invention may provide a method of controlling architecture in a cell aggregate produced from a population of cells in which one or more of the cells have a modified surface region comprising causing the one or more cells of the population with a modified surface region to cross-link with one or more other cells of the population and/or to one or more micro-particles via the modified surface region on the cell surface. The cells with the modified surface region may cross-link with other cells which do not have a modified surface region, or which have the same modified surface region, or which have a different modified surface region.

By controlling which cells and/or micro-particles are used, and/or the modification made to a region of the surface of the cells and/or micro- particles the architecture of the aggregate can be controlled to some extent.

The micro-particles used according to any aspect of the invention may all be of the same type or may be of two or more different types. In addition to modifying a region of the surface of a first cell in a population, the surface, or a region thereof, of a second cell, or any further cells, in the population may also be modified.

Cells with a modified surface region may be modified at one region or may be modified at more than one region. Cells may have more than one type of modified surface region. For example, different regions of the cell surface may have different modifications.

A region of a cell surface may be modified so that it will generally only cross-link with cells which carry the same surface modification. Alternatively, a region of a cell surface may be modified so that it will generally only cross-link with cells which have no modified surface region or have a different modified surface region and/or with a micro- particle. In a further alternative, cells with a modified cell surface region may also, or alternatively, be able to cross-link to micro-particles via the modification at that region, or via another part of the cell surface, the micro-particles may have the same or a different modification on their surface.

Preferably, in a method of the invention for controlling the architecture in a cell aggregate, one or more cells of the population have a first modified surface region, and one or more other cells of the population have a second modified surface region, wherein the cells with the first and second modified surface regions may be caused to cross-link via their modified surface regions to form an aggregate of cells. The first and second modified surface regions may be the same or different. If the first and second modified surface regions are the same, and cross-linking occurs via the modified surface regions, then when cross -linking occurs cells with the first modified surface region and cells with the second modified surface region may be able to cross-link to cells with the first modified surface region and cells with the second modified surface region. Alternatively, the first and second modified surface regions may be different, and may be used to cause the cells with the first modified surface region to cross-link only with cells with the first modified surface region, and cells with the second modified surface region to cross-link only with cells with the second modified surface region; or the modified surface regions may cause cells with the first modified surface region to cross-link only with cells with the second modified surface region, and cells with the second modified surface region to cross-link only with cells with the first modified surface region. Alternatively, cells with the first modified surface region may be able to cross-link with cells with the first and second modified surface regions, whereas cells with the second modified surface region are only able to cross-link with cells with the first modified surface region.

The above are just examples of how cross-linking may occur and how it may be used to control the architecture of a cell aggregate, the skilled man will appreciate that there are many alternatives to those described which also fall within the scope of the invention.

By designing cell specific interactions, that is, for example, by choosing the cell surface modification and/or the linker it is possible to influence which cells will cross-link with which other cells and/or micro-particles and to encourage homotypic or heterotypic aggregate formation, or to vary the ratio of heterotypic to homotypic interactions.

By controlling which cells cross-link to which other cells the architecture of the bioengineered tissue/aggregate can be controlled. The method of the present invention allows complex aggregate/tissue architectures to be produced by controlling which cell can cross-link to which other cell and/or micro-particle and tissues and "organoids" more akin to natural tissue or organ tissue can be produced.

In addition to having cell populations with one or more cell types which may have modified surface regions, the method of the invention may also include using micro-particles. Preferably the micro-particles have a surface which allows them to cross-link to cells and/or each other as required. The micro-particles may cross-link to cells via modified regions on the cell surface, or they may cross-link with naturally occurring groups on the cell surface. The micro-particles may be configured to cross-link with only one cell type, or they may cross-link with more than one cell type, or they may cross-link only with other micro-particles or a combination of the aforementioned.

Preferably if a region of the surface of one or more of the cells used in any method of the invention has been modified this means that it has been modified to be different to the same region on an unmodified cell. That is, the modified region is a region of the cell surface that is atypical for a cell of that cell type. Preferably the modified region of the cell surface comprises one or more groups on the cell surface which arise due to the modification which are not naturally found on the surface of a cell of that cell type, or are not naturally found in that concentration on a cell of that cell type - for example, the cell may be modified to over-express on its surface a group normally found in much lower levels on the cell surface. Preferably the surface modification made produces a group on the cell surface which can be used to cross-link the modified cell, via this group, to another cell or cells and/or micro-particles. The group may be an organic or an inorganic group. The other cell or cells to which the modified cell is cross-linked may or may not be modified on its cell surface. Cell surface modification may be achieved by chemically modifying the cell surface, by metabolically modifying the cell surface, by enzymatically modifying the cell surface or by genetically engineering a cell to result in a modified region on the cell surface, the skilled man will appreciate that this list is not exhaustive and that any other suitable method to modify the cell surface may be used.

A region of the surface of a cell may be chemically modified by chemically treating the cell to generate a non-natural group on the cell surface. This group may be referred to as a reactive group, and such reactive groups on the cell surface preferably react with other cells, and/or with micro-particles, and/or with an appropriate linker to crosslink cells and/or micro-particles together.

The chemically modified reactive group on a cell surface may be an aldehyde or a ketone group. For example, a region of the cell surface may be modified by selective oxidation of sialic acid residues found naturally on the surface of cells. Selective oxidation may be achieved by treating the cells with sodium periodate. Preferably the sodium periodate causes selective oxidation to produce reactive aldehydes on the cell surface. The aldehydes that result from this reaction are not normally found on the surface of cells.

Metabolic modification of a region of a cell surface may be achieved by culturing cells to be modified in the sugar N-levulinoylmannosamine

(ManLev). Culturing cells in ManLev results in expression on the cell surface of a ketone containing sialic acid oligosaccharide. Such oligosaccharides are not found naturally on the cell surface. ManLev methods are described in Jacobs et al in Methods Enzymol (2000) 327:260-275, Mahal et al in Science (1997b) 276: 1125-1128 and Yarema et al in J Biol Chem (1998) 273:31168-31179. Alternatively, cells may be treated to metabolically decorate the cell surface with azides, cells displaying azides on their surface can be cross- linked with a bis-triarylphosphine.

Enzymatic modification of the cell may be achieved by the use of certain sialyl- and fucosyltransferases and unnatural sugar donors as illustrated by the work of Herrler et al. (J Bio Chem 1992 267) and also that of Gross and Brossmer (Glycoconjugate J. 1995 12) .

Preferably all the cross-linked cells in an aggregate resulting from the method of the invention have first been modified to produce a modified region or group on the surface of the cell, the cells then being cross-linked to other cells and/or micro-particles via the modified groups or regions.

An "anchor-adapter-tag" system may be used to cross-link cells and/or micro-particles. In an anchor- adapter-tag system (as described in WO99/036107 or PCT/GB1999/000192, or in US6855329) an adapter can interact specifically and with high selectivity with an anchor molecule (present on the surface of a cell) and a tag simultaneously to cross-link two or more cells and/or micro-particles.

Biotin and avidin can be used in an adapter-anchor-tag system. Such a biotin avidin system does not require covalent binding. For example, the cell surface may be modified to carry a biotin molecule, alternatively, or in addition, the cell surface may be modified to carry an avidin molecule.

Cells carrying the biotin molecule will cross-link, non-covalently, to avidin. The avidin may be free or it may be bound to a cell and/or to a micro-particle. Alternatively, cells may be modified to carry avidin on their surface, and will then cross-link to free biotin and/or to biotin bound to a cell and/or to a micro-particle.

Cross-linking refers to the indirect or direct linking of one or more cells with one or more other cells and/or micro-particles. Preferably all cross-linked cells have modified regions on their surface. The cross-linking preferably occurs using covalent bonds. Preferably a first group or region on a first cell is cross-linked to a second group or region on a second cell or a micro-particle. The first and/or second group or regions may be modified groups or regions or they may be naturally occurring (unmodified) groups or regions .

Direct cross-linking occurs when a first group or region on a first cell and a second group or region on a second cell or on a micro-particle directly interact and cross-link without the need for an intermediary to effect the cross-linking. The first and second groups or regions may be the same or different. The first and/or second group or region may be modified groups or regions or they may be naturally occurring (unmodified) groups or regions.

Indirect cross-linking occurs when a first group or region on a first cell and a second group or region on a second cell or on a micro-particle are indirectly cross-linked via an intermediary which cross-links to the first group or region on the first cell and the second group or region on the second cell and/or microparticle. The first and second groups or regions may be the same or different. The first and/or second group or region on the cell surface may be modified or they may be naturally occurring (unmodified) groups or regions.

A linker molecule may be used to indirectly cross-link cells and/or micro- particles. Where a linker is used the linker is preferably covalently cross-linked to groups or regions on the cells and/or micro-particles. Preferably the linker cross-links with one or more modified regions or groups on one or more cell surfaces to cross-link one or more cells and/or micro-particles. The linker may also cross-link to another linker allowing more than one linker to be used to cross-link cells and/or micro-particles to form a cell aggregate.

