WO2021038038A1

WO2021038038A1 - Multi-cell culture device and kit

Info

Publication number: WO2021038038A1
Application number: PCT/EP2020/074073
Authority: WO
Inventors: Mohamed ALHAMDANI; Jörg HOHEISEL
Original assignee: Deutsches Krebsforschungszentrum
Priority date: 2019-08-30
Filing date: 2020-08-28
Publication date: 2021-03-04

Abstract

The present invention relates to a cell culture device comprising at least a first culture compartment and a second culture compartment, wherein said first culture compartment and the second culture compartment are intervened by a removable barrier; and to kits and methods related thereto.

Description

Multi-cell culture device and kit

An organ is a multi-cell system with highly dynamic events that are orchestrated in an interwoven manner. Investigating events driven or affected by interactions between different cell types, e.g. drug resistance/efficacy, cell signaling, protein expression, etc., in classical cell culture often is not sufficient. Also, for the challenges in biomedicine such as understanding the tumor microenvironment, immune surveillance or response to therapy, models mimicking tissues or even organs are investigated, but nonetheless still desirable. In consequence, drugs may work in cell culture, but are not necessarily effective when tested in animals or patients, since the drugs frequently lose efficacy or the animals/patients develop resistance.

Another issue that is making cell interaction models desirable is the increasing difficulties associated with using animals for testing. A more advanced cell culture system, which represents much better the cell composition of a tissue, could provide the preliminary biological data to come to a conclusion whether subsequent animal experiments are worthwhile and likely to yield useful data. In some areas, such a model may even serve as an alternative to animal testing and act as a surrogate for some animal studies.

To enable co-culture, cell culture inserts were developed which comprise a permeable membrane as the bottom and which can be inserted into multi-well plates in order to allow compounds produced by cell in the upper chamber to diffuse into the underlying well. As a variation, the same insert with larger pore sizes, which allow for cell migration into the underlying well, were provided. However, these inserts have the disadvantage that the cells in the insert have to grow on the membrane, which frequently is deleterious to cell adhesion. Moreover, the handling of the inserts is difficult and prone to contamination. In addition, pore size of the membrane is fixed and cannot be adapted during the analysis. Moreover, the interaction of only two cell entities can be studied (unless mixed cultures are being used, which reduces the control over the experimental parameters substantially), and the interaction cannot be blocked during the progress of the experiment. Lack of keeping the cell types separate before/after interacting does not permit to discriminate the affected from the affecter cells in current systems. Instead, investigators obtain data that represent a sum of the overall biological activity resulting from the various processes within the biological system.

There is, thus, a need still in the art of providing reliable means for co-culturing cells. In particular, there is a need to provide means and methods avoiding at least in part the drawbacks of the prior art as discussed above.

This problem is solved by devices, kits and methods with the features of the independent claims. Preferred embodiments, which might be realized in an isolated fashion or in any arbitrary combination are listed in the dependent claims.

Accordingly, the present invention relates to a cell culture device comprising at least a first culture compartment and a second culture compartment, wherein said first culture compartment and the second culture compartment are intervened by a removable barrier.

As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. Also, as is understood by the skilled person, the terms "a" and "an" refer to "one or more", i.e. are equivalent to "at least one". Further, as used in the following, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features, without restricting further possibilities. Thus, features introduced by these terms are optional features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment" or similar expressions are intended to be optional features, without any restriction regarding further embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other optional or non-optional features of the invention.

As used herein, the term "standard conditions", if not otherwise noted, relates to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e. preferably, a temperature of 25°C and an absolute pressure of 100 kPa; also preferably, standard conditions include a pH of 7. Moreover, if not otherwise indicated, the term "about" relates to the indicated value with the commonly accepted technical precision in the relevant field, preferably relates to the indicated value ± 20%, more preferably ± 10%, most preferably ± 5%. Further, the term "essentially" indicates that deviations having influence on the indicated result or use are absent, i.e. potential deviations do not cause the indicated result to deviate by more than ± 20%, more preferably ± 10%, most preferably ± 5%. Thus, “consisting essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of’ encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Preferably, a composition consisting essentially of a set of components will comprise less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1%, most preferably less than 0.1% by weight of non-specified component(s).

