Removable module for the culture of cells in a perfused device

Abstract

A cell culture insert is described herein which is intended for use with a perfused cell culture. The insert comprises a generally cylindrical retaining ring for retaining the insert in a cell culture well. The retaining ring has upper and lower openings formed by a central bore, which has a stepped profile so that a narrower portion of the bore is spaced from the lower opening. The insert also comprises a cell scaffold, a filter and a compression ring. The compression ring is sized and shaped to be received within the lower opening of the retaining ring. The cell scaffold and filter are received within the lower opening of the retaining ring and the cell scaffold is exposed to the upper opening of the retaining ring. The compression ring retains the scaffold and filter within the retaining ring against the narrower portion of the bore. A cell culture system comprising the one or more inserts, and a multiwell cell culture plate are also described herein.

Description

[0001] Removable module for the culture of cells in a perfused device

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a cell culture insert suitable for use in a perfused cell culture device. Preferably the insert is suitable for use in a multiwell cell culture plate.

[0004] BACKGROUND TO THE INVENTION

[0005] Cell culture inserts are used in conjunction with well plates for the growth and differentiation of various cell types. For some applications, it can be beneficial to have perfused culture medium circulating within the well, to replenish the medium while cellular waste and depleted medium are removed and also to provide fluid shear stresses as mechanical stimuli to the cells. For example, this process may be carried out to study responses of cells to drugs, or to investigate metabolism of cells or tissues. It may also be advantageous to be able to remove the cultured cells from the well for analysis (for example, by microscopy). The present invention is intended to provide a simple and effective means for culturing cells in a perfused cell culture system which permits such removal. The invention also provides a means of easily handling the modules which facilitates their use and removal when used in applications requiring the simultaneous study of a large number of conditions or simultaneous repetitions of the same condition within a multiwell cell culture plate.

[0006] SUMMARY OF THE INVENTION

[0007] According to an aspect of the present invention, there is provided a cell culture insert comprising: a generally cylindrical retaining ring, for retaining the insert in a cell culture well, and having upper and lower openings formed by a central bore with a stepped profile providing a narrower portion of said bore spaced from the lower opening; a cell scaffold; a filter; and a compression ring sized and shaped to be received within the lower opening of the retaining ring; wherein the cell scaffold and filter are received within the lower opening of the retaining ring, the cell scaffold being exposed to the upper opening of the retaining ring, and wherein the compression ring retains the scaffold and filter within the retaining ring against said narrower portion of the bore. This conformation permits the insert to be easily used in either a static or perfused cell culture. In particular, the scaffold and filter can be located within the bore from the lower opening, and the compression ring inserted so as to compress the scaffold and filter against the narrower bore portion. Cells can be seeded into the cell scaffold and the module used statically for cell culture. Cells can also be seeded into the cell scaffold, and culture medium initially perfused through the insert from the upper to the lower openings. This allows the cells to grow and attach to the scaffold, while the filter ensures cells remain colocated to the scaffold to increase residence time for attachment. Once cells have attached and settled onto the scaffold, perfusion may continue in either direction of circulation. The retaining ring may be removed at any point to allow analysis of the cells outside the culture well. Analysis of the cells can also be performed without removing the retaining ring by other means such as assessing sampled media for biomarkers.

[0008] The cell scaffold, filter, and compression ring are each preferably configured to have compatible shapes with the bore. Generally this will be circular, but in some embodiments there may be asymmetries provided in each of the components to permit a particular alignment to be achieved, if desired.

[0009] Preferably the retaining ring is configured to provide an interference fit with a cell culture well. In some embodiments, this is achieved by providing a screw thread on the exterior portion of the retaining ring. This may be pushed or rotated into a cell culture well, so as to engage with the internal walls of the culture well, and removed by reversing the action. In other embodiments, ribs or other protrusions may be provided on the exterior portion of the retaining ring. The retaining ring may be formed from a rigid biocompatible plastic material.

