WO2010038013A1

WO2010038013A1 - Cell culture vessel

Info

Publication number: WO2010038013A1
Application number: PCT/GB2009/002321
Authority: WO
Inventors: Stefan Przyborski
Original assignee: Re-Innervate Limited
Priority date: 2008-10-02
Filing date: 2009-09-29
Publication date: 2010-04-08
Also published as: GB0817990D0

Abstract

A cell culture vessel comprises first and second interengageable portions, the first portion comprising an upwardly extending wall defining upper and lower rims, and the second portion comprising an inwardly extending flange arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said rim and flange for receipt of a cell culture scaffold. In another aspect, a cell culture vessel comprises first and second interengageable portions which, when engaged, are arranged to receive a cell culture scaffold between them. The first portion comprises an upwardly extending wall defining an outwardly extending formation and the second portion comprises an upwardly extending formation arranged to cooperate with said outwardly extending formation when said portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

Description

CELL CULTURE VESSEL

The present invention relates to a cell culture vessel, particularly, but not exclusively, a cell culture vessel that can be used as a well insert in a cell culture plate.

The culturing of eukaryotic cells, particularly mammalian cells, is fundamental to a wide variety of biotechnological and medical methods. For example, the successful culturing of animal cell lines is crucial for the manufacture of viral vaccines; the production of therapeutic molecules, including hormones and proteins; and the establishment of in vitro experimental systems for examining the usefulness of potential therapeutic compounds.

Furthermore, in recent years much research has been focused on the development of tissue culture and tissue engineering as part of regenerative medicine technologies, which aim to replace or repair diseased or damaged body parts. Cell culture is a fundamental component of tissue culture and tissue engineering, as it establishes the basics of growing and maintaining cells ex vivo.

Many existing methods are used successfully to promote the expansion and, optionally, differentiation of cultured eukaryotic cells. Two basic types of culturing system can traditionally be used: the growth of cells on a solid, essentially impermeable support structure, such as a glass or treated plastic substrate, to provide a monolayer of cells; or unattached cell culture in which cells are grown in a liquid suspension culture.

However, it is well recognised that cells that make up tissues in the body possess a complex three-dimensional (3D) architecture which is markedly different from the flat monolayer structure of their cultured counterparts commonly grown on the two-dimensional (2D) surface of tissue culture plastic or glass substrates. The 3D structure of cells and interactions with their neighbours significantly influences their ability to grow and function. Thus in order to replicate more closely the in vivo function of cells, it is preferable to develop effective means of culturing cells in a 3D environment.

Evidence for the functional superiority of cells cultured on 3D supports compared to 2D monolayer cultures has created much interest in the fabrication of materials that promote the growth and function of cells in a more realistic manner. This has lead the applicants to develop an improved three- dimensional cell culture scaffold as described in the applicant's co-pending International Patent Application No. PCT/GB2007/001464 (Publication Number WO-2007/125288).

The term "scaffold" is used herein for clarity and convenience when referring to any type of support upon which the culturing of cells can take place, regardless of the physical form of the support (i.e. whether it is 2D or 3D). Accordingly, the term "scaffold" is used in relation to both conventional two-dimensional monolayer cultures and three-dimensional supports.

When it is desired to use a two- or three-dimensional cell culture scaffold the scaffold can be either placed in the bottom of a well defined by a cell culture plate, or a well insert, which is then located within the well of the cell culture plate.

If a two-dimensional scaffold is used, the well insert can be formed as an annular body defining upper and lower openings, with the two-dimensional scaffold connected to a lower rim of the annular body so as to close the lower opening. By virtue of the porous nature of the two-dimensional scaffold, cell growth nutrients can pass through the scaffold and throughout the cell culture medium in which the scaffold is immersed, but the size of the pores defined by the scaffold are not so large as to permit cells to flow out of the cell culture vessel.

Two methods currently exist for retaining a two-dimensional scaffold against a lower rim of a well insert. A first method employs a heat weld (or could employ some form of adhesive) to affix the scaffold to the lower rim, and a second employs a tight-fitting ring to clamp a peripheral section of the scaffold against an outer surface of the well insert. Both of these methods suffer from problems however when used with two-dimensional scaffolds, which are exacerbated when applied to three-dimensional scaffolds.

The conditions required to affix the scaffold to the lower rim of the well insert, e.g. intense localised heating, risk harming the delicate structure of the scaffold, with the potential for deleterious effects on the performance of the scaffold. Moreover, where a clamp-like ring is employed, the width of the scaffold must be increased to provide a peripheral section which can be folded up along the side of the outer surface of the vessel to be retained in place by the ring, which clearly wastes material. Systems of this kind have also been observed to provide an unreliable level of force to hold the scaffold in place, which can increase the chances of the scaffold moving during use, which in turns encourages the user to apply an increased clamping force that can tear or puncture the scaffold and significantly reduce its performance.

The above described problems relating to heat welding and the like are increased when wishing to use a three-dimensional scaffold rather than a two- dimensional scaffold. Moreover, the increased thickness and in-plane structural rigidity of many three-dimensional scaffolds as compared to two-dimensional scaffolds is likely to further reduce the reliability of the clamping action of the systems employing clamping rings and indeed may prevent such systems being used with many three-dimensional scaffolds.

An object of the present invention is to obviate or mitigate the aforementioned problems.

According to a first aspect of the present invention there is provided a cell culture vessel comprising first and second interengageable portions, the first portion comprising an upwardly extending wall defining an upper rim and a lower rim, and the second portion comprising an inwardly extending flange which is arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said lower rim and said flange for receipt of a cell culture scaffold.

In this way, the inwardly extending flange affords a suitable surface to support a cell culture scaffold adjacent to the lower rim of the wall of the first portion so that the scaffold is retained in the correct position for cell culture studies. By appropriately dimensioning the height of the space between the lower rim and the flange so that it approximately matches or is slightly less than the thickness of the cell culture scaffold, the scaffold can be reliably secured within the cell culture vessel using just the correct degree of physical force. This is beneficial because it obviates the need to use an adhesive, heat weld or the like, as in prior art devices, and also ensures that the scaffold is not subjected to undesirably high compressive forces, all of which might otherwise harm the structural integrity of the scaffold. It will be appreciated that this represents a particularly important advantage when relatively rigid (e.g. 3D) cell culture scaffolds are to be employed with the vessel of the present invention where any deformation of the rigid scaffold is likely to be highly detrimental to its structural integrity and in consequently the proper functioning of the scaffold.

According to a second aspect of the present invention there is provided a cell culture vessel comprising first and second interengageable portions which, when engaged, are arranged to receive a cell culture scaffold between said first and second portions, the first portion comprising an upwardly extending wall defining an outwardly extending formation, and the second portion comprising an upwardly extending formation which is arranged to cooperate with said outwardly extending formation defined by the wall of the first portion when said first and second portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

Providing the cell culture vessel with cooperating features to limit movement of the second portion relative to the wall of the first portion provides a safe and reliable means of controlling the interaction between the first and second portions which holds the scaffold between said first and second portions. This is advantageous because it ensures that the scaffold is not subjected to undesirably high compressive forces, which might otherwise harm the structural integrity of the scaffold.

A third aspect of the present invention provides a cell culture device comprising a cell culture vessel and at least two cell culture scaffolds, the cell culture vessel comprised of first and second interengageable portions engaged together to define a space within which is received the at least two cell culture scaffolds. In a preferred embodiment of this aspect of the present invention the cell culture vessel is in accordance with the first and/or second aspect of the present invention.

In this way, the vessel of the present invention can be used to safely and securely support a plurality of cell culture scaffolds of the same or different design without fear of causing damage to the delicate structure of the scaffolds. The vessel of the present invention is eminently suitable for use with two or more 2D scaffolds, two or more 3D scaffolds, or a combination of 2D and 3D scaffolds. This represents a potentially significant advance on the art since it will enable two or more cell culture scaffolds, possibly containing different cell types, to be securely retained against one another within a cell culture medium to facilitate studies of the interaction of one cell type with the other cell type.