The linker may be homofunctional, that is it binds to only one type of group or region on a cell or micro-particle surface. For example, a linker may only bind to azide groups or a region including azide groups.

Alternatively, the linker may be heterofunctional. Preferably heterofunctional linkers are capable of cross-linking with two or more different groups or regions on the surface of two or more cells and/or micro-particles. For example, one end of a heterofunctional linker may cross-link with a biotin group and the other may cross-link with an azide.

Preferably a heterofunctional linker is used to cross-link two or more cells each of which have been modified to display/express a different modified group or region on their surface. Alternatively, a heterofunctional linker may be used to cross-link two or more cells which naturally have different groups or regions on their cell surface. By using different cells with different cell surface groups or regions (whether natural or modified) and heterofunctional linkers the aggregation of cells can be controlled, and certain cells types can be arranged to interact predominantly with only specific other cell types and/or micro-particles. In this way complex three-dimensional architectures can be produced. In addition to cells, micro-particles may be cross-linked to each other and/or to cells using heterofunctional linkers. Linkers may be used to control the architecture of an aggregate, for example, a first cell type may have a first group or region, which may be natural or a surface modification, and a second cell type or micro-particle may have a second group or region, which may be natural or a surface modification, by using different linkers the architecture of an aggregate produced from these cells and/or micro-particles can be controlled. For example, a linker reactive only with the first group or region will be able to cross-link only cells of the first type to produce a homotypic aggregate. Whereas a linker reactive only with the second group or region will be able to cross-link with cells of the second type and/or micro-particles only to produce a homotypic aggregate of second cell and/or micro- particles. However a heterofunctional linker with a first reactive group able to cross-link to the first group or region on the first cell type, and a second reactive group able to cross-link to the second group or region on the second cell type and/or micro-particle, will be able to cross-link cells of the first type to cells of the second type and/or micro-particles to produce a heterotypic aggregate. In this embodiment the first and second reactive groups on the linker are different and the first and second groups or regions on the cells and/or micro-particles are different. By choosing the reactive groups on the linker and the groups or regions on the cells or micro-particles to use, and controlling the mix of these, tissue architecture can be controlled.

A linker may have two so-called reactive portions, each reactive portion being able to cross-link with a group or region on the surface of a cell or a micro-particle. Alternatively, the linker may have more than two reactive portions, making the linker able to react with more than two cells and/or micro-particles.

Preferably if a linker is used the linker is non-cytotoxic or non-cytostatic. Referring to the example discussed previously, in an anchor-adaptor-tag system using free avidin and/or free biotin, the free avidin and/or the free biotin, may act as linker molecule indirectly cross-linking cells or micro- particles which have biotin or avidin respectively on their surface. If however both the avidin and biotin are bound/expressed on a cell surface or a micro -particle surface then cross-linking via these groups would be direct.

The linker may include a spacer compound. The spacer compound may be hydrophilic, for example, a polysaccharide, peptide, dextran or polyalcohol such as polyethylene glycol (PEG) . A PEG spacer may be used to improve linker solubility and to improve linker degradation.

The linker may also be used to deliver an active agent such as a drug or growth factor, that is, a drug or growth factor could be incorporated into the linker, or trapped within the linker, and thus be delivered to the aggregating cells, and optional micro-particles.

The linker may also include a liposome. The liposome may act as a carrier of lipophilic substances, and allow lipophilic active agents to be included in an aggregate.

The linker may also be used to control the properties of the aggregate, for example the linker may be used to control the physiochemical properties, or the packing properties, such as porosity and proximity, of an aggregate of cells and/or micro-particles. The length of the linker molecule may help to determine the orientation/position/distance of cells and/or micro- particles. The further cells and/or micro-particles are away from each other the more open the aggregate structure, and therefore the more porous the aggregate. Preferably a linker used in the method of the invention is biodegradable. The term "biodegradable" as used herein, with reference to a linker and a scaffold, is intended to mean that the material dissolves or is broken down or fragmented within a period that is acceptable in the desired application. Preferably the linker biodegrades in less than or about five years, preferably between about one hour and about 5 years, more preferably between about one day and one year, and ideally between about one week and about one year.

Conveniently the rate of degradation is measured on exposure to a physiological saline solution of about pH 6.0-8.0 having a temperature between about 25°C and about 37°C, for example pH 7.0 at 3O⁰C, although other methods may be used. It will be appreciated that the size and shape of the sample may have some influence on the degradation rate and that tests may preferably be carried out on samples of a similar shape and size to those intended to be used in practice.

By programming the rate of linker degradation the cell aggregate/tissue structure arising from the interaction of cells and/or micro-particles using the linker can be maintained long enough to allow natural cell-cell interactions to occur.

In the case of the expression of a ketone or an aldehyde on the cell surface, the expression of which is typically not natural to that cell and is a surface modification, the cells may be cross-linked by adding a linker which reacts with the ketone or aldehyde. Preferably the cross-linking between the linker and the cell and/or the micro-particle is covalent. Preferably the linker can form a covalent bond with a ketone or an aldehyde group on the surface of at least two cells, or the surface of at least one cell and at least one micro-particle, thereby cross-linking the cells and/or micro-particles. Preferably, the linker comprises at least two hydrazide groups each hydrazide group being capable of covalently cross-linking to an aldehyde or a ketone on a cell or micro-particle surface. Preferably the linker is a dihydrazide, and so can cross-link with two aldehyde or ketone groups. The dihydrazide may be PEG-Ms- hydrazide.

The method of the invention may be used to aggregate cells of one or more cell types and the cells may be of any cell type, for example the cells may be from an animal, bacteria or yeast. Preferably the cells are human or non-human animal cells. Examples of cells which may be used include, but are not limited to, cells from human or non-human animal tissues such as bone, cartilage, muscle, liver, kidney, skin, neural, central nervous system or specialised cells such as placental, amnionic, chorionic or foetal cells, stem cells, chondrocytes or reprogrammed cells from other parts of the body such as apidocytes reprogrammed to become cartilage cells. Especially preferred are human or non-human animal adult or embryonic cells, or reprogrammed cells.

Preferably the method of the invention uses cells from a patient requiring the aggregate. The patient's own stem cells may be used. By using a patient's own cells this will reduce the risk of rejection of the aggregate by the patient's body and it will reduce the need to use immunosuppressive drugs which possess a number of side effects and may leave the patient more susceptible to disease.

Any method of the invention may be used with bacterial cells, for example to encourage aggregation in quorum sensing.

Any method of the invention may be used in or with any tissue, whether living, healthy, impaired, diseased, necrotising or dead, including bone and cartilaginous tissue. Examples of where the method of the present invention may be used include: spinal disc regeneration; autologous chondrocyte repair; spinal cord repair; cell delivery to the brain, especially for use in the treatment of Alzheimer's and Parkinson's diseases; regeneration of liver function; osteoarthritis; bone cavity filling; soft tissue augmentation for example in urology or breast augmentation following mastectomy; implantation of in vitro fertilised embryo; and regeneration of the pancreas or pancreas function in diabetes mellitus.

Any method to the invention may be performed in the presence of a tissue scaffold, the tissue scaffold may be provided in the form of micro- particles. The scaffold may be cross-linked to the cells and/or micro-particles of the aggregate, or it may be an independent structure around which the aggregate forms.

Known tissue scaffolds made from micro-particles include polyvinyl alcohol micro-particles (WO 00/23054); hydrogels including gelatin and alginate, CA2437250 and WO0040252 describe an alginate micro-particle scaffold and Payne et al (Biomaterials 2002 Nov 23 (22)) describes gelatin micro-particles; a particulate matrix which may be internally cross-linked (WO 99/11196); an open porous matrix or particulate material (PCT/GB02/02813) .

Other known tissue scaffolds include hydrogels which are not micro-particles, or water based polymers such as poly lactide-co-glycolide (WO 99/25391) . It may be possible to form micro-particles from PLG (Nichole R. Mercier et al. Annals of Biomedical Eng. 32 (3) 2004.)

An example of a porous scaffold, which may be used with the invention, is discussed in WO 2004/084968, which describes a scaffold which can be triggered to assemble into a porous matrix. Useful triggers include a change in pH and/or temperature, the introduction of a cross-linking, gelling or setting agent, the presence or absence of light, UV curing and/or a change to anaerobic conditions.

The method of the present invention may be used with any known scaffold.

If micro-particles are used in the invention they may be dispersed through the cell population, or they may form a core in a population of cells, or they may form separation barrier between two populations of cells, or they may form an outer shell around a population of cells. The micro- particles preferably provide some rigidity to the cell population and may aid the formation of a three-dimensional structure, while preferably not significantly impeding cell growth and expansion of the aggregate structure as it forms and grows.

