WO2024188751A1

WO2024188751A1 - Adjustable bottom platform for cell seeding into an open bottom well

Info

Publication number: WO2024188751A1
Application number: PCT/EP2024/055836
Authority: WO
Inventors: Dharaminder SINGH; David Hughes
Original assignee: Cn Bio Innovations Limited
Priority date: 2023-03-10
Filing date: 2024-03-06
Publication date: 2024-09-19
Also published as: GB2628007A; GB202303587D0

Abstract

A cell culture vessel comprises a first upper portion defining a culture volume, and a second lower portion in fluid communication with the first upper portion, defining a sleeve and piston. The piston is movable within the sleeve between a first closed position in which the fluid communication between the second lower portion and the first upper portion is closed; and a second open position in which the fluid communication is open. The vessel also includes a fluid circulation loop between the first and second portion to permit circulation of fluid between the first and second portions. When the piston is in the first closed position, the fluid circulation loop is also closed by the piston; and when the piston is in the second open position, the fluid circulation loop is also open.

Description

Adjustable bottom platform for cell seeding into an open bottom well

FIELD OF THE INVENTION

The present invention relates to products and systems for use in cell culture. In particular, embodiments of the invention relate to cell culture vessels, such as culture wells and multi-well plates, having an adjustable lower surface. Such vessels are believed to be advantageous in techniques relating to three-dimensional cell culture.

BACKGROUND TO THE INVENTION

Many biological protocols, for example, drug discovery, involve culturing and observing cells grown in tissue culture. Cell culture in flat plates has the disadvantage that cells which are constrained to an essentially two-dimensional environment may not exhibit behaviour or phenotype which accurately reproduces that of an in vivo three-dimensional environment. Furthermore, cell interactions influence the morphology, function and plasticity of a cell. Within the body most cell interactions are cell-cell interactions, however traditional in vitro biology is centred around cultures on flat plastic surfaces, which are planar or 2D; this favours cell-surface interactions. This cell-surface interaction often leads to flattened cells, with physiology and function different to the in vivo environment.

In order to obtain a three-dimensional cell culture, various techniques can be used. One such technique is so-called “hanging drop” tissue culture, in which a small volume of growth medium is suspended from a surface so as to allow gravity and surface tension to maintain the medium in a droplet. Culturing cells within this hanging drop allows growth in a three-dimensional environment, so potentially allowing observation of behaviour and phenotype without the constraints of two-dimensional cell culture. One use of hanging drop culture in particular is generation of spheroids, three-dimensional aggregates of multiple cells which are considered to more accurately model tissues and tumours, given the presence of both surface and internal cells, which may have different proliferation rates.

Other techniques for three-dimensional cell culture include the use of ultra-low adhesion (ULA) (or ultra-low attachment) surfaces. Although such ULA surfaces technically involve culturing cells on a two-dimensional surface, these surfaces are treated with a ULA material, for example, a hydrogel, which minimises cell attachment to that surface. The result is that cells preferentially adhere to one another, resulting in a three-dimensional spheroid or other aggregate.

Also in use are techniques such as culture within extracellular matrix hydrogel, or within porous three-dimensional scaffolds. However, the hanging drop method and ULA culture are favoured by the pharmaceutical industry, as they are relatively simple to implement and highly scalable, making them particularly suitable for drug screening.

However, three-dimensional cell cultures (and in particular, spheroids) produced using hanging drop or ULA methods typically suffer from a size limitation. For example for liver cells the diameter of the spheres produced can not exceed -350 pm without the core of the sphere becoming necrotic owing to insufficient mass transport of oxygen through the mass of cells (Nath, S. and Devi, G.R., 2016. Three-dimensional culture systems in cancer research: Focus on tumor spheroid model. Pharmacology & therapeutics). Studies have shown that the spheroid fabrication platforms directly influence the onset of hypoxia. (Schmitz, C., Potekhina, E., Belousov, V.V. and Lavrentieva, A., 2021. Hypoxia onset in mesenchymal stem cell spheroids: monitoring with hypoxia reporter cells. Frontiers in Bioengineering and Biotechnology). This limitation can be overcome to some degree by flowing cell culture medium through or around the spheroid or microtissue, which acts to improve mass transport. In regular multi-well ULA plates (eg 96 well, 384, etc) fluid flow past the spheroid is not possible as the spheroid is formed in the base of a solid well; likewise, fluid flow is at best difficult to implement in the hanging drop technique.

WO 2017/187680 to JTEC Corp describes a large scale cell culture system for subculturing and for translocation between spheroids and a liquid culture medium in a closed system using a vessel having a syringe-type structure.