The term "cell culture device" is understood by the skilled person to relate to any device permitting at least transient maintenance, preferably growth, of a cell type as specified elsewhere herein. The cell culture device may be of any material deemed appropriate by the skilled person. Preferably, the chemical composition of the various components of the cell culture device is of high biocompatibility, in particular to cultured cells, and preferably is of low chemical reactivity (inert). Preferably, the properties affecting cell adhesion are adjusted as required for the application intended. For example, the bottom of a compartment designed for adhesive cell growth preferably has high affinity for cell attachment; also preferably, the removable barrier and/or the porous membrane is inert or even repellent to cell attachment. Preferably, the cell culture device material(s) are also compatible with downstream analysis such as light and fluorescent microscopy and colorimetric assays thus, the cell culture device comprises, preferably consists of, polystyrene, polypropylene, glass, and/or polycarbonate. The different parts of the cell culture device need not necessarily be made of the same material; e.g. the porous membrane preferably is made from a suitable porous material as specified herein below; or the base of the cell culture device may e.g. be a glass microscope slide, while at least parts of the walls of the culture compartments may be of a different material, e.g. polystyrene, polypropylene, and / or polycarbonate. The cell culture device or parts thereof are preferably pretreated to facilitate or improve cell adhesion, e.g. by etching, coating with adhesion molecules, and the like. Preferably, the cell culture device is sterilizable, preferably by radiation sterilization, e.g. by electron beam, gamma radiation or neutron beam, and/or chemical sterilization, e.g. by ethylene oxide. More preferably, the cell culture device is sterilized.

The cell culture device comprises at least a first culture compartment and a second culture compartment. Preferably, the cell culture device comprises additional compartments, in particular culture compartments. Preferably, the cell culture device comprises three, four, five, or even more than five culture compartments, more preferably the cell culture device comprises three, four, or five culture compartments, still more preferably comprises three or four culture compartments, most preferably comprises three culture compartments. As used herein, the term "compartment" relates to a subdivision of the cell culture device capable of, preferably adapted to, holding a liquid, preferably cell culture medium. Accordingly, the term "culture compartment" relates to a compartment adapted for holding medium comprising cells, preferably for at least temporally maintaining, more preferably, culturing, cells. The geometry of the compartment is chosen by the skilled person according to the specific application envisaged, the cell type used, the volume required, and other parameters known to the skilled person. Preferably, compartments are attached to each other in an orientation that allows the combinatorial and free communication for each single cell type with cells in other culture compartments. The attachment can be side by side, back to back, triangular, circular, etc., depending on the number of cell types investigated and downstream analysis intended; preferred examples of arrangements are provided herein in the Examples. Preferably, the culture compartment is flask shaped, more preferably has the shape of a cell culture flask known in the art. Also preferably, the culture compartment has a cylindrical shape, as e.g. in multi-well plates, wherein the bottom of the cylinder preferably is essentially even for adherent cells, and is preferably round for suspension cells. Thus, in particular for adherent cells, preferably culture compartments having at least one essentially even surface are used while for non-adherent cells, culture compartments with rounded surfaces enhancing culture medium movement during agitation may be preferred. Preferably, each culture compartment has at least one opening, preferably for venting and/or pipetting. The opening preferably can be closed, e.g. by a cap or, in particular if a multi-well format is used, by a lid. The size of the culture compartment is selected by the skilled person according to the application envisaged; the volume of the culture compartment preferably is of from 5 1 to 10 mΐ, preferably of from 500 ml to 100 mΐ, more preferably of from 250 ml to 250 mΐ; wherein the term "volume of the culture compartment", as used herein, relates to the volume of culture medium usable in the culture compartment, which is about two thirds, preferably about half, more preferably a third, most preferably about one fourth, of the maximal (geometric) volume of the culture compartment. In the case of a culture compartment with at least one essentially even surface, the area of said surface is of from 1000 cm² to 1 mm², preferably of from 250 cm² to 10 mm², more preferably of from 100 cm² to 25 mm². The culture compartments of the cell culture device may have the same size (volume and/or area) or may have different sizes. Thus, preferably, at least one culture compartment has a size (volume and/or area) different from the remaining culture compartment(s); also preferably, all culture compartments have essentially the same size. Preferably, in particular in case large volumes are used, the cell culture device may comprise several flask-shaped compartments appropriately arranged, e.g. as shown herein in the Examples. For medium volumes, the cell culture device may correspond to a multi-well plate, preferably a 6-well, a 12- well, a 24-well, a 48-well, or a 96-well plate, in which at least parts of the walls separating the wells were replaced by a removable barrier and/or a porous membrane. Moreover, for small volumes, the whole cell culture device may be provided on a microscopic slide or part thereof.

