System for creating an oxygen gradient across a cell culture insert barrier

Abstract

The invention relates to cell culture devices suitable for co-culturing cells, particularly for co-culturing aerobic and anaerobic cells. The invention also relates to methods of co-culturing aerobes and anaerobes.

Description

[0001] System for creating an oxygen gradient across a cell culture insert barrier

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to products and systems for use in cell culture. In particular, embodiments of the invention relate to cell culture systems for co-culturing aerobic and anaerobic cells.

[0004] BACKGROUND TO THE INVENTION

[0005] Cell culture is a vital technique to assess the normal physiology and biochemistry of cells. It is also used in drug screening and development, and large scale manufacturing of biological compounds, for example, vaccines and therapeutic proteins. The major advantage of using cell culture for any of these applications is the consistency and reproducibility of results that can be obtained.

[0006] Cell co-culturing is used to study cell crosstalk between two or more types of cells. Since most tissues contain more than one type of cell it is important to evaluate their interaction in a culture environment to gain a better understanding of their interaction. There are two types of co-culturing: direct and indirect. While direct co-cultures involve cells being in direct contact with each other, indirect co-cultures involve different environments where there is a physical barrier between different populations of cells, allowing signalling and soluble factors to participate and trigger cell-cell interactions.

[0007] There has been growing interest in research on the human microbiome and its effect on human health, however, tools to study the microbiome are limited particularly for investigating the interaction between microbial cells and mammalian cells such as the intestinal epithelium which resides at the interface of the gut microbiota and plays a pivotal role in shaping the gut ecosystem. Owing to a lack of tractable co-culture systems, our biological understanding of host-microbe interactions remains elusive, with conflicting oxygen demands between mammalian cells and gut anaerobes making their co-culture difficult.

[0008] To date, the field mostly relies on using animal models; however, animal models are not representative of the human microbiome. Therefore, scalable in vitro methods are required which can utilise human cells and a human-relevant microbiome. Critically, many of the most important microbes that comprise the human microbiome are highly sensitive to the presence of oxygen and will only survive in a very low oxygen environment. As such, the study of these bacteria in vitro requires an environment where the oxygen level can be well controlled at a very low, or zero, value. Such a system must also be able to sustain human gut cells, which require an oxygen supply. A product that can produce such an environment is not currently available, despite growing demand for such a system. The present invention addresses this need.

[0009] SUMMARY OF THE INVENTION

[0010] The inventors have developed a cell culture device suitable for the co-culture of cells with different atmospheric requirements. In particular, the cell culture device of the present invention is suitable for the co-culture of aerobes and anaerobes through the generation of an oxygen gradient across the device so that interactions between cells with different oxygen requirements can be studied.

[0011] In one aspect of the present invention, there is provided a cell culture device comprising upper and lower well plate elements, said elements together defining at least one culture well, wherein the device further comprises a cell culture insert having apical and basal surfaces, said insert located within the culture well and separating said culture well into first and second apical and basal volumes; wherein at least a portion of said insert is gas permeable, to permit gas to exchange across the apical and basal surfaces between the apical and basal volumes; and wherein said upper and lower well plate elements each further comprise at least one opening extending between the interior and exterior of each respective element, to permit movement of gas between the interior and exterior of each respective element.

[0012] The upper and lower well plate elements may be considered as separate chambers, in view of the separation of the culture well into apical and basal volumes. Where there are multiple culture wells, it will generally be the case that the apical or basal volumes respectively of each well are connected via these elements or chambers into a single apical or basal headspace. In the present application, the upper and lower well plate elements may be referred to interchangeably as chambers. The apical and basal volumes are typically continuous with the upper or lower well plate elements respectively. In some embodiments there is provided a gas impermeable seal between the well plate elements of the culture well. That is, the elements may be divided by a gas impermeable barrier other than at the gas permeable portion of the cell culture insert, to ensure that gas exchange may only occur across that portion. The seal may be formed from a portion of the insert optionally in combination with, for example, an elastomeric O ring or similar, and / or a sealant gel.