A fourth aspect of the present invention provides a cell culture apparatus comprising a plurality of cell culture vessels connected together, at least one of said cell culture vessels being in accordance with the first and/or second aspect of the present invention.

A fifth aspect of the present invention provides a cell culture apparatus comprising a plurality of cell culture vessels, at least one of said cell culture vessels being in accordance with the first and/or second aspect of the present invention and the apparatus further comprising a cell culture plate associated with said plurality of cell culture vessels, said plate defining a reservoir for cell culture medium in fluid communication with at least two of said plurality of cell culture vessels.

A sixth aspect of the present invention provides a cell culture plate defining one or more wells for receipt of a cell culture scaffold or receipt of a well insert to support a cell culture scaffold, at least one of said wells possessing a volume for receipt of cell culture medium of at least around 10 ml.

A seventh aspect of the present invention provides a cell culture plate defining one or more wells for receipt of a cell culture scaffold or receipt of a well insert to support a cell culture scaffold, the plate further defining a reservoir for cell culture medium in fluid communication with at least one of said wells, wherein said at least one well and the reservoir possess a combined volume for receipt of cell culture medium of at least around 10 ml per well.

The fifth, sixth and seventh aspects of the present invention may be particularly useful when employing 3D cell culture scaffolds which have been found to impose greater metabolic requirements on the culture than conventional 2D scaffolds. This issue may be addressed by using the cell culture vessel of the present invention with one or more 3D scaffolds and a cell culture plate defining a reservoir in fluid communication with a plurality of wells, as in the fifth aspect of the invention, and/or a plate according to the sixth or seventh aspects of the invention which incorporates one or more wells which, optionally in combination with a reservoir, define a greater volume for cell culture medium than normal (normal volume is around 4 ml per well), and/or include wells of greater depth than normal (normal depth is around 12 to 15 mm per well).

Cell culture vessels in accordance with the first and/or second aspect of the present invention are eminently suitable for use with the cell culture plates according to the sixth and seventh aspects of the present invention. Further aspects of the present invention thus relate to apparatus comprised of a cell culture plate according to the sixth or seventh aspects of the present invention, in combination with a cell culture vessel according to the first and/or second aspects of the present invention.

It has been found that enabling scaffolds retained in two or more separate cell culture vessels to access the same reservoir containing cell culture medium can be advantageous. While the inventors do not wish to be bound by any particular theorem it is postulated that since the cells are growing in an avascular system, circulation of the medium may assist in supporting cell growth. Clearly, such circulation is aided by using apparatus according to the fifth and seventh aspects of the present invention, which might, for example, also incorporate some form of stirring means (e.g. a magnetic flea) to efficiently stir the medium throughout the reservoir.

With respect to the aspects of the present invention defined above it will be apparent to the skilled person that the cell culture vessel according to each aspect is suitable for use with any kind of cell or tissue culture substrate, such as a two- or three-dimensional cell culture scaffold. The design of the vessel of the present invention enables a cell culture scaffold of any desirable size and/or shape to be securely retained within the vessel without the risk of harming the structural integrity of the scaffold. As such, any desirable type of flexible or rigid scaffold may be used with the vessel of the present invention, including, but not limited to, textiles, fibres, foils, membranes and tissue samples.

The vessel according to each aspect is eminently suitable to be employed with a three-dimensional cell culture scaffold of the kind described in the applicant's co-pending International Patent Application No. PCT/GB2007/001464 (Publication Number WO-2007/125288).

Suitable scaffolds may comprise a high internal phase emulsion polymer (polyHIPE). A preferred material is a microcellular polymeric material, composed of, for example, a hydrophobic elastomer, such as 2-ethylhexyl acrylate, n-butyl acrylate or n-hexyl acrylate. The scaffold material may comprise a polyvinyl compound, such polystyrene or a polystyrene / divinylbenzene copolymer, and optionally further comprises a surfactant.

The scaffold used with the cell culture vessel of the present invention may be provided with a coating, for example a proteinaceous coating, and/or subjected to one or more types of surface treatment, such as gas plasma treatment, to improve cell attachment and adhesion during use.

Accordingly, a further aspect of the present invention provides a cell culture device comprising a cell culture vessel (preferably in accordance with the first and/or second aspect of the present invention) and a three-dimensional cell culture scaffold, the cell culture vessel comprised of first and second interengageable portions engaged together to define a space within which is received the three-dimensional cell culture scaffold. In another aspect, there is further provided a kit of parts to be assembled to provide a cell culture device, said kit of parts comprising a cell culture vessel and a three-dimensional cell culture scaffold, the cell culture vessel comprised of first and second interengageable portions engaged together to define a space within which is received the three-dimensional cell culture scaffold.

In relation to each of the above defined aspects of the present invention the first and second portions of the cell culture vessel may be releasably interengageable (i.e. capable of being engaged and disengaged any desirable number of times) or non-releasably interengageable (i.e. engageable once after which disengagement is not possible without risk of damage to one or both of the two portions). The two portions may be non-releasably engaged together in any appropriate manner, for example, by means of chemical bonding with the use of a suitable adhesive or the like and/or physical interlocking of suitably formed components. In a preferred embodiment, the two portions are non- releasably engaged by the application of a suitable amount of a substance, such as acetone, which dissolves a small area of the surface material of one or both of the two portions such that upon contact the dissolved material irreversibly bonds the two portions together. Such a system may be used in a "single-use" version of the vessel of the present invention, while a "multi-use" version may be offered where the two portions are releaseably interengageable.

Concerning the first aspect of the present invention it is preferred that the second portion of the cell culture vessel comprises an upwardly extending wall which is dimensioned so as to be located outwardly of the upwardly extending wall of the first portion when the first and second portions of the cell culture vessel are engaged together. Preferably the upwardly extending wall of the second portion is dimensioned to contact said upwardly extending surface of the first portion with an interference fit. In this way, the first and second portions can be retained together as a result of the interference fit without the need to apply a continuous external force.

The upwardly extending wall of the second portion of the cell culture vessel of the first aspect of the present invention is preferably shorter than the upwardly extending wall of the first portion. The upwardly extending wall of the second portion possesses a height that is preferably up to around 90 %, more preferably up to around 75 %, and still more preferably around 5 to 50 % of a height of the upwardly extending wall of the first portion. It is particularly preferred that a height of the upwardly extending wall of the second portion is around 20 to 40 % of a height of the upwardly extending wall of the first portion.

The upwardly extending wall of the second portion may define upper and lower parts, with said inwardly extending flange preferably extending from said lower part of said upwardly extending wall. A distance between opposite regions of an outer surface of the upper part of the upwardly extending wall of the second portion is preferably greater than a distance between opposite regions of an outer surface of the lower part of the upwardly extending wall. The outer surface of the upwardly extending wall of the second portion of the cell culture vessel preferably tapers outwardly with a substantially constant taper angle relative to the vertical over the full height of the wall. In a preferred embodiment of the vessel according to the first aspect of the present invention the upwardly extending wall of the second portion defines an annular wall, which is preferably of constant radius. Accordingly, it is preferred that the annular wall tapers radially outwardly from the lower part of the wall to the upper part of the wall.

It is further preferred that a distance between opposite regions of an outer surface of the upwardly extending wall of the first portion of the vessel at the upper rim is greater than a distance between opposite regions of said outer surface at the lower rim. The outer surface of the upwardly extending wall of the first portion preferably tapers outwardly with a substantially constant taper angle relative to the vertical over the full height of the wall.

The upwardly extending wall of the first portion preferably defines an annular wall, in which case, the annular wall preferably tapers radially outwardly from the lower rim to the upper rim.

The upper and lower rims of the first portion may define upper and lower openings to facilitate access to an interior cavity defined by the upwardly extending wall of the first portion. In which case, it is preferred that a diameter defined by said upper opening is greater than a diameter defined by said lower opening.

