CELL MIGRATION

Background of the Invention

The present invention relates to a method and apparatus for use in monitoring cell migration.

Description of the Prior Art The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Tissue engineering aims to replace organs and tissue damaged by injury or disease development. Typically replacement of damaged tissue involves the use of scaffolds, which provide the 3D structural organisation and mechanical support required for the developing tissue. Ideally, cells will attach, proliferate and differentiate within the scaffold and form the essential tissue and extracellular matrix (ECM). The scaffold can then biodegrade when the natural ECM has mechanical integrity.

Colonisation and spatial organisation of cells within scaffolds remains problematic due to issues of inadequate cell growth, inadequate nutrient transport, limitations in cell infiltration and spatial organisation. For example vascularisation and organisation of different cell types within complex tissues have virtually limited clinical applications to very simple nonvascular tissues of a single cell type.

Factors which influence cell colonisation and spatial distribution include scaffold pore organisation/geometry and material surface chemistry. Accordingly, it is desired to be able to identify optimal geometries and surface chemistries for particular cell types, allowing scaffolds to be generated which enable spatial organisation of different cell types thus permitting 3D tissue organisation.

Optimisation or identification of ideal scaffold pore organisation/geometry and surface chemistry is non-trivial and requires a deep understanding of cell migration in different circumstances. Cell migration is central to many normal and pathological processes including embryonic development, wound healing, inflammation and tumour metastasis. It requires integration of signalling events and changes in cellular architecture. It is a cyclical process in which a cell extends its protrusions at its front, is stabilised by the formation of adhesive complexes and then retracts at its trailing end, allowing the cell to advance over its substrate. Thus, the cell-substratum adhesiveness plays a significant role in determining the rate of migration.

A major function of the integrin family of receptors is to provide a physical connection between extracellular adhesion proteins and intracellular cytoskeletal/signalling molecules. AU integrins are non-covalently linked, heterodimeric molecules containing an alpha and a beta subunit. Both subunits are type 1 transmembrane proteins, containing large extracellular domains and mostly short cytoplasmic domains. Mammalian genomes contain 18 alpha subunits and to date 24 different alpha-beta combinations have been identified.

Integrins bind to a wide variety of ligands; their promiscuity can be explained by common modes of molecular interactions. There are four main classes reflecting the structural basis of the molecular interaction. These classes are RGD-binding integrins, LDV-binding integrins, A domain βl integrins and non-alpha A-domain-containing, laminin-binding integrins.

Although many ligands may share a certain subset of integrins, the rank orders of ligand affinity varies, presumably reflecting the precision of the fit between ligand structural basis and conformation with specific site.

Migration of cells in higher organisms is mediated by these adhesions, transmitting forces and signals necessary for the previously described cell motility. Whether cells migrate on a certain substratum and their respective speed depends on several variables related to integrin- ligand interactions. These include ligand surface density, integrin levels and binding affinity.

Maximal cell migration speed occurs at an intermediate ratio of cell- substratum adhesiveness to intracellular contractile force. This results in the ligand concentration promoting maximum migration speed decreasing reciprocally as integrin expression increases. Therefore, at low ligand concentrations, cell speed increases as integrin expression or binding affinity increases, whereas at high ligand concentration cell speed increases as integrin expression or binding affinity decreases. The maximum attainable cell speed remains the same, only the biphasic curve is shifted.

Obviously rates determined within these studies are cell, ligand and even integrin specific. Therefore to identify optimal levels of ligand- integrin combinations to direct lineage specific migration, a high throughput screening platform is required.

A variety of methods have been developed to study cell motility and wound restitution.

One example is the wound-healing assay, which is described in Blow, N., Cell Migration: Our Protruding Knowledge. Nature Methods, 2007. 4(7): p. 589-594, and Leonard R. Johnson and K.E. Barratt, Physiology of the Gastrointestinal Tract. Fourth ed. Vol. 1. 2006. 2018. The wound healing assay is one of the earliest developed methods to study directional cell migration in vitro and mimics cell migration during wound healing in vivo. The basic steps involve scraping a "wound" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the wound.

This approach is both simple, powerful and allows rapid analysis of alterations to cell lines, growth factors or regulation peptides. However, the wound healing assay has some inherent problems that confound results. For example, the surface is ambiguous due to the fact that when creating the wound the underlying matrix comprised of specific ligands or otherwise cell secreted matrix may be removed when performing the 'scrape'. Additionally, the thickness of the wound may vary along the length and if pictures are not taken at precisely the right location results may be highly skewed. The act of creating the wound destroys and damages cell of the initial wave front; changing the local cellular microenvironment. The cell confluence in the area where the scrape is created will affect results. Finally, wound closure involves the process of two cell wave fronts moving towards each other, making it difficult to study a single wave front and cell-cell interactions.

The fence assay, as described for example in Pratt et al., 1984; Am. J. Pathol. 117: 349- 354,involves cell cultures being placed on dishes with a removable ring or "fence" on a portion of surface area. Once cellular attachment occurs the fence is removed and migration - A -

into the cell-free zone is measured over time. Whist this resolves some of the problems involved with the wound healing assay, primarily by allowing migration over a virgin defined surface without removing bound cells, fence assays also suffer from a number of problems, including time-consuming setup and unbound cells floating into migration area affecting results.

A transwell migration assay, as described for example in U.S. Pat. Nos. 5,210,021 and 5,302,515, uses two chambers separated by a filter through which cells migrate. This allows chemotactic gradients to be established by placing a chemotactant in the upper or lower chamber (or both), which has the added advantage of discriminating between chemo-kinetic and chemotactic influences. The transwell migration assay requires cells to move through a filter of a nominal pore size, but this only gives a relative evaluation of cell migration and not a defined migration rate.

A microstencil assay approach is described in Poujade, M., et al., From the Cover: Collective migration of an epithelial monolayer in response to a model wound. Proceedings of the National Academy of Sciences, 2007. 104(41): p. 15988-15993. This uses a PDMS microstencil to replicate the wound healing assay. A stencil is deposited on the surface before plating of cells, which are then cultured until confluence is achieved, at which point the stencil is removed allowing them to migrate similar to the fence assay.

However, the surface that remains is ambiguous; it is not known whether a surface would remain after the stencil step or if a thin layer of polymer residue would remain.

Nie, F. -Q., et al., On-chip cell migration assay using microfluidic channels. Biomaterials, 2007, describe using a microfluidic channel created an on chip cell migration device. The device consists of a central chamber with three inlets. After cells are cultured within the central chamber, trypsin flows down two of the inlets removing cells from a portion of the channel, essentially creating a wound edge and allowing them to migrate into the vacant area. This method lacks the ability to control the surface chemistry of the substrate, thereby providing limited control. Traditional migration assays described above also have the added disadvantage of requiring both a large number of cells and an increased amount of reagents, in order to operate. Additionally, these methods are generally not suitable for scaling-up for high throughput screening.

Accordingly, migration devices conceived thus far do not provide the necessary platform to analyse 2D geometrical and surface chemistry effects.

Summary of the Present Invention

The present invention seeks to substantially overcome, or at least ameliorate, one or more disadvantages of existing arrangements.

In a first broad form the present invention seeks to provide a method for use in monitoring cell migration, the method being performed using apparatus including a reservoir and at least one cell channel having a first end in fluid communication with the reservoir and a second end in fluid communication with an opening, the method including: a) adding solution to the reservoir, the solution containing cells of interest to thereby position at least one cell adjacent the first end of the channel; and, b) adding media to the channel via the opening thereby causing at least one cell to migrate along the channel from the first end towards the second end.

Typically the method includes washing the reservoir prior to adding media to the channel.

Typically the method includes, adding the solution to the reservoir to establish a layer of bound cells over an inlet of the first end of the channel.

Typically the method includes washing the reservoir to thereby remove unbound cells.

Typically the method includes selecting the surface tension of the solution to thereby prevent cells entering an inlet of the first end of the at least one channel.

Typically the surface tension of the solution is selected in accordance with a size of the inlet.

Typically the method includes selecting solution properties in accordance with inlet properties to thereby prevent cells entering an inlet of the first end of the at least one channel. Typically the method includes using at least one channel having a channel geometry selected so as to simulate a cell migration pathway.

Typically the channel geometry includes at least one of: a) a channel shape; b) a channel length; c) a channel width; and, d) a channel angle.

Typically the method includes modifying the surface chemistry of the at least one channel prior to performing cell migration.

Typically the method includes adding material to at least part of the at least one channel to thereby modify the surface chemistry.

Typically the apparatus includes a plurality of channels, and wherein the method includes causing cells to migrate along a number of the plurality of channels.

Typically the method includes comparing cell migration for different ones of the channels.

Typically the apparatus includes a plurality of channels, and wherein the method includes adding media to a number of the plurality of channels via a common opening.

Typically the apparatus includes a plurality of channels, and wherein the method includes adding one or more soluble cell migration-modulating agents to a number of the plurality of channels.

Typically the apparatus includes a plurality of groups of channels, each group of channels having a common opening, and wherein the method includes comparing cell migration for different channels within a group of channels.

Typically the method includes measuring at least one of: a) a cell migration time; and, b) a cell migration rate. Typically the method includes applying a force to media in the opening thereby urging the media into the channel.

Typically the method includes applying a force to media using a centrifuge.

Typically the reservoir is a first reservoir and the opening includes a second reservoir, and wherein the method includes establishing a differential fluid depth between the first and second reservoirs.

In a second broad form the present invention seeks to provide apparatus for use in monitoring cell migration, the apparatus including: a) a reservoir for receiving a solution containing cells of interest; b) at least one cell channel having a first end in fluid communication with the reservoir and a second end in fluid communication with an opening for receiving media, wherein adding solution to the reservoir, the solution containing cells of interest to thereby position at least one cell adjacent the first end of the channel, and adding media to the channel via the opening thereby causes at least one cell to migrate along the channel from the first end towards the second end.

Typically the apparatus includes a plurality of channels, each channel having a first end in fluid communication with the reservoir.

Typically the apparatus includes a plurality of channels, and wherein at least some of the plurality of channels include a second end in fluid communication with a common opening.

Typically the apparatus includes a plurality of groups of channels, each group of channels having a common opening.

Typically the at least one channel has a channel geometry selected to as to simulate a cell migration pathway.

Typically the channel geometry includes at least one of: a) a channel shape; b) a channel length; c) a channel width; and, d) a channel angle.

Typically the at least one channel has a length of at least one of: a) 12 mm; and, b) 2 mm.

Typically the at least one channel has a width of less than at least one of: a) 0.2 mm; and, b) 0.5 mm.

Typically the apparatus includes a plurality of channels, and wherein at least some of the channels have respective channel geometries.

Typically the first end of the channel includes an inlet for connecting the first end to the reservoir.

Typically the inlet has a size selected to prevent cells in the solution entering the inlet when the reservoir is being filled.

Typically the inlet is provided in a body, the body including a hydrophobic material to prevent cells in the solution from entering the channel when the reservoir is being filled.

Typically the reservoir includes a reservoir opening for allowing solution to be provided to and removed from the reservoir.

Typically the reservoir includes: a) a base; b) an opening; and, c) side walls extending from the base to the opening.

Typically the reservoir includes four side walls.

Typically the opening is smaller in area than the base.