[0013] Preferably, the cell culture device comprises a gas supply connected to at least one opening of at least one well plate element. The gas supply may be a low oxygen gas to permit the introduction of a low oxygen atmosphere in at least one element of the device. For example, suitable low oxygen gases may comprise a mixture of nitrogen and carbon dioxide; or comprise primarily nitrogen (eg, 95% or more N2). There may be at least one sensor in at least one element of the device to monitor the environmental conditions; preferably said at least one sensor is an oxygen sensor. Said at least one sensor may be connected to a computer processing unit which can modify the amount of gas directed through the gas supply to the gas inlet of the cell culture device. In other embodiments, a similar computer processing unit may be present which is not connected to a sensor; this may be programmed to adjust the gas supply on a preset program, or in response to user adjustment.

[0014] Preferably the well plate elements of the cell culture device are detachable from one another, permitting the elements of the device to be replaced. In some embodiments, at least one well plate element of the device comprises (or is in the form of) a cell culture plate.

[0015] The cell culture device may comprise at least one population of aerobic and / or anaerobic cells. Examples of aerobic cells include but are not limited to mammalian, for example, human, cell types; for example, epithelial cells, leukocytes, hepatocytes and adipocytes. Examples of anaerobic cells include but are not limited to, yeast, protozoa, as well as Bacteroides, Lactobacillus, Clostridium and Fusobacterium species.

[0016] The invention further provides a method of culturing cells using the cell culture device described herein, comprising adding cells to the device and introducing a low oxygen gas through at least one opening of at least one well plate element, so that one volume has a low oxygen atmosphere compared to the other volume.

[0017] A low oxygen gas may be continuously administered through an opening on at least one well plate element. Alternatively, after a low oxygen gas is administered to at least one well plate element, all openings on said well plate elements of the device are sealed.

[0018] In some embodiments ambient gas in the environment is allowed to enter at least one well plate element through at least one opening of the cell culture device.

[0019] Preferably the apical volume of the device has a low oxygen atmosphere, and the basal volume of the device has an atmosphere with oxygen levels resembling the ambient environment.

[0020] In a preferred embodiment, at least two populations of cells are co-cultured. More preferably, at least one population of anaerobic cells and a least one population of aerobic cells are co-cultured.

[0021] In some embodiments at least one population of cells being co-cultured comprises microorganisms that populate the microbiome, preferably the microbiome present in the gastrointestinal tract.

[0022] In other embodiments at least one population of cells that is co-cultured are microorganisms selected from bacteria, fungi, viruses, archaea, protozoa, algae and combinations thereof.

[0023] Preferably the method comprises co-culturing at least one population of microorganisms and at least one population of mammalian cells, preferably human cells.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a sketch of the culture device of an embodiment of the invention where there are two well plate elements or chambers together defining culture wells comprising cell culture inserts. In this embodiment the apical volume of the device has a low oxygen environment and the basal volume of the device has oxygen levels resembling the ambient environment.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] Described herein is a cell culture device which is intended to have at least two well plate elements or chambers defining culture well(s) and methods of cell culture using said device.

[0027] The present invention is intended to facilitate the co-culture of cells.

[0028] In one embodiment, the invention is able to provide an environment for the co-culture of aerobic and anaerobic cells. Preferably, mammalian cells and microbial cells, and even more preferably human cells and cells derived from the human microbiome of the gastrointestinal tract are co-cultured.

[0029] The microbiome is the community of microorganisms (such as fungi, bacteria and viruses) that exists in a particular environment. The term is often used to describe the microorganisms that live in or on a particular part of the body such as the gastrointestinal tract, oral cavity, nasal cavity, or skin. The terms microbiome and microbiota are used interchangeably herein.