An inner surface of the upwardly extending wall of the second portion preferably defines a taper angle relative to the vertical which is at least similar to, more preferably substantially the same as, a taper angle relative to the vertical defined by the outer surface of the upwardly extending wall of the first portion.

In a particularly preferred embodiment of the first aspect of the present invention the upwardly extending wall of the first portion of the cell culture vessel defines an outwardly extending formation which is arranged to cooperate with an upper rim defined by the upwardly extending wall of the second portion when said first and second portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

Preferably said cooperation of the outwardly extending formation with the upper rim of the second portion determines a height of the space between said lower rim and said flange for receipt of the cell culture scaffold.

In this way, the cooperating features at least partially control the height of the space between the lower rim of the first portion and the flange of the second portion within which the cell culture scaffold is to be received. Providing this control enables the height of the space to be accurately matched to the thickness of the scaffold to be used, thereby allowing the scaffold to be securely retained within the cell culture vessel and avoiding the risk of harming the structure of the scaffold.

The second aspect of the present invention defined above provides a cell culture vessel comprising first and second interengageable portions, which define complementary formations which cooperate to at least partially control positioning of the second portion relative to the upwardly extending wall of the first portion.

With regard to the second aspect of the invention it is preferred that the upwardly extending wall of the first portion defines an upper rim and a lower rim, and the second portion comprises an inwardly extending flange which is arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said lower rim and said flange for receipt of the cell culture scaffold. As in the first aspect of the present invention, cooperation of the outwardly extending formation of the first portion with the upwardly extending formation of the second portion preferably determines a height of the space between said lower rim and said flange for receipt of the cell culture scaffold. As discussed above in relation to the first aspect of the present invention, providing the cell culture vessel with cooperating features to limit movement of the second portion relative to the wall of the first portion provides a safe and reliable means of controlling the height of the space between the lower rim and the flange so that the scaffold can be retained within the cell culture vessel using just the correct level of physical force. The need to use an adhesive, heat weld or the like, as in prior art devices, is thereby avoided and the scaffold is not excessively compressed, which might otherwise harm the structural integrity of the scaffold.

For reasons outlined above in relation to similar features of the first aspect of the present invention it is preferred that said cooperation of features of the second aspect of the present invention is effected by contact between a lower surface of the outwardly extending formation of the first portion and an upper rim of the upwardly extending formation of the second portion.

A height of the space within which the scaffold is to be received may take any appropriate value to suit a particular application. This flexibility in the application of the vessel of the present invention represents a significant advantage of the present invention. The height may be up to around 1000 microns, more preferably up to around 750 microns, and still more preferably up to around 500 microns. The height of the space may take any suitable minimum value from around 0 microns upwards, but the space may possess a height of at least around 25 microns or at least around 50 microns. As mentioned, the space between the lower rim of the first portion and the flange of the second portion may take any appropriate value, by way of example only the space may have a height of around 75 microns to around 500 microns, or around 100 microns to around 250 microns.

The cell culture vessel of the present invention may be designed for use with a two- or three-dimensional cell culture scaffold of any desirable thickness. As mentioned above, it is desirable that the height of the space to receive the scaffold is approximately the same as, or slightly less than, the thickness of the scaffold that it is desired to use in a particular application. Thus, it is preferred that the scaffold used with the vessel of the present invention possesses a thickness of up to around 1000 microns, more preferably up to around 750 microns, and still more preferably up to around 500 microns. The minimum thickness of the scaffold is at least around 1 or 2 monolayers of scaffold material, and is more preferably at least around 25 or 50 microns. One or more scaffolds of any desirable thickness may be employed with the vessel of the present invention. By way of example, the or each scaffold may be around 75 to around 500 microns thick, or around 100 microns to around 250 microns thick.

In a further preferred embodiment of the first and/or second aspect of the present invention the first and second portions of the cell culture vessel, when engaged, define a substantially vertical axis extending through a centre of said first and second portions, and at least one of the outwardly extending formation of the first portion and the upper rim of the second portion is configured such that the height of the space for receipt of the cell culture scaffold can be varied between at least two values by adjusting an angular displacement of the first portion relative to the second portion with respect to said substantially vertical axis.

This preferred feature of the vessel of the present invention enables a user of the device to simply and conveniently select a height for the space to suit a particular thickness of cell culture scaffold. In a preferred embodiment of the vessel of the first aspect of the present invention where both the first and second portions define generally annular bodies (i.e. the upwardly extending walls of the first and second portions are both annular), the angular displacement of the first portion relative to the second portion can be changed simply by rotating the first and/or second portion about the vertical axis passing through the centre of both portions before and/or after assembling the first and second portions together.

The first portion of the cell culture vessel of the present invention may incorporate any desirable number of outwardly extending formations of any suitable size and shape provided they could cooperate with the upper rim of the second portion in the manner described above. It is preferred that the first and second portions are provided with at least one, more preferably at least two, and most preferably three or more pairs of cooperating features, one feature from each pair being provided on the first portion of the vessel and the other feature from each pair being provided on the second portion of the vessel. Where two or more pairs of cooperating features are provided the pairs may be equi-angularly spaced or non-equi-angularly spaced about the periphery of their respective portion of the vessel.

The cooperation of the outwardly extending formation of the first portion with upper rim of the second portion is preferably effected by contact between a lower surface of the outwardly extending formation of the first portion and the upper rim of the second portion.

In a preferred embodiment the upper rim of the second portion is stepped, inclined and/or curved along a length of the rim such that at least two sections of the upwardly extending wall of the second portion possess different heights which thereby define at least two different values for the height of the space for receipt of the cell culture scaffold. Additionally or alternatively, the lower surface of the outwardly extending formation may be stepped, inclined and/or curved such that at least two sections of the outwardly extending formation possess different heights which thereby define at least two different values for the height of the space for receipt of the cell culture scaffold.

The upper rim of the second portion preferably defines one or more upwardly extending teeth which are arranged to contact a surface of the upwardly extending wall of the first portion and thereby provide an interference fit between said one or more teeth and said surface. In this way, the tooth or teeth increase the frictional contact area between the first and second portions and thereby increase the force required to separate the portions when engaged together so that they are more securely locked together. Said one or more teeth are preferably configured to cooperate with said outwardly extending formation defined by the upwardly extending wall of the first portion to control relative alignment of the first and second portions during engagement and/or provide a frictional contact between said one or more teeth and said outwardly extending formation. This is advantageous not only in terms of more securely locking the two portions together, but also in terms of ensuring accurate alignment of the two portions so as to precisely control the height of the space into which the one or more cell culture scaffolds are to be received.

The upper rim of the second portion and/or outwardly extending formation of the first portion may be provided with any desirable number of appropriate formations to define any suitable number of predetermined heights for the space between the lower rim of the first portion and the flange of the second portion in which the cell culture scaffold is to be retained.

In the specific embodiment of the first and second aspects of the present invention described below, the vessel is provided with three pairs of equi- angularly spaced cooperating formations, each pair consisting of two flared ribs extending radially outwardly from the side wall of the first portion and a set of steps of varying height in the upper rim of the second portion. Each set of steps consists of six steps, defining six different heights, from 0 microns up to 500 microns, for the space in which the scaffold is to be retained. In this way, the height of the space can be accurately predefined by aligning the appropriate step from each set with each pair of ribs. In this way, the risk of damaging a scaffold by applying to great a clamping force to retain the scaffold against the lower rim of the first portion is avoided. This arrangement also provides the user with the flexibility to easily select a height for the space which approximates the combined thickness of two or more scaffolds placed one on top of another, which allows the vessel to be used in studies to observe cell growth throughout relatively thick three-dimensional scaffolds without the expense of manufacturing thicker scaffolds, and also to study the behaviour of different cell types located in close proximity to one another in neighbouring scaffolds. In the sixth aspect of the invention which provides a cell culture plate defining one or more wells for receipt of a cell culture scaffold or receipt of a well insert to support a cell culture scaffold, in which at least one of said wells possesses a volume for receipt of cell culture medium of at least around 10 ml. It is preferred that said at least one well possesses a volume for receipt of cell culture medium of up to around 100 ml. More preferably said volume is around 20 to 90 ml, and still more preferably said volume is around 40 to 90 ml. Said volume for receipt of cell culture medium is most preferably around 50 to 80 ml, which represents an optimum range of volumes when using the plate with 3D scaffolds as scaffolds of this kind are up to around 20 times the volume of a conventional 2D scaffold used in conventional cell culture plates which possess a volume per well of around 4 ml. By using larger volume wells the cell growth medium does not require replenishing as often as when using a conventional plate in which the wells can only typically contain around 4 ml of cell growth medium.

