Filter for cell culture and cell culture platform thereof

Abstract

The present invention concerns a filter for use in a cell culture well, comprising: a ring sized to fit within the cell culture well; and a mesh attached to a planar side of the ring. The present invention also concerns a cell culture platform, comprising at least one cell culture well; a filter according to the invention wherein positioned within the at least one cell culture well, the filter partitions the at least one cell culture well into a first compartment and a second compartment; wherein the at least one filter is configured to allow fluid communication of a cell culture medium in both directions between both compartments and cell movement only from the first compartment to the second compartment. The invention belongs to the field of cell culture.

Description

[0001] Filter for Cell Culture and Cell Culture Platform Thereof

[0002] Technical Field of the invention

[0003] The present invention relates, in general terms, to a filter and a cell culture platform comprising the filter thereof.

[0004] Background

[0005] Cell culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. After cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. They need to be kept at body temperature (37 °C) in an incubator. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or rich medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, 02), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate to form an adherent culture as a monolayer (one single-cell thick), whereas others can be grown free floating in a medium as a suspension culture. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium, such as broth or agar.

[0006] Among the common manipulations carried out on culture cells are media changes, passaging cells, and transfecting cells. These are generally performed using culture methods that rely on aseptic technique. Aseptic technique aims to avoid contamination with bacteria, yeast, or other cell lines. Manipulations are typically carried out in a biosafety cabinet or laminar flow cabinet to exclude contaminating micro-organisms.

[0007] As cells undergo metabolic processes, acid is produced and the pH decreases. Often, a pH indicator is added to the medium to measure nutrient depletion. Accordingly, the cell medium needs to be changed. In the case of adherent cultures, the media can be removed directly by aspiration, and then is replaced. Media changes in non-adherent cultures involve centrifuging the culture and resuspending the cells in fresh media.

[0008] However, such techniques are highly dependent on the skill of the operator, and in many cases, the cells are damaged or aspirated away.

[0009] It would be desirable to overcome or ameliorate at least one of the above-described problems.

[0010] Summary of the invention

[0011] The present disclosure provides a filter for use in a cell culture well, comprising: a) a ring sized to fit within the cell culture well; and b) a mesh attached to a planar side of the ring.

[0012] In some embodiments, the ring is sized such that its diameter is about 0.1 mm to about 20 mm smaller than a diameter of the cell culture well.

[0013] In some embodiments, the ring is configured to frictionally engage an inner wall of a cell culture well.

[0014] In some embodiments, the ring is formed from an elastic material. In some embodiments, the ring is characterised by an inner diameter of about 1 mm to about 30 mm.

[0015] In some embodiments, the ring is characterised by an outer diameter of about 2 mm to about 35 mm.

[0016] In some embodiments, the ring is characterised by a wall thickness of about 1 mm to about 20 mm.

[0017] In some embodiments, the ring is characterised by a height of about 2 mm to about 15 mm.

[0018] In some embodiments, the ring comprises a recess or hole on at least one of its planar side.

[0019] In some embodiments, the mesh is formed from Nylon.

[0020] In some embodiments, the mesh is a rectangular mesh, wherein the mesh is characterised by a mesh size of about 20 pm by about 20 pm to about 900 pm by about 900 pm.

[0021] In some embodiments, mesh is characterised by a mesh size of about 20 mesh / inch to about 500 mesh / inch.

[0022] In some embodiments, the filter is positioned within the cell culture well such that it is substantially perpendicular to a longitudinal axis of the cell culture well.

[0023] In some embodiments, the filter is positioned within the cell culture well such that it is in contact with a bottom of the cell culture well.

[0024] In some embodiments, the filter is positioned within the cell culture well such that the mesh is adjacent to a bottom of the cell culture well.

[0025] The present disclosure also concerns a cell culture platform, comprising: a) at least one cell culture well; b) a filter as disclosed herein positioned within the at least one cell culture well, the filter partitions the at least one cell culture well into a first compartment and a second compartment. wherein the at least one filter is configured to allow fluid communication of a cell culture medium in both directions between both compartments and cell movement only from the first compartment to the second compartment.

[0026] In some embodiments, the cell culture platform further comprises a second filter, the second filter positioned on top of the filter and within the cell culture well.

[0027] In some embodiments, the second filter comprises a mesh with a larger mesh size than the filter.

