BACKGROUND TO THE INVENTION Field of the invention

The present invention relates to cell seeding methods, cell seeding devices and cell suspension shaping covers for use in cell seeding devices. Related art

Methods of cell seeding are used to apply cells to a substrate for attachment. Known methods of cell seeding include the "sessile drop method" and "open cell seeding" (also known as "free seeding").

The sessile drop method involves placing discrete drops of suspension of cells and media in a set location on the surface to be seeded, typically the centre of the surface to be seeded. The open cell seeding method involves slowly pipetting a suspension of cells and media onto a surface to be seeded, such as a petri dish containing a quantity of media, and allowing the cells to settle and attach to the substrate.

The present inventors have found that the open cell seeding method can lead to local variations in the density of the cells in the suspension on the substrate due to nonuniform density of the cells in the suspension of cells and media held in a pipette before the suspension is pipetted onto the cell seeding surface. These local cell density variations lead to a high standard deviation in cell distribution across the seeded surface. To try to minimise the impact of local variations in cell density across the cell seeding surface, when using the open cell seeding method an operator may move the cell seeding surface in a north-south and then east-west direction after the suspension is deposited on the cell seeding surface. Although this method may improve the uniformity of the density of the cells across the centre of the substrate, it causes cells to collect around the edge of the substrate and therefore still leads to a high standard deviation in cell distribution across the seeded surface. The sessile drop method also results in a high variation in cell distribution across the seeded surface due to the shape of droplets (deeper in the middle than at the sides) causing a higher density of cells in the middle of the droplets which causes aggregation of cells on the seeded surface.

Therefore, both of these known methods of cell seeding have the disadvantage of lack of control of cell distribution across the seeded surface. When it is desired to seed cells on hydrophobic surfaces, in practice only the sessile drop method can be used, because locating larger volumes of the cell suspension (as would be used for open cell seeding) on the surface to be seeded results in the cell suspension de-wetting off of the surface to be seeded, in some cases the de-wetting can be strong enough that the substrate floats to the top of the cell suspension. The sessile drop method overcomes these problems of the open cell seeding method, but has the problem of producing surfaces with an extremely non-uniform distribution of cells due to the high contact angle formed between the cell suspension and the hydrophobic surface. SUMMARY OF THE INVENTION

The present inventors consider that improved control over the seeding of cells across a cell seeding surface is of great importance for various applications. For example, in order to test differential adhesion across a surface having one or more gradients of various surface parameters such as chemical surface parameters or topographical surface parameters, it is important to provide a method allowing improved control over the seeding of cells across the surface.

The present invention aims to address at least one of these problems. The present invention seeks to improve control in the distribution of cells across a seeded surface. In particular, embodiments of the present invention seek to reduce the standard deviation in cell distribution across a seeded surface and increase the likelihood that the cells adhere evenly across a surface. In a general aspect of the invention, the present inventors have found that it is advantageous to shape the volume of the cell suspension applied to the surface to be seeded. In a first aspect the present invention provides a method for seeding cells at a cell seeding surface, including the steps:

(i) providing a cell seeding surface;

(ii) providing a flowable cell suspension;

(iii) locating a cell suspension shaping cover above the cell seeding surface to form a shaped space between the cell suspension shaped cover and the cell seeding surface; and

(iv) flowing the cell suspension into the shaped space,

so that the cell suspension is in contact with the cell seeding surface, the depth of the cell suspension across the cell seeding surface being defined and constrained by a corresponding depth of the shaped space.

The present inventors have found that this method of seeding cells allows the shape of the volume of cell suspension contacted with the cell seeding surface to be controlled by the shaped space formed between the cell suspension shaping surface and the cell seeding surface, this in turn provides improved control of the cell distribution across the cell seeding surface. The shape of the volume of the cell suspension contacted with the cell seeding surface may be selected to provide the desired cell distribution across the cell seeding surface. For example, to provide a uniform cell distribution across the cell seeding surface the cell suspension provided to the cell seeding surface should be constrained to have a constant depth across the cell seeding surface. For alternative cell distributions, e.g. graduated cell distributions, a corresponding depth distribution of the cell suspension can be provided by a suitable shaped space using the cell suspension shaping cover. Using the cell suspension shaping cover to form a shaped space for constraining the shape of the cell suspension also dramatically improves the distribution of cells seeded on a hydrophobic surface compared with the methods of the prior art. This is because the shaped space formed between the cell suspension shaping cover and the cell seeding surface forces the cell suspension to take the shape it defines, preferably a shape with constant height across the cell seeding surface, which prevents the cell suspension running off the hydrophobic surface, the substrate swimming (or floating) to the top of the cell suspension or droplets with a high contact angle from being formed.