It may be convenient to provide the larger volume well or wells by increasing the depth of the or each well as compared to a conventional cell culture plate well, which typically have depths of around 12 to 15 mm. Thus, in a preferred embodiment of the sixth aspect of the invention the at least one well possesses a depth of at least around 20 mm, and preferably, a depth of up to around 100 mm. The at least one well may possess a depth of around 25 to 30 mm, up to a depth of around 40 to 50 mm. Most preferably, the depth is around 25 to 35 mm.

The seventh aspect of the invention relates to a cell culture plate defining one or more wells for receipt of a cell culture scaffold or receipt of a well insert to support a cell culture scaffold, the plate further defining a reservoir for cell culture medium in fluid communication with at least one of said wells, wherein said at least one well and the reservoir possess a combined volume for receipt of cell culture medium of at least around 10 ml per well. The combined volume may be up to around 100 ml, more preferably around 20 to 90 ml, and still more preferably around 40 to 90 ml. Most preferably the combined volume for receipt of cell culture medium is around 50 to 80 ml. The plate may define a single well, or, more preferably defines a plurality of wells, such as 6, 12, 24 etc, and the reservoir is in fluid communication with at least two of said plurality of wells.

A further aspect of the present invention related to the first aspect of the present invention provides a kit of parts to be assembled to provide a cell culture vessel, said kit of parts comprising first and second interengageable cell culture vessel portions, the first portion comprising an upwardly extending wall defining an upper rim and a lower rim, and the second portion comprising an inwardly extending flange which is arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said lower rim and said flange for receipt of a cell culture scaffold.

Another aspect of the present invention that is related to the second aspect of the present invention provides a kit of parts to be assembled to provide a cell culture vessel, said kit of parts comprising first and second interengageable cell culture portions which, when engaged, are arranged to receive a cell culture scaffold between said first and second portions, the first portion comprising an upwardly extending wall defining an outwardly extending formation, and the second portion comprising an upwardly extending formation which is arranged to cooperate with said outwardly extending formation defined by the wall of the first portion when said first and second portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

A still further aspect of the present invention related to the fifth aspect of the present invention provides a kit of parts to be assembled to provide a cell culture apparatus comprising a plurality of cell culture vessels, at least one of said cell culture vessels being in accordance with the first and/or second aspects of the present invention and the apparatus further comprising a cell culture plate associated with said plurality of cell culture vessels, said plate defining a reservoir for cell culture medium in fluid communication with at least two of said plurality of cell culture vessels. While the cell culture vessel of the present invention is not limited to use just as a well insert in a cell culture plate, in a preferred embodiment of the above defined aspects of the present invention, the cell culture vessel is a well insert. Application of the vessel as a well insert may be facilitated by providing the second portion of the vessel in the preferred form of an annular body or ring which defines both the upwardly extending wall and radially inwardly extending flange, in which case the second portion preferably defines a central aperture over which the cell culture scaffold lies, during use, and through which cell culture nutrients and the like can flow into and out of the scaffold.

The various components of the cell culture vessel of the present invention can be manufactured from any desirable material, such as a plastics material. Many different plastics could be used, although it is preferred that both the first and second portions of the cell culture vessel of the present invention are formed from polystyrene or a polystyrene-containing plastic.

It will be appreciated that terms such as 'upper', 'lower', 'upwardly' etc have been used herein solely for the sake of convenience and clarity in defining the position, size and/or shape of features of the various components which form part of the different aspects of the present invention in relation to other features forming part of said components. Such terms should not be construed as limiting the orientation in which any component or group of components should be constructed or used relative to the horizontal and/or vertical.

The cell culture vessel according to the aspects of the invention set out above can be used in the culturing of a wide variety of different cell and tissue types. The type of cells cultured in the culture vessel can be dependent upon the nature of the cell culture scaffold employed.

However, it is anticipated that the cells that can be cultured in the vessels of the invention include prokaryotic (for example bacterial) and eukaryotic cells. The eukaryotic cell may be a mammalian cell; a plant cell; a fungal cell; or a slime mold.

The mammalian cell may be a primate cell, such as a human cell. The types of cells that can be cultured include epidermal keratinocytes; fibroblasts; epithelial cells; neuronal glial cells or neural cells; hepatocytes or hepatocyte stellate cells; mesenchymal cells; muscle cells; kidney cells; blood cells. The cell may be a cell derived from a tumour cell line.

The mammalian cell may also be a stem cell. The types of stem cells that can be cultured include haemopoietic stem cells; neural stem cells; bone stem cells; muscle stem cells; mesenchymal stem cells; epithelial stem cells; endodermal stem cells; embryonic stem cells; embryonic germ cells; embryonal carcinoma stem cells. The stem cell can be a pluripotent cell or a totipotent cell.

The cells that can be cultured in the cell culture vessel according to the aspects of the invention set out above can also be genetically modified.

The cells that are cultured in the cell culture vessel according to the aspects of the invention set out above can be used for a wide variety of purposes.

For example, the cells can be cultured as part of tissue culture and tissue engineering techniques that can be of use in regenerative medicine technologies. The vessels can be used to establish cell cultures as models systems for studying basic cell biology; the interactions between disease- causing agents and the cells; and the effects of the drugs on cells. The cultured cells can be used to study the effects of new drugs, cosmetics and chemicals on cellular viability in a wide-variety of cell types, including liver and kidney cell cultures. The cultured cells can be used for the monitoring of cancer cell and tissue development.

An embodiment of the present invention will now be described, by way of example only, with reference to the accompanying drawings in which: Figure 1 is a perspective view of a two-part cell culture vessel according to a first embodiment of the present invention;

Figure 2 is a top view of a first portion of the two-part cell culture vessel shown in figure 1 ;

Figure 3 is a bottom view of the first portion shown in figure 2;

Figure 4 is a side view of the first portion shown in figure 2;

Figure 5 is a sectional view of the first portion shown in figure 2 taken along line A-A of fig u re 4;

Figure 6 is a top view of a second portion of the two-cell culture vessel shown in figure 1;

Figure 7 is a bottom view of the second portion shown in figure 6;

Figure 8 is a side view of the second portion shown in figure 6;

Figure 9 is a sectional view of the second portion shown in figure 6 taken along line A-A of figure 8;

Figure 10 is a perspective view of a two-part cell culture vessel according to a second embodiment of the present invention;

Figure 11 is a top view of a second portion of the two-cell culture vessel shown in figure 10;

Figure 12 is a bottom view of the second portion shown in figure 11 ;

Figure 13 is a side view of the second portion shown in figure 11 ; Figure 14 is a sectional view of the second portion shown in figure 11 taken along line A-A of figure 13;

Figure 15 is a perspective view of a cell culture vessel according to the present invention used as an insert in a cell culture plate; and

Figure 16 is a perspective schematic illustration of a deep well cell culture plate according to an aspect of the present invention.