[0010] Preferably the retaining ring includes a shaped profile at the lower opening to engage with a corresponding profile provided on the compression ring. For example, the lower opening may include detents or notches which may engage with corresponding protrusions on the compression ring, as will be described. The remainder of the retaining ring may be received within the bore of the retaining ring.

[0011] In preferred embodiments, the stepped profile of the bore provides a narrower central section of the bore, with the sections adjacent both the upper and lower openings being wider. The bore may provide shoulders facing both the upper and lower openings. These serve multiple purposes. The lower-facing shoulders provide a portion of the bore against which the cell scaffold and filter can be urged by the compression ring, and retain these in place when the insert is in use. The upper-facing shoulders provide a portion of the bore where cells will not be cultured, and may be used as a ledge to add or remove cell culture medium from the insert (for example, by pipette) such that the risk of disturbing or dislodging the cells from the cell scaffold is reduced.

[0012] Preferably the cell scaffold comprises pores or other openings extending therethrough; these pores provide a region within which cells may settle and adhere during cell culture. The pores may be provided in a generally triangular repeating pattern, which provides a relatively high density of said pores. In preferred embodiments, the cell scaffold does not have pores in the peripheral or edge regions. These regions will engage in use with the bore of the retaining ring and / or the compression ring, so the absence of pores provides improved sealing against these components. The arrangement also has the advantage of restricting flow of cell culture medium to the region of scaffold having pores. The pores are preferably cylindrical.

[0013] In preferred embodiments the cell scaffold is formed of a biocompatible (for example, bio-inert) polymer. Preferably the polymer is polystyrene. The cell scaffold may be coated with a suitable extracellular matrix material, to enhance cell attachment. Preferably the scaffold is coated with collagen, although other materials may be used.

[0014] The filter will have pore sizes selected to retain cells on the scaffold side, while permitting culture medium to pass. The precise size will therefore depend on the application, although typical pore sizes may be, for example, 0.2, 0.5, 0.7, 1 , 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, or 10 pm. A preferred pore size is 5 pm.

[0015] The filter may be made of any suitable material, although polymer materials are preferred; for example, nylon, cellulose, polyethylene, PTFE, or PVDF. PVDF (polyvinylidene difluoride) is most preferred. The filters are typically nonwoven, and may be, for example, electrospun polymer fibres or a track-etched film.

[0016] In preferred embodiments, the filter is not bonded or otherwise adhered to the cell scaffold, instead being retained simply by the compression ring. However, in certain embodiments the filter may be bonded to the cell scaffold, to provide additional support to the filter; such bonding should be carried out in a manner which minimises potential damage to the filter and scaffold, and is biocompatible with cell culture.

[0017] In some embodiments the filter may comprise an additional support material; this may be particularly useful when the filter is relatively thin (for example, less than 0.3, 0.25, 0.2, 0.15, or 0.1 mm). The support material again may be any suitable material, but preferably is a non-filtering perforated sheet polymer material, to provide support without hindering filtration.

[0018] The compression ring is preferably generally cylindrical in shape, although as mentioned it may include ribs or protrusions to engage with the retaining ring. Preferably the compression ring and retaining ring engage in a non-rotatable relationship; this ensures that the insert as a whole is retained in the same relative position when the insert is rotated (for example, to insert or remove it from a cell culture well). The compression ring may further or alternatively comprise one or more (preferably at least three) crush ribs designed for an interference fit with the internal surface of the retaining ring. These crush ribs may be in addition to protrusions which match with detents in the retaining ring. In some embodiments, crush ribs may be provided on the retaining ring in addition to, or as an alternative to, such ribs on the compression ring.

[0019] The compression ring preferably includes one or more internal ribs to provide support to the scaffold and filter. In a preferred embodiment, the compression ring may comprise at least three radial internal ribs.