In the cell culture device, at least two culture compartments have a common interface, i.e. share a common surface delimiting their inner lumen. Preferably, in case the cell culture device comprises more than two culture compartments, each culture compartment comprises at least one interface to a common compartment and/or to at least one, preferably at least two, adjacent culture compartment(s). As used herein, the term "common compartment" relates to a compartment having a common interface with at least three, preferably all, culture compartments of the cell culture device. Preferably, the size (volume and/or area) of the common compartment is smaller than the size of the culture compartments, preferably is smaller than the smallest culture compartment. Preferably, the common compartment comprises an aqueous liquid, more preferably comprises cell culture medium. Preferably, the common compartment comprises no cells. Thus, the common compartment preferably does not comprise adaptations appropriate for cell adhesion; also, the common compartment may comprise agitation means, e.g. stirring means such as a stir bar. As will be understood by the skilled person, the compartment having a common interface with at least three, preferably all, culture compartments of the cell culture device may also itself be a culture department and may comprise cells.

Thus, preferably, in case the cell culture device has two culture compartments, these two culture compartments have a common interface. Also preferably, in case the cell culture device has three culture compartments, each of said three culture compartments has a common interface to a common compartment and/or has common interfaces to one or two adjacent culture compartments. The above applies to cell culture devices with four or more compartments mutatis mutandis. As used herein, the term "common interface" relates to a delimiting structural element comprising, preferably consisting of, a removable barrier and, optionally a porous membrane, both as specified herein below.

In the cell culture device, at least a first compartment and a second compartment are intervened by a removable barrier. The term "barrier" is used herein as relating to any means reversibly and/or temporarily preventing diffusional and/flow exchange between two compartments which would otherwise be enabled by an opening or by a porous membrane of the cell culture device. Thus, preferably, the barrier is a piece of material covering the opening or the porous membrane at least over the whole diffusion- or flow-enabling area. Preferably, the barrier is of a material considered inert for cell culture purposes and in particular does not enable adhesion of cells. Preferably, the barrier is of the same material as the walls of the compartment. The barrier of the cell culture device is removable; i.e., the barrier may be present in the manufactured state of the cell culture device and may be removed by the user at a time point deemed appropriate, thus enabling diffusion and/or flow over the porous membrane or opening which was prevented by the presence of the barrier. Preferably, at least one barrier is present in the cell culture device, more preferably all culture compartments are separated from all adjacent compartments by removable barriers. More preferably, the barrier is resealable, i.e. may be introduced or re- introduced between two compartments in order to stop diffusion between said compartments. In accordance, in case a resealable barrier is comprised in a cell culture device, the cell culture device in the manufactured state may lack one or more barriers, which may be inserted only before or during use. A resealable barrier may be provided by including a guiding means guiding the barrier, e.g. a barrier as specified herein above over a porous membrane or an opening to prevent diffusion or flow over the porous membrane or opening from occurring. For the guiding means, materials ensuring water-tight sealing while being inert for cell culture purposes are preferred, e.g. rubber, silicone, and the like. If required, fastening means may be further included. Preferred embodiments of a resealable barrier are shown herein in the examples. As will be understood by the skilled person, the resealable barrier, preferably, is adapted to enable more than one opening/resealing cycle. Preferably, a barrier may also be provided by a first and a second porous membrane arranged in essentially parallel orientation, the first porous membrane forming a delimitation of a first compartment and the second membrane forming a delimitation of a second compartment, and the first and the second porous membrane forming a slot; preferably, the distance between the first and the second porous membrane, i.e. the slot width, is of from 1 mm to 50 mm, in an embodiment of from 2 mm to 20 mm in such case. As the skilled person will understand, the slot filled with e.g. ambient air will form a closed barrier, while the slot being filled with liquid, e.g. cell culture medium, will form an open barrier enabling diffusion.

The term "intervening", as used herein, relates to a removable barrier and/or a porous membrane being spatially arranged to form or be comprised in the common interface between two compartments. Thus, preferably, the removable barrier and/or porous membrane is the material forming, partly or completely, the common interface between two compartments. Thus, preferably, unless a barrier is present, the porous membrane diffusibly connects the aforesaid compartments, i.e. enables diffusion of compounds between said compartments. In accordance, preferably, unless a barrier is present, the porous membrane is the only diffusion-limiting material between said compartments.