Typically the reservoir and at least one channel are formed from a moulded polymer material. Typically the surface chemistry of the at least one channel is modified prior to performing cell migration.

Typically the surface chemistry is modified using a material added to at least part of the at least one channel.

Typically the apparatus includes a centrifuge for applying a force to media in the opening thereby urging the media into the channel.

Typically the reservoir is a first reservoir and the opening includes a second reservoir.

Typically the first reservoir is a central reservoir, and the second reservoir an annular reservoir positioned radially outwardly from the central reservoir, the channels extending radially outwardly from the central reservoir to the outer reservoir.

In a third broad form the present invention seeks to provide a method of manufacturing apparatus for use in monitoring cell migration, the method including: a) creating a reservoir for receiving a solution containing cells of interest; and, b) creating at least one cell channel having a first end in fluid communication with the reservoir and a second end in fluid communication with an opening for receiving media.

Typically the method includes: a) selecting a channel geometry for simulating a cell migration pathway; and, b) creating the at least one channel in accordance with the selected channel geometry.

Typically the channel geometry includes at least one of: a) a channel shape; b) a channel length; c) a channel width; and, d) a channel angle.

Typically the method includes creating at least one of the reservoir and the at least one channel using a polymer moulding process. Typically the method includes: a) selecting a channel and reservoir arrangement; b) creating a mould using the selected arrangement; and, c) using the mould to create a first body later including the reservoir and at least one channel.

Typically the method includes creating the mould using at least one of an etching process and a photolithographic process.

Typically the method includes coupling the first body layer to a second substrate layer.

Typically the method includes coupling the first body layer to the second substrate layer using at least one of: a) adhesive coupling; and, b) thermal coupling.

Typically the method includes modifying the surface chemistry of at least part of the at least one aperture.

Typically the method includes adding material to at least part of the at least one channel to thereby modify the surface chemistry.

In a fourth broad form the present invention seeks to provide a method for use in monitoring cell migration, the method being performed using apparatus including a reservoir and at least one cell channel having a first end in fluid communication with the reservoir and a second end, the method including: a) adding solution to the reservoir, the solution containing cells of interest to thereby position at least one cell adjacent the first end of the channel; and, b) adding media to the channel thereby causing at least one cell to migrate along the channel from the first end towards the second end.

Typically the method includes applying a force to the media to thereby urge the media into the channel.

Typically the method includes applying a force to media using a centrifuge. In a fifth broad form the present invention seeks to provide apparatus for use in monitoring cell migration, the apparatus including: a) a reservoir for receiving a solution containing cells of interest; b) at least one cell channel having a first end in fluid communication with the reservoir thereby allowing cells of interest to be positioned adjacent the first end by providing solution containing cells of interest into the reservoir, and wherein adding media to the channel causes at least one cell to migrate along the channel from the first end towards the second end.

Typically the channel includes a second end in fluid communication with an opening thereby allowing media to be added to the channel.

In a sixth broad form the present invention seeks to provide a method of manufacturing apparatus for use in monitoring cell migration, the method including: a) creating a reservoir for receiving a solution containing cells of interest; and, b) creating at least one cell channel having a first end in fluid communication with the reservoir and a second end, and wherein cells of interest can be positioned adjacent the first end by providing solution containing cells of interest into the reservoir, and wherein adding media to the channel causes at least one cell to migrate along the channel from the first end towards the second end.

1

Brief Description of the Drawings An example of the present invention will now be described with reference to the accompanying drawings, in which: -

Figure IA shows a schematic plan view of an example of apparatus for use in monitoring cell migration;

Figure IB shows a schematic side view of the apparatus of Figure IA; Figure 2 is a flow chart of an example of a process for use in monitoring cell migration;

Figure 3A shows a schematic side view of a second example of apparatus for use in monitoring cell migration;

Figure 3B shows a schematic plan view of the apparatus of Figure 3 A;

Figure 3 C shows a schematic plan view of as modified version of the apparatus of Figure 3B; Figure 4 is a flow chart of a second example of a process for use in monitoring cell migration;

Figure 5 is a flow chart of an example of a process for use in manufacturing apparatus for use in monitoring cell migration; Figures 6 A to 6D are schematic plan views of example reservoir and channel arrangements;

Figures 7 A and 7H are schematic diagrams of the steps involved in creating a reservoir and channel arrangement;

Figure 8A is a schematic side view of a third example of apparatus for use in monitoring cell migration; Figure 8B is a schematic cross section view along the line A-A' of Figure 8 A;

Figure 8C is a schematic plan view of an example of the apparatus of Figure 8A including different channel geometries;

Figure 8D is a schematic plan view of an example of a well plate;

Figures 9A and 9B are schematic diagrams of a first example procedure for initiating cell migration using the apparatus of Figures 8 A and 8B;

Figures 1 OA to 1 OD are schematic diagrams of a second example procedure for initiating cell migration using the apparatus of Figures 8 A and 8B;

Figure 1OE is a schematic diagram of an example of a process for initiating channel filling using a centrifuge; Figure 11 shows photographs of an example of 3T3 cell migration within the multi- width channel apparatus;

Figure 12 is a graph of the wavefront velocity for example NIH 3T3 fibroblast cell migration rates for the apparatus of Figure 8;

Figure 13 is a graph of an example of NIH-3T3 Mouse Fibroblast migration rates within different width channels;

Figures 14A to 14D are phase contrast images of examples of NIH-3T3 Mouse Fibroblasts within channels having widths of 500μm, 200μm, 30μm and 20μm, respectively;

Figure 15 is a graph of an example of NIH-3T3 Fibroblast spreading area within channels of different widths; Figure 16 is a graph of an example of NIH-3T3 Fibroblast circularity within channels of different widths; Figure 17 is a schematic diagram of an example of cell migration within channels of different widths;

Figure 18 shows photographs of examples of channel contractions and expansions;

Figure 19 is a graph of wavefront velocity for example NIH 3T3 fibroblast cell migration rates for apparatus including contracting channels;

Figure 20 is a graph of wavefront velocity for example NIH 3T3 fibroblast cell migration rates for apparatus including expanding channels;

Figure 21 is a schematic diagram of an example design schematic for apparatus with tortuous channels; Figure 22 shows photographs of example channel tortuosities;

Figure 23 is a graph of wavefront velocity for example NIH 3T3 fibroblast cell migration rates along tortuous channels;

Figure 24 shows photographs of example junction configurations;

Figure 25 is a graph of wavefront velocity for example NIH 3T3 fibroblast cell migration rates along channel junctions;

Figures 26A to 26C show photographs of examples of cell migration at different channel junctions;

Figure 27 is a photograph of examples of 3T3 cell migration within example apparatus under a 1OX microscope objective; Figure 28 is a graph of wavefront velocity for example NIH 3T3 fibroblast cell migration rates in the apparatus of Figure 19;

Figure 29 is a graph of wavefront velocity for example Saθs2 human osteosarcoma migration rates in the apparatus of Figure 19;

Figure 30 is a graph of examples of the cell spreading area of NIH 3T3 Fibroblast and Saθs2 Osteosarcoma cell lines on various surfaces;

Figure 31 shows photographs of examples of NIH 3T3 fibroblast attachment to surfaces of

BSA ₅ collagen I, collagen I blocked with BSA, collagen IV and collagen IV blocked with

BSA.

Figure 32A is a graph of example relative hMSC migration rates on various functionalised ECM molecules; Figure 32B is a graph of example relative hMSC population doubling time on various functionalised ECM molecules;

Figure 33A shows example phase contrast images of hMSCs migration along 3 adjacent

200μm channels; Figure 33B is a schematic diagram of an example of a HA/CHI multilayer- PEG linker surface;

Figure 33C is an image of an example of Fibronectin surface staining before (left) and after

(right) cell migration;

Figure 34 shows example mesenchymal stem cell flow cytometry plots; Figure 35A is a graph of example integrin blocked hMSC migration rates on fibronectin over

48 hrs;

Figure 35B is a graph of example attachment percentages of integrin blocked hMSCs;

Figure 35C is a graph of example population doubling times of integrin blocked hMSCs on functionalised surfaces; Figure 36A is an image of a specific example of a well containing apparatus for use in monitoring cell migration;

Figure 36B is an image of apparatus of Figure 35 A with the channels having a fluidic connection to the central reservoir following centrifugation;

Figure 37A to 37D are example images of cell migration in the apparatus of Figure 35 A after 0, 24, 48 and 72 hours respectively;

Figure 38 is a graph of example cell wave front propagation in the apparatus of Figure 36A on various surfaces and with or without foetal bovine serum (FBS) in the medium; and,

Figure 39 is a schematic plan view of a fourth example of apparatus for use in monitoring cell migration.

Detailed Description of the Preferred Embodiments

An example of apparatus for use in monitoring cell migration will now be described with reference to Figures IA and IB.

In this example, the apparatus 100 includes a reservoir 110 and at least one channel 120, with two channels being shown in this example for the purpose of illustration only. Each channel 120 includes a first end 121 which is in fluid communication with the reservoir 110, whilst a second end 122 is provided in fluid communication with an opening 125.

The reservoir 110 may also include means for allowing a solution containing cells to be added into the reservoir 110. In one example this is achieved having an open reservoir, allowing solution to be added directly into an opening of the reservoir. Alternatively, inlet/outlet channels 111 may be provided coupled to respective ports 112, thereby allowing solution to be added into or withdrawn from the reservoir 110.

An example of a process for monitoring cell migration will now be described with reference to Figure 2.

In this example, at step 200, a solution containing cells of interest is added into the reservoir 110, either via the ports 112, or reservoir opening. The solution is added so that cells are provided near the first end 121 of the channel 120. This may arise simply due to solution being present in the region adjacent the first end 121, but more typically the cells are constrained in some manner.

Constraint of the cells can be achieved in any suitable manner, depending on the preferred implementation. In one example, constraint is imposed by surface energy inhibiting media flow into the channel(s). Surface energy can be dictated by choice of material, geometry or through the modification of the physical properties of either the air/solid/media interface or through an agent attached or otherwise immobilised (e.g., by physical adsorption or chemical binding) to the apparatus in the region of the first end 121.

The step of providing cells near the first end 121 may be performed so that a required number of cells are constrained in some manner. Thus, this may involve constraining the cells until a layer of cells has been established, although this is not essential.

At step 210 media is added to the channel 120 via the opening 125. This typically involves injecting media into the opening 125 so that the media flows along the channel 120 from the second end 122 to the first end 121. As the media reaches the first end 121, this causes one or more cells to migrate along the channel from the first end 121 towards the second end 122, in turn allowing the migration process to be monitored. Thus, the addition of the media will typically unbind the cells, allowing cell migration along the channel to commence.

Migration of cells within the channels 120 can then be monitored in any suitable manner, such as by visual observation, or through the use of an appropriate sensor capable of detecting the position of the cell. Thus, in one example, analysis can be performed solely through the use of a microscope, allowing visual inspection of the cells to be performed at designated time points. However, alternatively, a detector can be used, for example by attaching a fluorescent label to the cells, allowing the position of the cell to be detected.

Accordingly, the above described apparatus and method allow cells of interest to be provided in a reservoir with the cells being retained therein until it is desired to perform a cell migration monitoring procedure. At this point, media can simply be added into the channels, releasing cells from the reservoir, thereby initiating the cell migration process and allowing the migration process to be monitored.