The micro-particles used in any method of the invention are preferably made of a polymeric material. Examples of polymers usable in the present invention include poly (α-hydroxyacids) , polylactic or polyglycolic acids, poly-lactide poly-glycolide copolymers, poly-lactide or poly-glycolide polyethylene glycol (PEG) copolymers, polyesters, poly (ε-caprolactone) , poly (3-hydroxy-butyrate) , poly (s-caproic acid) , poly (p-dioxanone) , poly (propylene fumarate), poly (ortho esters) , polyol/diketene acetals addition polymers, poly anhydrides, poly (sebacic anhydride) (PSA) , poly (carboxybiscarboxyphenoxyphosphazene) (PCPP) , poly [bis (p-carboxyphenoxy) methane] (PCPM), copolymers of SA, CPP and CPM poly (amino acids) , poly (pseudo amino acids) , polyphosphazenes, derivatives of poly [(dichloro) phosphazene] , poly [(organo) phosphazenes] polymers, polyphosphates, polyethylene glycol polypropylene block co-polymers for example that sold under the trade name Pluronics™, natural or synthetic polymers such as silk, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides (including pectins), alginates, collagen, poly (amino acids) , peptides, polypeptides or proteins, copolymers prepared from the monomers of these polymers random blends of these polymers or mixtures or combinations thereof.

In a preferred embodiment polyesters of poly (hydroxy acids) such as poly- lactide acid (PLA) , polyglycolide acid (PGA), poly (lactic-co-glycolic) acid (PLGA) are used. These polymers have been approved for parenteral administration by the FDA. Because PLGA degrades via non- enzymatic hydrolysis in the initial stages, in vivo degradation rates can be predicted from in vitro data. PLGA degrades to lactic and glycolic acids, substances found naturally in the body.

The micro-particles may be at least partially hollow, absorbent or porous to allow the micro -particles to uptake a substance to be released at the target site. The drug may, for example, be entrapped within the polymer structure of the micro-particle. Alternatively, the drug or active agent may be included as part of a polymer chain which forms the micro-particle. For example, where sustained slow delivery of a substance to the target site is desired the micro-particles may encapsulate, be filled with or be impregnated with a substance which will either slowly be released by diffusion or will be released as the micro-particle is broken down by the body's natural metabolic processes. For example, polymers such as PLA, PGA and PLGA will undergo hydrolytic degradation in the presence of water. The ability to be able to protect substances from the action of the body and thereby delay or sustain their release is advantageous, for example where a slow release of a growth promoter or a tumour inhibitor is required, or, for example, in the case of delayed release, where a core of a substance representing a "stop" signal, such as a morphogenetic protein, is embedded within a particle for release only after a predetermined degradation period. Preferably, the micro-particles are co-formed with the substance to be released at the target site. In the most preferred embodiment a multilamellar technique is used so that a bolus or boli of substance becomes embedded within the micro-particle.

Alternatively, the micro-particles may be coated in a substance intended for immediate or very short-term release, for example, an antibiotic to prevent infection of the target site or an integrin target or other target ligands for cell receptors to promote adhesion of the surrounding cells to the matrix produced by the micro-particles. It is especially preferred that at least the outer portion of the matrix is so coated.

Examples of substances which can be in or on the micro-particles include epidermal growth factor (EGF) , nerve growth factor, insulin-like growth factor (IGF) , basic fibroblast growth factor (bFGF) , platelet derived growth factor (PDGF) , transforming growth factor-P and related growth factors, for example bone morphogenetic proteins (BMPs) , cytokines including interferons, interleukins, monocyte chemotactic protein- 1 (MCP-I) , oestrogen, testosterone, kinases, chemokinases, glucose or other sugars, amino acids, dopamine, amine-rich oligopeptides, such as heparin binding domains found in adhesion proteins such as fibronectin and laminin, other amines tamoxifen, cis-platin, peptides and certain toxoids. This list is intended as illustrative and is not intended to be limiting in any way. As described above, any substance such as a drug, hormone, enzyme, nutrient, or other therapeutic agent or factor or mixtures thereof may be in or on the micro-particle.

Preferably, the micro-particles have an average or mean dimension in the micro- or nano-metre range. For example, it is preferred that the micro- particles are between 500nm and lmm. Ideally, the micro-particles are of between 1 and 200μm. The ratio of cells to micro-particles is important in that micro-particles are not intended to form a large part of the aggregate. Preferably the ratio will be at least 50:50 cells :micro-particles. Preferably, the ratio between cells and micro-particles is of the order of 95:05 cells :micro- particles. More preferably, the ratio is 90:10 cells :micro-particles, more preferable is 80:20 cells: micro-particles. Ideally, the ratio is 70:30 cells :micro-particles.

Preferably the micro-particles and/or cells and/or scaffold are deliverable by a syringe, that is, they are injectable into the tissue or cavity where needed. Preferably the micro-particles are delivered with the cells as a slurry as described in Salem et al (Adv. Materials 2003) 15 No:3 210-213 and WO 03/000234. Once delivered the micro-particles or the scaffold material may self assemble in situ to form a scaffold. Preferably the scaffold produced is porous. The scaffold is intended to support the cells but not impede their establishment.

The injectable micro-particles/scaffold may be made of poly (lactic acid)- poly (ethylene glycol) -biotin (PLA-PEG-biotin) . Preferably in use the

PLA-PEG-biotin is co-injected or co-applied with free avidin at a concentration appropriate to cause cross-linking of the micro-particles/scaffold. Biotin and avidin are discussed merely by way of example and the skilled man will appreciate that alternative systems may be used to cross-link the micro-particles/scaffold.

The surface of the micro-particles may be configured so that they are complementary to the cross-linker employed in the method of the invention. Preferably the micro-particle can cross-link covalently with the cells to form an aggregate, they may be covalently cross-linked via a linker molecule. Preferably if a scaffold is used the scaffold is biodegradable.

The method of the invention may be performed ex-vivo that is outside the body, for example, for use in vitro in tissue models or to produce tissue to repair or replace damaged, diseased or excised tissues in vivo.

Alternatively, the method may be performed in vivo, for example at the site of tissue damage, disease or repair. If the method is performed in vivo the cells and/or micro-particles may be injected into the site where needed and aggregated in situ. If required a linker may also be injected.

If used the micro-particles are preferably injected with the cells, that is, using one syringe, thereby avoiding the need for seeding of the micro- particles/scaffold.

Any method of the invention may be used to fabricate cell sheets, and thereby form tissues that are composed of cell layers, for example, but not limited to, blood vessels, skin and urinary bladder tissue. Using the method of the invention these sheets or tissues may be formed rapidly, particularly if covalent bonds are used to crosslink the cells and/or micro-particles.

Use of the method of the invention to produce layers or sheets of cells, for example, layers of keratinocytes for use in skin applications, has the advantage over conventional methods of seeding cells onto biocompatible matrices in that how the cells aggregate can be controlled and cells with intimate cell to cell contact and thus having a high degree of mechanical strength can be used.

The method of the invention may also be used to form three-dimensional cell aggregates. For example, the method of the invention may be used to produce three-dimensional tissue/cell aggregates for use in artificial medical support systems such as bio artificial liver devices.

By performing the method of the invention in suspension rather that on a cell monolayer three-dimensional aggregates will form. This may be as a result of incubation in a vessel such as a spinner flask or a bioreactor system such as a rotary cell culture system.

Preferably the invention allows the production of functional liver tissue. In particular, it has been shown that hepatocytes, that are the functional units of the liver, rapidly lose functionality in in vitro culture. This functionality can however be improved by culturing the cells as aggregates (Riccalton-Banks et al Tiss Eng (9) 3 2003) . More specifically, the invention will allow non-parenchymal cells such as stellate cells and hepatocytes to be co-cultured as spheroids in vitro. Tissue produced this way may be useful to support or replace diseased or damaged tissue/organs, or for the screening of drugs and toxicology studies. More specifically, the invention will allow hepatocytes and non- parenchymal cells to be co-cultured as spheroids in vitro.

The method of the invention may be used with pancreatic β-cells. β-cells are the constituents of the islets of Langerhans and are responsible for insulin secretion, and cell aggregates made from β-cells may be used to treat diabetes.

Preferably once the cells are cross-linked they will begin to deposit the extracellular matrix needed for maintenance of the tissue. Ultimately the cells will cross-link using natural mechanisms and any linkers used will biodegrade. The method of the invention may be used with stem cells to produce embryoid bodies. Preferably the method of the invention allows embryoid bodies to be rapidly formed. By controlling the microenvironment of the embryoids and encouraging their development down a specific lineage, earlier formation of bone tissue is observed in addition to an increased cell population.

The skilled man will appreciate that all the preferred features discussed with reference to any method of the invention can be applied to all other aspects of the invention.