As the skilled person will understand, removal of the removable barrier will uncover an opening in common interface, e.g. a wall, between two compartments. Thus, in case a free opening is uncovered, components may freely diffuse, flow, and/or migrate between two compartments. Preferably, exchange of compounds between two compounds through the opening is restricted by the presence of a porous membrane covering the opening, preferably completely covering the opening. Thus, in the cell culture device, preferably, at least a first compartment and a second compartment are intervened by a porous membrane. The term "porous membrane", as used herein, relates to any material restricting the exchange of compounds and/or cells between two compartments. Thus, the porous membrane may in principle be made from the same material as the residual compartment walls, provided that pores as specified herein are provided. Preferably, the porous membrane comprises, more preferably consists of, polyethylene, polyamide, nylon, polycarbonate, polytetrafluorethylene, polyvinylidene fluoride, glass fiber, cellulose, nitrocellulose, and/or mixed cellulose esters. Preferably, the diameter of the pores in said porous membrane is about 0.1 pm, preferably about 0.5 pm, more preferably about 1 pm, most preferably about 5 pm. As used herein, pore diameter is indicated as average pore diameter for capillary pore membranes, e.g. track-etched membranes, and as equivalent pore diameter for non-capillary pore membranes. Methods for determining an equivalent pore diameter are known in the art, e.g. the bubble-point test. Also preferably, the porous membrane is permeable for molecules with a molecular mass of at most 100 Da, preferably at most 1 kDa, more preferably at most 10 kDa, still more preferably at most 100 kDa, most preferably at most 1 MDa. Preferably, the porous membrane has a pore density of at least 10⁴ /cm², preferably at least 10⁵ /cm², more preferably at least 10⁶ /cm², most preferably at least 10⁷ /cm². The thickness of the porous membrane may be any thickness deemed appropriate by the skilled person. Preferably, the porous membrane has a thickness of from 10 pm to 1 mm, preferably of from 20 pm to 500 pm, more preferably of from 3 pm to 250 pm, most preferably of from 50 pm to 200 pm. However, in particular for membranes with large pore size and in cases where diffusion of small compounds or active migration of cells shall be enabled, even thicker membranes, preferably with a thickness up to 2 mm, preferably 5 mm, most preferably up to 10 mm may be envisaged. Preferably, all porous membranes in the cell culture device have the same pore diameter. Also preferably, all porous membranes in the cell culture device have the same area. The porous membrane, preferably, is essentially flat; however, other geometries, such as curved or folded porous membranes may be used.

Advantageously, it was found in the work underlying that cell culture devices having two or more compartments connected via a porous membrane can be used for experimentally addressing a variety of questions regarding interaction of cells, thus enabling more life-like models for cell interaction studies, drug testing, and the like. Moreover, by including barriers into the design, temporal control over interaction and conduction of control experiments was improved. The definitions made above apply mutatis mutandis to the following. Additional definitions and explanations made further below also apply for all embodiments described in this specification mutatis mutandis.

The present invention also relates to a kit comprising a first culture compartment, a second culture compartment, and a fastening means, wherein at least one of said culture compartments comprises a removable barrier, and optionally a porous membrane, delimiting at least part of its circumference and wherein said fastening means is configured to fix said first and second culture compartments such that said removable barrier, and optionally said porous membrane, separates the lumen of the first culture compartment from the lumen of the second culture compartment. In a further embodiment, the invention relates to a kit comprising a first culture compartment, a second culture compartment, a fastening means, and a common compartment, wherein said common compartment comprises at least one removable barrier, and optionally a porous membrane, delimiting at least part of its circumference, wherein said first and second culture compartment comprise an opening at a position corresponding to the position of at least one removable barrier and, optionally porous membrane, of the common compartment, and wherein said fastening means is configured to fix said first and second culture compartments such that said at least one removable barrier, and optionally porous membrane, separates the lumen of the common compartment from the lumen of the first and/or second culture compartment.

The term “kit”, as used herein, refers to a collection of the aforementioned components. Preferably, said components are combined with additional components, preferably within an outer container. The outer container, also preferably, comprises instructions for carrying out a method of the present invention. Examples for such the components of the kit as well as methods for their use have been given in this specification. The kit, preferably, contains the aforementioned components in a ready-to-use configuration. The present invention also relates to the use of said kit in any of the methods according to the present invention.

Preferably, the components of the kit, when assembled, provide a cell culture device of the present invention. Preferably, the components of the kit provide flexibility as to which cell culture device shall be assembled: e.g. the kit may comprise a plurality of culture compartments comprising a porous membrane delimiting at least part of its circumference, and the fastening means may be adapted to allow for two, three, four, or even more of these culture compartments to be assembled, with the porous membranes together forming part of the walls of a common compartment as specified herein above. As will be appreciated, at least a baseplate of the common compartment and, optionally a lid thereof, will preferably additionally be required in case more than two culture compartments are used. As will also be appreciated, in case two culture compartments are assembled, one of the culture compartments may be devoid of a porous membrane.