[0028] In some embodiments, the first filter and the second filter are spaced apart by at least about 1 mm.

[0029] Brief description of the drawings

[0030] Embodiments of the present invention will now be described, by way of non-limiting example, with reference to the drawings in which:

[0031] Figure 1 : Schematic drawing of the filters ring inside a well with cavities during seeding phase (A) and spheroid (B). Filters can be use together or separately, d: distance between the two filters 0.1>d>3cm

[0032] Figure 2: Example of the filter of the invention A and different filter rings size for different culture vessel as I Bl DI and 24 Aggrewell plate.

[0033] Figure 3: Representative imaging without (A) and with (B) filter on the top of Aggrewells.

[0034] Figure 4: A: Organoids in aggrewell plate with Blocking Device before and after 1 medium exchange step (100% of the wells remained filled). B: Organoids in aggrewell plate without the blocking device before and after one medium exchange steps (80% wells remain filled on average). It requires about 5 to 8 medium exchange / washing steps to culture organoids, fix and stain them resulting to a final yield of (0.8)5 to 8= 15-30% without the blocking device and 100% with it.

[0035] Figure 5: An embodiment of the filter of the present disclosure.

[0036] Figure 6: An image of the filter in a cell culture well when in use. Figure 7: Two photos (A - B) of another embodiment of the filter of the present disclosure.

[0037] Detailed description of the invention

[0038] The present disclosure is predicated on the understanding that spheroids / organoids are cultured in different types of cell culture vessels. However, there can be some loss of organoids during their formation and during various manipulation steps (e.g. drug treatment, washing, etc).

[0039] In order to avoid this inconvenience, the present disclosure concerns a filter for use with a 3D cell culture well. The filter according to the invention can also be called blocking device. By placing the filter within the well and at a distance away from the bottom of the well, cells and / or organoids may be kept inside the well during the experiment. The filter may be sized to allow cells to pass through but not organoids. This allows a user to obtain homogeneous and stable spheroids / organoids throughout the various steps of the experiment up to imaging. In this regard, the filter disperses the cells that are seeded in the wells and lead to an homogeneous seeding of the cell number in each of the microwells. It enhances the reproducibility and the standardization of the initial cell number in each well. Further, the organoids may be cultured, treated (fixed) and imaged (immunostained) in the same culture well throughout the experiment. This makes it possible to follow the organoid through to imaging.

[0040] Accordingly, the present disclosure concerns a filter for use in a cell culture well, comprising: a) a ring sized to fit within the cell culture well; and b) a mesh attached to a planar side of the ring.

[0041] In the filter of the present disclosure, the mesh is attached to the ring such that it covers the ring hole. The size or diameter of the ring may be adjusted according to the size of the cell culture well. The size of the mesh may be adjusted according to the type of cells being cultured. The mesh can also be called filter cloth. Additionally, the ring breaks the flow of medium at each manipulation by reducing the shear force and lifting force. Thus, the medium is easily changed without any loss of organoids.

[0042] The ring is sized to fit within the cell culture well. In some embodiments, the ring is sized such that its diameter is slightly small than the diameter of the cell culture well. For example, the ring may have a diameter which is about 0.1 mm to about 20 mm smaller than the diameter of the cell culture well.

[0043] Accordingly, the ring is sized to be adjacent to an inner wall of the cell culture well. In some embodiments, the ring is sized such that it is spaced apart from the inner wall by about 0.5 mm to about 20 mm.

[0044] In some embodiments, the ring is configured to engage an inner wall of a cell culture well. In some embodiments, the ring is configured to frictionally engage an inner wall of a cell culture well.

[0045] The ring may be formed from an elastic material. For example, the ring may be formed from an elastic polymer. The elastic polymer may be a silicon, such as polydimethylsilane (PDMS). The elastic polymer may be rubber. Alternatively, the ring may be made from polyoxymetylene (POM). Such materials are resistance to wear and tear, and also has a high resistance to chemicals and hydrolysis.

[0046] The ring is sized to fit into a cell culture well. In this regard, the ring may be sized to fit in a well of a 6-well plate, 12-well plate, 24-well plate, 48-well plate, 96-well plate, 384- well plate, 1536-well plate and ibidi p-Dish 35 mm.