Furthermore, this confinement of the cell suspension improves the stability of the flowable cell suspension. This improved stability reduces the impact of movement of the cell seeding surface on the distribution of cells formed when the seeded cells attach to the seeded surface.

Optional features will now be set out, which may be applied, singly or in any combination, to the first aspect of the invention.

Preferably, step (iv) is carried out while the cell seeding surface is held substantially horizontal. It is preferred that during step (iv), the cell suspension flowing into the shaped space displaces gas (typically air but optionally another gas such as an inert gas) from the shaped space. Preferably, at least one vent means is provided. This may be provided between the cell suspension shaping cover and the cell seeding surface or the cell seeding surface support set out below. The vent means preferably provides a route for the displaced gas to leave the shaped space during step (iv).

It is noted that the present invention is considered not to be a bioreactor, as that term it understood in this technical field. Bioreactors typically require that a cell culture medium (liquid) is filled into a defined space and then a cell suspension is injected into the cell culture medium.

Preferably the cell suspension shaping cover is located above the cell seeding surface to define a shaped space with substantially constant depth across the surface of the cell seeding surface. As indicated above, in alternative embodiments, a non-uniform depth may be selected for the shaped space in order to provide a corresponding nonuniform (but controlled) cell distribution.

Optionally the cell seeding surface is supported on a cell seeding surface support. For example the cell seeding surface support may be an open dish such as a petri dish. When a cell seeding surface support is provided the cell seeding surface support may, along with the cell seeding surface and the cell suspension shaping cover, assist in retaining the cell suspension in the shaped space. The cell suspension may be held within the shaped space due to the surface tension of the cell suspension, which leads to a physical confinement of the cell suspension by the shaping cover and the cell seeding surface.

Optionally the cell suspension shaping cover is positioned a predetermined distance above the cell seeding surface. This may be done by, for example, providing the cell suspension shaping cover with at least one support member or providing a cell seeding surface support with at least one support member to support the cell suspension shaping cover above the cell seeding surface. Positioning the cell suspension shaping cover a predetermined distance above the cell seeding surface allows a shaped space with a predetermined depth to be formed. This in turn allows control over the cell distribution across the cell seeding surface.

Optionally the shaped space formed between the cell suspension shaping cover and the cell seeding surface defines a volume with a constant depth not greater than 3 mm, more preferably not greater than 2 mm, more preferably not greater than 1.5 mm and most preferably not greater than 1 mm.

The present inventors have found that the uniformity of cell distribution on a seeded surface can be improved by using a cell suspension shaping cover positioned to form a shaped space with a depth of 3 mm or less. Suitably the shaped space formed between the cell suspension shaping cover and the cell seeding surface defines a volume with a constant depth of not less than 0.1 mm, more preferably not less than 0.5 mm. The present inventors have found that it can be difficult to prepare a seeded surface if the depth of the shaped space is less than 0.5 mm. Therefore, a depth of 0.5 mm or more is preferred.

Suitably the shaped space formed between the cell suspension shaping cover and the cell seeding surface defines a volume with a substantially constant depth in the range of 0.1 mm to 3 mm, more preferably 0.5 mm to 3 mm, more preferably 0.5 to 2 mm, still more preferably 0.5mm to 1.5 mm and most preferably 0.5 to 1 mm across the surface of the cell seeding surface. In embodiments where the shaped space formed between the cell suspension shaping cover and the cell seeding surface defines a volume with a non-uniform depth, preferably the depth of the shaped space still lies in the range of 0.1 mm to 3 mm, more preferably 0.5 mm to 3 mm, across the surface of the cell seeding surface.

Preferably the method for seeding cells at a cell seeding surface comprises the step of (v) removing the cell suspension shaping cover. Suitably step (v) is carried out after cell adhesion of the cells to the seeded surface is complete. Step (v) is preferably carried out after at least 30 minutes after step (iv). Suitably step (v) is carried out after at least 1 hour after step (iv). Step (v) is suitably carried out after not more than 12 hours after step (iv), suitably after not more than 6 hours after step (iv). Removal of the cell suspension shaping cover allows free diffusion, for example, of oxygen and carbon dioxide to and from the attached cells which allows improved cell growth.