The term "fastening means" is known to the skilled person and the skilled person is well able to select appropriate means for the indicated purpose. Preferably, the fastening means or components thereof are included in the structure of the culture compartment; thus, the fastening means may be a snap-on connection. However, other fastening means, such as plug-in connectors, in particular comprising sealing components made from rubber or silicon, or screw connectors are also envisaged.

The present invention also relates to a method for co-culturing at least two, preferably at least three cell types, comprising culturing said cell types in culture compartments of a cell culture device according to the present invention, wherein at least one cell type is cultured separate from the other cell types, preferably wherein all cell types are cultured in separate culture compartments.

The method of the present invention, preferably, is an in vitro method. Moreover, it may comprise steps in addition to those explicitly referred to above; e.g. further steps may relate to providing cells for culturing and/or analyzing cells and/or their supernatants after co-culture.

The term "co-culturing" as used herein, relates to culturing at least two different cell types in a manner at least temporarily excluding direct physical contact between cells of the at least two types. Thus, preferably, the at least two cell types are at least initially applied to two different culture compartments of the cell culture device, although later cell migration is not necessarily excluded. Preferably, co-culturing comprises exposing a first cell type to diffusible factors produced, removed, and/or modified by a second cell type. Thus, preferably, an improved analysis of cell interactions is possible. The term "cell" is used herein in a broad sense and includes cells from all domains, i.e. eukaryotic cells, bacterial cells, and archeal cells. Preferably, in case the cells are bacterial cells, they are cells of infectious bacteria, more preferably bacteria causing disease in animals, preferably mammals, more preferably humans. Preferably, in case the cells are eukaryotic cells, they are plant cells, preferably cells from crop plants; more preferably, the eukaryotic cells are vertebrate or insect cells, more preferably are mammalian cells, in particular human, mouse, rat, Chinese hamster, dog, cat, llama, sheep, goat, pig, cow, donkey, or horse cells. More preferably, the cells are human cells. Also preferably, the cells comprise or are immune cells, e.g. T cells, B cells, dendritic cells, and/or macrophages, are connective tissue or skin cells, e.g. fibroblasts, and/or comprise or are tumor cells. As the skilled person will understand, the different culture compartments of a cell culture device may be used with different cells; e.g. for toxicity studies, infectious bacteria may be applied to one culture compartment, while human cells e.g. of the respiratory tract may be applied to a second compartment. The term "cell type" is used solely to differentiate between cells from a first type and cells from a second type; i.e., a cell type may be a cell from a given domain, family, genus, species, or strain, and two cell types may be from the same domain, family genus, species, or strain, provided that they are distinguishable.

In view of the above, the following embodiments are particularly envisaged:

Embodiment 1. A cell culture device comprising at least a first culture compartment and a second culture compartment, wherein said first culture compartment and the second culture compartment are intervened by a removable barrier.

Embodiment 2. The cell culture device of embodiment 1, wherein said device further comprises at least a third culture compartment, wherein said third culture compartment and at least one of said first and second culture compartments are intervened by at least one removable barrier.

Embodiment 3. The cell culture device of embodiment 1 or 2, wherein all adj acent culture compartments are intervened by one or more removable barriers.

Embodiment 4. The cell culture device of embodiment 2 or 3, wherein at least three, preferably all, culture compartments can be connected to a common compartment by one or more removable barrier(s).

Embodiment 5. The cell culture device of any one of embodiments 1 to 4, wherein said connection to further culture compartments is via a common compartment. Embodiment 6. The cell culture device of any one of embodiments 2 to 5, wherein at least one culture compartment and a first adjacent culture compartment are intervened by a first removable barrier and said at least one culture compartment and a second adjacent culture compartment are intervened by a second removable barrier, preferably wherein said firs tremovable barrier is non-identical to the second removable barrier.

Embodiment 7. The cell culture device of any one of embodiments 1 to 6, wherein at least one culture compartment has a size different from the remaining culture compartment(s). Embodiment 8. The cell culture device of any one of embodiments 1 to 7, wherein all culture compartments have essentially the same size.

Embodiment 9. The cell culture device of any one of embodiments 1 to 8, wherein at least one of said barriers is resealable.

Embodiment 10. The cell culture device of any one of embodiments 1 to 9, wherein at least two of said compartments are additionally separated from each other by a porous membrane. Embodiment 11. The cell culture device of any one of embodiments 1 to 10, wherein all culture compartments are separated from all other compartments by one or more porous membranes.

Embodiment 12. The cell culture device of any one of embodiments 1 to 10, wherein all porous membranes have the same area and/or the same pore diameter.