Figure 1 shows a first embodiment of a cell culture vessel 1 composed of first and second interengageable portions 2, 3. The two portions 2, 3 may be releaseably engageable such that they can be connected and subsequently disconnected as many times as desired, or the two portions 2, 3 may be non- releasably engageable, such that they can be connected together once but then cannot be disconnected. The two portions 2, 3 may be non-releasably engaged together in any appropriate manner, for example, chemically with the use of a suitable adhesive or the like and/or physically by the interlocking of suitably formed components (not shown in Figure 1). The two portions 2, 3 may be non- releasably engaged by applying a suitable amount of a substance, such as acetone, which dissolves a small amount of the surface material of one or both of the two portions 2, 3 such that when the two portions 2, 3 contact one another the dissolved material effectively welds the two portions 2, 3 together.

The first portion 2 (shown in detail in figures 2 to 5) has a generally annular body defined by an upwardly extending wall 4, which tapers radially outwardly from a lower rim 5 to an upper rim 6. The lower rim 5 thus defines a diameter which is smaller than a diameter defined by the upper rim 6.

The lower and upper rims 5, 6 define respective lower and upper openings to an internal chamber 7 within which can be retained cell culture media and the like. The second portion 3 of the cell culture vessel 1 of the present invention (shown in detail in figures 6 to 9) is in the form of a generally annular ring comprising an upwardly extending wall 8 defining lower and upper parts 9, 10, with a radially inwardly extending flange 11 extending from the lower part 9 of the wall 8.

The upwardly extending wall 8 tapers radially outwardly from the lower part 9 of the wall 8 to the upper part 10 of the wall 8. Thus, the lower part of 9 of the wall 8 defines a smaller diameter than the upper part 10 of the wall 8.

The angle at which the upwardly extending wall 8 of the second portion 3 tapers radially outwardly relative to a vertical axis X is substantially similar to the angle at which the upwardly extending wall 4 of the first portion 2 tapers radially outwardly relative to axis X. A diameter between diametrically opposite regions of an inner surface 12 of the second portion 3 is approximately equal to a diameter defined between diametrically opposite regions of an outer surface 13 of the upwardly extending wall 4 of the first portion 2. In this way, the second portion 3 is dimensioned so that it can be slid into contact with a lower part 14 of the wall 4 of the first part 2 in the direction of arrow Z (or the first portion 2 can be slid in a direction opposite to arrow Z into contact with the second portion 3) such that the upwardly extending wall 8 of the second part 3 radially overlies the lower part 14 of the upwardly extending wall 3 of the first part 2 with an interference fit. The first and second portions 2, 3 are then retained together by virtue of this interference fit without the need to apply any further external force.

When the second portion 3 is slideably engaged around the lower part 14 of the first portion 2 of the cell culture vessel 1 , a space (not shown) is defined between the lower rim 5 of the first portion 2 and an upper surface 15 of the radially inwardly extending flange 11 of the second portion 3. The space extends axially along axis X to define a height that is suitable to receive a cell culture scaffold (not shown) of a desired thickness. In the specific embodiment described herein, the space also extends uniformly radially outwardly from axis X to the inner surface 12 of the upwardly extending wall 8 of the second portion 3 such that the space has a cylindrical form approximately matching the form of a typical cell culture scaffold. Although it is possible to use the cell culture vessel 1 of the present invention to retain conventional two-dimensional cell culture membranes, the vessel 1 of the present invention is eminently suitable to safely and securely receive three-dimensional cell culture scaffolds, for example, of the kind described in detail in the applicant's co-pending International Patent Application No. PCT/GB2007/001464 (Publication Number WO-2007/125288).

Sliding movement of the second portion 3 in the direction of arrow Z into engagement with the lower part 14 of the wall 4 of the first portion 2 is controlled by the corporation of two pairs of features, one feature of each pair being provided by each of the first and second portions 2, 3. First, when a cell culture scaffold is retained on the upper surface 15 of the flange 11 of the second portion 3, upward movement of the second portion 3 in the direction of arrow Z is limited by contact of an upper surface of the scaffold contacting the lower rim 5 of the first portion 2. Second, an upper rim 16 of the upwardly extending wall 8 of the second portion 3 is stepped so as to define a plurality of sections of differing height which cooperate with radially outwardly extending ribs 17 connected to the first portion 2 to limit upward movement of the second portion 3 in the direction of arrow Z and also to define the height of the space within which the cell culture scaffold is received. It can be seen from figures 1 to 9 that the first portion 2 of the cell culture vessel 1 is provided with 3 equally spaced pairs of ribs 17 and that the second portion 3 is provided with 3 equally spaced sets of steps. Cooperation of the stepped upper rim 16 of the second portion 3 with the ribs 17 of the first portion 2 therefore ensures that the space between the lower rim 5 and the flange 11 possesses an appropriate height to receive a cell culture scaffold of a desired thickness.

When it is desired to use the cell culture vessel 1 of the present invention, the first and second portions 2, 3 and cell culture scaffold (not shown) should be assembled as follows. An appropriate scaffold is first selected based on the application in hand. The scaffold will have a pre-determined thickness which will need to be accommodated in the space between the lower rim 5 of the first portion 2 and upper surface 15 of the flange 11 of the second portion 3. The cell culture scaffold is first located within the second portion 3 so as to rest on the upper surface 15 of the flange 11. The second portion 3 carrying the scaffold is then slid into engagement with the lower part 14 of the upwardly extending wall 4 of the first portion 2 until the appropriate section of the stepped upper rim 16 of the second portion 3 contacts its respective outwardly extending rib 17 of the first portion 2. Provided the first and second portions 2, 3 have been engaged together in an appropriate manner, i.e. with the correct relative angular displacement of the second portion 3 relative to first portion 2 with respect to axis X, contact of the upper rim 16 with the ribs 17 should define a space of suitable height so that the inwardly extending flange 11 securely retains the cell culture scaffold against the lower rim 5 of the first portion 2 but does not exert too great a force which might otherwise compress the periphery of the scaffold and potentially harm the integrity of the structure of the scaffold.

If, having engaged the second portion 3 with the first portion 2, the scaffold is not securely retained between the flange 11 and the lower rim 5 of the first and/or second portion 2, 3 can be rotated about axis X so as to adjust the relative angular displacement of these portions 2, 3 so as to align a shorter section of the stepped rim 16 with the ribs 17, such that the second portion 3 can be slid further upwards in the direction of arrow Z to reduce the height of the space within which the scaffold is received. This process can be repeated as necessary until the scaffold is properly retained between the first and second portions 2, 3.

Figure 10 shows a second embodiment of a cell culture vessel 101 composed of first and second interengageable portions 102, 103. As in the first embodiment, the first and second portions 102, 103 may be releasably or non- releasably engageable. The general form and intended application of the second embodiment of a cell culture vessel 101 is the same as the first embodiment described in detail above with reference to figures 1 to 9. Reference numerals are used in figures 10 to 14 in relation to the second embodiment which are the same as those used to refer to corresponding features in the first embodiment save for being increased by 100.

As will be appreciated from figure 10, the shape and function of the first portion 102 of the second embodiment of the vessel 101 is the same as that of the first portion 2 of the first embodiment. Accordingly, the first portion 102 of the second embodiment will not be described further. The difference between the first and second embodiments of the vessel 1 , 101 resides in the design of the second portions 3, 103 of each vessel 1 , 101.

In the second embodiment, the second portion 103 defines a stepped upper rim

116 of generally similar design to the upper rim 16 of the second portion 3 of the first embodiment, but the upper rim 116 of the second portion now defines a plurality of upwardly extending teeth 122, one tooth 122 extending upwardly from the centre of each step. The width, x, of each tooth 122 approximates the distance, y, separating the ribs 117 within each pair. In this way, when the second portion 103 is brought into engagement with the first portion 102 and the step of desired height aligned with its respective set of ribs 117, the tooth 122 extending upwardly from the centre of the pre-selected step can slide between the ribs 117 and thereby aid proper alignment of the first and second portions 102, 103. It will also be appreciated that by appropriate selection of the width, x, of the teeth 122 so as to closely match the separation, y, between each pair of ribs 117, frictional contact can be achieved between the teeth 122 and the ribs

117 so as to increase the force required to separate the first and second portions 102, 103.