[0020] The compression ring is preferably formed of a deformable material, to allow it to seat snugly in the retaining ring. A preferred material is polypropylene.

[0021] Preferably the cell culture insert further comprises cells. In a preferred embodiment, the cells are mammalian cells, preferably human cells, and more preferably liver cells.

[0022] A further aspect of the invention provides a cell culture system comprising a multiwell cell culture plate and one or more inserts as described herein.

[0023] A still further aspect of the invention provides a kit of parts for assembling a cell culture insert as described herein. BRIEF SUMMARY OF THE DRAWINGS

[0024] Figure 1 shows an exploded view of a cell culture insert.

[0025] Figure 2 shows a side cross sectional view of a retaining ring for a cell culture insert.

[0026] Figure 3 shows a top view of a cell culture insert located in a cell culture well.

[0027] Figure 4 shows a section of the retaining ring illustrating an engaging tool

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention provides a cell culture insert intended for use with a perfused cell culture. An example of an insert 10 is shown in Figure 1. The insert 10 comprises a retaining ring 12 of generally cylindrical shape formed with a through bore 14 extending between upper 16 and lower 18 openings. The retaining ring 12 is shown in cross section in Figure 2, from which can be seen that the through bore 14 has a stepped profile, providing a narrower central portion 20 of the through bore, and upper 22 and lower 24 shoulders. The exterior surface of the retaining ring 12 is provided with a screw thread 26 which, in use, engages with a cell culture well. The lower opening 18 includes a pair of notches 28.

[0030] The other components of the insert 10 are received into the retaining ring 12 via the lower opening 18. In order, these are a cell scaffold 30, a filter 32, and a compression ring 34. The scaffold 30 includes a number of apertures or pores 44 extending through it, and sized to allow cells to enter the scaffold. The compression ring 34 includes a pair of tabs 36 which engage with the notches 28 of the retaining ring 12, and a number of crush ribs 38 which are received in an interference fit into the retaining ring 12. The compression ring 34 further includes a number of radial ribs 40 (in this example, three) which support the filter 32 and scaffold 30.

[0031] To assemble the insert 10, the scaffold 30 and filter 32 are placed into the lower opening 18 of the retaining ring 12, and the compression ring 34 inserted so as to urge the scaffold 30 and filter 32 against the lower shoulder 24. The crush ribs 38 ensure the compression ring 34 is snugly received within the retaining ring 12, while the tabs 36 and notches 28 engage to prevent relative rotation of the components. The insert 10 may then be placed within a cell culture well or removed as a single unit. Figure 3 shows a top view of the insert 10 within a culture well 42, showing also the scaffold 30 and the upper shoulders 22.

[0032] In general terms, the insert is intended to facilitate the culture of cells and their subsequent removal for analysis (e.g. microscopy). The insert permits the creation of cell tissue in tubule structures, designed to replicate the tubule structures found in liver organs. To create the tubule the module is used in combination with a cell perfusion system which perfuses the cell growth media through the central bore of the tubule. Once the tubule has been cultured, it can be subjected to various conditions of interest by adding reagents to the cell growth media. The insert may then be removed from the culture well and perfusing device to analyse the effect of the conditions on the cell tissue.

[0033] The insert comprises four components: a retaining ring 12, a scaffold 30, a filter 32, and a compression ring 34. The components are largely cylindrical in shape and are axially assembled to each other, in the order described above, prior to the introduction of any biological material or fluid. The insert can then be inserted into a culture well in the perfusion device where the insert is primed with cell growth media. The fluid volume in the top half of the retaining ring 12 is removed and replaced with a concentrated cell suspension using a pipette or similar apparatus. The fluid is then drawn through the scaffold 30 until a thin layer of fluid remains above the scaffold surface. As the fluid is perfused out of the insert, the cells in suspension remain within the scaffold pores 44, and are prevented from perfusing further into the system by the filter 32. Additional cell growth media is then added to replace the previously removed fluid and the insert continues to be perfused to promote attachment of the cells to the scaffold 30 and the formation of tissue. Once the cells have attached and tissues begin to form, the perfusion may then continue in either direction.