Embodiment 13. The cell culture device of any one of embodiments 10 to 12, wherein said porous membrane is permeable for molecules with a molecular mass of at most 100 Da, preferably at most 1 kDa, more preferably at most 10 kDa, still more preferably at most 100 kDa, most preferably at most 1 MDa.

Embodiment 14. The cell culture device of any one of embodiments 10 to 13, wherein the diameter of the pores in said porous membrane is about 0.1 pm, preferably about 0.5 pm, more preferably about 1 pm, most preferably about 5 pm.

Embodiment 15. The cell culture device of any one of embodiments 10 to 14, wherein said porous membrane comprises polyethylene, polyamide, nylon, polycarbonate, polytetrafluorethylene, polyvinylidene fluoride, glass fiber, cellulose, nitrocellulose, and/or mixed cellulose esters.

Embodiment 16. The cell culture device of any one of embodiments 10 to 15, wherein said porous membrane has a pore density of at least 10⁴ /cm², preferably at least 10⁵ /cm², more preferably at least 10⁶ /cm², most preferably at least 10⁷ /cm². Embodiment 17. The cell culture device of any one of embodiments 10 to 16, wherein said porous membrane has a thickness of from 10 pm to 1 mm, preferably of from 20 pm to 500 pm, more preferably of from 3 pm to 250 pm, most preferably of from 50 pm to 200 pm. Embodiment 18. The cell culture device of any one of embodiments 1 to 17, wherein said device is sterilizable, preferably is sterilized.

Embodiment 19. A kit comprising a first culture compartment, a second culture compartment, and a fastening means, wherein at least one of said culture compartments comprises a removable barrier delimiting at least part of its circumference and wherein said fastening means is configured to fix said first and second culture compartments such that said removable barrier separates the lumen of the first culture compartment from the lumen of the second culture compartment.

Embodiment 20 The kit of embodiment 19, wherein said fastening means is comprised in the first and/or the second culture compartment.

Embodiment 21. The kit of embodiment 19 or 21, wherein said fastening means further contributes to formation of a common compartment.

Embodiment 22. The kit of any one of embodiments 19 to 21, wherein said kit further comprises a porous membrane, preferably wherein said porous membrane is sealed by the removable barrier.

Embodiment 23. The kit of any one of embodiment s 19 to 22, wherein said removable barrier is resealable.

Embodiment 24. A method for co-culturing at least two, preferably at least three cell types, comprising culturing said cell types in culture compartments of a device according to any one of embodiments 1 to 18 or 25 to 26, wherein at least one cell type is cultured separate from the other cell types, preferably wherein all cell types are cultured in separate culture compartments. Embodiment 25. A cell culture device comprising at least a first culture compartment, a second culture compartment, and a third culture compartment, wherein at least two of said culture compartments are intervened by a removable barrier and/or a porous membrane. Embodiment 26. The cell culture device of embodiment 25, further having a feature of any one of embodiments 2 to 18.

Embodiment 27. A kit comprising a first culture compartment, a second culture compartment, and a fastening means, wherein at least one of said culture compartments comprises a removable barrier and/or a porous membrane delimiting at least part of its circumference and wherein said fastening means is configured to fix said first and second culture compartments such that said removable barrier and/or porous membrane separates the lumen of the first culture compartment from the lumen of the second culture compartment. Embodiment 28. The kit of embodiment 27, further comprising at least one further culture compartment.

Embodiment 29. The kit of embodiment 27 or 28, having a feature of any one of embodiments 20 to 23.

Embodiment 30. A kit comprising a first culture compartment, a second culture compartment, a fastening means, and a common compartment, wherein said common compartment comprises at least one removable barrier and/or a porous membrane delimiting at least part of its circumference, wherein said first and second culture compartment comprise an opening at a position corresponding to the position of at least one removable barrier and/or porous membrane of the common compartment, and wherein said fastening means is configured to fix said first and second culture compartments such that said at least one removable barrier/or porous membrane separates the lumen of the common compartment from the lumen of the first and/or second culture compartment.

Embodiment 31. The kit of embodiment 30, further comprising at least one further culture compartment.

Embodiment 32. The kit of embodiments 30 or 32, further having a feature of any one of embodiments 20 to 23.

All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

Figure Legends

Fig. 1 : Schematic representation of a bi-culture system for real time cell-to-cell communication; the connection frame is optional and may be replaced by other connection means or by a direct connection; A) device view; B) exploded view; c) as in B) but with additional porous membrane.

Fig. 2: Schematic representation of a tri-culture system with three culture compartments and a common compartment (A)) or without a common compartment (B)). Fig. 3: Schematic representation of a multi-culture system for two-cell-type co-culture.