According to another aspect, the invention provides an aggregate of cells comprising a population of cells wherein at least one cell is cross-linked to at least one other cell and/or micro-particle. Preferably the at least one cell is covalently cross-linked to at least one other cell and/or micro-particle. Preferably each of the cross-linked cells has a chemical modification on its surface.

The aggregate may be homotypic - containing only one cell type. Alternatively, the aggregate may be heterotypic and may contain two or more cell types. Both homotypic and heterotypic aggregates may also comprise micro-particles.

The aggregate may comprise in the order of tens of cells, more preferably in the order of hundreds of cells, more preferably in the order of thousands of cells, or more preferably in the order of millions of cells.

According to another aspect, the invention provides an aggregate obtainable by any method of the invention. According to a further aspect, the invention provides a method of treatment of a human or non-human animal comprising introducing a cell aggregate according to the invention into to or onto target tissue of a human or non-human animal in need of treatment.

According to another aspect, the invention provides a method of treatment of a human or non-human animal comprising performing the method of the invention in vivo in a human or non-human animal. Preferably the cells and/or micro-particles are injected into a target tissue of a human or non-human animal in need of treatment and caused to cross-link.

Preferably at least some of the injected cells have a chemically modified region on their surface which is used to cross-link the cells.

Preferably a linker is also injected if needed. Preferably micro-particles are also injected which can form a scaffold in vivo. Alternatively, other scaffold material may also be injected.

Preferably the cells and/or micro-particles are covalently cross-linked.

According to another aspect, the present invention provide a method of cross-linking cells to an existing tissue structure comprising applying one or more cells to an existing tissue structure and causing the applied cells to cross-link to one or more cells of the existing tissue structure.

Preferably the applied cells have a chemically modified region on their cell surface which is used to cross-link the cells to the existing tissue structure. The cells of the existing tissue structure may also have a chemically modified region on their cell surface which is used in the cross-linking. Preferably the applied cells are covalently cross-linked to cells of the existing tissue structure.

Linker molecules may be used to cross-link the cells.

According to another aspect, the invention provides an aggregate comprising a population of cells wherein at least one cell is covalently cross -linked to another cell for use as a medicament.

According to another aspect, the invention provides an aggregate according to the invention for use as a medicament.

According to another aspect, the invention provides the use of an aggregate according to the invention for the treatment of a disease.

According to another aspect, the invention provides the use of an aggregate according to the invention in the preparation of a medicament for the treatment of a disease.

Diseases or other conditions treatable with the cell aggregates of the invention include, but are in no way limited to Alzheimer's disease, Parkinson's disease, osteoarthritis, burns, spinal disk atrophy, cancers, hepatic atrophy and other liver disorders, bone cavity filing, regeneration or repair of bone fractures, diabetes mellitus, ureter or bladder reconstruction, prolapse of the bladder or the uterus, IVF treatment, muscle wasting disorders, atrophy of the kidney, organ reconstruction and cosmetic surgery.

According to another aspect, the invention provides a method of cosmetic treatment of a human or non-human animal comprising performing the method of the invention in vivo in an animal or introducing a cell aggregate according to the invention into to or onto target tissue of a human or non-human animal in need of treatment.

The cosmetic treatment may be, but is not limited to, breast enhancement, lip and eye implants, and any other form of cosmetic implantation.

According to another aspect, the invention provides a method of testing the toxicity of a chemical comprising applying the chemical to a cell aggregate according to the invention and observing the effect on the aggregate.

According to a further aspect, the invention provides the use of an aggregate according to the invention in a diagnostic test kit.

Preferably the test kit includes instructions to use the aggregate in toxicology studies.

Preferred embodiments of the invention will now be described by way of example with reference to the accompanying drawings in which:

Figure IA - shows schematically an example of the components used to covalently cross-link cells of the same type;

Figure IB - shows schematically the covalent cross-linking of cells of the same type using the components of Figure IA in a layered two-dimensional manner;

Figure 1C - shows schematically the cross-linking of cells of the same type using the components of Figure IA to form a three-dimensional structure; Figure 2 - show schematically the generation of a two cell-type aggregate with defined architecture using triarylphosphine- and azide-functionalised cells;

Figure 3 - shows schematically the configuration of an alternative heterotypic aggregate to that depicted in Figure 2;

Figures 4A to 41 - shows schematically alternative aggregate configurations/architectures according to the invention;

Figure 5 - illustrates the synthetic route to PEG-bis-hydrazide;

Figure 6 - illustrates the synthesis of an alternative hydrazide to that depicted in Figure 5;

Figure 7 - illustrates the synthetic routes to a triarylphosphine and azide cross-linking polymers;

Figures 8A to 8D - illustrates various alternative chemistries which can be used to covalently cross-link cells. Figure 8 A depicts an aldehyde or ketone cross-linking with a hydrazide compound.

Figure 8B depicts an aldehyde or ketone cross-linking with an amino-oxy compound. Figure 8 C depicts an aldehyde or ketone cross-linking with a thiosemicarbazide compound. Figure 8D depicts cross-linking via a Diels-Alder reaction;

Figures 9A to 9D - Figure 9A is a schematic representation of the aggregation process using cells with a modified surface region, namely they have a biotin molecule of their surface, and free avidin is used as a linker to cross-link the cells. Figure 9B shows representative phase contrast images demonstrating the difference between natural and engineered (10 μg/ml avidin) aggregation of L6 myoblast cells. Scale bars = 100 μm. Figure 9C illustrates the effect of avidin concentration on the mean apparent area of 3T3 fibroblast and L6 myoblast aggregates after one hour in spinner flask culture. Data are shown as mean ± SEM. Figure 9D shows the effect of culture time on the mean apparent area of 3T3 fibroblast and L6 myoblast aggregates formed using 10 μg/ml avidin in spinner flask culture. Data are shown as mean ± SEM.

Figures 1OA to 1OG - illustrate the engineered adhesion of heterotypic cell types to produce stratified and 3-D aggregate structures. Figure 1OA to 1OD show seeding of streptavidin-coated 3T3 fibroblasts, labelled with CellTracker™ green, onto untreated (Figure 10A) or biotinylated (Figure 10B) L6 myoblast monolayers. Scale bars = 400 μm. Figure 1OC shows the number of adhered streptavidinated 3T3s was between 15- and 30-fold greater on biotinylated monolayers compared to untreated cells. Data are shown as mean ± SEM. Figure 1OD shows engineered adhesion did not affect the formation of natural cell-cell contacts, demonstrated by fibroblast spreading after 24 hours in co-culture.

Scale bar = 100 μm. Figure 1OE shows a schematic representation of layered, heterotypic 3-D aggregation utilizing free surface avidin groups on a homotypic aggregate to selectively attach a second cell type. Figure 1OF shows a cryostat section of a heterotypic aggregate of L6 myoblast (red/dull light grey) and 3T3 cells (green/bright light grey) . L6 myoblast aggregates were formed in spinner flasks and, upon addition of biotinylated 3T3s, were cross-linked into a multi-aggregate structure. Scale bar = lOOμm. Figure 1OG shows a fluorescence microscopy image of a layered heterotypic L6 myoblast (green) and 3T3 (red) aggregate.

Biotinylated L6 myoblasts were aggregated using FITC-avidin and diluted before addition of biotinylated 3T3s to reduce the aggregate cross-linking shown in Figure 1OF, enabling a cell layering effect. Scale bar = 100 μm.

Figure 1OF and 1OG are difficult to appreciate in black and white.

Figure 1OF shows a mottled effect of red L6 cells and green 3T3 cells, and Figure 1OG shows a core of green L6 cells and outer edge of red 3T3 cells.

Figures HA and HB - show the effect of enhanced seeding of keratinocytes onto model skin wound beds via engineered adhesion. Figure HA shows the effect of cell and surface modification on the adhesion of keratinocytes to collagen-coated dishes. Collagen (Coll) and keratinocyte (Kc) surfaces were unaltered, biotinylated (Bio) or avidinated (Av) and the number of adhered cells counted in random microscope fields at different time periods post seeding. Data are shown as mean ± SEM; * indicates P < 0.05 and ** indicates P < 0.005 as determined by un-paired t-tests. Figure HB shows the number of viable, adhered cells after 30 and 60 minutes when unmodified or biotinylated keratinocytes were seeded onto the ventral surface of unmodified or avidinated de- epidermalized human dermis (DED) . Cell number was assessed by the MTT-ESTA assay. Data are shown as mean ± SEM; *** indicates P < 0.001 as determined by un-paired t-tests.