WO 2018/015561 to Celyad describes a cell processing device for performing independent concurrent processing of a plurality of cell preparations, the cell processing device comprising:(l) a cell processing station; and(ll) a plurality of cell processing modules, wherein the plurality of cell processing modules engage and communicate with the cell processing station; wherein each of the plurality of cell processing modules comprises discrete processing compartments defined within, the processing compartments comprising:(i) a reagent pack, the reagent pack comprising one or more reagent vessels; and (ii) one or more fluidic cartridges, each fluidic cartridge comprising one or more cell processing compartments and a cell incubation compartment, wherein each of the processing compartments is in fluid communication with each other.

CN 101974423 to Univ Beihang describes a quasi-physiological pulsating flow environment arterial blood vessel tissue engineering reactor, comprising a liquid storage device, a liquid driver and a blood vessel tissue dynamic culture chamber which are sequentially connected through a fluid pipeline to form a fluid circulation loop; and a pulsation generator.

US 2008/0213819 to Besson-Faure et al describes a device for preparing a body fluid for a bacteriological analysis thereof comprising a container provided with a chamber wherein a piston is mobile between an opening position and a closing position, the position of the piston determining whether separate volumes within the chamber are able to communicate.

It is among the objects of embodiments of the present invention to provide alternative products and devices for three-dimensional cell culture; particular embodiments are intended to provide products and devices for three-dimensional cell culture which permit fluid flow through or around the cultured cells. Embodiments of the invention provide products and devices which reduce cell-surface interactions, by permitting three- dimensional cell culture.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a cell culture vessel comprising: a first upper portion defining a culture volume, comprising a volume for receiving a cell culture medium and cells; a second lower portion in fluid communication with the first upper portion, defining a sleeve and piston, the piston being movable within the sleeve between a first closed position in which the fluid communication between the second lower portion and the first upper portion is closed; and a second open position in which the fluid communication is open; and a fluid circulation loop between the first and second portion to permit circulation of fluid between the first and second portions; wherein, when the piston is in the first closed position, the fluid circulation loop is also closed by the piston; and when the piston is in the second open position, the fluid circulation loop is also open.

The culture vessel of the invention therefore permits initial growth of a three-dimensional cell culture in a closed vessel, with the piston in the closed position. As the culture increases in size, the piston can be opened, and circulation of culture medium and/or other nutrients established through the vessel and around the cell culture. This enhances cell growth compared with a culture without circulation. The closed piston initially allows culture growth on a surface as the cell culture is smaller; once the size has increased (through cells adhering together and producing extracellular matrix) to the point where the piston may be opened, the larger cell cluster will be too large to enter the sleeve, and hence will be retained in the upper portion of the culture vessel.

The upper portion is generally conical or otherwise in the shape of a well, with walls converging toward the lower part of the upper portion. This configuration helps draw cells together and allows cell-cell attachment to take place. The piston and sleeve may be located at the lowest part of the upper potion, thereby ensuring that any well contents are in contact with the piston and associated flow path.

In the closed position, the first upper portion and the piston preferably together define a cell culture surface. The cell culture surface may be formed directly from elements of the culture vessel (for example, a plastic-walled vessel), or may be formed in part indirectly, for example, from a cell culture scaffold disposed within the vessel (for example, an extracellular matrix scaffold, which may be a natural ECM scaffold ora synthetic scaffold, or any other suitable material). The cell culture surface is preferably an ultra low attachment (ULA) material; for example, this may take the form of a coating applied to the cell culture vessel wall and/or to a surface of the piston. In a preferred embodiment only the piston may include a ULA material; this reduces manufacturing costs, while retaining the advantages of the use of a ULA material. The skilled person will be aware of suitable materials such as those routinely used in cell culture work.

Use of a ULA material allows cells to rest on (but not adhere to) a ULA surface during initial cell seeding and growth, affording cells time to adhere to each other or the sides of a potential scaffold enabling the formation of microtissues and three-dimensional cultures (eg, spheroids, organoids etc). Following this the ULA surface is removed, by moving the piston to the second, open, position, both reducing the surfaces available for cells to rest on (and hence reducing cell-surface interactions), and creating a flow path which allows cell culture medium to circulate around or through the spheroid or other cell cluster. To aid changing of the cell culture medium or retrieval of the cultured cells, the piston and associated ULA surface may be returned to the original position, to close the fluid flow path.