Preferably the step (iv) of flowing the cell suspension into the shaped space includes injecting the cell suspension into the shaped space. Suitably the cell suspension is injected through an aperture in the cell suspension shaping cover. Alternatively, the cell suspension may be injected through a space formed between the cell suspension shaping cover and the cell seeding surface.

Suitably the volume of the shaped space is selected to require minimum liquid handling, e.g. a volume that can be inserted into the shaped space in one attempt using a pipette or another injection device such as a syringe. The injection of the cell suspension into the shaped space or the provision of an aperture through which cell suspension may be delivered into the shaped space also limits the amount of liquid handling necessary.

Preferably the volume of cell suspension introduced into the shaped space is less than or equal to the volume of the space between the cell seeding surface and the cell suspension shaping cover (i.e. the shaped space). Over-filing of the shaped space can lead to cell suspension spilling out of the shaped space which may cause wicking of cell suspension from within the shaped space. This wicking of cell suspension out of the shaped space may result in a non-uniform depth of cell suspension across the cell seeding surface which leads to non-uniform distribution of cells across the seeded surface and a reduction in the number of cells seeded on the surface as the suspension flows outside of the intended area of confinement. The "volume of cell suspension introduced into the shaped space" referred to here is intended to define the total volume of cell suspension flowed into the shaped space during step (iv).

Preferably, during step (iv), the flow of cell suspension into the shaped space is substantially non-turbulent. This assists in uniformity of cell seeding because turbulence in the flow of cell suspension is considered to be deleterious to achieving uniformity of cell seeding.

Preferably the aperture has a diameter of 5 mm or less, preferably smaller than 3mm, more preferably smaller than 2mm and still more preferably smaller than 1 mm.

Suitably the diameter of the aperture is designed for compatibility with labware, for example a 100μΙ to 1 ml pipette tips.

Optionally the aperture has a diameter not less than 0.5 mm.

Optionally the aperture in the cell suspension shaping cover is tapered towards the shaped space in use. Optionally the aperture is configured to form a Luer fitting with a complimentary injection device, for example the aperture may be configured, e.g. threaded, to screw into a complimentary injection device. Optionally the cell suspension shaping cover is connected to an injection device.

The cell seeding surface may be a device, e.g. a medical device, or a structured or any other flat surface suitable for cell growth.

Suitable cell seeding surfaces may have topographic or chemical heterogeneities that influence local surface wettability such that even seeding would be difficult to achieve otherwise. Examples of suitable cell seeding surfaces having topographic

heterogeneities are the surfaces described in WO 2010/094944. It is also possible to use cell seeding surfaces with micron-scale topographical features as structured cell seeding surfaces. Suitably the cell seeding surface is a flat surface with a surface height variation (e.g. Rmax) of less than 200 μηι. Therefore the structured surfaces referred to above are considered to be flat cell seeding surfaces. Optionally the cell seeding surface is a smooth surface with substantially no topographical features. Alternatively, where the cell seeding surface is not flat, the cell suspension shaping cover is preferably shaped to counter the curvature or shaping of the surface to create a shaped space with a constant depth across the cell seeding surface.

The cell seeding surface can be any surface on which cells may be seeded. Optionally the cell seeding surface comprises a biocompatible material, for example

polycarbonate, polymethylmethacrylate, or poly ε-caprolactone. However, other biocompatible polymers may be used. Furthermore, other biocompatible materials such as metals and ceramics may also be used.

The cell suspension shaping cover may be made of any material. In some

embodiments the cell suspension shaping cover is made of a permeable material to allow oxygen to reach the cell seeding surface.

Suitably the cell seeding surface has an area of at least 1 mm ² . Cell seeding surfaces with an area less than 1 mm ² may be difficult to handle. Optionally the cell seeding surface has an area of at least 1 cm ² . Suitably the cell seeding surface has an area of 300 cm ² or less, optionally an area of 75 cm ² or less. This upper limit is due to the mechanical stability of the cell suspension shaping cover and the substrate. Above this limit the depth of the shaped space may vary across the cell seeding surface which results in non-even seeding across the surface.