[0034] The retaining ring 12 provides the means of assembling and disassembling the insert 10 from the perfusion device. The preferred shape of the retaining ring 12 is a hollow cylinder with an external thread 26 which provides the means of retention in the culture well of the perfusion device. The retaining ring 12 may be screwed and unscrewed using a rotary tool which engages with surfaces of the retaining ring 12 to facilitate its removal by external means, and avoid potential damage to the cells. An example of a portion of a tool 44 with the retaining ring 12 is shown in Figure 4. Once the insert has been removed from the perfusion device, the tool 44 may also be optionally used to handle the insert by remaining attached to the tool. A notch 46 or series of notches within the inner bore 14 of the retaining ring engages with a sprung clip 48 within the tool to allow the retaining ring to be mechanically retained on the tool. The tool may also incorporate means to facilitate the disassembly of the module by allowing the compression ring to be pushed out of the module assembly so as to make the scaffold containing the cell tissue accessible. These features reduce the need for additional tools for handling, manipulating, or disassembling the module thus reducing the probability of contamination with external biological material or chemicals.

[0035] A notch 28 or series of notches engages with the tab features 36 on the compression ring 34 to ensure that the compression ring 34 remains rotationally locked to the retaining ring 12. This prevents the generation of torque forces on the scaffold 30 and filter 32 during assembly and removal of the insert 10 from the perfusion device. The cell media is perfused through the central bore of the cylinder 12. At the scaffold interface, the cross- sectional flow area is limited to the area of the scaffold 30 containing the pores 44, thanks to the lower shoulder 24 against which the scaffold 30 is urged by the compression ring 34. Additional flat surfaces formed by the upper shoulder 22 within the cylinder 12 permit the addition or removal of cell media using a pipette without risk of contacting or fluid shearing the cell tissue. Lastly, the retaining ring 12 includes a recess open at the lower end 18 to accept the scaffold 30, filter 32, and compression ring 34. The contacting surfaces seal against the scaffold 30 by means of pressure to allow the perfused fluid to flow through tissue structures rather than around it. The internal volume of the cylinder 12 between the scaffold surface and top face is sized to house the quantity of concentrated cell suspension fluid required to correctly seed the scaffold at a given cell density.

[0036] The scaffold 30 provides the main structure for the attachment of the tissue. The preferred shape of the scaffold 30 is a flat surface containing a number of cylindrical pores 44 perpendicular to the scaffold surface. The pores 44 are primarily located in the centre of the scaffold and are surrounded by a flat area without pores which functions as the sealing face against the retaining ring 12. The thickness of the scaffold 30 and size of the pores 44 are tailored to replicate the size of tubules found in liver organs. To encourage entrainment of the cells into the pores 44, the pores 44 are evenly distributed across the centre area of the scaffold 30 and arranged in a repeating triangular pattern so as to minimise the amount of non-pore surface area presented to the flow. The preferred material for the scaffold 30 is polystyrene, however, alternative materials may be used if consideration is given to their biocompatibility and the quantity and nature of any extractable substances which may leach into the cell growth media. To promote the adhesion of cells to the scaffold 30, the surfaces of the scaffold are coated with collagen to provide an extracellular matrix. Alternative extracellular matrix materials may be preferred depending on the cell type or experiment to be conducted.