By controlling the migration pathway, through the use of a channel, this removes a large number of degrees of freedom that arise in unconstrained cell migration, as occurs for example in fence assays. This in turn allows different factors that influence the cell migration process, such as channel geometry and surface chemistry, to be controlled, so that their influence on cell migration can be readily established.

Thus, channels having a variety of different geometries, such as different channel widths, lengths, shapes or angles, can be used to study the impact of channel geometry on cell migration. Additionally, or alternatively, the surface chemistry of the channels may be altered, for example by adding material into the channel to influence the surface chemistry of the channel, which in turn influences cell migration.

In one example, the apparatus is provided with a number of different channels, allowing cell migration to be performed in each of the different channels substantially simultaneously. This in turn allows direct comparison of cell migration in each of the channels. Accordingly, by suitable configuration of the channels, for example by providing channels with different geometries or different surface properties, this allows the impact of such factors on cell migration to be easily assessed. Alternatively, this can be used to establish average cell migration characteristics across a number of channels, thereby taking into account inherent variations that may arise, for example due to different amounts of media in the respective channels.

The apparatus can also be created relatively simply, for example by moulding a suitable polymer material, such as poly (dimethylsiloxane) (PDMS). This in turn allows the apparatus to be created rapidly and cheaply, thereby allowing the apparatus to be easily used to study a wide range of factors that influence cell migration.

Additionally, whilst providing additional control over cell migration than is achievable with the prior art, the above described technique can also reduce the number of cells and amount of reagents required in order to operate, as compared to prior art techniques, thereby resulting in a device that is cheaper to operate.

It will be appreciated that the apparatus and method is therefore suitable for use in a wide of different applications, such as designing scaffold configurations and chemistries to assist in would healing and tissue regeneration applications.

A second example of apparatus for use in cell migration will now be described with reference to Figures 3A and 3B.

In this example, the apparatus 300 includes a reservoir 310 and a number of channels 320 formed in a body 340. The channels and reservoirs may be formed in any suitable manner, but in one example this is achieved by moulding a first material layer 341, thereby defining the reservoir 310 and channels 320, before attaching a second material layer 342 to thereby define provide a base for the body 340.

In this example, the channels are arranged in three respective groups designated 320A, 320B, 320C. As shown, each of the channel groups 320A, 320B, 320C includes three individual channels 320, each of which are in fluid communication with a respective opening 325 A, 325B, 325C, so that the channels 320 within each group 320A, 320B, 320C are all connected to the same opening 325 A, 325B, 325C. In this example, the reservoir 310 includes a base 311, side walls 312 and an opening 313 allowing solution to be inserted into and removed therefrom. Notably, in this example, the side walls 312 slope inwardly from the base 311 towards the opening 313, so that the opening has a smaller surface area than the base 311. This prevents shadows falling on the channels 320, which can in turn impact on the visualisation of the cell migration process.

It will be appreciated that the above-described apparatus can be used in a similar manner to the apparatus described above with respect to Figures IA and IB. However, in this example, providing a common opening 325A, 325B, 325C for each group 320A, 320B, 320C ensures that media is added into each channel 320 within a group 320A, 320B, 320C simultaneously, thereby allowing direct comparison of cell migration along each of the channels. This example thus permits replicates of experimental conditions and controls to be run simultaneously, or allows a plurality of different experimental conditions to be assessed at the same time.

For example, cell migration may be inherently chaotic, such that even under substantially identical conditions, cells will migrate at a variety of rates. By performing multiple cell migrations simultaneously under the same conditions, this allows a mean and variance for the cell migration to be established relatively easily.

Alternatively, each of the channels within a single group can have different channel properties, such as different channel geometries, or different channel surface properties. This allows direct comparison of the affect of different channel properties to be easily performed.

It will also be appreciated that each of the openings 325A, 325B, 325C may have media added simultaneously, thereby further enhancing the ability to perform direct comparisons. Thus, it will be appreciated that this could allow all of the channels within a single group to have common migration parameters, with channels in different groups having different migration parameters.

The apparatus 300 may also include inlets 321 A provided at the first end 321 of the channels 320. This can be used in restricting the ability of the cells to enter the channels 320, to thereby assist in positioning cells in the reservoir 310, in the vicinity of the first end 321 of the channels 321, as will be described in more detail below.

It should be noted that the term "inlet" is used for the purpose of explanation only, and is not intending to be limiting. In particular, the term "inlet" is used generally to refer to a region where the channel and reservoir meet, and as result, any function attributable to an "inlet" could be performed by configuration of either the reservoir or channel. Thus, for example, functionality described as being provided by an "inlet" could simply be provided by appropriate shaping of the channel, in which case the "inlet" is a notional element having dimensions equal to the channel.

An example of a process for monitoring cell migration using the apparatus of Figures 3 A and 3B will now be described in more detail with reference to Figure 4.

In this example, at step 400 at least a required solution surface tension is determined. The required surface tension is selected to allow a confluent cell monolayer to be established at the inlets 32 IA. The surface tension will therefore typically depend on at least a size of the inlet 32 IA. Accordingly, the process of determining the surface tension may involve determining the size of the inlets 32 IA and using this to select the surface tension based on a known relationship.

Additionally, and/or alternatively, the ability of the solution and hence cells to enter the inlet may depend on other inlet properties such as the hydrophobicity of the inlet and the surrounding material in which the inlet is provided, as well as other solution properties such as the solution viscosity. Accordingly, whilst inlet size and solution surface tension may be the main factors involved in establishing the confined cells, other solution and inlet properties could also be taken into account.

In one example, the surface tension can be selected based on the Young— Laplace equation, which is a nonlinear partial differential equation that describes the capillary pressure difference sustained across the interface between two static fluids, such as water and air, due to the phenomenon of surface tension. This allows for calculation of boundaries for channel size and contact angle, and is used to select channel material properties such that the channel capillary pressure is greater than the head pressure generated by the medium contained in the reservoir.

At step 410 a solution containing cells of interest is created. This typically involves adding the cells to a suitable media and then optionally adding one or more surface tension agents for modifying the surface tension of the solution. The surface tension agents can be of any suitable form, such as surfactants or alcohols for reducing surface tension, or surface tension enhancers such as dissolved inorganic salts.

At step 420 the reservoir 310 is filled with solution by injecting or otherwise adding the solution into the reservoir 310 via the opening 313. As there is minimal fluid resistance in the reservoir 310 and the pressure is not substantial enough to overcome the surface tension and urge the solution into the inlets 32 IA, the reservoir 310 fills thereby establishing the cell monolayer across the inlets 321A.

At step 430 the reservoir is washed to remove any unbound cells. It will be appreciated that this may be performed to reduce the number of cells in the reservoir, thereby limiting the number of cells that migrate along each of the channels 320, as well as to reduce cell migration into the reservoir 310.

It will be appreciated that in this example the cells are therefore bound by the surface tension of the solution. However, any other suitable binding mechanism may be used. Thus, in one non-limiting example, a binding agent, such as a receptor may be provided at the inlets 321 A so that the cells bind to the receptor.

Illustrative binding agents to which cells can bind include proteinaceous molecules (e.g., specific receptors or cell receptor recognition sequences [e.g., RGD], antigen-binding protein, and peptides), nucleic acid (e.g., single nucleotides or nucleosides, oligonucleotides, polynucleotides and single- and higher-stranded nucleic acids), lipids, phospholipids, or a combination thereof. In some embodiments, the binding agent is an antigen binding protein, representative examples of which include polyclonal, monoclonal, chimeric, single chain, Fab fragments, and Fab antibodies. In other embodiments, the binding agents are selected from proteins and peptide sequences normally found in the extracellular matrix that support cell attachment and function (e.g., collagens including types I, II, III and IV ₅ integrins, laminins, fibronectin, vitronectin). In illustrative examples of this type, peptide sequences are attached as short functional sequences (e.g., RGD) or the functional sequences may be contained within longer peptide sequences (e.g., x-RGD-x). Examples of peptide sequences include, but are not limited to, all of the known integrin binding sequences, including RGD, EILDV, LDV, LDVP, IDAP, PHSRN, SLDVP, and IDSP.

At step 440 the channels 320 are backfilled with media via the openings 325. The media might be any suitable form of media, such as water, or another liquid. This typically simply involves adding media to the openings 325A, 325B, 325C allowing the media to propagate along the length of the channels. Complete filling of the channels is not required although may be beneficial to help ensure each channel has a similar amount of media, which in turn avoids fluid levels influencing cell migration differently in each channel.

As the backfilling occurs and media reaches the first end 321 of the channels 320, the media will release one or more of the bound cells. The release mechanism will depend on the manner in which cells are bound. For example, this may simply involve disturbing the cells thereby releasing the cells from the influence of surface tension of any remaining solution. Thus, the cells could be constrained merely by a lack of media in the channel, which thereby prevents the cells migrating along the channel. In this instance, the cells are effectively released by the presence of media in the channel, which makes the channel surfaces available as an attachment surface for the cells, thereby allowing the cells to commence migration. However alternatively, the media may be required to displace the cells from a receptor to which they are bound, which could be achieved in a variety of manners, such as by providing a material in the media which binds to the receptors in preference to the cells, thereby displacing and releasing the cells.

Accordingly, it will be appreciated that the term bound should be interpreted broadly to cover any manner in which the cells are in some way confined to the reservoir, with the term release covering any mechanism for allowing the cells to commence migration.

At step 450 the cell migration process is monitored. In one example this is performed via visual inspection, in which case the body 340 is produced from substantially transparent material, allowing direct observation of the cell migration, for example using a microscope. In one example, the channels 320 are arranged such that each of the channels 320 within a respective group 320A, 320B, 320C is visible within the microscope field of view at all times. It will be appreciated that this is particularly advantageous as it allows simultaneous monitoring of cell migration in each of the three channels in the group. However, any suitable arrangement may be used.

For example, this could involve using cell-permeate cytoplasmic fluorescent label to allow particle image velocimetry (PIV) to be performed, which can in turn provide information regarding cell displacement, direction, velocity and coordination of cell movements.

An example of the process for manufacturing apparatus for use in monitoring cell migration will now be described with reference to Figure 5.

Conventional microfluidic devices are typically fabricated in materials such as glass and silicon, and similar techniques can be used to manufacture the apparatus. However, such arrangements are typically time consuming and expensive to manufacture, and have limited biocompatibility, non natural stiffness and valve incompatibility.

Accordingly, for the purposes of the current example, the apparatus is manufactured using a polymer-based material, which is generally inexpensive and easy to manipulate, allowing custom apparatus to be rapidly and cheaply manufactured. In particular, this allows channels to be moulded or embossed rather than using etching as is required in manufacturing glass and silicon based devices. This also allows the devices to be sealed thermally or by using adhesives. Whilst any suitable polymers may be used, in one example, the polymer is poly (dimethylsiloxane) (PDMS), which is a widely used silicone based organic polymer known for its unusual rhelogical properties. It has many advantages over other polymers and conventional materials including: • It is inexpensive, flexible and cures at low temperatures;

• It is optically transparent down to 230 run, and is therefore compatible with numerous optical methods for detection ( e.g. UV/ Vis absorbance and fluorescence); • It is compatible with biological studies as it is impermeable to water, non-toxic to cells and gas permeable, and can also be implanted in vivo;

• Features on the microscale can be reproduced with high fidelity in PDMS by replica moulding; • It can be deformed reversibly and sealed reversibly to itself (and other materials);

• Surface chemistry can be controlled; and,

• Its elastomeric, allowing it to conform to smooth, non-planar surfaces, and release from delicate features of a mould without damage.