Note that in some embodiments of the invention, the flow path as such may not actually be used; cells may be cultured in the vessel without active circulation of culture medium. Movement of the piston to the open position retains the advantage of reducing available surfaces for cells to rest on. It may not be necessary to incorporate circulation of culture medium to achieve at least some benefits of the invention. In some embodiments in which the flow path is not used, the fluid circulation loop may be arranged to be closed regardless of the position of the piston; or may be absent or removed from the culture vessel altogether. However, it is preferred to retain the fluid circulation loop to allow for some movement of fluid within that loop as the piston is opened or closed; this might reduce currents within the vessel which may impact the cultured cells.

The cell culture surface may include patterning (for example, grooves, spots, etc) to guide cell culture formation and/or growth. The patterning may be physical and/or chemical in nature.

The culture vessel may further comprise one or more seals to close any gaps between the piston and the sleeve. The seals may be located on the piston or the sleeve, or both; but preferably the seals are located on the sleeve. The seals may be in the form of one or more O-rings. In a preferred embodiment, at least two seals are provided, located on either side of the location where a fluid circulation loop connects to the lower portion.

The culture vessel may further comprise a pump arranged to circulate fluid between the first and second portions via the fluid circulation loop. Alternatively, in other embodiments, the fluid circulation may be gravity-driven from the first upper portion to the second lower portion. In yet further embodiments, the fluid circulation loop need not be driven at all, such that there is no active circulation of culture medium. In some embodiments, the fluid circulation loop may be a closed loop; in others at least some portion of the loop may be open - that is, either open to the atmosphere, which may allow oxygen to be adsorbed into the cell culture medium in the loop; or open in the sense that the loop does not comprise a continuous stream of fluid - for example, in part the loop may be represented by a series of volumes of fluid rather than a continuous flow.

The culture vessel may further comprise an actuator arranged to move the piston between the first and second positions (and, optionally, may locate the piston at one or more additional positions). Any suitable form of actuator may be used; for example, mechanical, pneumatic, hydraulic, or magnetic.

In preferred embodiments, the culture vessel may comprise a plurality of culture volumes, sleeves, and pistons, with each volume being associated with a respective sleeve and piston. That is, the culture vessel may effectively replicate the format of a multi well plate, with multiple culture wells each with an associated piston. The fluid flow arrangements may include appropriate manifolds and so forth to allow for fluid circulation individually within multiple wells.

Aspects of the invention further provide a method of culturing cells, the method comprising: providing a cell culture vessel according to the invention, wherein the piston is located in the first position; culturing cells in a culture medium in the culture volume while the piston is in the first position; moving the piston to the second position, and maintaining the cell culture in the culture volume.

In other aspects of the invention, the piston arrangement may be useful to provide mechanical stimulation to a cell culture; for example, by moving the piston between positions so as to gently contact the cell culture at intervals.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects of the invention will now be described by way of example only and with reference to the accompanying drawings, in which: Figure 1A and 1 B show a sketch of a culture vessel in accordance with an embodiment of the invention, illustrating in particular the piston in first and second positions;

Figure 2A and 2B show a sketch of a culture vessel in accordance with a further embodiment of the invention, illustrating in particular the piston in first and second positions.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a cell culture vessel which is intended to be particularly suitable for use in culturing larger-volume cell clusters, such as spheroids or organoids, in three- dimensional culture. There are currently a number of options for cell culture of spheroids and organoids, but each of these suffers from one or more disadvantages.

At the most basic, cells can of course be grown in two-dimensional cell culture on tissue culture plastics. This produces cells adhered to a continuous bottom surface of the culture vessel, which do not form three dimensional cultures. Cultures of cells adhered to a surface often display features which are unlike physiological conditions. For example, this may produce flattened cells, and can negatively affect the plasticity of cells (that is, whether they differentiate).

One possible way to avoid the problems of two dimensional cell culture is the use of so- called “hanging drop” methodologies, in which cells are cultured in a droplet of medium suspended from a support. This ensures that the cells do not seed onto a bottom surface, so providing true three dimensional culture, but the droplet and physics set a maximum size for the culture, so limiting the options in terms of seeding size. Further, this is a relatively labour intensive and time consuming process. It is also difficult to change the culture medium without potentially disrupting the spheroid. Further, the hanging drop method is sensitive to mechanical shocks; and reduces options for potential flow of medium throughout the culture.

A further approach is the use of ULA culture plates, which are coated to reduce or prevent cell adhesion to the surface. Regular ULA plates allow spheroid formation, but not the application of flow through the culture. Further, this can be an inefficient methodology for spheroid formation, resulting in non-uniform spheroid size across wells; and as with hanging drops, renders it difficult to change the culture medium. Embodiments of the present invention are intended to address one or more of these shortcomings of alternative methods. In some embodiments, this is achieved by providing a movable lower surface to a culture well. The movable surface may be left in position during early culturing of cells while the cell mass is relatively small. A ULA coating on the surface can reduce or prevent cell adhesion to the surface, thereby encouraging three-dimensional growth of the culture.