The flowable cell suspension is preferably an aqueous solution containing cells to be seeded. Suitably the cell suspension comprises a suitable cell culture media. A suitable cell culture media is a solution that allows the particular cells in the cell suspension to survive and adhere. Optionally the cell culture media comprises serum and/or growth factors. The cell suspension may contain any type of cells for seeding on a substrate. Suitably the cell suspension comprises cells other than human embryonic stem cells. Optionally the cell suspension comprises self-signalling cells such as immune cells or stem cells. It has been observed that the cell density can be used to control cell response of self-signalling cells (Lu, H., Guo, L, Wozniak, M. J., Kawazoe, N., Tateishi, T., Zhang, X., & Chen, G. (2009), "Effect of cell density on adipogenic differentiation of mesenchymal stem cells", Biochemical and Biophysical Research Communications, 38/(3), 322-327 doi: 10.1016/j.bbrc.2009.01.174). The method and device of the present application allows the growth of self-signalling cells and the cell responses to be better investigated. The present inventors have found that the method and device of the present application allow for easy repetition of this work. The improved homogeneity of cell seeding provided by the method of the present invention has been observed to affect the ability of stem cells to retain differentiation potential.

In a second aspect the present invention provides a cell suspension shaping cover for defining a shaped space for constraining a cell suspension to be applied to a cell seeding surface, the cell suspension shaping cover comprising:

a cell suspension contact surface; and an aperture to allow delivery of a cell suspension through the cell suspension shaping cover to the shaped space formed in use.

Optional features described above with respect to the first aspect may be applied, singly or in any combination, to the second aspect, and vice versa.

In use, the cell suspension shaping cover may be located above a cell seeding surface so that a shaped space is formed between the cell suspension contact surface and the cell seeding surface. Using the cell suspension shaping cover to control the shape of a volume of cell suspension contacted with a cell seeding surface provides improved control of the cell distribution across the cell seeding surface. Optionally the cell suspension contact surface has an area of at least 1 cm ² . Optionally the cell suspension shaping cover has at least one support member to support the cell suspension shaping cover above the cell seeding surface such that the cell suspension contact surface is located a predetermined distance above a cell seeding surface in use.

Optionally the or each support member is configured to rest on the cell seeding surface in use. The or each support member provides the advantage that the cell suspension shaping cover may be easily and quickly located above a cell seeding surface to define a shaped space between the cell seeding surface and the cell suspension shaping cover that may be filled with a cell suspension. As discussed above, preferably the shaped space formed between the cell seeding surface and the cell suspension shaping cover has a constant depth across the surface of the cell seeding surface. Therefore, preferably the cell suspension shaping cover is configured to provide in use a shaped space with a constant depth across the cell seeding surface. Preferably the cell suspension contact surface of the cell suspension shaping cover is smooth and/or flat.

When the cell suspension shaping cover comprises at least one support member, suitably the or each support member is configured such that when the cell suspension shaping cover is positioned over a cell seeding surface the cell suspension contact surface is aligned with the cell seeding surface so that a shaped space with a constant depth across the cell seeding surface is formed.

Suitably the at least one support member is configured to support the cell suspension contact surface a predetermined distance above a cell seeding surface in use.

Preferably the at least one support member is configured to support the cell suspension contact surface a predetermined distance of no greater than 3 mm, more preferably no greater than 2 mm, more preferably no greater than 1.5 mm, more preferably no greater than 1 mm and most preferably no greater than 0.75 mm above a cell seeding surface in use. Optionally the aperture is situated in the centre of the cell suspension shaping cover. In other embodiments an off-centre aperture is preferred.

It is preferred that the cell suspension shaping cover is substantially rigid. This is intended to require that in normal use the cell suspension shaping cover substantially will not flex, bulge or otherwise deform in response to the flow of cell suspension into the shaped space.

The cell suspension shaping cover may be produced by any means suitably to provide a cell suspension shaping cover as described herein, for example a suitable cell seeding surface can be formed by injection moulding, cutting a sheet to size and attaching support members or CNC machining. The cell suspension shaping cover can be formed from any sufficiently hard and stiff polymer, metal, ceramic, glass or similar material that can sustain a flat, or other desired shape, over the area of the shaped space required.