However, other materials can be used in the manufacturing process, in which case the following process will be modified as required in accordance with appropriate manufacturing techniques. Thus, for example, the device could be manufactured from glass and silicon with the channel shape being provided in the silicon, which is then bound to the glass substrate. Alternatively, other materials such as tissue culture grade polystyrene or the like could be used.

In this example, at step 500 a channel geometry is determined. Variation of the channel geometry allows investigation to be performed into the effect of geometry on cell migration. Channels can be altered to allow variations in channel width, height, length, angle, as well as the presence of any contractions, expansions, tortuosities and channel junctions (various angles). In one example channels typically have a width of less than 0.5 mm and more typically in the region of 0.2 mm, whilst channel lengths are typically at least 2 mm, and in one example at least 12 mm.

Selection of channel geometry may be achieved in any one of a number of manners and will typically depend on the migration properties being investigated. Thus, for example, if the cell migration is be analysed to provide an insight into the effect of scaffold internal geometry on the colonisation of space by seeded or recruited cells within a wound healing scaffold, then a geometiy mimicking the internal structure of the scaffold can be used. Typically, if channel geometry is being investigated, then it will be typical to provide a number of different channel geometries on the single device. It will also be appreciated that whilst the current example focuses on the production of 2D channel geometries, this is not essential, and in some examples it may be desired to fabricate 3D channels to recreate the specific geometry of a scaffold. This will be achieved via a polymer reverse template technique.

The geometry selection can also be influenced by whether it is desired to investigate whether effects are due to the surface chemistry of the channels, in which case it would be typical to provide an arrangement including a number of channels with identical geometries. In this case, if the geometry is not of interest in the investigation, typically straight channels similar to those shown in Figures 3A and 3B, are used. Example channel geometries are described in more detail below.

At step 510, a channel and reservoir arrangement is determined. In the example of Figure 6 A, a rectangular reservoir 610 is used, with a number of channels 620 extending from the reservoir on one or more sides. This allows migration of cells in a number of channels to be monitored relatively easily. However, any suitable arrangement may be used and so, for example, a circular central reservoir 660 may be provided with channels 670 extending radially outward therefrom, as shown in Figure 6B. An alternative example using multiple channels coupled to a common opening is shown in Figures 6C and 6D.

At step 520 a mask is created based on arrangement, with an etching processing being used at step 530 to etch a mould from the mask. In one example, this can be performed using a computer aided design package, allowing the reservoir and channel arrangement to be designed. Once completed, the design can be printed onto a transparency film which then serves as a photomask in contact photolithography to produce a positive relief of photoresist (usually a photo-curable epoxy, SU-8) on a silicon wafer. After dissolving the unpolymerised photoresist, a positive relief of the channel structure is left on the wafer, forming a 'master' can be used to cast the PDMS devices. This is shown in more detail in Figures 7A to 7H.

In this example, a transparency 700 is printed using a suitable printer, such as a high resolution commercial image setter. The transparency 700 is then positioned on a photoresist material 710 provided on a silicon substrate 720, and exposed to radiation 730, as shown in Figure 7B. Exposing the photoresist material 710 cures the exposed material, so that uncrosslinked photoresist can be removed using suitable etching, such as acid etching, thereby leaving the mould remaining as shown in Figure 7D.

In one example, multiple layers of photoresist can be applied and exposed in turn, allowing complex multi-layer geometries to be provided, as shown in Figures 7E and 7F.

At step 540 the channel and reservoir arrangement is formed by casting the polymer material against the master creating the negative of the master, as shown at 740 in Figure 7G. If PDMS is used, the PDMS is cured in an oven at 60 ⁰ C for 1 hr and then peeled from the master, as shown in Figure 7H, to thereby form the first layer 341 of the apparatus 300.

As part of this process, channel inlets and openings, can be punctured into the PDMS and the channels are sealed against glass or tissue culture plastic by exposing the surface of the polymer to oxygen plasma.

At step 550 the first layer 341 is coupled to a second substrate layer 342, such as a glass substrate to thereby form the apparatus, and this may be achieved using thermal bonding, adhesive or the like.

Following this, the surface properties of the channels and/or reservoir may be modified in some embodiments, for example, by providing a surface-modifying agent in the channels at step 560, whose migration-modulating properties are desired to be assayed. The manner in which this is achieved will depend on the intended use of the cell migration monitoring, and the particular types of cells being examined. An apparatus of the invention according to these embodiments provides assays for qualitatively and/or quantitatively determining the migration (e.g., random movement as well as attraction or repulsion) of cells under control conditions and in response to surface modifying agents.

In some embodiments, the surface-modifying agents are covalently or non-covalently associated with the surface of the channels and/or reservoir using procedures well known to those of skill in the art. Non-limiting examples of surface-modifying agents include sugars, proteins (e.g., extracellular matrix proteins such as collagen, laminins, fibronectin, vitronectin, osteopontin, thromospondin, intercellular adhesion molecule- 1 (ICAM-I), ICAM-2, proteoglycans such as chondroitin sulphate, von Willebrand factor, entactin, fibrinogen, tenascin, mucosal adressin cell adhesion molecule (MAdCAM-I), C3b, and MDC (metalloprotease/disintegrin/cysteine-rich) proteins), nucleic acids, specific receptors and cell receptor recognition sequences (e.g., cadherein, immunoglobulin superfamily, selectin, mucin and integrin binding sequences such as RGD, EILDV, LDV, LDVP, IDAP, PHSRN ₅ SLDVP, GRGDAC, and IDSP)). Surface-modifying agents may also be selected from chemotactic compounds (e.g., compounds that stimulate directed cell migration in response to a gradient) such as, but not limited to, For-Nle-Leu-Phe-Nle-Tyr-Lys-DTPA, For-Met- Leu-Phe-Pu-DTPA, For-Nle-Leu-Phe-Lys-DTPA, For-Nle-Leu-Phe-Lys(NH ₂ )-DTPA, For- Met-Leu-Phe-Lys-DTPA, For-Met-Leu-Phe-D-Lys (NH2)-DTPA, Ac-Nle-Leu-Phe- Lys(NH ₂ )-DTPA as described for example in EP 0398143; and chemokinetic compounds (e.g., compounds that stimulate cell migration that is not gradient or directionally dependent), representative examples of which include: IL-8, GCP -2, Gro α, Gro β, Gro γ, ENA-78, PBP, MIG, IP-IO, I-TAC, SDF-I (PBSF), BLC (BCA-I), MIP-Ia, MlP-lβ, RANTES, HCC-I, -2, -3, and -4, MCP-I, -2, -3, and -4, eotaxin-1, eotaxin-2, TARC, MDC, MIP-3α (LARC), MIP- 3β (ELC), 6Ckine (LC), 1-309, TECK, lymphotactin, fractalkine (neurotactin), TCA-4, Exodus-2, Exodus-3 and CKβ-11.

For example, when analysing surfaces that encourage tissue specific migration, the goal is to identify the surface chemistry that will optimise cell migration for the migration/proliferation of a given cell type. In the example of scaffolding for wound healing, this typically involves generating defined surfaces that promote the specific migration of chondrocytes (i.e., cartilage), osteoblasts (i.e., bone) and the interfacial link between the two cell types.

In this regard, bone and cartilage are connective tissue, which bear the mechanical loads related to body weight and to locomotion. Bone and cartilage are required to form tight contact with each other; an interfacial link of mineralised cartilage is formed adjacent to the bone tissue surface. This layer is crucial for the load transfer between the two different materials. Even though bone and cartilage are distinct in composition, the interface between them is not distinct. Traditional tissue engineering efforts have failed to recreate this interface due to the requirement of multiphasic cellular architecture and associated weight bearing issues (i.e. inferior mechanical properties and stress shielding caused by different mechanical properties at the interface). Accordingly, by identifying surfaces that increase motility of these cell lines a network of desired tissue could be produced.

The present invention is not limited to any particular cells of interest whose migration characteristics are desired to be assayed. Indeed, the use of a variety of cells is contemplated, non-limiting examples of which include: stem cells including, but not limited to, haemopoietic stem cells, neural stem cells, bone stem cells, muscle stem cells, mesenchymal stem cells, epithelial stem cells, endodermal stem cells, pluripotent embryonic stem cells, and pluripotent embryonic germ cells; myoblasts; neutrophils; lymphocytes; mast cells; erythroblasts; osteoblasts; osteoclasts; chondrocytes; basophils; eosinophils; adipocytes; neurons; adrenomedullary cells; melanocytes; epithelial cells; endothelial cells; hepatocytes,; lung cells; renal cell; and precursors respectively thereof; tumour cells, illustrative examples of which include: melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas, brain and testes. Other suitable cells include known research cells and cell lines including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, COS, etc. In some embodiments, the cells form a substantially homogeneous population of cells. In other embodiments, the cells form a heterogeneous culture and include cell types that are known to migrate together, or where one cell type migrates towards another (e.g., cells of the immune system).

In some embodiments, the cells of interest are selected from mesenchymal stem cells (MSC) derived osteoblasts and MSC derived chondrocytes, as well as naive MSCs. MSCs are progenitors of all connective tissue (including bone, cartilage, fat, muscle) and have been isolated from bone marrow and other tissues. MSCs can differentiate into different tissues in an in vitro setting, depending on the culture medium and culture conditions;

• MSC derived osteoblasts- MSC differentiation into osteoblast involves addition of induction/differentiation medium which includes dexamethasone, ascorbate 2- phosphate and β-glycerophosphate.

• MSC derived chondrocytes- The tradition method of MSC-chondrocyte differentiation involves a very dense cell culture system, called micropellets medium supplemented with dexamethasone, ascorbate 2-phosphate and TGF-B a. This can provide information as to which surfaces MSCs are most motile and colonize a scaffold the fastest, while examining whether cell genotype/phenotype authenticity markers are retained on different surfaces (CD 105, CD90 etc.).

In these embodiments, the surfaces chosen to perform an initial screening on the three cell types include, but are not restricted to, ECM molecules Collagen I, collagen IV, laminin and fibronectin. Due to their integrin /ligand binding systems and in vivo ECM synthesis other molecules will be investigated such as, Collagen Type II (articulated cartilage ECM contains 60%), hydroxyapatite (main component within bone).

Ligand density can also be varied as it has been shown previously that maximal cell migration speed will occur at intermediate ratio of cell-substratum adhesiveness. Accordingly, optimal concentrations for individual ligand/integrin combinations can be examined, so true comparison can be made between surfaces. Bound ligand density will be varied by varying the solution concentration between 100 μg/ml - 1 μg/ml.

Using surface characterisation techniques also allows determination of the optimal binding method and surface ligand density identifying the true amount of protein on the surface. Literature has demonstrated that the amount of protein on a surface is a key factor of binding affinity and therefore migration speed. Therefore, in one example, the surface can be modified by adding and then quantifying an amount of protein on the surface (defined concentration or gradient). This has been performed previously by either protein labelled with a radio-isotope, mixing a fluorescence collagen IV product (Oregon Green) sold by molecular probes into the surface of choice and reading the relative intensity or by labelling the surface with a Europium (Eu) or Samarium (Sm) chelate which have unique fluorescence properties.