[0047] In some embodiments, the ring is characterised by an inner diameter of about 1 cm to about 30 cm. For example, the ring may have an inner diameter of about 1 mm to about 28 mm when used with a 6-well plate, about 1 mm to about 14 mm when used with a 12- well plate, about 1 mm to about 12 mm when used with a 24-well plate, about 1 mm to about 2 mm when used with a 96-well plate, or about 1 mm to about 25 mm when used with a 35 mm p-Dish.

[0048] In some embodiments, the ring is characterised by an outer diameter of about 2 mm to about 35 mm. In some embodiments, the ring is characterised by an outer diameter of about 1 .55 cm. For example, the ring may have an outer diameter of about 31 mm to about 33 mm when used with a 6-well plate, about 17 mm to about 18 mm when used with a 12- well plate, about 15 mm to about 16 mm when used with a 24-well plate, about 3 mm to about 4 mm when used with a 96-well plate, or about 27 mm to about 28 mm when used with a 35 mm p-Dish.

[0049] In some embodiments, the ring is characterised by a wall thickness. The wall thickness is the distance between the inner diameter and the outer diameter. The wall thickness may be about 1 mm to about 20 mm. It was found that when the wall thickness is in this range, the filter is sturdy enough to be inserted within the cell culture well without deformation and tearing of the mesh. For example, the ring may have a wall thickness of about 1 mm to about 15 mm when used with a 6-well plate, about 1 mm to about 9 mm when used with a 12-well plate, about 1 mm to about 7 mm when used with a 24-well plate, about 1 mm to about 2 mm when used with a 96-well plate, or about 1 mm to about 13 mm when used with a 35 mm p-Dish.

[0050] In some embodiments, the ring is characterised by a height of about 2 mm to about 15 mm. The height of the ring corresponds to the depth of the well. The height thus allows the mesh to be spaced apart from the bottom of the well. In some embodiments, the ring is characterised by a height of about 3 mm. It was found that when the height is in this range, the filter is sturdy enough to be inserted within the cell culture well without deformation and tearing of the mesh. Further, the mesh may be spaced such that the filter does not disturb or affect the growth of the organoid. For example, the ring may have a height of about 2 mm to about 11 mm when used with a 6-well plate, about 2 mm to about 12 mm when used with a 12-well plate, about 2 mm to about 10 mm when used with a 24-well plate, about 2 mm to about 6 mm when used with a 96-well plate, or about 2 mm to about 5 mm when used with a 35 mm p-Dish.

[0051] In some embodiments, the ring comprises a groove or hole on at least one of its planar side. The groove may extend partway or at least halfway through the height of the ring. The hole may be a through-hole, which runs substantially perpendicular to the ring's planar side. The groove or hole allows a user to insert or remove the filter into the cell culture well by using tweezers (Figure 7). The ring comprises a planar side (or first planar side). The other planar side is thus the second planar side. The mesh is attached to the first planar side of the ring as shown in Figure 5. The attachment may be via the wall thickness. The attachment may be by an adhesive, such as NOA (Norland Optical Adhesive 73) or epoxy. In particular, the adhesive may be biocompatible.

[0052] In some embodiments, the mesh substantially overlaps with the ring across its wall thickness. It was found that this allows the mesh to be securely fixed to the ring and provides a homogenous surface when contacted with the bottom of the well.

[0053] The mesh size may be configured based on the type of cells being cultured and if organoids are being formed. For example, stem cells have a size of about 10 pm, thus a mesh size of about 50 pm by about 100 pm may be used. After 1stday of culture, the organoids that are formed have a size about 50 pm to about 60 pm, and the 6thday the size reaches around 200 pm. Thus, the mesh size of about 50 pm by about 100 pm is sufficient to maintain the organoids within the cell culture well.

[0054] In some embodiments, the mesh is a screen mesh. In some embodiments, the mesh is a rectangular mesh. In other embodiments, the mesh is formed from a woven fabric or a nonwoven fabric. The fabric may be Nylon, or in particular Nylon 66.

[0055] In some embodiments, mesh is characterised by a mesh size of about 20 pm by about 20 pm to about 900 pm by about 900 pm. The mesh size may be chosen according to the expected size of the organoids. For example, for organoids that will be about 80 pm after 24 hours of culture, a mesh size of 50 pm x 100 pm may be used, or a mesh size of 50 pm x 50 pm may be used.