Once the cultured spheroid has grown to a larger size, the movable surface is moved to a second position, which primarily has the effect of reducing the surface area available for cell-surface interactions. In certain embodiments, the movement secondarily opens a fluid flow circuit around the well, allowing medium to circulate within the well. The initial growth phase with the movable surface in place allows the spheroid to grow to a larger size such that it will be retained in the well once the surface is moved.

A first embodiment of the invention is illustrated in Figures 1 A and 1 B. Here, a cell culture vessel 10 is illustrated schematically; this may represent a single well in a multi-well plate. The vessel 10 includes an upper portion 12 forming the body of the well and defining in part a culture volume. The lower surface of the culture volume (and of the well) is defined by an upper face 14 of a piston 16. The upper face 14 of the piston 16 is coated with a ULA coating. The piston 16 is located within a sleeve 18 which together define a lower portion 20 of the vessel 10. The sleeve 18 includes a narrowing 22 at a lower edge to form a stop against which the piston 16 may rest, and below the stop is a pneumatic tube 32. Corresponding stops (not shown) may also be provided at the upper part of the sleeve 18 to limit movement of the piston 16. The sleeve 18 further includes a connection to a fluid flow circuit 24 joining the sleeve 18 with the upper portion 12. (Only a part of the full fluid flow circuit 24 is illustrated; the circuit may include other elements, such as a pump, a reservoir, and access ports to allow addition or removal of medium and/or other additives.) The upper part of the fluid flow circuit 24 is shown here as an open spillway 26 leading from the upper portion of the well, but in other embodiments the circuit may be formed of closed fluid transport vessels. The sleeve 18 also includes upper 28 and lower 30 O rings or other seals, which form a fluid-tight seal between the piston 16 and sleeve 18 when in the correct position, as well as between the piston 16, sleeve 18, and fluid flow circuit 24. Figure 1A shows the culture vessel 10 in the initial, closed configuration, for early spheroid 34 growth. Here the piston 16 may be urged into the closed position by applying pneumatic or other force via the pneumatic tube 32; the piston 16 seals against the O rings 28, 30 and prevents fluid circulation around the vessel 10. The upper surface 14 of the piston 16 provides a lower surface to the culture well, and supports the initial growth of the seeded spheroid 34.

Once the spheroid 34 has grown to a sufficient size, the piston 16 may be opened - see Figure 1 B. This can be achieved by applying a vacuum force to the pneumatic tube 32, so urging the piston 16 downwards to rest against the lower stop 22. In this position, the lower O ring 30 maintains a seal against the piston, but the fluid flow circuit 24 is now opened, and culture medium can be circulated between the upper 12 and lower 20 portions of the culture vessel 10. The spheroid 34 at this time is too large to enter the opening defined by the upper part of the sleeve 20, such that the spheroid remains in the upper portion 12 of the vessel 10 while receiving circulated medium. This allows sufficient oxygenation to allow further growth, and provides a three-dimensional space with reduced cell-surface interaction for cell culture to occur.

Culture medium may be circulated by means of a pump mechanism, or may be gravity driven. Where a pump is present, this may be operationally connected to the piston such that operation of the two is coordinated. In a gravity driven system, actuation of the piston could be used itself to stop or start flow.

To aid changing of the cell culture medium or retrieval of the spheroid/microtissues at the end of growth, the piston 16 may be returned to its original position to close the fluid circulation loop, and fluid removed from the vessel.

Actuation of the piston may be pneumatic, electromagnetic or by direct application of external force, as required. The ULA surface may be created by surface modification or addition of a second material, in line with conventional understanding of ULA culture vessels. The ULA surface may be patterned to guide the growth and formation of spheroids.

The pistons can be arrayed, one piston per well to allow all wells of a multi-well plate to experience flow. An alternative embodiment of the invention is shown in Figures 2A and 2B, again in open and closed configurations. The vessel 110 here is generally similar to that of Figs 1 A and 1 B, but further includes a three dimensional scaffold 136 being formed with a number of through holes 138. The scaffold may be any suitable three-dimensional cell scaffold material; for example, extracellular matrix material, hydrogel, matrigel, and the like. It will also be noted that the vessel 110 does not take a curved well shape unlike the above embodiment, but rather is essentially flat-bottomed. With the piston 116 in the closed position, the scaffold 136 and piston 116 form a number of closed wells, within which cells may seed and begin to form an organelle or tubule through the holes 138. Once the piston 116 is opened (Figure 2B) the scaffold remains in place, but culture medium can be circulated through the cellular tubules. This can be beneficial in modelling organs which rely on exchange of materials through a tube; for example, kidney, liver, etc. The arrangement of the culture vessel also allows for circulating immune cells to be added to the culture, to support normal functioning of the cultured microtissues within the scaffold, where relevant. These immune cells can completely circulate around the system without becoming trapped, enabling immune invasion studies and similar. Circulation could also enable metastasis studies following the culture of cancer cells.