Accordingly, the above described manufacturing technique allows the apparatus to be manufactured rapidly and cheaply in accordance with a range of different channel geometries and surface characteristics. It will be appreciated however that a wide variety of manufacturing techniques could be used including etching, photolithographic or molding processes, and that the above described examples are not intended to be limiting. In some embodiments, the apparatus and methods are used to identify agonists or antagonists of cell binding to the surface modifying agents, to thereby provide modulators of cell migration. For example, a candidate compound can be added to the medium in a respective channel (e.g., via a corresponding opening) to assay the ability of a cell to migrate. If net migration of the cell is enhanced in the presence of the candidate compound, then the compound is an agonist of cell migration. Conversely, if the candidate compound inhibits or otherwise reduces cell migration, then the compound is an antagonist of cell migration. In these embodiments, it is generally desirable to provide separate reservoirs for each agonist/antagonist experiment, to avoid cross contamination. Whilst this could be achieved using separate apparatuses, in one example, the reservoir 310 can include dividing walls 350, to divide the reservoir into three separate regions 310A, 3 IB, 310C. This avoids cross contamination, whilst allowing the tests to be performed via a single apparatus, which can assist with visual comparison of the results.

Candidate compounds encompass numerous chemical classes. In certain embodiments, they are organic molecules, suitably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons. Candidate compounds will typically comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, suitably at least two of the functional chemical groups. The candidate compounds may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate compounds are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate compounds of interest also include peptide and protein agents, such as antibodies or binding fragments or mimetics thereof, e.g., Fv, F(ab') ₂ and Fab.

Candidate compounds can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

In related embodiments, the apparatus and methods of the present invention are used to identify which molecule(s) (e.g., integrins) on the surface of the migratory cells mediate or otherwise promote migration of the cells on a given surface. For example, an integrin will test positive for cell migration on a surface of interest (i.e., bearing one or more surface modifying agents) if an antibody specific to the integrin is capable of inhibiting migration of the cell on that surface. Conversely, an integrin will test negative for cell migration on the surface of interest if the antibody is incapable of inhibiting cell migration.

In other embodiments, the apparatus and methods of the present invention are used to identify modulators of chemotaxis or fugetaxis (i.e., movement of a migratory cell away from an agent source, for example, towards a lower concentration of agent), to thereby provide agents that modulate directed cell movement. In non-limiting examples of this type, a test compound is introduced into a reservoir adjacent to the opening of a respective channel or provided in the form of a porous or nonporous material that releases the test compound into the channel in a controlled fashion, to provide a gradient of test compound into the channel. If the test compound is chemotactic, the net migration of the cells in the channel will be towards the reservoir port or porous/non porous material. Conversely, if the test compound is fugetaxic, the net migration of the cells in the channel will be away from the reservoir port or porous/non porous material. In these embodiments, it is generally desirable to provide separate reservoirs for each test compound experiment, as shown for example in Figure 3 C, to avoid cross contamination.

In specific embodiments, the test compound is formulated for controlled release in the form of a matrix, which is suitably placed in a location within the channel. Non-limiting examples of which include polymer matrices such as but not limited to chitosan, chitosan-alginate, poly(N-isopropylacrylamide) hydrogels, lipid microspheres, copolymers of polylactic and polyglycolic acid, dextran hydrogels, and poly(ethylene glycol) hydrogels. (See, e.g., Zambito et al. ₅ Acta Technol. Et Legis Medicamenti 14(1):1-11 (2003); Bhopaktar et al., Advances Chitin Sci. 5:166-170 (2002); Zhuo et al., J. Polymer Sci. 41(1):152-159 (2002); Del Curto et al., Proceedings of the 28th Symposioum on Controlled Release of Bioactive Materials, San Diego, Calif., 2:976-977 (2001); Hu et al., J. Drug Targeting 9(6):431-438 (2001); Lambert et al., J. Controlled Release 33(1):189-195 (1995); Hennink et al., J. Controlled Release 48(2,3):107-114 (1997); and Zhoa et al., J. Pharm. Sci. 87(l l):1450-1458 (1998). In some embodiments, the matrix further comprises an extracellular matrix component (e.g., collagen, vitronectin, fϊbronectin or laminin). A variety of test compounds may be provided in the matrix, including, but not limited to, polypeptides, carbohydrates, amino acids, and small organic compounds.

An example of alternative apparatus for use in monitoring cell migration will now be described with reference to Figures 8 A to 8D.

In this example, the apparatus includes a well 800, having well side walls 801 and a well base 802. A body 840 is provided on the base 802, the body 840 including side walls 841 that extend away from the body 840 and well base 802, to thereby define a first reservoir 810 and an annular second reservoir 825. The body also includes multiple channels 820 extending from the first reservoir 810 to the second reservoir 825. The channels include first and second ends 821, 822, where the channels 820 meet the first and second reservoirs 810, 825, respectively. Whilst four channels are shown in this example, this is for the purpose of illustration only, and in practice any number of channels may be provided.

In one example, the first reservoir 810 is a central substantially circular reservoir, and the second reservoir 825 is an annular reservoir positioned radially outwardly from the central reservoir, with the channels 820 extending radially outwardly from the central reservoir to the outer reservoir. However, this is not essential and other suitable arrangements may be used.

In use, the reservoirs 810, 825 are adapted to receive a solution containing cells of interest, and/or media, thereby allowing cell migration along the channels 820 to be performed in a manner similar to that described above. In one example, either or both of the reservoirs 810, 825 may be open, allowing fluid such as cell solution or media to be provided directly into the reservoirs 810, 825, for example using a syringe, pipette, or the like. In another example however, fluid could be introduced into the reservoirs 810, 825 using other suitable techniques, such as introduction via a microfluidic channel or similar. In this arrangement, one or both of the reservoirs 810, 825 would be connected to a corresponding port or other opening via a microfluidic channel, in an arrangement similar to that of the inlet/outlet channels 111 and respective ports 112, described above with respect to Figure IA. This allows fluid to be introduced into the respective reservoir 810, 825 via the corresponding port. It will be appreciated that in this example, the respective reservoir 810, 825 could therefore be closed, which would be achieved for example by having a lid or other similar enclosure in place. Thus, for enclosing the reservoir 825, a lid or other member could extend between the well side walls 801 and the side walls 841, whilst closure of the reservoir 810 could be achieved by a lid or other member extending between the well side walls 801.

In any event, the first reservoir 810 can perform a function similar to the reservoir 110 of Figures IA and IB, whilst the second reservoir 825 can perform a function similar to the openings 125. However, this is not essential, and the process may alternatively be reversed, with cell solution being placed in the second reservoir 825 and media in the first reservoir 810. In a further alternative, cell solution may be placed in both the first and second reservoirs 810, 825, thereby allowing cell migration to occur in both directions along the channels 820. In any event, it will therefore be appreciated that the term opening as used in previous examples could encompass a reservoir.

As in the previous examples, the body 840 and side walls 841 can be formed of any suitable material, such as silicone, PDMS, or the like. The body 840 and side walls 841 may be formed from individual elements coupled together or may be manufactured integrally, for example through a suitable molding process, or the like. The use of the side walls 841 is not essential. However, by providing the side walls 841, this increases the volume of the first and second reservoirs 810, 825, which can assist in the filling of the reservoirs with cell solution and/or media.

In one example, the channels are substantially identical, as shown in Figure 8B. However, this is not essential, and alternatively the channels can have different arrangements thereby allowing comparative studies of the effect of channel arrangement on cell migration to be easily performed. Thus, in one example, the channels 820 can have different geometries, as shown by the channels 820A ₅ 820B, 820C, 820D in Figure 8C. Additionally and/or alternatively, the channels 820 can have different surface chemistries, or the like.

The use of a well 800 for containing the body 840, allows the apparatus to be replicated a number of times on a well plate 850, an example of which is shown in Figure 8D. This allows multiple cell migration experiments to be performed substantially simultaneously, as will be described in more detail below.

In one example, operation of the well based apparatus is substantially as for the apparatus described above with respect to Figures IA and IB. Thus, in this example, a solution containing cells of interest is initially added to the first reservoir 810, with the solution and cells being constrained in the first reservoir 810, as shown in Figure 9 A. Constraint of the cells can be achieved in any suitable manner, for example through selection of suitable material for the body 840, channel geometry, size of opening 821, or through the modification of the physical properties of either the air/solid/media interface or through an agent attached or otherwise immobilised (e.g., by physical adsorption or chemical binding) to the apparatus. In one specific example, this is due in part to the hydrophobic nature of the PDMS body 840.

In any event, constraining the cells results in the formation of a defined cell wave front at the first end 821 of the channels 820. As the cell wave front is only in contact with air at this point it remains a fully defined surface, not modified by cellular secretions. In one example, cells are permitted to attach to the surface inside the first reservoir, such as the base 802, for a set time period, such as 4 hours or overnight, thereby ensuring the cells are suitably constrained prior to commencing cell migration.

Following this, media is added to the second reservoir 825 so that the media flows along and fills the channels 820, as shown in Figure 9B. As the media reaches the first end 821, this causes one or more cells to migrate along the channel from the first end 821 towards the second end 822, in turn allowing the migration process to be monitored. Thus, upon addition of the media to the reservoir 825, the media flows along the channel 820, causing the cells to unbind and commence migration. In an alternative example however, addition of media to the second reservoir 825 may not be sufficient to trigger the migration process. For example, the media can be constrained in the second reservoir 825 until some additional triggering mechanism is initiated. An example of this will now be described with reference to Figures 1OA to 1OE.

In this example, a solution containing cells of interest is initially added to the first reservoir 810, with the solution and cells being constrained in the first reservoir 810, as shown in Figure 1OA. This is substantially as described above with respect to Figure 9 A.

Next, media is added to the second reservoir 825. In this example, the media is constrained in the second reservoir 825, as shown in Figure 1OB, so that the channels 820 remain air filled. This can be achieved using any suitable mechanism, such as through appropriate selection of a size of the opening 822, the hydrophobic nature of the PDMS body 840, or the like.

Once the reservoirs 810, 825 are filled, fluid can be selectively added or removed from the reservoirs 810, 825 to ensure a differential depth d of fluid in the first and second reservoirs 810, 825, as shown in Figure 1OC. It will be appreciated that addition or removal of fluid may not be required if a differential depth exists after initial filling of the reservoirs. However, in one specific example, all excess solution can be removed from the first reservoir 810, thereby leaving only the constrained cells within the first reservoir 810.

The differential height can then be used to initiate the migration event by inducing flow of fluid along the channels. In one example, this is achieved through the use of a centrifuge, arranged to urge the liquids in the reservoir towards the base 802. An example of this arrangement is shown in Figure 1OE. In this example, the wells 800 are coupled to a rotor 1000, arranged to rotate about an axis 1001, in the direction of the arrow 1002, thereby urging fluid in the reservoirs 810, 825 in the direction of the arrows 1003. The differential height of the fluids generates a relative force, thereby urging fluid from the reservoir 825 along the channels 820, displacing the air contained therein. This results in at least the channels 820 being filled with media from the second reservoir 825. In the event that the first reservoir 810 was emptied of excess solution prior to the centrifuging process, the first reservoir 810 will also typically contain media from the second reservoir 820. In any event, once the channels are filled with media, this again initiates a cell migration process.