[0030] The term cell culture refers to the cultivation of cells outside a living organism under controlled conditions, for example, temperature, pH, nutrient, and waste levels. Both eukaryotic and prokaryotic cells can be subjected to cell culture.

[0031] Primary cell culture refers to culturing cells that have been directly obtained from a multicellular organism.

[0032] Secondary cell culture refers to culturing immortalised cell lines.

[0033] Immortalised cell lines have acquired the ability to proliferate indefinitely either through a random mutation or through genetic manipulation of a cell to express a gene that induces immortalisation such as human telomerase reverse transcriptase or SV40 T- antigen. The term co-culture refers to a cell cultivation set-up in which at least two populations of cells are cultured with some degree of contact between them. Co-culture of cells is fundamental in studying cell-cell interactions. There are two forms of co-culture known in the art, namely direct and indirect co-culture.

[0034] Direct co-culture refers to culture of at least two populations of cells with direct physical contact between them.

[0035] Indirect co-culture refers to culture of at least two populations of cells together, but with a physical barrier between them so that they are not in direct contact with each other. Although not in direct physical contact with each other, cells may communicate via release of signalling molecules such as cytokines and extracellular vesicles.

[0036] The present invention is suitable for both primary and secondary cell culture as well as co-culture of both primary and secondary cells.

[0037] Figure 1 shows a cell culture device 10 according to the present invention, formed from a first 1 and second 2 chamber, which two chambers of the device together define a culture well 9. Figure 1 shows a device with two cell culture wells, however in alternative embodiments the device may comprise multiple wells (for example, in a multiwell plate format having, eg, 12, 16, 20, 24, 48 etc wells).

[0038] Figure 1 shows the device 10 with two culture inserts 5, each insert having an apical 3 and a basal surface 4. However, the device can comprise any number of inserts, the skilled person would be able to modify the number of inserts in the device to suit their specific application.

[0039] Cell culture inserts are well known in the art. Examples of cell culture inserts include, but are not limited to, transwell inserts, snapwell inserts, netwell inserts, and falcon inserts.

[0040] Cell culture inserts are used in the art in concert with carrier plates. Cell culture inserts can be placed in multiple positions, facilitating surface height and required media volume to be adjusted. Different pore sizes of inserts can be selected for different applications. The skilled person would understand how to select an appropriate insert depending on the cells they are culturing and the precise application the device is being used for. Referring to Figure 1 , the cell culture insert or inserts 5 are located within a cell culture well 9 of the device 10 separating said culture well into apical 3 and basal 4 volumes. According to figure 1 , the apical volume 3 is located in the first chamber 1 of the device and the basal volume 4 is located in the second chamber 2 of the device 10.

[0041] With reference to figure 1 , at least a portion 11 of the insert 5 is gas permeable, to permit gas exchange across the apical and basal surfaces between the apical 3 and basal 4 volumes. In one aspect of the invention the portion of the insert that is gas permeable comprises a membrane, preferably a semi-permeable membrane.

[0042] A semi-permeable membrane is a membrane that will allow certain substances to pass through it while restricting the passage of others. In the art a semi-permeable membrane is frequently used to separate different substances. Semi-permeable membranes have pores that facilitate the passage of some substances (whether, eg, molecules, or whether larger particles or biological materials such as cells), whilst others are too large to fit through the pores of said membrane. The rate of diffusion through a semi-permeable membrane depends on several factors including but not limited to the molecular weight of a compound, physical size of materials or pores, temperature, and the concentration of substances on either side of the membrane.

[0043] According to figure 1 , there is a gas impermeable seal 7 between the first 1 and second 2 chambers of the device. Figure 1 shows said gas impermeable seal 7 between a portion of the first 1 and second 2 chambers of the device 10, alongside a gas permeable portion 11 of the insert 5 of the cell culture device 10.

[0044] Suitable gas impermeable seals include but are not limited to seals comprising rubber, plastic or silicone.