Thus, embodiments of the invention allow cells to be seeded with a bottom ULA surface, allowing them to form microtissues/spheroids, and then allow for the spheroids to be cultured long term under fluid flow of eg culture medium (without the presence of the bottom surface). It will be appreciated that if the piston were not present and cells were seeded into a well/scaffold with an open bottom they would simply fall through the hole, and not form spheroids/microtissues.

After the cells have formed spheroids/microtissues the flow can be applied without the need to handle or disturb the cells. Some prior art systems may require cells to be formed in cell culture plates and then manually transferred to flow systems.

The removal of the bottom surface coupled with fluid flow allows immune cells to be circulated around the spheroid/microtissue, where relevant.

The adjustable bottom surface can reduce/remove the need to use hydrogels/protein based ECM to support the formation of spheroids/microtissues, which can be a source of variability in other systems when made from natural materials and can make the handling of spheroids/microtissues difficult.

In some embodiments, the piston feature can also be used to provide mechanical stimulation to the microtissue throughout culture - for example, by repeatedly raising and lowering the piston to either cause direct physical stimulation, or to urge current flow through the culture medium. This can be important for the correct function of mechanosensory cells.

The piston feature could also be adapted in some embodiments to handle the spheroids/microtissues, for example raising them above the level of the medium and out of the well.

The present invention therefore enables new configurations for three dimensional cell culture where large numbers of spheroids/microtissues can be created and then exposed to flow, without the need for transfer to new systems or manual handling. This technology is independent of the type of cell and organ being modelled so has wide potential application.

Claims

CLAIMS:

1 . A cell culture vessel comprising: a first upper portion defining a culture volume, comprising a volume for receiving a cell culture medium and cells; a second lower portion in fluid communication with the first upper portion, defining a sleeve and piston, the piston being movable within the sleeve between a first closed position in which the fluid communication between the second lower portion and the first upper portion is closed; and a second open position in which the fluid communication is open; and a fluid circulation loop between the first and second portion to permit circulation of fluid between the first and second portions; wherein, when the piston is in the first closed position, the fluid circulation loop is also closed by the piston; and when the piston is in the second open position, the fluid circulation loop is also open.

2. A cell culture vessel according to claim 1 wherein the upper portion is generally conical or otherwise in the shape of a well, with walls converging toward the lower part of the upper portion.

3. A cell culture vessel according to any preceding claim wherein the piston and sleeve are located at the lowest part of the upper portion.

4. A cell culture vessel according to any preceding claim wherein in the closed position, the first upper portion and the piston together define a cell culture surface.

5. A cell culture vessel according to claim 4 wherein the cell culture surface further comprises a cell culture scaffold disposed within the vessel.

6. A cell culture vessel according to claim 4 or 5 wherein the cell culture surface comprises an ultra low attachment (ULA) material.

7. A cell culture vessel according to any of claims 4 to 6 wherein the cell culture surface comprises patterning to guide cell culture formation and/or growth; optionally wherein the patterning is physical and/or chemical in nature.

8. A cell culture vessel according to any preceding claim wherein the piston comprises a surface having a ULA material.

9. A cell culture vessel according to any preceding claim further comprising one or more seals between the piston and the sleeve.

10. A cell culture vessel according to claim 8 wherein the seals comprise one or more O-rings.

11. A cell culture vessel according to any preceding claim further comprising a pump arranged to circulate fluid between the first and second portions via the fluid circulation loop.

12. A cell culture vessel according to any preceding claim wherein the fluid circulation loop is a closed loop.

13. A cell culture vessel according to any of claims 1 to 11 wherein at least some portion of the fluid circulation loop is open.

14. A cell culture vessel according to any preceding claim further comprising an actuator arranged to move the piston between the first and second positions.

15. A cell culture vessel according to any preceding claim wherein the culture vessel comprises a plurality of culture volumes, sleeves, and pistons, with each volume being associated with a respective sleeve and piston.

16. A method of culturing cells, the method comprising: providing a cell culture vessel according to any preceding claim, and wherein the piston is located in the first position; culturing cells in a culture medium in the culture volume while the piston is in the first position; moving the piston to the second position, and maintaining the cell culture in the culture volume.