Accordingly, in the above example, cells of interest are provided into the first reservoir 810 with the cells being retained therein until it is desired to perform a cell migration monitoring procedure. At this point, media is placed in the second reservoir 825, before the migration event is triggered by performing centrifuging of the well, thereby initiating the cell migration process and allowing the migration process to be monitored.

It will be appreciated that this is particularly advantageous when utilising a well plate 850 to allow multiple migration events to be simultaneously monitored across a number of different wells. In particular, this allows the reservoirs 810, 825 of different wells 800 to be appropriately filled without the migration event being initiated. This allows the required conditions for each migration event to be established without timing constraints regarding the provision of fluid to the reservoirs 810, 825, which is particularly helpful if, for example, different media is being used across different wells 800. In any event, once the second reservoir 825 of each well 800 is prepared, migration across each of the wells 800 can be initiated simultaneously by placing the well plate 850 in a centrifuge. This allows a comparative analysis to be performed across multiple wells 800.

It will be appreciated that alternative triggering mechanisms could be used. For example, positive pressure could be applied to the media in second reservoir, for example through the use of a pressurisation system or the like. Alternatively, negative pressure could be applied to the first reservoir to draw fluid through the channel from the first reservoir. Accordingly, the use of the centrifuge is not intended to be limiting. It will also be appreciated that similar triggering mechanisms could also be used with the apparatus described in Figures IA and IB and 3 A to 3 C, and that the description with reference to the apparatus of Figures 8 A to 8D is also not intended to be limiting.

In a further variation, the use of an external trigger to urge media into the channel could be used to allow cell migration to be performed in a modified manner. In particular, in the majority of the above applications, the channels are back filled from the second end, via either an opening or reservoir. However, this is not essential, and alternatively, the channels could be filled by media from the first end. In the arrangement, the first end of the channel is configured to prevent fluid flow along the channel in absence of any urging force. This allows the constrained cells to be established in the reservoir adjacent to the first end of the channel. Following this, media can be placed in the reservoir and then subsequently urged into the channel, for example using the centrifuge technique described above. The relative size of the channel and cells, means that this can be achieved without unduly disturbing the constrained cells, allowing migration to be achieved substantially as described above. It will be appreciated that in order for this to function correctly, the channel would still typically be in fluid communication with an opening, for example at the second end, thereby allowing air in the channel to be displaced by the entering media, thereby ensuring the channel is filled with media.

A number of experiments further describing example methods and apparatus will now be described.

For the purpose of these examples, the reservoir and channel arrangement was designed using CAD and printed on a transparency at a 4800 DPI Resolution. The wafer substrate 720 is pre-treated by rinsing with DI water and IPA, before being baked at 150 - 200 ⁰ C for 15 minutes.

The wafer is then spin coated with SU-8-100 resin to produce a coating of approximately 100 μm. The resin is soft baked to evaporate the solvent and density the resist film ready for exposure, which may be achieved through multiple stage baking, such as baking at 65 ⁰ C for 10 minutes, at 95 ⁰ C for 30 minutes and 65 ⁰ C for 5 minutes.

The wafer is then exposed, for example for five minutes, using an appropriate radiation source. Following exposure a post exposure bake is performed to selectively cross link the exposed portions of film, by baking at 65 ⁰ C for 1 minute, at 95 ⁰ C for 10 minutes and at 65 ⁰ C for 2 minutes.

The wafer is developed by immersion and agitation in PGMEA for 15 minutes, before being rinsed and then dried with a nitrogen gas flow. The PDMS polymer is then applied to the mould. To achieve this, PDMS and PDMS cross linking agent can be mixed at a suitable ratio, such as 10:1, with the mixture then being degassed under vacuum. The mixture is applied to the mould and degassed, before being baked at 80 ⁰ C for 20 minutes.

The PDMS layer is then removed from the mould and any required openings added, for example by cutting the reservoir out of the material. The resulting PDMS body is then mould was then cut and peeled from the SU-8 mould.

The PDMS layer can then cleaned before being bonded to a substrate layer 342, such as a cleaned glass slide to form the finished device.

A surface coating for the device can then be prepared. For investigating cell migration in would healing scaffolds, Ligand Solutions can be prepared and injected into main chamber and migration channels. The solution is typically at 4 ⁰ C overnight or 37 ⁰ C for two hours. The solution is then removed and the apparatus rinsed with PBS, and then subsequently allowed to dry at 4 ⁰ C. If blocking is required, the process can be repeated using a 2% solution of BSA in PBS.

For this example, the cell culture is formed from a high density cell suspension of 500,000 cells/ ml. Medium for NIH/3T3 cells were low glucose DMEM with 10 % serum supreme, while Saos 2 were culture in DMEM/F12 with 10% serum supreme. Cells can be seeded at a density of 1,000,000 cells per reservoir and incubated at 37 ⁰ C, 5 % CO ₂ for 4 hrs.

The cells can also be optionally stained by fixing the cells in a solution of 4% paraformaldhyde, incubated at room temperature for 20 minutes. Cells are then, stained with P.I (1 :20) and incubated at room temperature for 20 minutes.

Once the cells are established, media can be exchanged and cells rinsed with culture medium. Migration channels can then be backfilled allowing the cell wave front to progress down the channel. During this process, the apparatus can be incubated at 37 ⁰ C, 5 % CO ₂ , with the apparatus being removed from the incubator at selected time points to allow cell migration to be monitored. This is achieved by imaging the apparatus on an upright microscope, with the images being analysed using Image J. Channel Geometry

Example experiments investigating the effect of channel geometry will now be described. The cell line chosen for this purpose was NIH-3T3 mouse fibroblast. The following examine different channel sizes, expanding and contracting channels, tortuous channels and channel junctions. These all represent geometric variables present in all TE scaffolds.

When investigating the channel size, apparatus including multiple channel is used, with the channels having a variety of channel widths. In one example, widths were varied from 50μm, lOOμm, 200μm and 500μm. Figure 11 shows, as an example, the view of 3T3 cell migration within the multi-width channel device under a 1Ox microscope objective taken at the indicated time intervals of Ohrs, 6hrs, 12hrs, 30hrs. The results of the 3T3 migration experiment using this device can be seen in Figure 12.

Even though a large range in channel width was investigated, it is apparent that the wave front speed does not vary with channel width. With all rates being statistically equivalent using a standard students T-test at a 95% confidence interval.

All of the migration speeds found within the study were between 9 and 10 um/hr and statistically equivalent. This is not unexpected as when a cell wave front hits the channel, cells encompass the entire width of the channel regardless of size. Therefore when proliferating and migrating each cell along the channel proceeds at approximately the same rate, resulting in the same rate for different channel widths. This could possibly be different with a smaller channel width, more relevant to the width of a cell (for example below 50μm).

Accordingly, a further study was performed to further understand the effect of channel size on cell migration, and in particular to examine channel widths varying from 500μm to lOμm. In this example, the experiment was performed substantially as described above with varying channels widths of 500μm, 200μm, lOOμm, 50μm, 40μm, 30μm, 20μm and lOμm over an untreated glass surface.

The results of the experiment are shown in Figure 13, which highlights that the migration rate is significantly affected by channel width. In particular, from 500μm to 50μm the migration rate is statistically equivalent at approximately 1 Oμm/hr, thereby supporting the conclusions of the earlier study. However, as the cell wave front propagates into smaller channel sizes, the migration rate decreases significantly to 3.66μm/hr, 3.02μm/hr and 2.35μm/hr within channel widths of 40μm, 30μm and 20μm, respectively. This trend continues until a lOμm wide channel, where the cells cannot penetrate into the channel and the migration rate is effectively zero.

Figures 14A to 14D are images of cells migrating within channels having widths of 500μm, 200μm, 30μm and 20μm, respectively. These highlight that within channels having a width of less than 50μm there is a change in cell morphology. Within the smaller channels the fibroblasts lose their traditionally cobble stone morphology and taken on a more elongated phenotype. Therefore, cell size and circularity were investigated further.

Examination of the spreading area within the different channel widths, as shown in Figure 15, highlights that all spreading areas were found to be approximately 600μm ² . However, there were notable differences found in the circularity of the cells within the smaller channels, as shown by the result in Figure 16. In particular, from 500μm to 50μm channels the cell circularity was approximately 0.6, whereas in smaller channels under 50μm the circularity was significantly reduced to approximately 0.4. This is indicative of the change in cell morphology from the standard 'cobblestone' appearance to a more elongated shape. Therefore it appears that the change in cell morphology results in a decrease in migration rate.

It is known from literature that cell migration is a force balance between the force generated at the cells leading edge by extending protrusions and forming adhesions and the force at the rear of the cells, as a result of rear adhesions and cell-cell contact. Accordingly, the above results indicate that within smaller width channels, due to the physical constraint, the cells cannot form multiple leading or lateral protrusions that would usually be formed. This reduces the amount of force that can be generated at the cells leading edge, which in turn results in a decrease in migration rate, as highlighted in Figure 17.

In another example, apparatus was constructed in which channel contractions and expansions were presented to the cells. The apparatus included five different contractions and expansions; channels contracted from 200μm to lOOμm and similarly expansions were from lOOμm to 200μm. This was achieved over a number of lengths to provided different scenarios, including;

• 10 mm - ratio lOμm laterally per lmm forward

• 5 mm - ratio 20μm laterally per lmm forward • lmm - ratio lOOμm laterally per lmm forward

• 500um - ratio 200μm laterally per lmm forward

• 250um - ratio 400μm laterally per lmm forward

Example channel contractions and expansions are shown in Figure 18. Results of the 3T3 migration for contracting channels are shown in Figure 19, with expanding channel results being shown in Figure 20. In both examples, error bars represent the standard deviation of the mean. In this example, all of the cell wave front speeds, whether an expansion or contraction, and over any length, were all statistically equivalent using a student t-test. All rates were between lOum/hr and 11.8 um/hr.

In contracting channels this arises due to the entire channel front being encompassed by cells, with only those without contact inhibitions proliferating. For expanding channels, cells adjacent to the wall spread more to encompass the vacant area, thereby maintaining wave front velocity. It is possible that the expansion is not biologically relevant to the cells due the size of the channel.

Using traditional wound healing scaffold manufacturing techniques often results in scaffolds with significantly varying levels of tortuosity. An example of the effect of tortuous curves on cell migration and the increase in time associated with infiltrating a tortuous channel compared to that of a linear channel will now be described.

In this example, channels are designed with a deviation of 600μm, with the channel deviating 200μm from either side of the 200μm channel, with the period (difference between peaks) of the oscillations changing to present different relevant tortuosities. These include 4mm, 2mm, lmm, 0.5 mm. Figure 21 is a schematic diagram of an example design schematic for apparatus with tortuous channels, while Figure 22 shows photographs of example tortuosities. The results of the NIH 3T3 fibroblast cell migration rates down tortuous channels are shown in Figure 23, with error bars represent the standard error of the mean. In this example, it can be seen that the tortuosities tested did not statistically influence migration rates (95% confidence interval using a student's t-test). However as the tortuosity increases, the path length increases and therefore the cell infiltration into the 'scaffold' decreases. This resulted in approximately 2.5 times the cell infiltration into the 4mm period path compared to the 0.5mm period path.