Fig. 4: Schematic representation of a multi-culture system comprising four culture compartments and a common compartment.

Fig. 5: Wound healing assay of pancreatic stellate cells (PSCs). (A) A typical scratch is shown. (B) PSCs that were cultured alone in serum free medium after 24 hrs. Basically all cells had died. (C) PSCs incubated under identical conditions but with connection to the PT45P1 cancer cell line in another compartment. Although grown in serum-free medium, the PSCs were alive and had grown, covering the scratch area after 24 hrs.

The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

Example 1 : Embodiments of the device

As shown in Fig. 1 A, B), the cell culture system 110 may comprise two culture compartments 112, 114 essentially having the shape of a culture flask and connected via an optional connection frame 118 and comprising removable barrier 116. Connection frame 118 may be adapted to permit re-insertion of removable barrier 116 to close connection between culture compartments 112, 114.

As shown in Fig. C, the cell culture system 110 may additionally comprise a porous membrane 120, which may limit exchange of constituents between culture compartments 112, 114 to diffusion and/or active migration, e.g. depending on membrane and pore size.

Fig. 2 shows exemplary arrangements of a device with a first culture compartment 112, a second culture compartment 114, and a third culture compartment 122, to form a common compartment 124 (A), or not forming a common compartment 124 (B). As will be appreciated, according to A), the removable barrier 116 and optionally the porous membrane 120 would be placed between the lumens of the culture compartments 112, 114, 122 and the lumen of the common compartment 124. The arrangement schematically shown in Fig. 3 provides three identical two-culture compartment 112, 114 arrangements; in contrast to the device according to Fig. 1, additionally a common compartment 124 is provided.

As schematically shown in Fig. 4, also a four-culture compartment device can be provided. Similar construction principles may be used to provide devices with even more compartments. As will be understood, also more than one common compartment may be provided; and one culture compartment may be connected to two or more independent common compartments.

Example 2

The tumor microenvironment of pancreatic cancer represents a good model to explain the device. In pancreatic ductal adenocarcinoma, tumor cells compose only a small fraction of the tumor mass while stromal cells such as activated pancreatic stellate cells and immune cells dominate the tumor with up to 90% of the tumor mass. Two cell lines are used as representative of tumor microenvironment of pancreatic cancer: the primary pancreatic cancer cell line PT45P1 and pancreatic stellate cells (PSCs). Pancreatic cancer cells are well known to take part in the activation and increased proliferation of PSCs (Thomas & Radhakrishnan (2019), Mol Cancer 18:14.). To demonstrate this with the present invention, the PSC and PT45P1 cell lines were cultured in a two-compartments vessel designated as (Actri) control vessel. The PSC were cultured in the first compartment named Actrii; PT45P1 were cultured in the other compartment named A_ctri2. Similarly, another two-compartment vessel was used with the two cell lines to serve as a test vessel (B_test), in which PSCs were cultured in the first compartment named B_ctrii and PT45P1 were cultured in the other compartment named B_ctri2. In all vessels, the two cell lines grew under identical conditions using Dulbecco's Modified Eagle's Medium (DMEM) containing and 10% fetal bovine serum (FBS) in a 37°C incubator with standard flow of O2 and CO2 gas. The cell lines PSC and PT45P1 in both A and B were allowed to grow until 90% confluence, while completely separated by a closed gate that prevented molecular communication between PSC and PT45P1. The cell lines in all four compartments were washed three times with phosphate buffered saline while gates continued to be closed. The cell lines in all compartments were fed with fresh DMEM medium without FBS and kept in culture for 3 hours to synchronize cell growth. A scratch assay was conducted with the PSC cells by drawing a line with a sterile pipette tip (Fig. 5 A)). Cells were washed once with PBS and mounted again with fresh DMEM without FBS. Cells were allowed to grow for additional 24 hours under the same conditions with the gate still closed (Fig. 5B)) or opened (Fig. 5 C)). As shown, PSC cells without contact to PT45P1 cells died, whereas PSC cells with contact survived and grew to close the scratch.