[0056] In some embodiments, mesh is characterised by a mesh size of about 20 mesh / inch to about 500 mesh / inch. In some embodiments, the mesh size is about 40 mesh / inch to about 500 mesh / inch, about 60 mesh / inch to about 500 mesh / inch, about 80 mesh / inch to about 500 mesh / inch, about 100 mesh / inch to about 500 mesh / inch, about 120 mesh / inch to about 500 mesh / inch, about 140 mesh / inch to about 500 mesh / inch, about 160 mesh / inch to about 500 mesh / inch, about 180 mesh / inch to about 500 mesh / inch, about 200 mesh / inch to about 500 mesh / inch, about 250 mesh / inch to about 500 mesh / inch, about 300 mesh / inch to about 500 mesh / inch, about 350 mesh / inch to about 500 mesh / inch, or about 400 mesh / inch to about 500 mesh / inch.

[0057] In some embodiments, mesh is characterised by a mesh size of about 20 pm to about 900 pm.

[0058] In some embodiments, the filter comprises a second mesh attached to the second planar side of the ring. The mesh may have the same mesh size, or may have a different mesh size relative to the first mesh. The two meshes are thus separated by a distance provided by the ring height.

[0059] The filter thus sits within the cell culture well as the ring is sized to be slightly smaller than the diameter of the inner wall. In some embodiments, the filter is configured to frictional ly engage an inner wall of a cell culture well via the ring.

[0060] In use, the filter is positioned within the cell culture well such that it is substantially perpendicular to the longitudinal axis of the well. The filter may be positioned at a bottom of the well such that the second planar side of the ring is in contact with the bottom of the well. The mesh is thus spaced apart from the bottom of the well, where the cells may adhere.

[0061] Alternatively, the cell culture well may comprise cavities, or microwells at the bottom of the well. In this instance, the filter may be positioned at the bottom of the well such that the first planar side of the ring (and hence the mesh) is adjacent to the bottom of the well. In other embodiments, the first planar side (and hence the mesh) is in close proximity with the bottom of the well. In other embodiments, the first planar side (and hence the mesh) is in contact with the bottom of the well (Figure 6). The second planar side is thus facing the opening of the well. This arrangement allows cells to settle to the bottom and organoids to form within the cavities. The arrangement also avoid the release of organoids / spheroids. The filter allows for a change in culture medium without any loss of organoids.

[0062] When the cell medium is required to be changed, a substantial portion of the cell medium may be removed and fresh cell medium added back to fill the well. For example, in a 24 well plate, 2 ml of medium may be used for the cell / organoids culture. Half of the medium may be removed by pipetting 1 ml above the filter and 1 ml of new media is added. The filter breaks the (turbulent or laminar) flow of the medium as it is removed or added back in, thus at least reduce the force acting on the cells or organoids. This helps maintain their viability. The filter also prevents the cells or organoids from being removed together with the medium.

[0063] The filter may also be used with adherent cell lines. Some adherent cells may be loosely attached to the cell culture vessel. In such cases, the filter may be used to prevent the flow and the loss of cells during medium exchange.

[0064] The present disclosure also concerns a cell culture platform, comprising: a) at least one cell culture well; b) a filter as disclosed herein positioned within the at least one cell culture well, the filter partitions the at least one cell culture well into a first compartment and a second compartment. wherein the at least one filter is configured to allow fluid communication of a cell culture medium in both directions between both compartments and cell movement only from the first compartment to the second compartment.

[0065] Thus, after the cell have settled to the bottom of the cell culture well or the organoids formed, they are prevented from escaping the cell culture well. Due to the porosity of the filter, cell medium may be exchanged.

[0066] In some embodiments, the cell culture platform further comprises a second filter, the second filter positioned on top of the first filter and within the cell culture well. The first filter may be a trap filter, to prevent the loss of organoids when cell medium is changed. The second filter may be homogeneity filter, to allow a homogenous dispersion of cell seeding. The space between these two filters allows the cells to be dispersed more homogenously over the whole surface. The second filter acts to further reduce the forces acting on the cells and / or organoids. It allows for homogeneous cell seeding and homogeneous organoid size across the wells, and allows for keeping of organoids / spheroids inside the well from the beginning till the end of the cell culture.