The 2D tortuous migration provides a general idea of infiltration into a tortuous path; however it is not a true representation of tortuosity within a scaffold, as the time and length scales are vastly different. Tortuosity within 3D dimensional scaffold extends in greater direction with more intricacy; it is simply not possible to replicate the 3D "hair pin" morphology of many scaffold pores in a 2D model, and accordingly, alternative arrangements of 3D channels may be used.

Within a conventional scaffold there will be multiple junctions between channels. Accordingly, an example of the effect of a junction or an intersection of multiple channels on cell wave front movement will now be described.

For the purpose of this example, devices are fabricated with channels having perpendicular (90 degree), 45 degree, 30 degree and 180 degree (full turn) junctions. Figure 24 shows photographs of example junction configurations.

Straight line migration was analysed before, within and after cells passed through the channel. Figure 25 is an example of NIH 3T3 fibroblast cell migration rates down channel junctions, with error bars represent the standard error of the mean.

Although all rates are statistically equivalent to that recorded in previous experiments, there was an array of different responses with various cell wave fronts reacting differently to the junction. In some examples, once cells were within the junction, they would simply expand radial as seen in Figure 26 A. However, in some channels the junctions were ignored and cells expanded directly down the channel as shown in Figure 26B, while in other channels cells expanded laterally proceeding down the channel in a single wave front as shown in Figure 26C. Accordingly, the geometries described above, there is little influence of geometry on cell migration, with migration rates being between 9-12μm/hr. However, for other channel dimensions, such as those in which the feature size/cell size ratio is comparable to that of a cell, then additional effects will occur. To achieve this, a more advanced manufacturing technique could be used, such as a chrome mask, allowing geometric features on the scale of a cell, approximately l-30μm. Additionally, analysis into tortuous channels and channel junctions can be performed in real-time scale to fully analyse the cell behaviour.

Surface Chemistry

Surface chemistry is important for cell attachment, growth, motility and therefore colonisation. A number of examples of the impact of surface chemistry will now be described.

In one example, the apparatus is used to examine the cell wave front migration rates of two cell lines over various surfaces. Cell lines chosen were NIH-3T3 mouse fibroblast and Saθs2 human osteosarcoma. Surface chosen were: 1. Untreated Glass (positive control) - glass was cleaned using a piranha solution

(hydrogen peroxide and ammonia) to remove any organic residue off substrate.

2. Bovine Serum albumin (BSA) (negative control) - Albumin is the most abundant plasma protein in mammals, it serves as a blood osmotic pressure regulator. It is commonly used in science for its binding properties, mammalian cells do not have integrins for albumin and therefore cells can not adhere to it.

3. Collagen Type I - Collagen type I is one of the major ECM molecules within mammals.

4. Collagen Type I backfilled with BSA- BSA due to it relatively small size is used to fill empty sites on the surface so FBS protein within media do not adsorb. 5. Collagen Type IV - Collagen type IV is a main component within the basal membrane.

6. Collagen Type IV backfilled with BSA.

7. Foetal Bovine Serum (FBS) - FBS is commonly used in cell culture to maintain cells so they can survive, grow and divide. It contains a rich variety of proteins including many ligands such as fibronectin. 8. FBS backfilled with BSA

Figure 27 is an example of 3T3 cell migration within example apparatus under a 1OX microscope objective, with the results of NIH 3T3 fibroblast and Saθs2 human osteosarcoma migration on functionalised surfaces within the apparatus being shown in Figures 28 and 29 respectively.

In these examples, the highest NIH 3T3 fibroblast migration rate of approximately 11 μm/hour was observed on surfaces composed of untreated glass, collagen I, FBS and FBS blocked with BSA (all statistically equivalent using a student's t-test, 95% confidence interval). The collagen IV surfaces generated an intermediate migration rate, which differed from any other surface in this study (P<0.05). Surfaces treated with BSA or collagen I or IV surfaces blocked with BSA produced the slowest migration rates, of approximately 5 μm/hour (all statistically equivalent using a 95 % confidence interval student t-test).

Overall, recorded cell migration rates for NIH 3T3 fibroblast are generally slower than those quoted in the literature. For example, NIH 3T3 cells have been previously observed to migrate at rates of 40 μm/hour, while mouse embryonic fibroblasts have been shown to have a migration rate of approximately 50 μm/hour, as described for example in Wang, H.B., et al., Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(20): p. 11295-11300 and Webb, K., V. Hlady, and P.A. Tresco, Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage- dependent cells on model surfaces. Journal of Biomedical Materials Research, 2000. 49(3): p. 362-368.

By contrast, the observed migration rate of approximately 11 μm/hour is due to the cell wave front velocity emanating from a confluent cell monolayer in the apparatus, while the above previously observed rates are from observing cell migration in a non confluent and uninhibited environment (i.e. individual cells moving).

From Figure 29, it is notable that the maximal migration rates for SaOs ₂ cells were observed on surfaces having compositions which were either untreated glass, collagen I, collagen IV, collagen IV blocked with BSA, FBS and FBS blocked with BSA (all statistically equivalent). Migration rates were reduced relative to these surfaces when on surfaces treated with either BSA or collagen I blocked with BSA (P<0.05). It should also be noted that Collagen I was statistically different to Collagen IV and Collagen IV blocked with BSA. Interestingly, blocking collagen IV surfaces with BSA did not inhibit Saθs2 migration in the same way that it did with NIH 3T3 cells.

The maximal migration rate noted for SaOs ₂ human osteosarcoma was approximately 5.5 μm/hour, in contrast to the 11 μm/hour observed for the NIH 3T3 fibroblasts. This contrast is not surprising given that the doubling time for the SaOS2 cells is approximately 43 hours while the NIH 3T3 doubling time is approximately 20 hours. However as the doubling time is an important part of cell migration this and other migration characterisations will be discussed in more detail below.

To assess the effect of cell size on cell wave propagation, manual cell measurements were performed on each surface within the apparatus. Figure 30 shows the spreading area of NIH 3T3 Fibroblast and Saθs2 Osteosarcoma cell lines on various surfaces. It can be seen that the cell spreading area follows the same trend as the cell wave front speed. Slower rates are seen to correspond with the smallest spreading area, intermediate rates with intermediate spreading area and faster rates corresponding to the largest spreading area. It was determined that cell spreading area was equivalent for both cell types on surfaces having compositions of Collagen I, Collagen IV, FBS and FBS blocked with BSA. 3T3 fibroblast cells were spread significantly more on untreated, BSA and Collagen I blocked with BSA surfaces while SaOs ₂ osteosarcoma cells spread significantly more on Collagen IV blocked with BSA.

Furthermore, circularity varied between the surfaces in a manner that was reflected by the speed of migration. Cells cultured on substrates that resulted in slower migration were more rounded, while cells cultured on substrates that resulted in faster migration were more elongated (results not shown).

If cell migration rate was assumed to be solely a product of surface area and proliferation rate (i.e. if cells are assumed immobile, velocity = surface area x proliferation rate), the wave speed velocity would be around a factor of 6 lower than that actually observed. Clearly this mechanism alone is insufficient to yield the observed velocity and would indicate that cell mobility is necessary to describe cell wave front movement.

Mathematical modelling of cell migration is well established and is essential to understanding experimental migration. Fisher's equation (Equation 1.1) has been used extensively to represent experimental data from wound healing assays. This equation describes the behaviour of a cell population as a combination of random cell motion and logical proliferation (proliferation up to a maximal density). Fisher's equation predicts that, under certain conditions the cell migration front takes the form of a travelling wave of fixed shape and constant velocity .

The first term represents cell migration and the second term logistical growth, defined as reduced growth as the cell population increases, where c (cells. cm ^"2 ) is the cell density, D (cmls ^"1 ) is the random motility coefficient (diffusion coefficient), R (s ^"1 ) is the unrestricted growth rate and C _max is the maximum cell density at confluence. As proliferation rate is a foremost aspect of cell migration, the doubling time of NIH 3T3 fibroblasts and SaOS2 human osteosarcoma on the different surfaces was experimentally determined.

As seen in Table 1, which shows experimentally determined doubling times of 3T3 fibroblasts and Saθs2 osteosarcoma cell lines on various surfaces (n =3), all doubling times of the individual cell lines were statistically equivalent to each other (students t-test - 95% confidence) and similar to that published within literature, with the doubling time for the Saθs2 cells being approximately 39 hours while the NIH 3T3 doubling time was approximately 21 hours.

Table 1

Surface NIH-3T3 Doubling Time (Hrs) SaOs 2 Doubling Time (Hrs)

Untreated 21.4 ± 1.7 37.8 ± 4.4

BSA 23.3 ± 1.9 40.1 ± 4.9

Collagen I 22.1 ± 2.1 39.4 ± 2.4

Collagen I W/BSA 21.0 ± 1.5 39.1 ± 3.7

Collagen IV 20.9 ± 1.9 38.0 ± 4.4 Surface NIH-3T3 Doubling Time (Hrs) SaOs 2 Doubling Time (Hrs)

Collagen IV W/BSA 19.3 ± 1.8 37.1 ± 2.8

FBS 20.3 ± 0.5 40.2 ± 2.9

FBS W/BSA 22.6 ± 2.0 38.6 ± 2.7

Average 21.4 ± 2.8 38.8 ± 3.1

Random diffusion coefficients can be estimated based on measured cell wave front velocities, measured proliferation rates and assuming half confluence (similar to that published within literature). Following the mathematical progression performed by Takamizawa et al and Maini et al, this approximates to: V = 2. ^y ]Dλ (1.2) where v is cell wave front velocity (cm.s-1) and λ = In2/R.

The calculated values are tabulated below in table 2, which shows experimentally determined estimates of the diffusion coefficient for 3T3 fibroblasts and Saθs2 osteosarcoma cell lines on various surfaces.

Table 2

Surface NIH-3T3 Estimated Diffusion Saos 2 Estimated Diffusion Coefficient (Cm ² S ^'1 ) Coefficient (Cm ² S ^'1 )

Untreated 2.5 ± 0.2 x 10 ^"y l.O ± O.l x 1O* BSA 0.8 ± 0.3 x lO ^"9 0.4 ± 0.1 x l0 ^"9 Collagen I 2.6 ± 0.2 x l0 ^"9 0.8 ± 0.1 x l0- ⁹ Collagen I W/BSA 0.5 ± 0.01 x 10 ^"9 0.3 ± 0.7 x l0- ⁹ Collagen IV 1.6 ± 0.2 x 10 ^"9 1.4 ± 0.2 x 10 ^"9 Collagen IV W/BSA 0.6 ± 0.1 x 10 ^"9 1.7 ± 0.3 x 10 ^"9 FBS 2.6 ± 0.2 x lO- ⁹ 1.0 ± 0.2 x l0- ⁹ FBS W/BSA 2.5 ± 0.4 x IQ ^'9 0.8± 0.2 x l0 ^'9

Estimated motility coefficients derived for both cell lines correlated well with values from literature for similar cell lines and surfaces. For example, neonatal rat osteoblasts on various peptide motif (RGDS, RDGS) modified glass substrates have been reported to have diffusion coefficient ranging from 1.22-2.33 xlO ^"9 Cm ² S ^"1 . Difference in cell motility on different surfaces using human peritoneal mesothelial cells have also been established. A diffusion coefficient of 4.17 xlO ^"9 cmV ¹ on an untreated surface was estimated while on a collagen IV surface it was nearly double that at 9.18xlO ^"9 Cm ² S ^"1 . It should be noted that diffusion coefficients an order of magnitude higher have been reported on glass, typically using the highly motile Leukocyte cell lines.