Figures 12 A to 12E - show engineered aggregation accelerates embryoid body (EB) formation from murine embryonic stem cells and stimulates osteogenic differentiation. Figure 12A shows phase contrast comparison of natural and engineered aggregation of mES cells into embryoid bodies over a 3 day period. Scale bars =

200 μm. Figure 12B shows confocal immunofluorescence (FITC) images of myosin heavy chain (MHC) and cadherin-11 expression in 3-day-old natural and engineered EBs. Scale bars = 5O-Um. Figure 12C and Figure 12D show phase contrast comparison of bone nodules resulting from naturally formed (Figure 12C) and engineered (Figure 12D) EBs. Scale bars = 20O-Um. Figure 12E shows the number of bone nodules per Petri dish following dissociation of natural or engineered EBs and their subsequent growth for 21 days in osteogenic medium. Data are shown as mean ± SEM; * indicates P < 0.05, ** indicates P < 0.005 and *** indicates P < 0.001 as determined by un-paired t-tests.

Figures 13 A to 13E - show chemoselective modification of collagen-coated culture plates and human epidermal fibroblasts via sodium periodate treatment. Figure 13A to 13D show fluorescence images of collagen-coated tissue culture plastic following different chemical treatments. Wells were untreated (Figure 13A) , incubated with FITC-avidin only (Figure 13B), treated with PBS, biotin hydrazide and FITC-avidin (Figure 13C) , or treated with sodium periodate, biotin hydrazide and FITC-avidin (Figure 13D) . Figure 13E shows fluorescence image of primary human epidermal keratinocytes following periodate oxidation, biotin hydrazide ligation and FITC-avidin treatment. Cells treated with biotin hydrazide and FITC-avidin without prior periodate oxidation exhibited no fluorescence; and

Figures 14A and 14B - illustrate an aggregate comprising cells and micro-particles. Covalent Cross-Linking of Homotypic Cells

Figures IA to 1C illustrate schematically the covalent cross-linking of cells of the same type to produce a homotypic aggregate. Initially a population of homotypic cells are treated with sodium periodate which causes the selective oxidation of natural sialic acids on the surface of the cells to generate aldehydes on the cell surface. Novel aldehyde-reactive polymers (linkers) are then employed to covalently cross-link the chemically modified cells which display an aldehyde on their surface to one another. More specifically, the linker used is a novel bis-hydrazide cross-linking reagent incorporating poly (ethylenegly col) (PEG) between the two reactive groups. The PEG acts as a spacer and improves solubility and biodegradation of the linker. When this linker is incubated with periodate-treated cells (cells with chemically modified surface) the reaction of the hydrazide groups with cell surface aldehydes facilitates cell cross-linking by the formation of hydrazone bonds (Figure IA). Depending on the culture conditions employed, this strategy can be used to generate different types of tissue architecture. Under static culture conditions with adherent cells, stratified cell sheets can be fabricated (Figure IB), whereas with rotary, microgravity culture, three-dimensional cell "organoids" are possible (Figure 1C) .

After the assembly of multicellular aggregates the cross-linked structures can be allowed to mature under appropriate conditions. Natural deposition of extracellular matrix components by the cells then provides increasing structural integrity and a framework for continued cellular growth, while the cross-linker polymers, or linkers, biodegrade over time. Covalent Cross-Linking of Heterotypic Cells

The organization of cells within tissues is complex, with multiple cell types arranged in specific architectures and heterotypic cell-cell interactions are essential for correct tissue function and integrity. The bis-hydrazide strategy described above with reference to homotypic cell cross-linking can be employed with a mixture of cell types, but the resulting aggregate would consist of randomly arranged cross-linked cells. To generate a defined spatial organization within a mixed cell population heterofunctional polymer linker cross-linking reagents are used. In this example each heterofunctional polymer possesses a terminal hydrazide functionality to enable ligation to periodate-oxidized cell surfaces as well as a PEG linker and a heterofunctional, reactive chemical head group. In this example the aggregation of two cell types with controlled architecture necessitates that two different, mutually reactive heterofunctional linkers are used. To achieve this cross-linking polymers that utilize a modified Staudinger ligation reaction (Saxon and Bertozzi (2000) Science 287, 2007-2010) are used (Figure 2) . The cross-linkers are ligated to each of the two cell populations via hydrazone bonds described above and illustrated in Figure 2, such that cell type I has linker A attached and cell type II has linker B attached. On mixing the cells, the terminal triarylphosphine group of linker A, on cell type I, reacts with the azide group of linker B, on cell type II, generating an amide bond and covalently cross-linking cell type I to cell type II. In addition, these chemical functionalities are not reactive towards other cell surface chemical moieties, ensuring that specific cell-cell ligation occurs according to the predetermined scheme. This strategy can be expanded further, using multifunctional cross-linking species, for example by the presentation of the mutually-reactive head groups on a dendrimer-type construct. Thus, progressively more complicated biomimetic aggregate architectures can be produced. By modifying the surface chemistry of mammalian cells and inducing aggregate formation as described it is possible to closely manipulate and organise the aggregate size and architecture. Complex architectures can be formed in a heterotypic configuration as shown in Figure 2. it is also possible to construct a "layer" aggregate by controlling the location of specifically labelled cells. For example, as shown schematically in Figure 3, a second cell type (hashed circles) can be arranged to surround an inner homotypic aggregate of cells of a first cell type (open circles) . In an alternative embodiment the first and/or second cells types could be replaced with micro-particles.

Alternative Aggregate Configurations/Architectures

Figures 4A to 41 show schematically alternative aggregate configurations or architectures.

Figure 4A shows a first cell type (A) with a modified surface region and a second cell type (B) with an unmodified surface, the modifications made to cell type A encourage cell type A to directly cross-link with other cells of cell type A only; and not to cross-link with cell type B. Thereby producing a homotypic aggregate of cross-linked cells of cell type A only. By way of contrast Figure 4B shows a first cell type (A) with a modified surface region and a second cell type (B) with an unmodified surface, in this example the modification to the surface of cell type A encourages cell type A to directly cross-link only with cells of cell type B to produce a heterotypic aggregate.

Figure 4C shows a first cell type (A) with a first modified surface region and a second cell type (B) with second modified surface region, wherein both surfaces have the same modification and cells types A and B can directly cross-link with themselves and each other producing a heterotypic aggregate with homotypic pockets. Figure 4D shows a first cell type (A) with a first modified surface region and a second cell type (B) with a second modified surface region, wherein the first and second surface modifications are different and dictate that cells of type A can only interact with cells of type B and vice versa.

Figure 4E illustrates the use of cross-linkers and shows a first cell type (A) with a first modified surface region and a second cell type (B) with a second modified surface region, wherein both surfaces have the same modification and cells types A and B can be indirectly cross-linked to each other and themselves using a linker. Figure 4F shows a first cell type (A) with a first modified surface region and a second cell type (B) with a second modified surface region, wherein the first and second surface modifications are different and a heterolinker molecule can be used to indirectly cross-link cells of type A with cells of type B via these modifications .

The skilled man will appreciate that with the correct linker the principles relating to direct cross-linking can equally apply to indirect cross-linking. It will also be appreciated that the principles illustrated can equally apply to populations of only one cell type and to populations of two or more cell types, although no embodiments with more than two cell types are illustrated.

The skilled man will also appreciate that although not illustrated with reference to all possible permutations the cells may be replaced by micro- particles, or micro-particles may be included as well as the cells. By way of example Figure 4G shows a first cell type (A) with a modified surface region and a micro-particles (cross-hatched) with a group on the surface which can cross-link directly with the modified surface region of cell type A to produce an' aggregate of micro-particles and cells.

Figure 4H shows a first cell type (A) with a first modified surface and a second cell type (B) with a second modified surface, wherein the first and second surface modifications are the same. A micro-particle

(cross-hatched) with a third surface group are also illustrated. A heterolinker molecule is used to indirectly cross-link cells of type A and cells of type B via their surface modifications with the micro-particles to form a heterotypic aggregate. Cells types A and B however do not interact via these modifications .

Finally, in this series of examples, Figure 41 shows cells cross-linked directly as discussed with reference to Figure 4B in which a heterotypic aggregate of cells of cells types A and B is produced. In this embodiment free micro-particles (cross-hatched) are entrapped within the aggregate, but are not cross-linked to the cells. The micro-particles provides support to the cell aggregate.

The skilled man will appreciate that there are many further ways in which the cross-linking of cells of the same type or of different types, and/or micro-particles can be controlled which have not been illustrated, the arrangements illustrated are merely to demonstrate just some of the arrangements that could be achieved according to the method of the invention.

Synthesis of the Z?zs-Hydrazide Cross-Linker

The ^/5-hydrazide cross-linker discussed with reference to Figure 1 is synthesised from commercially available PEG diacid by formation of the diacid chloride and subsequent reaction with hydrazide as depicted in Figure 5.

Synthesis of an Alternative Hydrazide Cross-Linker

Figure 6 illustrates a mechanism for the synthesis of an alternative hydrazide linker to VEG-bis -hydrazide depicted in Figure 5.