[0045] Figure 1 shows that the first 1 chamber includes at least one opening 6 between the interior and exterior of the chamber, wherein each opening permits the movement of gas between the interior and exterior of said chamber. The second chamber 2 may be considered as having corresponding openings allowing for gas movement, formed here by the gap between the first and second chambers, although in other embodiments a distinct opening may be formed in the second chamber 2. In figure 1 a preferred embodiment of the invention is shown where each chamber has two openings. However, in some embodiments each chamber may comprise at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten openings. Preferably all openings are sealable. Figure 1 shows a first opening 6 of the first chamber 1 of the device is used as a gas inlet, and a second opening is used as a gas outlet. Any gas composition can be supplied to the inlet that is suitable for the application for which the device is being used and the cell populations being cultured. During use, as shown in figure 1 , at least one opening is used a gas outlet where gas is able to leave the device 10 after entering through the gas inlet.

[0046] In a preferred embodiment a low oxygen, or oxygen free gas is supplied through the gas inlet.

[0047] Non limiting examples of low oxygen gases that may be supplied through at least one opening of the device in the context of the present invention include but are not limited to nitrogen, carbon dioxide and hydrogen; these gases may be administered alone or in combination. Preferably, a combination of nitrogen, carbon dioxide and hydrogen is supplied through at least one opening of the device. More preferably, a combination of approximately 80% nitrogen, 10% carbon dioxide and 10% hydrogen is supplied through at least one opening of the device. Alternatively, a combination of 5% hydrogen and 95% nitrogen is supplied through at least opening of the device, or a combination of 85% nitrogen, 10% carbon dioxide and 5% hydrogen is supplied through at least one opening of the device. In some embodiments, any combination of nitrogen, carbon dioxide and hydrogen (or any other gas as required by the user) may be supplied through different openings of the cell culture device either simultaneously or in turn.

[0048] In one embodiment a gas, preferably a low oxygen or oxygen free gas is continuously supplied through at least one opening of the first chamber of the device, creating a dynamic low oxygen atmosphere.

[0049] In an alternative embodiment, after a gas, preferably a low oxygen or oxygen free gas is supplied through at least one opening of the first chamber of the device, all openings on the first chamber are sealed, creating a static low oxygen atmosphere.

[0050] In the context of the present invention a dynamic atmosphere is an atmosphere with continuous gas flow altering the precise composition of the atmosphere (or indeed maintaining the composition stable against biological perturbation). In contrast a static atmosphere remains constant, other than through biological effects of any cultured cells within the atmosphere. In the context of the present invention a static atmosphere is an atmosphere where all openings are sealed after gas is administered so that the atmosphere is not replenished. The skilled person would understand that the composition of either the dynamic or static atmosphere may change over time according to the respiration of cultured cells.

[0051] With reference to figure 1 at least one opening of the second chamber 2 of the device 10 is used to facilitate the inlet of ambient gas in the environment into the cell culture device 10. In preferred embodiments, this ambient gas is not pumped or otherwise actively transported into the opening, but is allowed to passively equilibrate with the surrounding atmosphere. In this way, the device 10 as a whole may be placed into an enclosing chamber which may have a different “ambient” atmosphere from the normal atmosphere. In other embodiments, active pumping of gas may be used to provide a given atmosphere to the second chamber 2.

[0052] In the present invention, providing a gas, preferably a low oxygen or oxygen free gas, to the first chamber 1 of the cell culture device and providing a different atmosphere (whether actively or passively) to the second chamber 2 generates an oxygen gradient between the apical and basal volumes of the device across the culture insert. In the described embodiment, the apical volume 3 has a lower oxygen level than the basal volume 4, as a consequence of the apical volume 3 having a low oxygen atmosphere and the basal volume 4 having an atmosphere with oxygen levels resembling the ambient environment.

[0053] The term ambient environment as used herein refers to the gaseous environment surrounding the exterior of the cell culture device, and hence may not necessarily be identical to the earth’s atmosphere.