Accordingly, preferably the Young's modulus of the material from which the cell suspension shaping cover is made is at least 1 GPa, more preferably at least 2 GPa, more preferably at least 5 GPa, more preferably at least 10 GPa, and more preferably at least 20 GPa. Many glass compositions, for example, have a Young's modulus of 50 GPa or higher. Typical polycarbonates of use in the present invention have a Young's modulus of 2 GPa or higher. The actual rigidity of the cell suspension shaping cover can therefore be ensured by selecting a suitable material and a suitable thickness for the cell suspension shaping cover.

Preferably the cell suspension shaping cover is configured to fit within a Petri dish or a multi dish plate. Suitably the cell suspension shaping cover has a diameter of 13-2000 mm. Optionally the cell suspension shaping cover is shaped to fit inside six-well culture plates.

The optional and preferred features of the first aspect also apply, either singly or in any combination, to the second aspect. In a third aspect the present invention provides a cell seeding device comprising a cell suspension shaping cover according to the second aspect.

Optionally the cell seeding device also comprises a cell seeding surface support.

Optionally the cell seeding surface support comprises a cell suspension shaping cover support to support the cell suspension shaping cover a predetermined distance above the cell seeding surface or the cell seeding surface support. The optional and preferred features of any one aspect can also apply, either singly or in any combination, to any of the other aspects. Furthermore, features disclosed in the context of a product may also apply to a method as a corresponding method step, and vice versa. Further optional features of the present invention are set out below. BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention and information illustrating the advantages and/or implementation of the invention are described below, by way of example only, with respect to the accompanying drawings in which:

Figure 1 is a schematic cross-section through a cell suspension shaping cover being an embodiment of the present invention;

Figure 2 is a schematic cross-section through a cell seeding device being an embodiment of the present invention;

Figure 3 is a graphical plot showing the distribution of cells across surfaces seeded using different seeding methods;

Figure 4 is a graphical plot showing the measurement of cell density surfaces seeded using different seeding methods; Figure 5 shows a scan produced by a flatbed scanner of a cell seeded surface produced by the method of the present invention;

Figure 6 shows a scan produced by a flatbed scanner of a cell seeded surface produced by the sessile drop method of the prior art;

Figure 7 shows a scan produced by a flatbed scanner of a cell seeded surface produced by open cell seeding method of the prior art; Figure 8a is a heat map showing cell density per field of view for a seeded surface produced by the sessile drop method of the prior art;

Figure 8b is a heat map showing sum of the local standard deviation in cell density over four neighbouring fields for a seeded surface produced by the sessile drop method of the prior art;

Figure 9a is a heat map showing cell density per field of view for a seeded surface produced by the open cell seeding method of the prior art; Figure 9b is a heat map showing sum of the local standard deviation in cell density over four neighbouring fields for a seeded surface produced by the open cell seeding method of the prior art;

Figure 10a is a heat map showing cell density per field of view for a seeded surface produced by the method of the present invention;

Figure 10b is a heat map showing sum of the local standard deviation in cell density over four neighbouring fields for a seeded surface produced by the method of the present invention; and

Figure 1 1 is a graph showing the standard deviation in cell density across a cell seeding surface formed by three methods differing in the height of the shaped space in which the cell suspension is located. Figures 12-16 show a cell seeding apparatus according to a modified embodiment of the invention.

Figure 12 shows a plan view of a cell seeding apparatus.

Figure 13 shows a side view of a cell suspension shaping cover of Figure 12.

Figure 14 shows a side view of a cell seeding surface of Figure 12. Figure 15 shows the cell suspension shaping cover lifted from the cell seeding surface in the apparatus of Figure 12.

Figure 16 shows the cell suspension shaping cover placed on the cell seeding surface in the apparatus of Figure 12.

Figures 17 and 18 show, respectively, the cell distribution accuracy and the cell density accuracy, in the form of quantitative analysis of the data in the heat maps of Figures 8a-10b, with an n of approximately 250,000 per condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS. FURTHER OPTIONAL FEATURES OF THE INVENTION

Figure 1 shows an example of a cell suspension shaping cover 10 having a cell suspension contact surface 12, an aperture 14 to allow delivery of a cell suspension through the cell suspension shaping cover 10 to a shaped space (not shown) formed in use.