Synthesis of the Triarylphosphine and Azide Cross-Linkers

Heterofunctional polymer cross-linking reagents discussed with reference to Figure 2 are synthesised using commercially available starting materials as depicted in Figure 7. Synthesis of the appropriately functionalized triarylphosphine head group (3) for linker A is achieved using well-established methodologies. Coupling of this intermediate via standard carbodiimide based protocols with the Boc protected amino-PEG- hydrazide (8) affords the desired linker A (5) . Intermediate (8) itself can be readily synthesized from the commercially available Fmoc-PEG amino acid succinimide ester (6) . Treatment with tert-butyl carbazate affords the orthogonally-protected intermediate (7) which following Fmoc removal, reveals the primary amine required for acylation. This common intermediate (8) is transformed into linker A as previously described, or coupled with chloroacetic anhydride to generate (9) , which upon reaction with sodium azide and Boc deprotection affords the desired linker B (11) . PEGylated compounds are soluble in most organic solvents and water, but can be selectively precipitated from ethers. This allows an efficient means of purification of these compounds and thereby permits the use of excess reagents to drive reactions to completion, since all by-products and unreacted material can be removed by simple washing/trituration and filtration. Terminal hydrazide functionality enables ligation to periodate-oxidised cell surfaces as well as a PEG linker and a heterofunctional, reactive chemical head group.

Experimental Data Showing Non-Covalent Cross-linking of Homotypic Cells

Figures 9A to 9D represent the engineered cell aggregation of a population of cells using surface modification and non covalent cross- linking.

The cells used with reference to Figures 9 A to 9D were subjected to mild sodium periodiate oxidation to generate aldehyde groups on terminal sialic acid resides (Lenten et al (1971) J Bio Chem 246 1889-1894). These aldehyde groups were then used to chemoselectively ligate biotin hydrazide to the cells via a covalent hydrazone bond. Exploiting the high binding affinity constant between avidin and biotin, and the presence of four biotin binding sites per avidin molecule, allowed suspensions of biotinylated cells to rapidly cross-link into multicellular aggregates upon the addition of avidin as depicted shematically in Figure 9A.

Suspensions of 3T3 fibroblasts or L6.G8.C5 (L6) myoblasts were biotinylated and incubated in spinner flasks with medium containing between 0 and 40 μg/ml avidin. After one hour the extent of aggregate formation was assessed by sampling the suspensions and measuring the apparent area of aggregates in random microscopic fields. Both cell types exhibited concentration-dependent aggregate formation, peaking at mean apparent areas of 3818±992μm² and 3392+192μm² respectively, at lOμg/ml (Figure 9C) . When the avidin concentration was increased above this the aggregate area declined, this is likely as a result of cell surfaces becoming saturated with avidin and thereby eliminating free biotin groups with which cross-linking could occur.

The kinetics of the aggregation using medium containing 10 μg/ml avidin was studied, and both cell types were observed to rapidly aggregate into large multicellular structures, with the key aggregation occurring within the first hour (Figure 9C) . The general observed trend was an increase in aggregate size over time, resulting from random collisions between aggregates and non-aggregated cells. Importantly, there was little or no aggregation of untreated cell suspensions within the same period of time, indicating that surface engineering rapidly increased aggregation

(Figure 9D) .

Experimental Data Showing Formation of Heterotypic Cell Structures

Many tissues contain multiple cell types whose spatial arrangement is vital to the tissue's function. Replicating this organization in vitro is a challenge. Engineered cross-linking was applied to mimic stratified and 3-D tissues and thereby generate structures with heterotypic cells in a defined architecture. By artificially promoting adhesion between heterotypic cell types, 3T3 fibroblasts were stratified onto L6 myoblast monolayers. This was achieved by biotinylating an L6 monolayer and subsequently seeding a 3T3 suspension that had been biotinylated and saturated with streptavidin (Figure 1OA and 10B) . Fibroblast cell number was 15-fold greater than on control monolayers immediately after seeding, and, although density fell slightly with time, there were between 18- and 30-fold more 3T3s than on control monolayers after up to 72 hours (Figure 10C) .

Immediately after seeding, the fibroblasts appeared rounded but, within 24 hours, they had spread on the monolayers, indicating that the artificial cell-cell adhesion process had not affected the establishment of natural cell contacts (Figure 10D).

To engineer a 3-D aggregate with a defined heterotypic cell architecture, a homotypic aggregate was initially constructed and then a second cell type was incorporated, using avidin molecules accessible on the surface of the cells in the homotypic aggregate that could be utilized to specifically adhere a second, biotinylated, cell type (Figure 10E) . To demonstrate this, L6 aggregates were formed in spinner flasks and mixed with biotinylated 3T3s to construct a myoblast core surrounded by a fibroblast layer. This initially proved ineffective, resulting in a heterotypic structures where the myoblast aggregates were themselves cross-linked by the biotinylated fibroblasts, creating a marbling effect of fibroblasts through the assembly (Figure 10F) . To avoid this, and promote specific fibroblast adhesion, the density of aggregates in the culture was reduced. This greatly reduced aggregate cross-linking, while enabling fibroblast adhesion to occur on the L6 aggregate surfaces (Figure 10G).

Cell seeding in skin wound models

In patients with extensive skin loss due to burns injuries or for the treatment of chronic nonhealing ulcers, the use of autologous laboratory- expanded keratinocytes to supplement skin grafting now has a place in wound management. However, this cell transfer is challenging due to the inflammatory response within the wound bed, and strategies to enhance this process would be beneficial. Using the method of the invention engineered keratinocyte adhesion was employed to deliver cells to model wound beds in vitro. Initially keratinocyte adhesion to collagen-coated tissue culture plastic was studied, as collagen is the predominant protein in the dermis. By oxidizing the sialic acid moieties naturally present in collagen by periodate treatment, the ECM protein was decorated with biotin and engineered adhesion of surface-modified keratinocytes was studied (Figures 13A to E) . In comparison to natural adhesion, the adhesion of biotinylated human epidermal keratinocytes to avidinated collagen was significantly greater at all time-points measured, ranging from a 164-fold increase in cell number at 5 minutes to 5 -fold at 35 minutes (Figure HA). All other combinations of cell and substrate modification were comparable with natural adhesion kinetics with the exception of biotinylated keratinocytes on collagen and unmodified keratinocytes on avidinated collagen, both at the 35 minute time-point. The respective increase and decrease in cell number in comparison with the control was minor, especially when compared to biotinylated keratinocytes on avidinated collagen. Surprisingly, it was observed that there was no increased adhesion of avidinated cells to biotinylated collagen.

This system was then extended to incorporate de-epithelialized acellular human dermis (DED), a more complex and biomimetic wound bed model (Chakrabarty et al (1999) BrJ. Dermatol. 141, 811-823) . Human fibroblasts were seeded on the reticular side of DED to replicate in vivo skin architecture and cultured for 24 hours prior to seeding experiments . After mild oxidation and avidination, the papillary surface of the DED was seeded with human epidermal keratinocytes within a metal ring. After 30 or 60 minutes, the DED was washed and the number of adhered, viable keratinocytes assessed by an MTT-ESTA assay, using fibroblast- only DED as a blank. At both time-points, the number of adhered epidermal keratinocytes was greater when adhesion was artificially engineered in comparison to the natural seeding process (Figure HB) . This difference was highly significant at 30 minutes (P = 0.0002) , falling just below significant (P = 0.0506) at one hour. Overall, these data demonstrate that, by engineering keratinocyte surfaces and the wound bed to induce artificial adhesion, efficiency of cell transfer can be increased.

Differentiation of Embryonic Stem Cells

To utilize engineered aggregation in a 3-D system, the effects on murine embryonic stem (mES) cells were studied. In suspension culture in the absence of leukaemia inhibitory factor (LIF), mES cells naturally aggregate into embryoid bodies (EBs), triggering their differentiation into a rudimentary embryo containing cells of the three germ layers (Keller (1995) Curr. Opin. Cell. Biol. 7, 862-869). Cell-cell interactions underpin this phenomenon, so whether accelerating EB formation by engineering such contacts would affect differentiation was considered. Undifferentiated mES cells were proliferated on a feeder layer in the presence of LIF, then suspended by trypsinization. The cells, in the absence of LIF, were then allowed to naturally differentiate and form EBs in suspension, or were biotinylated and cultured in suspension with avidin-containing medium. Unmodified mES cell cultures progressed from a suspension of single cells to embryoid bodies within 3 days. However, in the engineered cultures, multicellular aggregates rapidly formed on addition of avidin and subsequently coalesced into EBs that appeared denser and more heterogeneous than the corresponding, naturally-formed EBs (Figure 12A) . To investigate whether the rapid establishment of cell- cell contacts in engineered EBs affected the differentiation of mES cells, osteogenic development was considered. This is an attractive target for tissue repair strategies employing stem cells. As bone is derived from the mesoderm, mesodermal development in 3-day-old embryoid bodies by immunostaining with an antibody against myosin heavy chain (MHC) was examined. To specifically determine whether osteogenic differentiation was occurring, the expression of cadherin-11, a cell adhesion molecule implicated in mesodermal differentiation and bone formation (Kawaguchi et al (2001) J. Bone Miner. Res. 16, 260-269) was examined. In comparison to naturally-formed EBs, engineered mES cell aggregates exhibited an increased level of immuno staining for both MHC and cadherin-11 (Figure 12B), suggesting that artificially promoting cell-cell adhesion accelerates osteogenic differentiation of mES cells. To quantify the osteogenic potential of natural and engineered EBs, the differentiation of their constituent mES cells into mineralized bone nodules was examined. EBs of varying ages were dissociated by trypsinization and cultured in osteogenic medium for 21 days, after which time the number of bone nodules was determined (Buttery et al (2001) Tissue Eng. 7, 89-99) . Cells from both types of EB differentiated into osteoblasts and formed bone nodules (Figures 12C and 12D) . However, when the number of nodules was compared, significant differences between ES cells derived from natural and engineered EBs were apparent. Cells plated in osteogenic medium immediately after suspension from a feeder layer, with no chance to form EBs, produced very few bone nodules. From 1 day-old EBs, however, several hundred bone nodules were observed per dish, peaking at day two and declining until day five (Figure 12E) . These data are in contrast to conventional mES differentiation approaches, where EBs are often cultured for 5 days before dissociation and secondary plating (Conley et al (2204) Int. J. Biochem. Cell Biol. 36, 555-567) , suggesting that this time period may reduce the yield of the target cell type.