[0054] A low oxygen environment as used herein refers to an environment comprising less oxygen than that of the ambient environment. Said low oxygen environment may have an oxygen level decreased by at least or up to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90 or 95% or more compared to the ambient environment. The low oxygen environment may include 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less oxygen by volume.

[0055] In one aspect of the invention the oxygen levels within the device can be varied by changing at least one gas being supplied through at least one opening on at least one chamber of the device, and / or changing the flow rate at which gas is being administered to the device and / or opening or closing at least one opening on at least one chamber of the device.

[0056] In one embodiment, the oxygen conditions in one chamber of the device can be controlled by a single gas supply.

[0057] In one embodiment of the invention at least one chamber, preferably all chambers of the device comprise a sensor, wherein preferably said sensor is an oxygen sensor. The oxygen sensor detects oxygen levels in the chamber and the signal is preferably processed through a computer processing unit to alter the amount of gas being administered to the chamber, to meet the oxygen requirements set by the user.

[0058] The cell culture device of the present invention may also optionally comprise additional sensors to detect pH, temperature, nitrogen, hydrogen, carbon dioxide, gas flow rate or any other sensor known in the art with applications in cell culture.

[0059] In one aspect of the invention a user may administer a treatment to cultured cells. In embodiments, a treatment may be administered via at least one opening in at least one chamber. Alternatively, the device may include a removable closure which can be removed to allow access to the cell culture. Said treatment may be selected from one or more of a drug, protein, carbohydrate, lipid, bioactive compound, metabolite, vitamin, nutrient, nucleic acid, cytokine, chemokine or any other treatment suitable for use in cell culture. In one embodiment, at least one treatment may be administered once, or periodically depending on the needs of the user. In another embodiment different treatments may be administered through different openings on the cell culture device, in some embodiments different treatments may be administered to different culture wells compared to a treatment being administered to an additional culture well of the device. “Treatment” here may refer to a potentially clinically or physiologically relevant treatment (eg, a candidate drug), or may more generally refer to a set of given culture conditions, with different “treatments” being different specific culture conditions.

[0060] In one embodiment, the chambers of the device are detachable, facilitating the chambers to be replaced.

[0061] Referring to figure 1, the basal chamber 2 of the device is here formed of a cell culture plate 8.

[0062] Cell culture plates are commercially available in a variety of sizes, in single and multiwell formats. Cell culture plates are available with pre-treated surfaces to facilitate the adhesion of anchor-dependent cells or untreated to support the culture of suspension cells.

[0063] Common cell culture plate surface pre-treatments include but are not limited to collagen, laminin, fibronectin, vitronectin, matrigel, poly-l-lysine, poly-ornithine and gelatine.

[0064] In some embodiments the cell culture plate used in the invention may be a perfusion system; that is, a culture plate which allows and facilitates perfusion of cell culture medium to cell cultures within the wells of the plate. Various examples of this are known in the art.

[0065] The skilled person would be able to select a cell culture plate with an appropriate surface and number of wells to meet their needs regarding the cells being cultured, and the specific application.

[0066] In a preferred embodiment, the cell culture device of the present invention can be mounted in a commercially available cell culture incubator known in the art.

[0067] The present invention is suitable to be scaled up to allow multiple experiments to be performed in parallel in a single device, or by the use of multiple devices concurrently.

[0068] The present invention has applications in investigating interactions and communication between aerobes and anaerobes, particularly between cells that populate the microbiome and mammalian cells. In some applications the present invention is used to investigate interactions between cells that populate the microbiome of the gastrointestinal tract and mammalian cells, preferably human cells. In some embodiments, the present invention is used to investigate interactions between at least one population of microorganisms selected from bacteria, fungi, viruses, archaea, protozoa, algae and combinations thereof and mammalian cells, preferably human cells.