Figure 2 illustrates an example of a method of seeding cells at a cell seeding surface. A cell seeding surface 18 is provided on which cells 22 are to be seeded. A cell suspension shaping cover 10 is located above the cell seeding surface 18 to form a shaped space 20. The cell suspension shaping cover 10 is supported above the cell seeding surface by resting the support members 16 of the cell suspension shaping cover 10 on the cell seeding surface 18. A flowable cell suspension containing cells 22 is inserted into the shaped space 20 through aperture 14 in the cell suspension shaping cover 10. The cell suspension may be inserted into the shaped space 20 by injection from an injection device through the aperture. The aperture 14 may be Luer- lock sized and form a Luer-lock connection with a complimentary injection device such as a syringe. In an alternative embodiment, the cell suspension may be inserted directly into the shaped space 20 without the need for aperture 14, e.g. from the side of the assembly of the cell seeding surface 18 and the cell suspension shaping cover 10. Once the cell suspension is inserted into the shaped space 20 the cell suspension is constrained by the cell seeding surface 18 and the cell suspension contact surface 12 within this space. Once the cells 22 have attached to the cell seeding surface 18 the cell suspension shaping cover 10 may be removed from the cell seeding surface 18. In the embodiment illustrated by Figure 2 the shaped space 20 formed between the cell suspension shaping cover and the cell seeding surface 18 has a constant depth across the cell seeding surface, suitably the depth is between about 0.1 mm and 2 mm. The present inventors have found that for depths greater than 2 mm or less than 0.1 mm the uniformity of the cell distribution across a seeded surface is less reliably achieved than when depths within this range are used.

The cell seeding device may have a cell seeding surface support (not shown), for supporting the cell seeding surface, and a cell suspension shaping cover 10. The cell suspension shaping cover 10 may be configured to fit inside the cell seeding surface support, for example an open dish such as a petri dish. The cell seeding surface support may have an internal rim on which the cell suspension shaping cover may be supported. The cell suspension shaping cover 10 may have a circular shape, however other shapes are possible. Figs. 12-16 show a cell seeding apparatus according to a modified embodiment of the invention.

Fig. 12 shows a plan view of a cell seeding apparatus 200. Fig. 13 shows a side view of a cell suspension shaping cover 110. Fig. 14 shows a side view of a cell seeding surface 118. Cell suspension shaping cover 110 has three support member 1 16 for contact with the cell seeding surface 118. Cell suspension shaping cover 1 10 has aperture 1 14 extending through the full thickness of the cell suspension shaping cover 110. Fig. 15 shows the cell suspension shaping cover 1 10 lifted from the cell seeding surface 118, in perspective view. Fig. 16 shows the cell suspension shaping cover 110 placed on the cell seeding surface 118, in perspective view, forming a shaped space 120 between the cell suspension shaping cover 1 10 and the cell seeding surface 1 18, by virtue of the support members 1 16 contacting the cell seeding surface 118.

A flowable cell suspension containing cells is inserted into the shaped space 120 through aperture 1 14 in the cell suspension shaping cover 1 10. The cell suspension may be inserted into the shaped space 20 by injection from an injection device through the aperture. The aperture 14 may be Luer-lock sized and form a Luer-lock connection with a complimentary injection device such as a syringe. As shown in Figs. 12-16, the aperture may have a tapered shape.

Vent means is provided in the assembly in the form of the gap existing between the cell seeding surface 118 and the cell suspension shaping cover 110.

Once the cell suspension is inserted into the shaped space 120 the cell suspension is constrained by the cell seeding surface 118 and the cell suspension shaping cover 110. Once the cells have attached to the cell seeding surface 118 the cell suspension shaping cover 1 10 may be removed from the cell seeding surface 1 18.

Example 1

This example compares the seeding method of the present invention with the "sessile drop method" and the "open cell seeding" method. For each method endothelial cells were seeded onto a square (24 mm x 24 mm) flat polycarbonate injection cell seeding surface formed by injection moulding using a nickel shim.

A cell suspension of 10 000 Rat Lung Capillary Endothelial cells (LE2) ml ^"1 was created, the expected number of cells per 0.5 ml of cell suspension was 5000.

For the seeded surface prepared using the sessile drop method, a 0.5 ml drop of the LE2 cell suspension was placed on the cell seeding surface. The cells seeding surface was left in a flow hood for 1 hour to allow the endothelial cells to settle and attach to the cell seeding surface before the cell seeding surface was transferred into a dish with 3 ml of fresh media. For the seeded surface prepared using the open cell seeding method (also known as free seeding) 0.5 ml of the LE2 cell suspension was mixed with 3 ml of media to form a cell suspension of even cell dispersion. The cell suspension was then pipetted on top of the cell seeding surface. The cell seeding surface was left in a flow hood for 1 hour to allow the cells to settle and attach to the cell seeding surface before the cell seeding surface was transferred into a dish with 3 ml of fresh media.