[0067] The second filter may have a different mesh size, or a same mesh size as the first filter. For example, the first filter may have a mesh size of about 50 pm by about 100 pm. The second filter may have a mesh size of about 100 pm by about 100 pm. In some embodiments, the first filter and the second filter are spaced apart by at least about 1 mm. In other embodiments, the spacing is at least about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, or about 10 mm.

[0068] In some embodiments, the second filter is configured as the first filter.

[0069] To sum up, our approach allows to disperse cells homogenously during seeding and trap spheroids / organoids inside the same compartment from the beginning to the end of experiment.

[0070] It will be appreciated that many further modifications and permutations of various aspects of the described embodiments are possible. Accordingly, the described aspects are intended to embrace all such alterations, modifications, and variations that fall within the spirit and scope of the appended statements.

[0071] Throughout this specification and the statements which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0072] Throughout this specification and the statements which follow, unless the context requires otherwise, the phrase "consisting essentially of", and variations such as "consists essentially of" will be understood to indicate that the recited element(s) is / are essential i.e. necessary elements of the invention. The phrase allows for the presence of other nonrecited elements which do not materially affect the characteristics of the invention but excludes additional unspecified elements which would affect the basic and novel characteristics of the method defined.

[0073] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0074] REFERENCES LIST

[0075] 1 . filter 2. well

[0076] 3. ring

[0077] 4. mesh

[0078] 5. inner wall 6. longitudinal axis

[0079] 7. bottom

[0080] 8. cavity

[0081] 9. hole

[0082] 10. single cell 11. organoid

[0083] 12. ring thickness d. distance between two filters

Claims

CLAIMS1. A filter for use in a cell culture well, comprising: a) a ring sized to fit within the cell culture well; and b) a mesh attached to a planar side of the ring.

2. The filter according to claim 1 , wherein the ring is sized such that its diameter is about 0.1 mm to about 20 mm smaller than a diameter of the cell culture well.

3. The filter according to claims 1 or 2, wherein the ring is configured to frictionally engage an inner wall of a cell culture well.

4. The filter according to any one of claims 1 to 3, wherein the ring is formed from an elastic material.

5. The filter according to any one of claims 1 to 4, wherein the ring is characterised by an inner diameter of about 1 mm to about 30 mm.

6. The filter according to any one of claims 1 to 5, wherein the ring is characterised by an outer diameter of about 2 mm to about 35 mm.

7. The filter according to any one of claims 1 to 6, wherein the ring is characterised by a wall thickness of about 1 mm to about 20 mm.

8. The filter according to any one of claims 1 to 7, wherein the ring is characterised by a height of about 2 mm to about 15 mm.

9. The filter according to any one of claims 1 to 8, wherein the ring comprises a groove or hole on at least one of its planar side.

10. The filter according to any one of claims 1 to 9, wherein the mesh is formed from Nylon.

11. The filter according to any one of claims 1 to 10, wherein the mesh is a rectangular mesh, wherein the mesh is characterised by a mesh size of about 20 pm by about 20 pm to about 900 pm by about 900 pm.

12. The filter according to any one of claims 1 to 11 , wherein mesh is characterised by a mesh size of about 20 mesh / inch to about 500 mesh / inch.

13. The filter according to any one of claims 1 to 12, wherein the filter is positioned within the cell culture well such that it is substantially perpendicular to a longitudinal axis of the cell culture well.

14. The filter according to any one of claims 1 to 13, wherein the filter is positioned within the cell culture well such that it is in contact with a bottom of the cell culture well.

15. The filter according to any one of claims 1 to 14, wherein the filter is positioned within the cell culture well such that the mesh is adjacent to a bottom of the cell culture well.

16. A cell culture platform, comprising: a) at least one cell culture well; b) a filter according to any one of claims 1 to 15 wherein positioned within the at least one cell culture well, the filter partitions the at least one cell culture well into a first compartment and a second compartment; wherein the at least one filter is configured to allow fluid communication of a cell culture medium in both directions between both compartments and cell movement only from the first compartment to the second compartment.

17. The cell culture platform according to claim 16, wherein the cell culture platform further comprises a second filter, the second filter positioned on top of the filter and within the cell culture well.

18. The cell culture platform according to claim 17, wherein the second filter comprises a mesh with a larger mesh size than the filter.

19. The cell culture platform according to claim 17 or 18, wherein the first filter and the second filter are spaced apart by at least about 1 mm.

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