Example 3 A three cell line system composed of culturing macrophages (RAW264.7), pancreatic cancer cells (PT45P1) and stellate cells (PSC) can be used as a model for a more than two cell line setup. As an example, the system is used to study the mechanism of drug resistance to Gemcitabine in PT45P1 under the influence of either RAW264.7, PSC or both simultaneously. In this regard, one vessel of three compartments (Testo) and two vessels (Teste and Teste) of a two compartment setup are used. In all three vessels, PT45P1 is cultured in one compartment. In the three compartment vessel, RAW264.7 and PSC are cultured in the other two compartments; in the vessels of two compartments, RAW264.7 or PSC are cultured in the second compartment, respectively. As a control, a single normal cell culture vessel is used to culture PT45P1 under no influence from any cells (CtrU). All cells are cultured as described in example 1. During treatment mode, PT45P1 cells are treated with a defined dose of the anticancer drug Gemcitabine in TestD, Teste, Teste_, and CtrU for a specified period of time during which gates are opened to allow the exposure of PT45P1 to both PSC and RAW264.7 (TestD), RAW264.7 only (Teste), PSC only (Test_B), and no exposure to other cells at all (CtrU). Assays such as cell apoptosis, cell proliferation, cell cycle, gene expression, and the like can be conducted on the target cells (PT45P1) to evaluate the direct impact of the other cells on drug resistance of the targets cells.

Reference signs

110 cell culture device

112 first culture compartment 114 second culture compartment

116 removable barrier

118 fastening means, e.g. connection frame 120 porous membrane

122 third culture compartment 124 common compartment

126 fourth culture compartment

Claims

1. A cell culture device (110) comprising at least a first culture compartment (112) and a second culture compartment (114), wherein said first culture compartment (112) and the second culture compartment (114) are intervened by a removable barrier (116).

2. The cell culture device (110) of claim 1, further comprising at least a third culture compartment (122), wherein said third culture compartment (122) and at least one of said first and second culture compartments (112, 114) are intervened by at least one removable barrier (116).

3. The cell culture device (110) of claim 1 or 2, wherein all adjacent culture compartments (112, 114) are intervened by one or more removable barrier(s) (116).

4. The cell culture device (110) of claim 2 or 3, wherein at least three, preferably all, culture compartments (112, 114, 122) are connected to a common compartment (124) by one or more removable barriers (116).

5. The cell culture device (110) of any one of claims 1 to 4, wherein at least two of said compartments are additionally separated from each other by a porous membrane (120).

6. The cell culture device (110) of any one of claims 1 to 5, wherein all culture compartments (112, 114) are separated from all other compartments by one or more porous membranes (120).

7. The cell culture device (110) of claim 5 or 6, wherein at least one of said removable barriers (116) is resealable.

8. The cell culture device (110) of any one of claims 1 to 7, wherein the diameter of the pores in said porous membrane (120) is about 0.1 pm, preferably about 0.5 pm, more preferably about 1 pm, most preferably about 5 pm.

9. The cell culture device (110) of any one of claims 1 to 8, wherein said porous membrane (120) comprises polyethylene, polyamide, nylon, polycarbonate, polytetrafluorethylene, polyvinylidene fluoride, glass fiber, cellulose, nitrocellulose, and/or mixed cellulose esters.

10. The cell culture device (110) of any one of claims 1 to 9, wherein said porous membrane (120) has a thickness of from 10 pm to 1 mm, preferably of from 20 pm to 500 pm, more preferably of from 3 pm to 250 pm, most preferably of from 50 pm to 200 pm.

11. A kit

(i) comprising a first culture compartment (112), a second culture compartment (114), and a fastening means (118), wherein at least one of said culture compartments (112, 114) comprises a removable barrier (116) delimiting at least part of its circumference and wherein said fastening means is configured to fix said first and second culture compartments (112, 114) such that said removable barrier (120) separates the lumen of the first culture compartment (112) from the lumen of the second culture compartment (114); or

(ii) comprising a first culture compartment (112), a second culture compartment (114), a fastening means (118), and a common compartment (124), wherein said common compartment (124) comprises at least one removable barrier (116) delimiting at least part of its circumference, wherein said first and second culture compartments (112, 114) comprise an opening at a position corresponding to the position of at least one removable barrier (116) of the common compartment (124), and wherein said fastening means (118) is configured to fix said first and second culture compartments (112, 114) such that said at least one removable barrier (116) separates the lumen of the common compartment (124) from the lumen of the first and/or second culture compartment (112, 114).

12 The kit of claim 11, wherein said fastening means (118) is comprised in the first and/or the second culture compartment (112, 114).

13. The kit of claim 11 or 12, wherein said fastening means (118) further contributes to formation of a common compartment (124).

14. The kit of any one of claims 11 to 13, wherein said kit further comprises a porous membrane (120), preferably wherein said porous membrane (120) is sealed by a removable and optionally resealable barrier (116).

15. A method for co-culturing at least two, preferably at least three cell types, comprising culturing said cell types in culture compartments of a device according to any one of claims 1 to 18, wherein at least one cell type is cultured separate from the other cell types, preferably wherein all cell types are cultured in separate culture compartments.