For the seeded surface prepared using the method of the present invention a cell suspension shaping cover was placed on top of the cell seeding surface to form a shaped space at the surface of the cell seeding device, 0.5 ml of the LE2 cell suspension in media (the volume of media was such that the total volume of cell suspension and media fitted within the shaped space formed when the shaping cover was placed on top of the cell seeding surface) was pipetted through an aperture in the cell seeding device. In this Example 0.47 ml of cell suspension was pipetted into the shaped space because this was the approximate calculated volume of the shaped space. The cell suspension was held in the shaped space fixed by the positioning of the cells suspension shaping cover above the cell seeding surface. The cell seeding surface was left in a flow hood for 1 hour to allow the cells to settle and attach to the cell seeding surface. The cell suspension shaping cover was removed from the cell seeding surface and the cell seeding surface was transferred into a dish with 3 ml of fresh media.

The efficiency of these three seeding methods in evenly distributing cells across the cell seeding surface was compared by measuring the total cell count and the localised distribution of cells across the cell seeding surface by averaging over three samples formed by each method. The results for the surfaces formed using the three different methods are shown in Table 1. For each sample 144 images were captured in a noncontiguous array. The average cell count for the whole sample was found by summing the total number of cells in 144 images for each sample. However, as the images were captured in a non-contiguous array averaging the cell count in each of the 144 single images gives a more reliable idea of the coverage of cells. As a border appears to exist around the free seeding and seeding device samples, the most robust analysis is achieved by excluding a 1 mm border around the sample to discount, for example, interfacial effects. Table 1

Table 1 shows that the sessile drop method resulted in the highest average number of cells successfully seeded onto the cell seeding surface. However, the seeded surface formed using the sessile drop method shows a large variation in cell distribution across the seeded surface.

Figure 3 is a graph showing the cell count across a cross-section through the centre of a 24 mm cell seeding surface sample formed by each of the methods described above. Bars 40 show the cell count at different points across the sample surface formed by the method of an embodiment of the present invention, bars 42 show the cell count at different points across the sample surface formed by the sessile drop method and bars 44 show the cell count at different points across the sample surface formed by the open cell seeding method. Lines 46, 48 and 50 show the standard deviation in the number of cells seeded at a particular location by the methods of the present invention, the sessile drop method and the open cell seeding method respectively.

From Figure 3 it can be seen that the cells seeded by the sessile drop method follow the distribution of fluid across the cell seeding surface. The method of the present invention shows an improved cell distribution (lower standard deviation) over the free seeding method. However the method of the present invention shows a large improvement in the number of cells attaching to the cell seeding surface over the free seeding method. This is because in the free seeding method cells are free to "fall off" the cell seeding surface during the settling period.

Figure 4 is a graph showing the standard deviation of the cell density across the centre of the seeded area (i.e. the cell seeding surface excluding a 1 mm border around the sample) for each of the cell seeding surfaces prepared according to Example 1. The cell density across each of the samples was assessed by local density of Coomassie staining. Figure 4 shows that the standard deviation of the cell density was lower for the seeded surface prepared according to the present invention than the seeded surfaces prepared by the open cell seeding method or the sessile drop method.

For each of the three cell seeding methods described above, the volume of cell suspension and additional media used to seed cells was adjusted to allow the same number of cells to be seeded for each of the different methods. The method of the present invention allows an even height and density of cell suspension across a seeded surface independent of the user's level of experience. Additionally the method of the present invention allows the cell seeded area to be confined to the size of the volume of cell suspension inserted into the shaped space. The total size of the cell seeded area can be adjusted by adjusting the volume of cell suspension inserted into the shaped space. The cell seeded surface formed by the method of the present invention has a uniform cell density rather than a cell density distribution that follows the shape of the drop of cell suspension deposited on the cell seeding surface, with more cells towards the centre of the drop.

Example 2

Samples were prepared in the same way as Example 1 , but instead LE2 cells were seeded at 10 000 cells per sample and the cells were allowed to adhere for 4 hours. The prepared samples were then stained with coomassie blue. The samples were then scanned on a flatbed scanner in order to gain a general impression of the variation in cell coverage yielded by each seeding methodology. The 2.2 cm x 2.2 cm scans produced are shown in Figures 5, 6 and 7. These figures clearly show the difference in cell seeding distribution using the different methods.