When cells from natural and engineered EBs were compared, however, the number of bone nodules derived from EBs at each time-point was greater than from engineered EBs except at time zero, suggesting that engineered adhesion enhances the ability to induce the osteogenic differentiation of mES cells. Aggregation of Micro-Particles and Cells

An aggregate of cells and micro-particles was made between PLGA micro-particles and 3T3 fibroblast cells. PLGA micro-particles were prepared using Homogenizer at 13000rpm for 120 seconds. The PLGA micro-particles were then treated with plasma polarization instrument to add nitrogen atoms (amine groups) on their surface. Then the micro- particles were modified by the addition of biotin moieties on their surface and then aggregated using FITC- Avidin, so they acquired a green colour under a fluorescene lamp. 3T3 fibroblasts were stained red with Cell-Tracker™, oxidized with periodate oxidase, then modified with biotin. The micro-particles and 3T3 cells were then aggregated by mixing the treated PLGA micro-particles with the biotinylated 3T3 fibroblasts.

Figures 14A and 14B show fluorescence microscopy images of aggregates of the micro-particles and the cells. Where there is fluorescence in the images it is a mottled mix of red cells and green microparticles.

METHODS

Cell culture

Cell lines

3T3 Swiss-albino fibroblasts (ATCC, #CCL-92) and L6.G8.C5 myoblasts (ECACC, #92121114) were routinely cultured in DMEM supplemented with 10 % FCS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B.

Primary keratinocytes: Primary human keratinocytes cultures were derived from split-thickness skin obtained during breast reduction and abdominoplasty procedures using an adaptation of the method of Rheinwald and Green (Gumbine (1996) Cell 84, 345-357) as described previously (Stahl et al (2004) Biophys. Res. Commun. 322, 684-692). All patients gave informed consent for skin that was not used in surgery to be used for research purposes. Briefly, pieces of skin were incubated overnight in 0.1% trypsin solution and the cells isolated from the dermal/epidermal junction were cultured in Keratinocyte-SFM (Invitrogen, #17005) at 37⁰C in a 5% CO₂ humidified atmosphere and used at passage 1 or 2.

Primary fibroblasts: Primary human dermal fibroblasts were derived from split-thickness skin obtained from consenting patients as described above. Skin samples were washed in PBS, finely minced using a scalpel and digested in 0.5 % collagenase solution. The digest was centrifuged, the cell pellet resuspended in fibroblast culture medium (DMEM supplemented with 10 % FCS, 2 mM L-glutamine, 100 units/ml penicillin, 10 μg/ml streptomycin and 625 ng/ml amphotericin B) and cells plated in 75 cm² flasks. The fibroblasts were maintained at 37 ⁰C in a 5 % CO₂ humidified atmosphere and used at passage 7.

Embryonic stem cells: Murine ES (mES) cells from the CEE Iine3 were maintained on a mitomycin C inactivated fibroblast feeder layer (SNL cell line) in DMEM supplemented with 10 % FCS, 50 U/ml leukaemia inhibitory factor (LIF), 0.1 μM 2-mercaptoethnaol, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B.

Cell aggregation by biotinylation and avidin cross-linking

Cells were grown to confluence in 75 cm² flasks, suspended by brief trypsinization, pelleted and washed with room temperature PBS. Cells were then resuspended in 1 mM sodium periodate solution in PBS at 4°C, incubated in the dark at 4°C for 5 minutes, diluted with PBS and pelleted. After washing with biotin buffer (PBS; pH 6.5) , the cells were incubated in 5 mM biotin hydrazide solution in biotin buffer for 90 minutes at room temperature with constant agitation. After washing twice with PBS, 6 x 10⁶ cells were transferred to spinner flasks with avidin-containing culture medium (total volume 20 ml), operating at 85 rpm. To assess the extent of aggregation, 1 ml samples were removed and transferred to Petri dishes. Phase contrast images of random fields were analyzed using Leica QWin software to determine the apparent aggregate area. Mean aggregate area ± SEM was calculated using the data from ten images per experimental condition.

Layering of heterotypic cell types

L6 myoblasts were seeded into fibronectin-coated 24 well plates and grown until 95-100 % confluent. Wells were washed with PBS and incubated in the dark for 5 minutes at room temperature with PBS or 1 mM sodium periodate in PBS. After washing twice with biotin buffer (PBS with 0.1 % FCS; pH 6.5), cells were incubated for 90 minutes at room temperature with 5 mM biotin hydrazide in biotin buffer. Cells were then washed twice with avidin buffer (PBS with 0.1 % FCS) and incubated with HBSS until 3T3 fibroblast seeding. 3T3 fibroblasts were grown to confluence in fibronectin-coated 75 cm² flasks, washed with PBS then oxidized and biotinylated as described for L6 myoblasts. After washing twice with avidin buffer, cells were suspended by trypsinization and washed twice more with avidin buffer. The cells were then incubated twice for 15 minutes each at room temperature with a 5 μg/ml solution of streptavidin in avidin buffer, washed twice more with avidin buffer and then stained with CellTracker™ Green (Molecular Probes) according to the manufacturer's instructions. The cells were then washed twice with HBSS and the density adjusted to 5 x 10⁵ cells/ml. HBSS was removed from the L6 monolayers and 0.5 ml of the 3T3 suspension added per well. The buffer volume was adjusted to 2 ml and the cells incubated at 37 ⁰C for 30 minutes, after which time the buffer was aspirated and the wells washed with HBSS. Images of each well were obtained using phase contrast and fluorescent microscopy, and the HBSS then replaced with culture medium. Plates were returned to the incubator and wells imaged after 24, 36, 48 and 72 hours. For each time-point, the mean cell number ± SEM was determined by counting the cells in six random fields for each condition.

Preparation of de-epidermized dermis (DED)

Split thickness skin was harvested from consenting patients during breast reduction and abdominoplasty procedures and samples were immersed in glycerol/PBS (1 :1) for 4 hours, glycerol/PBS (85: 15) for 4 hours and in

100 % glycerol for 16 hours. The skin was patted dry with absorbent paper towels, placed into autoclave bags and sterilized by exposure to

15% ethylene oxide in CO₂ for 30 minutes at 55 ⁰C. After sterilisation, the skin samples were left at room temperature for a minimum of 3 days to allow aeration and dissipation of residual ethylene oxide gas. To rehydrate the skin, samples were immersed in PBS and incubated for 48 hours at 37 ⁰C. The PBS was then removed and the samples incubated at

37 ⁰C for between 16 and 18 hours in 1 M NaCl solution. After this time, the epidermis could be peeled or gently scraped off. The resulting DED was rinsed several times with PBS and soaked in keratinocyte-SFM for a minimum of 48 hours before cell seeding. Seeding of primary keratinocytes on collagen or DED

Collagen-coated 24 well plates or DED were oxidized, biotinylated and avidinated where necessary as described for L6 monolayers. Human keratinocytes were washed with PBS then oxidized and biotinylated as described above. For avidination, keratinocytes were then incubated for 15 minutes at room temperature with 5 μg/ml avidin, washed with avidin buffer, suspended by trypsinization and pelleted. The cells were then treated once more with 5 μg/ml avidin and washed with avidin buffer before being suspended in culture medium. For collagen-coated plate experiments, cells were plated in 0.5 ml of medium at a density of 5 x 10" cells/well and wells washed with PBS after various time-points. Wells were imaged using phase contrast microscopy and keratinocytes were counted in three random fields to determine the mean ± SEM of adhered cells. For DED experiments, 2 x 10⁵ primary human fibroblasts were seeded onto the reticular side of the sample within a metal ring and cultured for 24 hours before seeding of keratinocytes, 2 x 10⁵ of which were seeded within a metal ring onto the papillary side of the DED. After 30 or 60 minutes, the DED was washed with PBS and an MTT-ESTA assay performed.