[0069] Cell interactions that may be studied in the context of the present invention include but are not limited to, release of signalling molecules such as cytokines, extracellular vesicles, chemokines, cell migration and formation of cell-cell contacts,

[0070] In some applications, the present invention can be used in drug screening and drug discovery to simultaneously assess the effect of a drug on at least two populations of cells. In a preferred embodiment the present invention has applications in screening drugs for the treatment and / or prevention of diseases associated with the microbiome, as the present invention facilitates simultaneous study of the effect of a drug on microbial cells and mammalian cells.

[0071] In other applications the present invention can be used to investigate the effect of disease on at least two populations of cell simultaneously, preferably on microbial cells and mammalian cells, even more preferably on microbial cells found in the microbiota and human cells.

[0072] In other applications the present invention can be used to investigate the formation of biofilms by microbial cells and the effect on at least one other population of cells, preferably mammalian cells.

Claims

CLAIMS:

1. A cell culture device comprising upper and lower well plate elements, said elements together defining a culture well, wherein the device further comprises a cell culture insert having apical and basal surfaces, said insert located within the culture well and separating said culture well into first and second apical and basal volumes; wherein at least a portion of said insert is gas permeable, to permit gas to exchange across the apical and basal surfaces between the apical and basal volumes; and wherein said upper and lower well plate elements each further comprise at least one opening extending between the interior and exterior of each respective element, to permit movement of gas between the interior and exterior of each respective element.

2. A cell culture device according to claim 1 comprising a gas impermeable seal between the well plate elements of the culture well.

3. A cell culture device according to claim 1 or 2 comprising a gas supply connected to at least one opening of at least one well plate element.

4. A cell culture device according to any of claims 1 to 3 wherein at least one well plate element is filled with a low oxygen atmosphere.

5. A cell culture device according to any of claims 1 to 4 wherein at least one well plate element comprises a sensor, wherein preferably said sensor is an oxygen sensor.

6. A cell culture device according to claim 5 wherein the sensor is connected to a computer processing unit which can modify the amount of gas directed through the gas supply to the gas inlet.

7. A cell culture device according to any of claims 1 to 6 wherein the well plate elements are detachable.

8. A cell culture device according to any of claims 1 to 7 wherein at least one well plate element comprises a cell culture plate.

9. A cell culture device according to any preceding claim comprising aerobic and / or anaerobic cells.

10. A method of culturing cells using a cell culture device as defined in any of claims 1 - 9, comprising adding cells to the device and introducing a low oxygen gas through at least one opening of at least one well plate element, so that one volume has a low oxygen atmosphere compared to the other volume.11 . A method of culturing cells according to claim 10 wherein a low oxygen gas is continuously administered through an opening on at least one well plate element.

12. A method of culturing cells according to claim 10 wherein after the low oxygen gas is administered to at least one well plate element, all openings on said well plate element of the device are sealed.

13. A method of culturing cells according to claim 10 wherein ambient gas in the environment is allowed to enter at least one well plate element through at least one opening of the cell culture device.

14. A method of culturing cells according to any of claims 11 to 13 wherein the apical volume of the device has a low oxygen atmosphere, and the basal volume of the device has an atmosphere with oxygen levels resembling the ambient environment.

15. A method of culturing cells according to any of claims 11 to 14 wherein at least two populations of cells are co-cultured.

16. A method of culturing cells according to claim 15 wherein at least one population of anaerobic cells and a least one population of aerobic cells are cocultured.

17. A method of culturing cells according to claim 15 wherein at least one population of cells that is co-cultured comprises microorganisms that populate the microbiome, preferably the microbiome present in the gastrointestinal tract.

18. A method of culturing cell according to claim 15 wherein at least one population of cells that is co-cultured are microorganisms selected from bacteria, fungi, viruses, archaea, protozoa, algae and combinations thereof.

19. A method of culturing cells according to claim 15 wherein at least one population of microorganisms and at least one population of mammalian cells, preferably human cells are co-cultured.

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