An improved anaerobic cell reactor

Abstract

The invention relates to cell reactor (CR) device adapted to simulate anaerobic conditions in vitro, preferably simulation of anaerobic intestinal conditions in vitro. The cell reactor (CR) comprises a housing (HOU), the housing HOU) defining a vessel and wherein said vessel comprises a first and second compartment (CO, CO') divided by a gas-permeable support (SUP). The first compartment (CO) comprises an inlet (IL) and an outlet (OL) to provide a flow of a deoxygenized liquid (MED) within said first compartment (CO), wherein the gas-permeable support (SUP) is selected from one of a) a porous membrane made from a semi-rigid polymer with track etched pores, such as polyethylene or polycarbonate; or b) a hydrophilic support comprising silicone and hydrogel, and wherein the housing (HOU) is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment, such as polycarbonate.

Description

[0001] AN IMPROVED ANAEROBIC CELL REACTOR

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a cell reactor device adapted for anaerobic simulation of in vitro biologic processes, such as for in vitro simulation of anaerobic intestinal processes.

[0004] BACKGROUND OF THE INVENTION

[0005] In vitro simulation of cell layers within a cell reactor CR or cell reactor is known from the prior art.

[0006] For various reasons, known cell reactor CRs are not suitable for anaerobic simulation over prolonged periods of time, such as for more than 72 hours. Further, known cell reactor CRs are not designed for production of adequate amounts of, and easy harvesting of e.g. biofilms, post-simulation. Thus, known cell reactor CRs / cell reactors are not suitable for drug absorption studies due to a small absorption area and a lacking physiological (realistic) biofilm.

[0007] Even further, known cell reactor CRs are not suitable for microscopy during simulation.

[0008] Hence, an improved cell reactor CR would be advantageous, and in particular a more efficient and / or reliable cell reactor CR for anaerobic simulation would be advantageous.

[0009] OBJECT OF THE INVENTION

[0010] It is a further object of the present invention to provide an alternative to the prior art.

[0011] In particular, it may be seen as an object of the present invention to provide a cell reactor that solves the above mentioned problems of the prior art with biofilm harvesting, microscopy and anaerobic simulations for prolonged periods of time.

[0012] SUMMARY OF THE INVENTION

[0013] Thus, the above described object and several other objects are intended to be obtained in a first aspect of the invention by providing a cell reactor device adapted to simulate anaerobic conditions in vitro, preferably simulation of anaerobic intestinal conditions in vitro, the device comprising:

[0014] -a housing, the housing defining a vessel, the vessel comprising a first and second compartment divided by a gas-permeable support, the first compartment comprising an inlet and an outlet to provide a flow of a deoxygenized liquid within said first compartment, wherein the gas- permeable support is selected from one of:

[0015] -a porous membrane made from a semi-rigid material, such as a polymer, with track etched pores, such as polyethylene or polycarbonate, or -a hydrophilic support comprising silicone and hydrogel, and wherein the housing is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment.

[0016] The invention is particularly, but not exclusively, advantageous for obtaining an anaerobic environment for the simulation of anaerobic conditions during cell growth, cell layer growth and the study of anaerobic microorganisms, anaerobic bacteria.

[0017] In particular, the invention is advantageous for simulation of in-vivo conditions within the gut, such as gut bacteria, orally ingested drugs and the like, which are to at least pass through the gut and / or be absorbed in the gut.

[0018] Further, the invention is particularly, but not exclusively, advantageous for obtaining an anaerobic environment for the study and development of orally ingested drugs where anaerobic bacteria is presented, such as simulating a pathologic gut environment and the interactions of said pathologic gut environment with drugs or other therapeutic substances.

[0019] In particular, the invention is advantageous for obtaining an anaerobic environment for the growth and resilience of e.g. epithelial cell layers in an anaerobic environment.

[0020] It is an aim of the invention, to provide an improved cell reactor wherein the gas- permeable support, i.e. the porous membrane is made from a semi-rigid material with track etched pores or the hydrophilic support comprising silicone and hydrogel; prevents bacteria to pass through said gas-permeable support. In the context of the present invention, deoxygenized is to be understood as the lack of oxygen, i.e. wherein oxygen has been removed, subtracted or extracted and to sustain such an environment throughout the simulation. In particular, the invention is suitable for sustaining an oxygen free, i.e. anaerobic environment.

[0021] In the context of the present invention, in vitro is to be understood as a process or study of a biological event taking place outside a living organism. Particularly, the present invention attempts to solve existing problems with sustaining an anaerobic environment similar to that of some anaerobic biological environments, outside a true biological environment.