Figure 5 shows the uniform cell distribution across the samples produced by the method of an embodiment of the present invention.

Figure 6 shows samples produced by the sessile drop method. These samples show a large variation in cell distribution across the sample. A large number of cells are seeded in the centre of the sample and the number of cells seeded decreases away from the centre.

Figure 7 shows samples produced by the free seeding method. As for the samples produced by the sessile drop method the samples produced by this method also have a cell distribution that decreases away from the centre of the sample, although this is less marked than the cell distribution formed using the sessile drop method.

Example 3

Samples were prepared in the same way as Example 1 , except that after the samples were left to adhere for one hour and fresh media was added the samples were then incubated at 37°C for 12 hours and then the media was removed. Each of the samples was then stained with DNA stain to allow the cell seeded surfaces to be imaged using a fluorescence microscope. The extra incubation time compared to Example 1 was to ensure the cells were sufficiently attached for the staining procedure.

An array of fluorescence microscope images was taken across each cell seeded surface in a 10 x 10 matrix. Each image covered an area of approximately 0.9 mm x 0.6 mm and these images were spaced so as to capture the full 24 mm x 24 mm surface area. Figures 8a, 9a and 10a are heat maps which display the number of cells in each section of the 10 x 10 matrix using each captured image for the cell seeded surfaces produced by the sessile drop seeding method, the open cell seeding method and the method of the present invention respectively. Figures 8b, 9b and 10b show the sum of the local standard deviation of cell density over 4 neighbouring sections of the 10 x 10 matrix for the cell seeded surfaces produced by the sessile drop seeding method, the open cell seeding method and the method of the present invention respectively.

Figures 8a to 10b show that the samples prepared according to the present invention have a more uniform cell distribution than the samples prepared according to the sessile drop and open cell seeding methods of the prior art.

Figures 17 and 18 show, respectively, the cell distribution accuracy and the cell density accuracy, in the form of quantitative analysis of the data in the heat maps of Figs. 8a- 10b, with an n of approximately 250,000 per condition. The error bars indicate the standard deviation of data. The asterisks indicate statistical significance, calculated using a paired t-test with p <0.001. Example 4

This example compares the cell distribution across cell seeding surfaces seeded using the method of the present invention described above, for shaped spaces of different depths.

The cell suspension was prepared according to Example 1. Petri dishes with a diameter of 60 mm were provided as the cell seeding surfaces. Three different cell suspension shaping covers were provided with support members configured to support the shaping covers 0.75 mm, 3 mm and 5 mm respectively above the cell seeding surface. This provided shaped spaces with three different depths (0.75 mm, 3 mm and 5 mm) across the cell seeding surfaces. Cell suspension and media was pipetted into the three different shaped spaces. The volume of media was controlled to prevent overfilling of the shaped space. The cell seeding surfaces were left in a flow hood for 1 hour to allow the cells to settle and attach to the cell seeding surface.

A rectangular area of 4cm x 4cm in the centre of each cell seeding surface was used to compare the uniformity of the cell seeded surfaces produced from shaped spaces with different depths. Figure 11 shows the standard deviation in cell density across this rectangular area for each of the three types of cell seeding surface. Sampling a rectangular area in the centre of each sample excludes regions of cell seeding surface surrounding the support members of the cell suspension shaping covers. Figure 1 1 shows that the standard deviation of cell density across the central seeded area increases as the depth of the shaped space increases. This indicates that as the depth of the shaped space increases the tendency of uneven seeding rises.

Example 5

A cell seeding surface was prepared according to Example 1 using the method of the present invention, except that 3 μΙ of MDCK cell suspension was inserted into the shaped space, the shaped space having a constant depth across the cell seeding surface of 0.75 mm. The area of cell seeding surface covered by the volume of cell suspension inserted into the shaped space had a diameter of 1 mm. The seeded surface produced according to this Example was found to have a uniform cell density across the seeded surface with a diameter of 1 mm. These examples show that the method and device of the present invention provide a very significant improvement in the uniformity of cell distribution and cell adhesion across cell seeding surfaces. The above embodiments have been described by way of example. On reading this disclosure, modifications of these embodiments, further embodiments and modifications thereof will be apparent to the skilled person and as such are within the scope of the invention.