In addition to the increased frictional contact between the teeth 122 and the ribs 117, the provision of upwardly extending teeth 122 around the full extent of the upper rim 116 of the second portion 103 has the effect that, that as the two portions 102, 103 are brought into contact, a radially inner surface 123 of each tooth 122 contacts an outer surface 113 of an upwardly extending wall 104 of the first portion 102 so as to significantly increase the contact area between the first and second portions 102, 103 as compared to the first embodiment of the vessel 1 described above. In this way, the two portions 102, 103 are more securely locked together and the force required to subsequently disengage the two portions 102, 103 is increased. In variants to the second embodiment where it is intended that the first and second portions 102, 103 are non-releasably engageable, a relatively small amount of adhesive, solvent (e.g. acetone) or the like may be applied to some or all of the teeth 122 to provide the necessary chemical interaction to bond the two portions 102, 103 together.

As can be seen with reference to figure 15, the cell culture vessel 1 , 101 of the present invention is suitable for use as a well insert in a conventional cell culture plate 18 defining a plurality of wells 19. The first portion 2, 102 of the cell culture vessel 1 , 101 of the present invention is provided with a plurality of radially outwardly extending flanges 20, 120 at an upper part 21 , 121 of the upwardly extending wall 4, 104 of the first portion 2, 102 so as to support the cell culture vessel 1 , 101 within one of the wells 19 defined in the cell culture plate 18. Once the vessel 1 , 101 of the present invention has been loaded with a cell culture scaffold as hereinbefore described the vessel 1, 101 can then be used in the same way as a conventional cell culture well insert within the cell culture plate 18.

The cell culture vessel 1 , 101 may also be used with a modified cell culture plate (not shown) in which two or more vessels 1 , 101 are suspended in a common cell culture medium reservoir. That is, rather than each vessel 1 , 101 being supported within its own separate well, two or more vessels 1 , 101 may be in fluid communication with a plate which defines a common reservoir for cell culture medium.

The cell culture vessel 1 , 101 may also be used with a modified cell culture plate of the kind illustrated in figure 16. The plate 24 shown in figure 16 is in accordance with a further aspect of the present invention and includes a plurality of wells 25, which are deeper, i.e. possess a larger dimension 'a', than wells in conventional cell culture plates. The plate 24 shown in figure 16 has a typical 6-well layout, but it will be appreciated that it may define any appropriate number of wells, for example 12, 24, 48 or more.

The plate 24 may be used with cell culture vessels 1 , 101 as inserts to be supported within each well 25, in which case each vessel 1 , 101 supports one or more 2D or 3D cell culture scaffolds, or the vessels 1 , 101 may be omitted such that one or more cell culture scaffolds (2D, or more preferably 3D scaffolds) are located directly within one or more of the wells 25.

By virtue of using deeper wells 25, a tissue scaffold located within each well 25 can be exposed to a greater volume of cell culture medium than in conventional cell culture plates. In this way, the need to replenish cell culture medium during cell growth can be reduced or eliminated, thereby providing a more stable growth environment for the cells and reducing the amount of manual input required. It will be appreciated that this is a particular advantage when using the plate 24 with 3D scaffolds which can, for example, have a volume of up to around 20 times that of a conventional 2D scaffold and so may require up to around 20 times the volume of growth medium. In a conventional cell growth plate this can necessitate very regular monitoring and replenishment of growth medium, with the attendant disadvantages in terms of the lack of stability of the growth environment and increased manual input. Such problems can be overcome by using a deep-well plate 24 of the kind depicted in figure 16 since each well 25 can be appropriately dimensioned to provide the required increase in volume of growth medium. The embodiment illustrated in figure 16 employs deeper wells 25, but it will be appreciated that the increase in volume can be achieved by increasing one or more dimensions of a well in a conventional plate, such as the diameter of the well alone or in combination with an increase in depth of the well. A further alternative method to increase the volume of growth medium available to support cell growth is to connect one or more wells to a reservoir which can contain appropriate additional volumes of growth medium. The volume increase can therefore be achieved by modifying a conventional plate to incorporate a reservoir containing the additional growth medium in which the wells are of essentially conventional dimension, or the increase in volume can be achieved by making the wells deeper (as in figure 16) and/or wider, or a combination of a reservoir and larger wells can be employed.

It will be appreciated from the foregoing description that the cell culture vessel 1 , 101 of the present invention provides a relatively simple and convenient means for reliably supporting a three-dimensional cell culture scaffold within a cell culture medium but without damaging the structural integrity of the scaffold.

Numerous modifications can be made to the embodiments of the invention described above without departing from the underlying inventive concept and that these modifications are intended to be included within the scope of the invention.

The first and second portions 2, 3, 102, 103 of the cell culture vessel 1, 101 of the present invention have been provided with three sets of cooperating features (i.e. steps in rim 16, 116 and ribs 17, 117) to control the height of the space within which the cell culture scaffold is retained during use. It will be apparent to the skilled person that any number of cooperating features of this kind can be employed, such as just a single set of steps and a single outwardly extending formation to cooperate with said steps. Alternatively, 2, 4, 5 or more sets of cooperating features could be provided to suit a particular application. Moreover, each pair of cooperating features do not need to be identical, rather a first pair of cooperating features could define a first set of heights for receipt of cell culture scaffolds and a second pair of features could define a second set of heights that is different to the first set.

It will also be appreciated that the upper rim 16, 116 of the second portion 3, 103 may define curved and/or inclined sections supplementing or substituting some or all of the stepped sections to cooperate with outwardly extending formations defined by the first portion 2, 102. Furthermore, the outwardly extending formations (e.g. ribs 17, 117) of the first portion 2, 102 may take any desirable form, provided that they are appropriately positioned and dimensioned to cooperate with suitable features of the second portion 3, 103 to control the height of the space for receipt of the scaffold. By way of example, a first set of ribs 17, 117 could be provided a first distance from the lower rim 5, 105 of the first portion 2, 102, and a second set of ribs 17, 117 could be provided a different height from the lower rim 5, 105 of the first portion so as to define two different heights or sets of heights for the space within which the scaffold is to be retained.

One or more radially extending ribs or notches (not shown) may be defined by one or both of the first and second portions 2, 3, 102, 103 to provide a locking function to hold the two portions 2, 32, 3, 102, 103 together following engagement. In one preferred embodiment it is envisaged that the, or each, rib or notch on one portion 2, 3, 102, 103 may engage with a complementary formation, such as a groove or recess (not shown), on the opposite portion 2, 3, 102, 103. In a further embodiment, axially offset ribs or notches are defined by both portions 2, 3, 102, 103 which pass over one another during engagement of the two portions 2, 3, 102, 103 and may provide an audible 'click' to signify that the two portions 2, 3, 102, 103 are securely engaged together.

It will also be evident to the skilled person that the general form of the first and second portions 2, 3, 102, 103 does not have to be annular but could take any appropriate size and/or shape. For example, the upwardly extending walls 4, 8, 104, 108 of the first and second portions 2, 3, 102, 103 could be composed of four side walls of similar width such that the first and second portions 2, 3, 102, 103 define square cross sections.

Control of the height of the space between the lower rim of the first square portion and the inwardly extending flange of the second square portion could then be controlled in a similar way as the specific embodiment described above, but when it was desired to adjust the height of the space within which the scaffold is to be retained, the rotation of the second portion relative to the first portion would then be limited to one of four positions. Accordingly, it will be appreciated that the first and second portions could be provided with any appropriate number of side walls defining any desirable cross section which would then limit relative angular displacement of the first and second portions to a maximum number of positions defined by the number of side walls of each portion. Providing the first and second portions 2, 3, 102, 103 of the vessel 1, 101 with single continuous annular side walls defining circular cross sections is advantageous because the nature of the side wall does not limit the relative angular location of the second part 3, 103 relative to the first part 2, 102.