[0022] In the context of the present invention, gas permeable is to be understood as the penetration of a permeate, i.e. a fluid such as a liquid, gas, or vapor, through a solid. In this context, the solid is the gas permeable support, i.e. a support enabling the growth of a cell layer; and that allows gases, in particular but not limited to oxygen, to pass through it from a side opposite the cell layer and into the cell layer. The permeability of a gas through a material is dependent on the temperature of the interaction as well as the characteristics of both the material and the gas component. In the context of the present invention, the gas permeability is defined within typical ranges of atmospheric pressure and / or at or near room temperature. In some embodiments of the invention, the device and the gas permeable support may be adapted to be gas permeable under extreme conditions, such as extreme or atypical pressure conditions or at extreme temperatures, high or low.

[0023] In the context of the present invention, a cell reactor is to be understood as a cell reactor CR suitable for the growth of cells on a membrane or support structure within a controlled environment. Thus, it is to be understood, that the housing is adapted for containing the membrane or gas-permeable support between the two compartments; and shielding a simulated environment within the compartment and on the support, from any undesired influence or contaminants from a surrounding environment. In the context of the present invention, porous is to be understood as having pores. Further, in the context of the present invention, the pores may be adapted to ensure, that only gases may be transported through said pores.

[0024] In the context of the present invention, track etched is to be understood as pores of a nano scale size.

[0025] In a preferred embodiment of the invention, the track etched pores are created with an ion treatment, such as a heavy ion treatment, essentially damaging parts in the gas-permeable support, caused by passing high-energy ions.

[0026] I another preferred embodiment, the track etched pores are created by a chemical etching process.

[0027] In some embodiments, the ion treatment and chemical etching may be combined, to provide the track etched pores.

[0028] In the context of the present invention, the liquid may be a cell culture medium. The medium may comprise nutrients or other relevant ingredients to accommodate the specific environment to simulate. It is further to be understood, that the liquid is not an aspect of the present invention, but the treatment of the liquid, i.e. the deoxygenation of the liquid, may be an aspect of the invention, in specific embodiments. Thus, the liquid is preferably a medium simulating the gut content or in other ways supporting bacterial growth under physiological conditions.

[0029] In a preferred embodiment of the invention, the housing is made from a transparent polymer material, such as polycarbonate, thus enabling for microscopy through a surface of the housing.

[0030] In the context of the present invention, hydrophilic is to be understood as relating to, or having a strong affinity for water. The hydrophilic support may be selected from one or more of the following materials: poly-2- hydroxyethyl methacrylate (PHEMA), poly(ethylene glycol) (PEG), poly(ethylene glycol) methacrylate (PEGMA). It is further to be understood, that the gas-permeable support is to be biocompatible, to support prolonged simulations, such as for more than 72 hours, such as for more than a week, such as for more than a month or such as for years.

[0031] The porous membrane may be selected from a suitable polymer, such as polydimethylsiloxane, tris-(trimethyl-silyl-propyl-methacrylate) or similar material.

[0032] In a preferred embodiment, the gas-permeable support is transparent to improve microscopy of the cell layer or any elements within the liquid within a compartment, adjacent to said cell layer.

[0033] In the context of the present invention, semi-rigid material is to be understood as sufficiently rigid to support a cell layer and at least a flow of fluid on one side of the porous membrane.

[0034] In some embodiment, a flow of fluid is provided on both a first and second side of the gas-permeable support. Further, the flow direction on one side may be opposite to the flow direction on a second side of the gas-permeable support.

[0035] In a preferred embodiment of the invention, the pores of the porous membrane are substantially perpendicular to a surface plane of said porous membrane. This embodiment is particularly advantageous for ensuring a uniform transparency of the porous membrane. In some embodiments, the pores are further uniformly or substantially uniformly distributed on the porous membrane. These embodiments are particularly advantageous for microscopy purposes, such as for studying the cell layer or a biofilm on said cell layer.

[0036] In another preferred embodiment the porous membrane is substantially transparent to improve microscopy, including phase contrast microscopy, which requires a defined ring of light underneath the material to be observed. In yet another preferred embodiment, the hydrophilic support has a thickness of between 0,1 and 2,0 mm, more preferred between 0,2 and 0,8 mm, even more preferred between 0.5 and 3 mm. This embodiment is particularly advantageous for ensuring, that gas may move through said hydrophilic support while still being able to support the cell layer and endure the flow over said cell layer without breaking or rupturing.