Assessment of keratinocytes cell number using the MTT assay

DED samples were submerged in a 0.5 mg/ml solution of MTT in PBS and incubated at 37 ⁰C for 40 minutes. The stain was then eluted by agitation for 30-60 seconds in 2-ethoxyethanol, and the optical density of the eluent measured at 540 nm, with a protein reference at 630 nm subtracted. Wells containing DED + fibroblasts, without keratinocytes, were used as blank samples. Three replicates for each condition were measured and the mean ± SEM determined. Differentiation of mES cells

Cells were lightly trypsinized and plated into bacteriological grade Petri dishes in culture medium without LIF at a density of 6 x 10⁵ cells/ml. The absence of LIF and a feeder layer resulted in the formation of free- floating embryoid bodies. To promote osteogenic differentiation, EBs at different stages of culture were dissociated by treatment with 0.25 % trypsin/1 mM EDTA and resuspended in α-modified Eagle's medium containing 15% FCS, 50 μg/ml ascorbic acid, 10 mM β- glycerophosphate, 1 μM dexamethasone, 50 U/ml penicillin and 50 μg/ml streptomycin. Cells were plated into 6 well culture plates at 1 x 10⁵ cells per well and cultured for 21 days with the medium replaced every 2 to 3 days. Bone nodules per well were then quantified and the mean + SEM of three wells determined per EB time-point. For engineered EB formation, mES cells were oxidized and biotinylated in suspension as described above and aggregation induced by incubation in 1 ml of 10 μg/ml avidin solution. After incubation for 10-15 minutes, the resulting aggregates were transferred to Petri dishes containing mES medium without LIF and cultured for different time-points to assess EB formation. Immuno staining was performed as described below.

Immunostaining of embryoid bodies.

EBs were fixed for 30 minutes at room temperature with 4 % (w/v) paraformaldehyde in phosphate buffer, then permeabilized for 45 minutes in 10 mM PBS containing 0.2 % (w/v) Triton X-100. After washing three times for 5 minutes each with PBS, non-specific antibody binding was blocked by incubating the EBs for 20 minutes in a 3 % (v/v) solution of rabbit serum in PBS. Excess serum was then blotted and EBs were incubated overnight at 4 ⁰C in a solution of polyclonal goat antibody against cadherin-11 or MHC (Santa Cruz Biotechnology, #sc-6463 and #sc-12117) diluted 1:25 in PBS with 0.2 % BSA and 0.1 % NaN₃. The EBs were then washed three times for 10 minutes each with PBS and incubated for 1 hour at room temperature with FITC-conjugated anti-goat IgG diluted 1:400 in PBS with 0.2 % BSA and 0.1 % NaN₃. EBs were then washed three times for 10 minutes each with PBS and imaged by fluorescence confocal microscopy.

Claims

1. A method of aggregating a population of cells comprising causing one or more cells in the population to be cross-linked with one or more other cells in the population and/or one or more micro-particles added to the population of cells.

2. A method according to claim 1 in which the one or more cells in the population are caused to be covalently cross-linked to one or more other cells in the population and/or one or more micro-particles added to the population of cells.

3. A method according to claim 1 or claim 2 wherein the population comprises in the order of hundreds of cells or more.

4. A method according to any preceding claim wherein the population of cells comprises cells of only one cell type.

5. A method according to any of claims 1 to 3 wherein the population of cells comprises of two or more cell types.

6. A method according to any preceding claim wherein the surface of the one or more cells has a modified region.

7. A method according to claim 6 wherein the modified region of the cell surface of the one or more cells is used to cross-link the one or more cells to the one or more other cells and/or the one or more micro-particles.

8. A method according to claim 7 wherein the one or more other cells also have a modified region on their cell surface which is used in the cross-linking.

9. A method according to any of claims 5 to 8 wherein substantially all the cells in the population have modified cell surface regions.

10. A method according to any of claims 5 to 9 wherein the cell surface has been modified by one or more of the followings means chemical modification, metabolic modification, enzymatic modification and genetic engineering of the cell to produce a modified region on the cell surface.

11. A method according to any of claims 5 to 10 wherein the modification on the cell surface is selected from the group comprising an aldehyde, a ketone, an azide, biotin and avidin.

12. A method according to any of the preceding claims in which one or more cells are cross-linked to one or more other cells and/or micro-particles via naturally occurring groups on the cell surface.

13. A method according to claim 12 in which one or more cells are cross-linked via a naturally occurring group on one cell and a modified cell surface region on another cell.

14. A method according to any preceding claim wherein the cells and/or micro-particles are cross-linked directly.

15. A method according to any of claims 1 to 13 wherein the cells and or micro-particles are cross-linked indirectly.

16. A method according to claim 15 wherein a linker is used to cross-link indirectly one or more cells and/or micro-particles.

17. A method according to claim 16 wherein the linker is cross-linked to one or more modified regions on the surface of one or more modified cells.

18. A method according to any of claims 16 or 17 wherein the linker is homofunctional.

19. A method according to any of claims 16 or 17 wherein the linker is heterof unctional .

20. A method according to any preceding claim wherein cells in the population are selected from the group comprising human or non-human animal, bacteria or yeast cells.

21. A method according to claims 20 wherein the cells are human or non-human animal cells selected from the group comprising cells from human or non- human animal bone, cartilage, muscle, liver, kidney, skin, neural, and central nervous system, and specialised cells such as placental, amnionic, chorionic and foetal cells, stem cells, chondrocytes and reprogrammed cells from other parts of the body such as apidocytes reprogrammed to become cartilage cells.

22. A method according to any preceding claim performed in the presence of a tissue scaffold.

23. A method according to any preceding claim wherein the one or more micro-particles are made of one or more polymers selected from the group comprising poly (α-hydroxy acids) , polylactic or polyglycolic acids, poly-lactide poly-glycolide copolymers, poly-lactide or poly-glycolide polyethylene glycol (PEG) copolymers, polyesters, poly (ε-caprolactone) , poly (3-hydroxy-butyrate) , poly (s-caproic acid) , poly (p-dioxanone) , poly (propylene fumarate) , poly (ortho esters) , polyol/diketene acetals addition polymers, poly anhydrides, poly (sebacic anhydride) (PSA) , poly (carboxybiscarboxyphenoxyphosphazene) (PCPP), poly [bis (p- carboxyphenoxy) methane] (PCPM) , copolymers of SA, CPP and CPM poly (amino acids) , poly (pseudo amino acids) , polyphosphazenes, derivatives of poly [(dichloro) phosphazene] , poly [(organo) phosphazenes] polymers, polyphosphates, polyethylene glycol polypropylene block co-polymers for example that sold under the trade name Pluronics™, natural or synthetic polymers such as silk, elastin, chitin, chitosan, fibrin, fibrinogen, polysaccharides (including pectins), alginates, collagen, poly (amino acids), peptides, polypeptides or proteins, copolymers prepared from the monomers of these polymers random blends of these polymers and mixtures or combinations thereof..

24. An aggregate of cells comprising a population of cells wherein at least one cell is covalently cross-linked to at least one other cell.

25. An aggregate of cells comprising a population of cells wherein at least one cell has a modified surface region which is cross-linked via the modified surface region to another cell and/or a micro-particle.

26. An aggregate obtainable by the method of any of claims 1 to 23.

27. An aggregate according to any of claims 24 to 26 for use as a medicament or for the treatment of a disease.

28. A method of treatment of a human or non-human animal comprising introducing a cell aggregate according to any of claims 24 to 27 into or onto target tissue of an animal in need of treatment.

29. A method of treatment of a human or non-human animal comprising performing the method of any of claims 1 to 23 in vivo in a human or non-human animal including injecting cells into a target tissue of a human or non-human animal in need of treatment.

30. A method of cosmetic treatment on a human or non-human animal comprising performing the method of any of claims 1 to 23 in vivo in an animal or introducing a cell aggregate according to any of claims 24 to 27 into to or onto target tissue of an animal in need of treatment.

31. A method according to claims 30 wherein the treatment is selected from the group comprising breast enhancement, lip implants, eye implants, and any other form of cosmetic implantation.

32. A method of testing the toxicity of a chemical comprising applying the chemical to a cell aggregate according to any of claims 24 to 27 and observing the effect on the aggregate and its function.

33. A diagnostic test kit comprising a cell aggregate according to any of claims 24 to 27.

34. A kit according to claim 33 including instructions to use the aggregate in a toxicology study.

35. A method of aggregating a population of cells substantially as herein described with reference to the figures.

36. A cell aggregate substantially as herein described with reference to the figures.