The configuration of the vessel of the present invention also enables a plurality of cell culture scaffolds to be received at any one time within the space defined between the flange 11 , 111 of the second portion 3, 103 and the lower rim 5, 105 of the first portion 2, 102. By appropriate selection of the relative angular displacement of the second portion 3, 103 relative to the first portion 2, 102, the space between the flange 11, 111 and the lower rim 5, 105 can be of a height approximating twice the thickness of a particular cell culture scaffold or the thickness of two different types of cell culture scaffold when placed on top of one another, in which case two scaffolds could be retained within the space without harming the structural integrity of the scaffolds. The vessel 1 , 101 of the present invention can therefore be used to safely and securely support any desirable number of cell culture scaffolds of the same or different design without fear of causing damage to the delicate structure of the scaffolds. This enables two or more three-dimensional cell culture scaffolds, possibly containing different ceil types, to be securely retained against one another within a cell culture medium, thereby enabling the proliferation of one cell type into the other second cell type to be monitored in a three-dimensional environment. Moreover, the design of the vessel 1 , 101 of the present invention enables one or more three-dimensional cell culture scaffolds to be combined with one or more two- dimensional cell culture scaffolds.

While the embodiment of the present invention described above includes a single cell culture vessel, it will be apparent to the skilled person that two or more of such cell culture vessels can be connected together so as to define an array of cell culture vessels which could be located within a corresponding array of cell culture wells defined in a conventional cell culture plate. For example, six cell culture vessels according to the specific embodiment of the present invention described above could be connected via appropriate extensions of their upper rims 6 (in a similar fashion to radially outwardly extending flanges 20) to connect the six vessels together in a 3 x 2 arrangement corresponding to that of a conventional 6-wel! cell culture plate. It will be appreciated that the present invention is not limited to use with a 6-well plate and that it may be used with any desirable plate arrangement, such as a plate defining 12, 24, 48 or more wells.

Claims

1. A cell culture vessel comprising first and second interengageable portions, the first portion comprising an upwardly extending wall defining an upper rim and a lower rim, and the second portion comprising an inwardly extending flange which is arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said lower rim and said flange for receipt of a cell culture scaffold.

2. A cell culture vessel according to claim 1 , wherein the second portion comprises an upwardly extending wall which is dimensioned so as to be located outwardly of the upwardly extending wall of the first portion when the first and second portions of the cell culture vessel are engaged together.

3. A cell culture vessel according to claim 2, wherein said upwardly extending wall of the second portion is dimensioned to contact said upwardly extending surface of the first portion with an interference fit.

4. A cell culture vessel according to claim 2 or 3, wherein the upwardly extending wall of the second portion is shorter than the upwardly extending wall of the first portion.

5. A cell culture vessel according to claim 2, 3 or 4, wherein the upwardly extending wall of the second portion defines upper and lower parts, and said inwardly extending flange extends from said lower part of said upwardly extending wall.

6. A cell culture vessel according to claim 5, wherein a distance between opposite regions of an outer surface of the upper part of the upwardly extending wall of the second portion is greater than a distance between opposite regions of an outer surface of the lower part of the upwardly extending wall.

7. A cell culture vessel according to any one of claims 2 to 6, wherein the upwardly extending wall of the second portion defines an annular wall.

8. A cell culture vessel according to any one of claims 2 to 7, wherein the upwardly extending wall of the first portion defines an outwardly extending formation which is arranged to cooperate with an upper rim defined by the upwardly extending wall of the second portion when said first and second portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

9. A cell culture vessel according to claim 8, wherein said cooperation of the outwardly extending formation with the upper rim of the second portion determines a height of the space between said lower rim and said flange for receipt of the cell culture scaffold.

10. A cell culture vessel according to claim 9, wherein said height is up to around 1000 microns.

11. A cell culture vessel according to claim 9, wherein said height is around 75 microns to around 500 microns.

12. A cell culture vessel according to any one of claims 8 to 11 , wherein the first and second portions, when engaged, define a substantially vertical axis extending through a centre of said first and second portions, and at least one of the outwardly extending formation of the first portion and the upper rim of the second portion is configured such that the height of the space for receipt of the cell culture scaffold can be varied between at least two values by adjusting an angular displacement of the first portion relative to the second portion with respect to said substantially vertical axis.

13. A cell culture vessel according to any one of claims 8 to 12, wherein said cooperation is effected by contact between a lower surface of the outwardly extending formation of the first portion and the upper rim of the second portion.

14. A cell culture vessel according to claim 13, wherein the upper rim of the second portion is stepped, inclined and/or curved along a length of the rim such that at least two sections of the upwardly extending wall of the second portion possess different heights which thereby define at least two different values for the height of the space for receipt of the cell culture scaffold.

15. A cell culture vessel according to claim 13 or 14, wherein the lower surface of the outwardly extending formation is stepped, inclined and/or curved such that at least two sections of the outwardly extending formation possess different heights which thereby define at least two different values for the height of the space for receipt of the cell culture scaffold.

16. A cell culture vessel according to any one of claims 8 to 15, wherein the upper rim of the second portion defines one or more upwardly extending teeth which are arranged to contact a surface of the upwardly extending wall of the first portion and thereby provide an interference fit between said one or more teeth and said surface.

17. A cell culture vessel according to claim 16, wherein said one or more teeth are configured to cooperate with said outwardly extending formation defined by the upwardly extending wall of the first portion to control relative alignment of the first and second portions during engagement and/or provide a frictional contact between said one or more teeth and said outwardly extending formation.

18. A cell culture vessel according to any preceding claim, wherein a distance between opposite regions of an outer surface of the upwardly extending wall of the first portion at the upper rim is greater than a distance between opposite regions of said outer surface at the lower rim.

19.A cell culture vessel according to any preceding claim, wherein the upwardly extending wall of the first portion defines an annular wall.

20. A cell culture vessel according to any preceding claim, wherein the upper and lower rims of the first portion define upper and lower openings to facilitate access to an interior cavity defined by the upwardly extending wall of the first portion.

21. A cell culture vessel according to claim 20, wherein a diameter defined by said upper opening is greater than a diameter defined by said lower opening.

22.A cell culture vessel according to any preceding claim, wherein the first and second portions are releasably interengageable.

23.A cell culture vessel according to any one of claims 1 to 21 , wherein the first and second portions are non-releasably interengageable.

24. A kit of parts to be assembled to provide a cell culture vessel, said kit of parts comprising first and second interengageable cell culture vessel portions, the first portion comprising an upwardly extending wall defining an upper rim and a lower rim, and the second portion comprising an inwardly extending flange which is arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said lower rim and said flange for receipt of a cell culture scaffold.

25.A cell culture vessel comprising first and second interengageable portions which, when engaged, are arranged to receive a cell culture scaffold between said first and second portions, the first portion comprising an upwardly extending wall defining an outwardly extending formation, and the second portion comprising an upwardly extending formation which is arranged to cooperate with said outwardly extending formation defined by the wall of the first portion when said first and second portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

26.A cell culture vessel according to claim 25, wherein the upwardly extending wall of the first portion defines an upper rim and a lower rim, and the second portion comprises an inwardly extending flange which is arranged to underlie the lower rim of the first portion when said first and second portions are engaged so as to define a space between said lower rim and said flange for receipt of the cell culture scaffold.

27.A cell culture vessel according to claim 26, wherein said cooperation of the outwardly extending formation of the first portion with the upwardly extending formation of the second portion determines a height of the space between said lower rim and said flange for receipt of the cell culture scaffold.

28.A cell culture vessel according to claim 27, wherein said height is up to around 1000 microns.

29.A cell culture vessel according to claim 27, wherein said height is around 75 microns to around 500 microns.

30. A cell culture vessel according to any one of claims 27 to 29, wherein the first and second portions, when engaged, define a substantially vertical axis extending through a centre of said first and second portions, and at least one of the outwardly extending formation of the first portion and the upwardly extending formation of the second portion is configured such that the height of the space for receipt of the cell culture scaffold can be varied between at least two values by adjusting an angular displacement of the first portion relative to the second portion with respect to said substantially vertical axis.