[0037] In an advantageous embodiment of the invention, the porous membrane has a thickness of between 0.1 and 100 pm, preferably between 5 and 80 pm, even more preferably between 8 and 90 pm, most preferably between 10 and 50 pm. This embodiment is particularly advantageous for ensuring, that gas may move through said porous membrane while still being able to support the cell layer and endure the flow over said cell layer without breaking or rupturing.

[0038] In another advantageous embodiment of the invention, the flexible support is a chemical blend or a co-polymer or an interpenetrating polymer network. This embodiment is particularly advantageous for providing a hydrophilic and biocompatible surface for cell growth.

[0039] In yet another advantageous embodiment of the invention, the hydrophilic support comprises between 10 and 40% hydrogel.

[0040] In a preferred embodiment of the invention, the inlet and outlet comprises connectors, said connectors adapted to provide a fluidic connection with associated conduits, said connectors being non-permeable or substantially non- permeable to at least oxygen from a surrounding atmospheric environment. This embodiment is particularly advantageous for providing a cell reactor which is easy to use and wherein it is easy to ensure a sealed environment during simulation.

[0041] In another preferred embodiment, the second compartment is enclosed and sealed within walls of the housing and the gas-permeable support respectively. This embodiment may be advantageous to provide a cell reactor which is ready to use, such as wherein the second compartment is pre-filled with a fluid suitable for supporting a cell layer. In some embodiments, the fluid pre-filled is specific to a specific type of cell layer. In yet another preferred embodiment, the second compartment has an opening to an outside environment and / or atmospheric air, the gas-permeable support providing a gas-permeable sealing between the first compartment and said opening. This embodiment may be particularly advantageous for providing a cell reactor which enables for gas permeability from an outside environment, exclusively through the gas-permeable support.

[0042] In an advantageous embodiment of the invention, a fluid comprising oxygen is contained within the second compartment. This embodiment may be advantageous to provide a cell reactor which is ready to use, such as wherein the second compartment is pre-filled with oxygen, which may be suitable for a cell layer and wherein the operator does not need to control the environment within the second compartment. In some embodiments, the second compartment may further be pre-filled with a fluid comprising nutrients, enabled to move into the cell layer through the gas-permeable support by e.g. diffusion or facilitated diffusion.

[0043] In other advantageous embodiments of the invention, an oxygen-releasing chemical or solid, e.g. a chemical or solid that releases oxygen when pH declines, such as when cells metabolizes and releases CO2, which decreases pH, is contained within the second compartment. This embodiment may be advantageous to provide a cell reactor which is ready to use, such as wherein the second compartment is pre-filled with the oxygen-releasing chemical or solid, which may be suitable for a cell layer and wherein the operator does not need to control the environment within the second compartment.

[0044] In another advantageous embodiment of the invention, the second compartment comprises an inlet and an outlet adapted to provide a flow of a fluid through said second compartment. This embodiment may be particularly advantageous for providing a flow over the gas-permeable support on a side opposite the side of the cell layer. In some embodiments, the flow may be provided in cycles or with a varying pressure, to simulate e.g. peristaltic movements. It is to be understood, that the gas-permeable membrane is to be made of a thickness and durability enabled to withstand e.g. a partial pressure one each of the sides respectively. In yet another advantageous embodiment of the invention, at least a portion of the housing is transparent. In some embodiments, the housing is fully made of a transparent polymer, such as polycarbonate.

[0045] In a preferred embodiment, the porous membrane is made from polyester.

[0046] In another preferred embodiment, the porous membrane has a thickness between 8 and 14 pm.

[0047] In yet another preferred embodiment, the porous membrane is tissue culture treated.

[0048] In yet another preferred embodiment, the porous membrane has a pore size of between 0.2 and 0.6 pm.

[0049] In yet another preferred embodiment, the porous membrane has a porosity of between 0.2 and 1.0 %, more preferably between 0.4 and 0.8 %.

[0050] In an advantageous embodiment, the porous membrane is a 12 pm thick tissue culture treated transparent polyester (PET) membrane with a pore size of 0.45 pm and a porosity of 0.6%.

[0051] In a second aspect, the invention relates to a system adapted to simulate anaerobic conditions in vitro, preferably anaerobic intestinal conditions, the system comprising:

[0052] -the cell reactor device according to the first aspect of the invention,

[0053] -a container comprising a nutrient liquid,

[0054] -a deoxygenation device adapted to remove or reduce any oxygen from the nutrient liquid,

[0055] -a pump, the pump fluid ically connected to at least the container and the first compartment, the pump adapted to create a flow of the nutrient liquid through the first compartment. This aspect is particularly, but not exclusive advantageous for obtaining an anaerobic environment for the growth and resilience of e.g. epithelial cell layers in an anaerobic environment of the cell reactor. It is further to be understood, that in theory, it is the bacteria which requires an anaerobic environment for the simulations to occur at near-in-vivo conditions. Nevertheless, the inventors have hypothesized, that the cell layer may be grown more physiologically true, if subjected to anaerobic conditions or at varying conditions switching between near-anaerobic and aerobic conditions.