[0037] The filter 32 provides a mean of retention for the cells preventing them from being perfused past the scaffold 30 in the scaffold seeding process. To achieve this, the filter 32 must be in intimate contact with the scaffold surface, so as to prevent cells in suspension from sitting between the filter 32 and scaffold 30. A variety of filter types and materials may be chosen to suit the required application; specific consideration should be given to filter pore size (and its distribution) versus the size of the cells in use, the biocompatibility of the filter material, the quantity and nature of the extractables from the filter material, and the degree of absorbance or binding of the cell growth media, its components, or any reagents subsequently added to the cell growth media for the purpose of the experiment. In this particular example the filter 32 is not bonded to the scaffold surface so the filter thickness must be selected to provide sufficient flexural stiffness so as to minimise its displacement when it is subjected to the perfused flow pressure. Where the filter thickness cannot be easily increased, additional non-filtering components made from perforated sheet materials may be added behind the filter 32 to provide additional stiffness without impeding the fluid perfusion. The preferred filter material for this particular example is a 5pm electrospun polyvinylidene fluoride (PVDF) filter, 0.16mm in thickness without a stiffening component.

[0038] The compression ring 34 serves to channel the fluid passing through the scaffold 30 and filter 32 back into the perfusion device through a central aperture. The preferred shape for the compression ring 34 is a hollow cylinder. The central aperture may contain several ribs 40 on the filter contacting surface to ensure that the filter 32 and scaffold 30 remain in intimate contact when assembled and as an additional means of filter support when the insert is subject to perfused flow. The number, shape, distribution, and thickness of the ribs should be selected to minimise the surface area in contact with the filter 32 to reduce the number of pores in the scaffold 30 that are obscured, partly or fully, from the perfused flow. The present example has three ribs extending radially from the centre of the compression ring 34, 0.3mm in thickness. To facilitate assembly, several crush ribs 38 are located on the periphery of the compression ring 34. The crush ribs 38 are designed to interfere with the radial surfaces of the recess in the retaining ring 12 when the insert is assembled. Their purpose is to provide a temporary means of retention for the scaffold 30 and filter 32 when the insert 10 is located outside of the perfusion device so that the insert 10 may be handled as a single component. The number of crush ribs, their shape, and the level of interference is dependent on the materials of the retaining ring 12 and compression ring 34 and the manner of their manufacture. The present example includes three cylindrical ribs 38 distributed radially with 0.1mm interference on the retaining ring recess. The preferred material for the compression ring 34 is polypropylene. The external cylindrical surface of the compression ring 34 also features several tabs 36 designed to engage with a matching notch 28 on the retaining ring 12 to prevent rotational movement between the retaining ring 12 and compression ring 34. This prevents the generation of torsional forces on the scaffold 30 and filter 32 during assembly and disassembly of the insert 10 into the perfusion device. The top and bottom surfaces of the compression ring 34 are intended to provide fluidic seals against the filter 32 and perfusion device to prevent the flow from bypassing the scaffold pores 44. These surfaces are flat to match the surfaces on the scaffold 30 and retaining ring 12 in terms of size and location. The compressive force required to maintain these seals is generated by the thread 26 of the retaining ring 12 once it is assembled into the perfusion device. Lastly, the compression ring 34 is designed to be symmetrical between the top and bottom halves. The symmetry of this component removes the need for the user to identify the top or bottom half thus simplifying the assembly of the component into the insert.

[0039] In some embodiments, alternatives to a thread 26 may be used. For example, snap clips and removable superstructures attached to the cell culture device may be included with in the perfusion device to provide a suitable compression force to seal the compression ring 34 against the perfusion device.

[0040] In other embodiments, the insert components may be designed so that the compression ring 34 houses the recess which contains the scaffold 30, filter 32, and retaining ring 12. In this embodiment the direction of the crush ribs 38 would need to be inverted or relocated to the compression ring 34 to achieve a similar effect. Optionally, the thread 26 could also be relocated to the compression ring 34 and the insert assembled in the opposite direction. However, this would require the perfusion device to achieve the seal against the external sealing face of the compression ring.