31. A cell culture vessel according to any one of claims 27 to 30, wherein said cooperation is effected by contact between a lower surface of the outwardly extending formation of the first portion and an upper rim of the upwardly extending formation of the second portion.

32.A cell culture vessel according to claim 31 , wherein the upper rim of the second portion is stepped, inclined and/or curved along a length of the rim such that at least two sections of the upwardly extending formation of the second portion possess different heights which thereby define at least two different values for the height of the space for receipt of the cell culture scaffold.

33.A cell culture vessel according to claim 31 or 32, wherein the lower surface of the outwardly extending formation is stepped, inclined and/or curved such that at least two sections of the outwardly extending formation possess different heights which thereby define at least two different values for the height of the space for receipt of the cell culture scaffold.

34.A cell culture vessel according to any one of claims 26 to 33, wherein the upper rim of the second portion defines one or more upwardly extending teeth which are arranged to contact a surface of the upwardly extending wall of the first portion and thereby provide an interference fit between said one or more teeth and said surface.

35.A cell culture vessel according to claim 34, wherein said one or more teeth are configured to cooperate with said outwardly extending formation defined by the upwardly extending wall of the first portion to control relative alignment of the first and second portions during engagement and/or provide a frictional contact between said one or more teeth and said outwardly extending formation.

36.A cell culture vessel according to any one of claims 26 to 35, wherein a distance between opposite regions of an outer surface of the upwardly extending wall of the first portion at the upper rim is greater than a distance between opposite regions of said outer surface at the lower rim.

37.A cell culture vessel according to any one of claims 26 to 36, wherein the upper and lower rims of the first portion define upper and lower openings to facilitate access to an interior cavity defined by the upwardly extending wall of the first portion.

38.A cell culture vessel according to claim 37, wherein a diameter defined by said upper opening is greater than a diameter defined by said lower opening.

39.A cell culture vessel according to any one of claims 26 to 38, wherein the upwardly extending formation of the second portion defines upper and lower parts, and said inwardly extending flange extends from said lower part of said upwardly extending wall.

40. A cell culture vessel according to claim 39, wherein a distance between opposite regions of an outer surface of the upper part of the upwardly extending formation of the second portion is greater than a distance between opposite regions of an outer surface of the lower part of the upwardly extending formation.

41. A cell culture vessel according to any one of claims 25 to 40, wherein the upwardly extending wall of the first portion defines an annular wall.

42.A cell culture vessel according to any one of claims 25 to 41 , wherein the upwardly extending formation of the second portion is dimensioned so as to be located outwardly of the upwardly extending wall of the first portion when the first and second portions of the cell culture vessel are engaged together.

43.A cell culture vessel according to claim 42, wherein said upwardly extending formation of the second portion is dimensioned to contact said upwardly extending surface of the first portion with an interference fit.

44.A cell culture vessel according to any one of claims 25 to 43, wherein the upwardly extending formation of the second portion is shorter than the upwardly extending wall of the first portion.

45.A cell culture vessel according to any one of claims 25 to 44, wherein the upwardly extending formation of the second portion defines an annular wall.

46.A cell culture vessel according to any one of claims 25 to 45, wherein the first and second portions are releasably interengageable.

47.A cell culture vessel according to any one of claims 25 to 45, wherein the first and second portions are non-releasably interengageable.

48.A kit of parts to be assembled to provide a cell culture vessel, said kit of parts comprising first and second interengageable cell culture portions which, when engaged, are arranged to receive a cell culture scaffold between said first and second portions, the first portion comprising an upwardly extending wall defining an outwardly extending formation, and the second portion comprising an upwardly extending formation which is arranged to cooperate with said outwardly extending formation defined by the wall of the first portion when said first and second portions are engaged so as to limit movement of the second portion relative to the upwardly extending wall of the first portion.

49. A cell culture device comprising a cell culture vessel and at least two cell culture scaffolds, the cell culture vessel comprised of first and second interengageable portions engaged together to define a space within which is received the at least two cell culture scaffolds.

50. A cell culture device according to claim 49, wherein the cell culture vessel is in accordance with any one of claims 1 to 23 or any one of claims 25 to 47.

51. A cell culture apparatus comprising a plurality of cell culture vessels connected together, at least one of said cell culture vessels being in accordance with any one of claims 1 to 23 or any one of claims 25 to 47.

52.A cell culture vessel according to any preceding claim, wherein the cell culture scaffold is a three-dimensional cell culture scaffold.

53.A cell culture device comprising a cell culture vessel and a three- dimensional cell culture scaffold, the cell culture vessel comprised of first and second interengageable portions engaged together to define a space within which is received the three-dimensional cell culture scaffold.

54.A cell culture device according to claim 53, wherein the cell culture vessel is in accordance with any one of claims 1 to 23 or any one of claims 25 to 47.

55.A kit of parts to be assembled to provide a cell culture device, said kit of parts comprising a cell culture vessel and a three-dimensional cell culture scaffold, the cell culture vessel comprised of first and second interengageable portions engaged together to define a space within which is received the three-dimensional cell culture scaffold.

56.A cell culture apparatus comprising a plurality of cell culture vessels, at least one of said cell culture vessels being in accordance with any one of claims 1 to 23 or any one of claims 25 to 47 and the apparatus further comprising a cell culture plate associated with said plurality of cell culture vessels, said plate defining a reservoir for cell culture medium in fluid communication with at least two of said plurality of cell culture vessels.

57.A kit of parts to be assembled to provide a cell culture apparatus comprising a plurality of cell culture vessels, at least one of said cell culture vessels being in accordance with any one of claims 1 to 23 or any one of claims 25 to 47 and the apparatus further comprising a cell culture plate associated with said plurality of cell culture vessels, said plate defining a reservoir for cell culture medium in fluid communication with at least two of said plurality of cell culture vessels.

58.A cell culture vessel substantially as hereinbefore described with reference to figures 1 to 9, 10 to 14 and/or 15 of the accompanying drawings.

59.A cell culture plate defining one or more wells for receipt of a cell culture scaffold or receipt of a well insert to support a cell culture scaffold, at least one of said wells possessing a volume for receipt of cell culture medium of at least around 10 ml.

60. A cell culture plate according to claim 59, wherein said at least one well possesses a volume for receipt of cell culture medium of up to around 100 ml.

61. A cell culture plate according to claim 59, wherein said at least one well possess a volume for receipt of cell culture medium of around 50 to 80 ml.

62. A cell culture plate according to any one of claims 59 to 61 , wherein said at least one well possesses a depth of at least around 20 mm.

63.A cell culture plate according to claim 62, wherein said at least one well possesses a depth of up to around 100 mm.

64. A cell culture plate according to any one of claims 59 to 61 , wherein said at least one well possesses a depth of around 25 to 40 mm.

65. A cell culture plate defining one or more wells for receipt of a cell culture scaffold or receipt of a well insert to support a cell culture scaffold, the plate further defining a reservoir for cell culture medium in fluid communication with at least one of said wells, wherein said at least one well and the reservoir possess a combined volume for receipt of cell culture medium of at least around 10 ml per well.

66. A cell culture plate according to claim 65, wherein said at least one well and the reservoir possess a combined volume for receipt of cell culture medium of around 50 to 80 ml per well.

67. A cell culture plate according to claim 65 or 66, wherein said at least one well possesses a depth of at least around 20 mm.

68. A cell culture plate according to claim 65 or 66, wherein said at least one well possesses a depth of around 25 to 40 mm.

69. A cell culture plate according to any one of claims 65 to 68, wherein the plate defines a plurality of wells and the reservoir is in fluid communication with at least two of said plurality of wells.

70. A cell culture plate substantially as hereinbefore described with reference to figure 16 of the accompanying drawings.

71. A cell culture apparatus comprising one or more cell culture vessels in accordance with any one of claims 1 to 23 or any one of claims 25 to 47, and a cell culture plate in accordance with any one of claims 59 to 70.