3T3 cells within the current study showed higher diffusion coefficient compared with the SaOs 2 cells. The diffusion coefficents follow the same statistical trend as previous migration rates. This shows definitively that 3T3 cells diffuse/migrate fastest over untreated glass, collagen I, FBS and FBS blocked with BSA, intermediate coefficient are produced on collagen IV and slowest values on surfaces treated with BSA or collagen I or IV surfaces blocked with BSA. While SaOs 2 cells again diffused fastest on surfaces having compositions which were either untreated glass, collagen I, collagen IV, collagen IV blocked with BSA, FBS and FBS blocked with BSA with values reduced on surfaces treated with either BSA or collagen I blocked with BSA. This demonstrates that statistically different migration surfaces can be produced within the apparatus and methods described above.

The observed cell migration rates were not always consistent with literature results, for example, the effect of BSA blocking on collagen as a migration surface. These unexpected results may arise due to the stability and composition of the tailored surfaces. As described previously, surfaces were generated by adsorbing matrix proteins onto a glass substrate. This method, which is the most commonly utilised for functionalizing surfaces with proteins, relies on the electrostatic attraction between the protein and substrate. If this attraction is weak it may be possible for proteins contained in culture medium to displace, or competitively desorb these matrix proteins and redefine the surface. It is also possible for proteins in the culture medium to bind on top of or in gaps formed by the adsorbed matrix proteins, thus providing an alternative cell attachment surface.

To provide insight into the nature of surfaces and possible confounding affects associated with serum contained in the medium, a cell attachment/spreading assay was performed in the absence and presence of serum. Surfaces were generated as described previously and cells cultured on these surfaces for 4 hours.

The results shown in Figure 31 are for NIH 3T3 fibroblast attachment to surfaces of BSA, collagen I, collagen I blocked with BSA, collagen IV and collagen IV blocked with BSA. Cell attachment and spreading was performed using serum containing and serum-free medium. Observation suggests that the presence of serum significantly enhances cell attachment and spreading. However, blocking with BSA is not effective in preventing the binding of serum proteins to the surface and the subsequent attachment of cells. Serum proteins are either displacing BSA or binding to the BSA monolayer in such a way as to enable cell attachment. In either event, this demonstrates that if such studies are not performed in either serum-free medium or using a non fouling surface in conjunction with covalently bound ligands, then the presence or absence of serum proteins may have a significant impact on the observed biological outcome rather than the intended ligand.

A further study was then performed to examine migration mechanisms of Mesenchymal Stem Cells over a variety of defined surfaces.

Human mesenchymal stem/stromal cells (hMSCs) are an adult stem cell population that can be readily differentiated into cells such as osteoblasts, chondrocytes and adipocytes. This capability and their relative ease of isolation and expansion in culture make them a promising cell source for tissue engineering. However, cell colonisation of tissue engineered constructs has been problematic. The interaction between the construct surface and cell is paramount in encouraging specific cell growth and migration.

Accordingly, the device outlined above was modified to provide channels having surfaces functionalised with extracellular matrix (ECM) proteins (collagen I, collagen IV ₃ fibronectin and laminin).

In this study bone marrow derived hMSCs characterised by CD29+, CD44+, CD49a+, CD73+, CD90+, CD105+, CD146+, CD166+, CD34- and CD45- cells and tri-lineage potential (adipocyte, chondrocyte and osteoblast) were used. The surfaces investigated were based on a non-fouling hylaronic acid / chitosan multilayer, using a polyethylene glycol (PEG) linker to attach a specific protein, including collagen I, collagen IV, fibronectin and laminin at 50μg/ml. A schematic representation of the functionalised surface is shown in Figures 33B and 33C, with images of the cells being shown in Figure 33A.

Integrin expression was analysed using flow cytometry and results displayed are the average of 3 hMSCs donors. Figure 32A shows the resulting hMSC migration rates, whilst Figure 32B shows the population doubling times (PDs) on the various ECM functionalised surfaces. All migration rates were statistically equivalent, at a rate of approximately 16 μm/hr, with the exception of fibronectin which was significantly slower, at a rate of 9 μm/hr (ANOVA- p<0.05). hMSC PDs were significantly lower on fibronectin (50 hrs) and greatest on laminin (70 hrs), with all other surfaces being statically equivalent (PD of approximately 60 hrs). It is known that migration rates are directly related to ligand-integrin interactions and accordingly, hMSCs migration mechanisms over fibronectin were further investigated through characterisation of the integrin expression of hMSCs.

In particular, Fibronectin interacts predominantly through integrin sub-units αv, βi and β ₃ ; as hMSCs express high levels of P ₁ and αy, as shown in Table 3 and Figure 34, which show mesenchymal stem cell integrin expression and flow cytometry plots ranked between - and ++++ expression,, respectively.

Table 3

Accordingly, these particular sub-units were further investigated through antibody blocking experiments. Cell migration rates, PDs and cell attachment were analysed with the aforementioned integrin sub-units blocked.

Figure 35 A shows the results of Integrin blocked hMSC migration rates on fibronectin over 48 hrs (n=18, 22, 20), where n is the number of channels measured. Figure 35B shows an attachment percentage of integrin blocked hMSCs, (n=3), whilst Figure 35C shows the population doubling times of integrin blocked hMSCs on functionalised surfaces, (n =3).

These results highlight that with P ₁ blocked hMSC migration rates were significantly higher (ANOVA- p<0.05) than those with αy blocked (or control cells), at a rate of approximately 15μm/hr compared to lOμm/hr. Cell attachment was consistent with migration results: 70% of control cells and αy blocked cells attached after 4 hrs, whereas only 40% of βi blocked cells attached (ANOVA- p<0.05). In contrast, PDs were equivalent over all conditions, remaining unchanged at approximately 50 hrs.

Thus, these results indicate that P ₁ rather than αy is the principle integrin sub-unit to facilitate attachment of hMSCs to fibronectin coated substrates.

In any event, this demonstrates the ability of the above described cell migration apparatus to test the impact of surface chemistry on cell migration.

The above described studies were performed using apparatus similar to that shown in Figures 3A to 3C. Accordingly, a further study was performed using apparatus similar to that shown in Figures 8 A to 8D.

In this example, devices were fabricated having channel widths of 100 μm and 200 μm, and heights of 100 μm. Channel molds were formed using standard SU8 and silica wafer methods. The body 840 was formed from a 1.5 mm thick layer of PDMS, made over the channel mold, with the side walls 841 being provided using a plastic tube. Apparatus was formed in each well of a 24 well plate, which was then sterilized with 70% ethanol and washed with sterile water.

In this study, evaluation of B16 cells surfaces were pre-treated with 300 μl of 50 μg/ml fibronectin, collagen I or collagen IV (all human, sourced from Becton Dickinson, Franklin Lakes, NJ) or with 100 μg/ml bovine serum albumin (BSA) (Sigma- Aldrich, Sydney, Australia). Control surfaces had not been previously coated with any protein. The culture medium was high glucose DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% foetal bovine serum (Invitrogen). Cells were seeded at 200,000 cells per ml in 20 μl aliquots. Devices were placed in a 5% CO ₂ incubator and cells permitted to attach to the surfaces for 4 hours. Once cell attachment was confirmed, 300 μl of culture medium was placed into each second reservoir 825 and the majority (~80%) of medium removed from the first reservoir 810. Devices were centrifuged for one minute at 1000 x G to backfill the channels.

Figure 36A is an image of a single well, with Figure 36B showing the channels having a fluidic connection to the first reservoir following centrifugation.

Figures 37A to 37D show the progression of the cell wave front down the channels in such a device at 0, 24, 48 and 72 hours, highlighting that cell migration follows a predictable and reproducible pattern within the device.

In Figure 38 the results of cell wave front propagation rate on various surfaces and with or without foetal bovine serum (FBS) in the medium is considered. This highlights that the migration rate is dependent on the surface and the presence of FBS over the first 12 hours.

However after 24 hours cell wave fronts move at a similar rate on all surfaces when FBS is present in the medium. In the first 12 hours cell wave front propagation is significantly (p <

0.01) reduced on bovine serum albumin (BSA) surfaces and in the absence of soluble FBS in the medium. At 24 hours the absence of FBS in the medium noticeably compromises the health of the Bl 6 cells, and at 48 hours many cells in the FBS-free medium appear to longer be viable.

Cell migration appears to be slower over the first 12 hours, but this is likely an artefact of the addition of cool medium to the culture system during the channel backfilling process.

In any event, this highlights the ability of the apparatus of Figures 8A to 8E to allow cell migration to be accurately and easily monitored.

In particular, the use of the centrifugal backfilling method allows the establishment of a confluent cell monolayer in the first reservoir, and the subsequent establishment and controlled release of a cell wave front at the microchannel entrance. This method allows the investigation of cell migration rates on non-fouled surfaces.

The results indicate that migration rates ultimately (after >24 hours) are similar, which is an indication of how critical it is to maintain a defined surface prior to the cell migration event (especially when the culture medium contains serum). The results, which include plus or minus serum, can also be considered as an example of the assessment of soluble factors in the device.

In a further variation, the channels can be filled with a gel, and through the use of a chemo attractant in either the first or second reservoir, this can be used to establish a soluble gradient through the channel. This allows for the observation of cells moving through a tissue-like environment in response to a soluble signal.

An example of this is shown in Figure 39. In this example, the apparatus 3900 includes a well body 3901 defining a first reservoir 3910 and a body 3940 defining a second reservoir 3925. In this example, multiple channels 3920 are provided extending through the body 3940 between the first reservoir and second reservoirs 3910, 3925.

In use, the cell solution is placed in one of the first and second reservoirs 3910, 3925, with the chemo attractant being provided in the other reservoir 3925, 3910, thereby establishing a chemotactic gradient within gel provided in the channels 3920. As shown, in this example, the channels 3920 are provided in groups of the three channels, with each group of channels having a respective length. This is not essential, but advantageously allows different chemotactic gradients to be established between the different groups of channels 3920. This allows a comparative study of the impact of chemotactic gradients on cell migration to be performed.

It will be appreciated from the above, that the cell migration apparatus and method can be used for the analysis of surface chemistry effects on cell migration, and in particular to generate statistically different migration surfaces.

Thus, the above described cell migration apparatus and method provide a cheap device that is easy to design and manufacture and that allows a range of different influencing factors in cell migration to be analysed.

In particular, the cell migration apparatus is particularly suited to investigating cell migration properties in would healing scaffolds, and preliminary experiments highlight the ability of the apparatus to identify factors influencing cell migration in such circumstances. However, it will be appreciated that the apparatus and method could be used in any situation in which it is desired to investigate factors influencing cell migration.

Persons skilled in the art will appreciate that numerous variations and modifications will become apparent. All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope that the invention broadly appearing before described.