[0041] A further variant may alter the relationship between the scaffold and the filter gap. The example described above relies on the rigidity of the scaffold 30 and filter 32 to eliminate gaps between these two components. Bonding methods such as adhesives or welding (e.g. laser, ultrasonic, heat) may be used to achieve a similar effect reducing or eliminating the need for mechanical rigidity. These processes may interact and degrade the collagen coating where it is applied before the bonding process. Furthermore, any adhesives must also remain biocompatible, have minimal amounts of extractable substances, and cause minimal absorbance or binding of cell growth media, its components, or of any experimental reagents used.

[0042] A further variant may allow multiple modules to be linked by means of a rigid connecting element to allow their simultaneous removal from the device. The retaining means, a screw thread or otherwise, for the linked modules may be present on a single module, several, or all of the modules. Where some of the modules are not provided with a retaining means, their retention is achieved through the rigid connecting element. This facilitates the removal of large numbers of modules quickly where a number of conditions or repetitions are required to be studied.

Claims

CLAIMS:

1. A cell culture insert comprising: a generally cylindrical retaining ring, for retaining the insert in a cell culture well, and having upper and lower openings formed by a central bore with a stepped profile providing a narrower portion of said bore spaced from the lower opening; a cell scaffold; a filter; and a compression ring sized and shaped to be received within the lower opening of the retaining ring; wherein the cell scaffold and filter are received within the lower opening of the retaining ring, the cell scaffold being exposed to the upper opening of the retaining ring, and wherein the compression ring retains the scaffold and filter within the retaining ring against said narrower portion of the bore.

2. The cell culture insert according to claim 1 , wherein the cell scaffold, filter, and compression ring are each configured to have compatible shapes with the bore.

3. The cell culture insert according to any preceding claim, wherein the retaining ring is configured to provide an interference fit with a cell culture well.

4. The cell culture insert according to any preceding claim, wherein a screw thread is provided on the exterior portion of the retaining ring.

5. The cell culture insert according to any preceding claim, wherein the retaining ring includes a shaped profile at the lower opening to engage with a corresponding profile provided on the compression ring.

6. The cell culture insert according to claim 5, wherein the lower opening of the retaining ring includes detents or notches which engage with corresponding protrusions on the compression ring.

7. The cell culture insert according to any preceding claim, wherein the stepped profile of the bore provides a narrower central section of the bore, with the sections adjacent both the upper and lower openings being wider.

8. The cell culture insert according to claim 7, wherein the bore provides shoulders facing both the upper and lower openings.

9. The cell culture insert according to any preceding claim, wherein the cell scaffold comprises pores or other openings extending therethrough.

10. The cell culture insert according to claim 9, wherein the cell scaffold does not have pores in the peripheral or edge regions.

11. The cell culture insert according to claim 9 or claim 10, wherein the pores are cylindrical.

12. The cell culture insert according to any preceding claim, wherein the cell scaffold is formed of a biocompatible polymer.

13. The cell culture insert according to any preceding claim, wherein the cell scaffold is coated with an extracellular matrix material.

14. The cell culture insert according to any preceding claim, wherein the filter is not bonded or otherwise adhered to the cell scaffold.

15. The cell culture insert according to any preceding claim, wherein the compression ring and retaining ring engage in a non-rotatable relationship.

16. The cell culture insert according to any preceding claim, wherein the compression ring comprises one or more crush ribs designed for an interference fit with the internal surface of the retaining ring.

17. The cell culture insert according to any preceding claim, wherein the compression ring includes one or more internal ribs to provide support to the scaffold and filter.

18. The cell culture insert according to claim 17, wherein the compression ring comprises at least three radial internal ribs.

19. The cell culture insert according to any preceding claim, wherein the compression ring is formed of a deformable material.

20. The cell culture insert according to any preceding claim, wherein the cell culture insert further comprises cells.

21. The cell culture insert according to claim 20, wherein the cells are mammalian cells.

22. A cell culture system comprising a multiwell cell culture plate and one or more inserts according to any preceding claim.

23. A kit of parts for assembling a cell culture insert according to any one of claims 1 to

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