[0056] In the context of the present invention, deoxygenation is to be understood as the removal, subtraction or extraction of oxygen. In particular, the deoxygenation device is suitable for deoxygenation of fluids, such as liquids, wherein the oxygen content of said fluid is reduced, removed or substantially removed.

[0057] In a preferred embodiment of the invention, the deoxygenation device is adapted to deoxygenize the liquid flowing to the cell reactor, and wherein the deoxygenation device comprises:

[0058] -a conduit comprising an inner lumen, a first end and a second end, the conduit comprising a gas permeable wall, the inner lumen adapted to contain and transport the liquid from said first end to said second end, wherein the gas permeable wall is enveloped in a fluid, the fluid being : -substantially void of oxygen, or

[0059] -having an affinity for oxygen higher than that of the liquid, the deoxygenation device enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid.

[0060] In an alternative embodiment, the deoxygenation device comprises:

[0061] -a conduit comprising an inner lumen, a first end and a second end, the conduit comprising a gas permeable wall, the inner lumen adapted to contain and transport the liquid from said first end to said second end, wherein the gas permeable wall is immersed in a fluid within a vessel, the fluid being : -substantially void of oxygen, or

[0062] -having an affinity for oxygen higher than that of the liquid, the deoxygenation device enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid, and wherein the deoxygenation device is adapted to be connected to the cell reactor, to provide deoxygenized liquid, such as cell culture medium, to the cell reactor.

[0063] In another alternative embodiment, the deoxygenation device comprises:

[0064] -a two-lumen conduit comprised of an inner lumen and an outer lumen surrounding the inner lumen, the inner and outer lumen separated by a first wall and wherein the outer lumen is defined by an outer wall, the inner lumen further comprising a gas permeable wall section; the inner lumen adapted to contain and transport the liquid from a first end to a second end of the conduit, and wherein at least oxygen transport is enabled from the inner lumen to the outer lumen by the second lumen being adapted to contain a fluid, the fluid being:

[0065] -substantially void of oxygen, or

[0066] -having an affinity for oxygen higher than that of the liquid, the deoxygenation device enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid, and wherein the deoxygenation device is adapted to be connected to the cell reactor, to provide deoxygenized liquid, such as cell culture medium, to said cell reactor.

[0067] In the context of the present invention, it is to be understood, that a two-lumen conduit may be manufactured with stringers or other type of radially extending walls to ensure structural integrity and the position of the inner lumen substantially within the centre of the outer lumen.

[0068] In yet another alternative embodiment, the deoxygenation device comprises:

[0069] -a conduit comprising an inner lumen, a first end and a second end, the conduit comprising a gas permeable wall, the inner lumen adapted to contain and transport the liquid from said first end to said second end, wherein at least the gas permeable wall is contained within a sealed container vessel, the sealed container being:

[0070] -substantially void of oxygen, or

[0071] -further containing a fluid with an affinity for oxygen higher than that of the liquid, the deoxygenation device enabling oxygen to move from the liquid to the sealed container to deoxygenize said liquid, and wherein the deoxygenation device is adapted to be connected to an associated cell reactor CR, to provide deoxygenized liquid, such as cell culture medium, to said cell reactor CR.

[0072] In some embodiments, the system may fit onto a support plate or within a box, which can be carried by a use, as a plug-in / turn key solution.

[0073] In the context of the deoxygenation device, gas permeable is to be understood as the penetration of a permeate, i.e. a fluid such as a liquid, gas, or vapor, through a solid. In this context, the solid is the gas permeable wall, i.e. a wall that allows gases, in particular but not limited to oxygen, to pass through it. The permeability of a gas through a material is dependent on the temperature of the interaction as well as the characteristics of both the material and the gas component. In the context of the present invention, the gas permeability is defined within typical ranges of atmospheric pressure and / or at or near room temperature. In some embodiments of the invention, the system and the gas permeable wall may be adapted to be gas permeable under extreme conditions, such as extreme or atypical pressure conditions or at extreme temperatures, high or low.

[0074] In the context of the present invention, enveloped is to be understood as surrounded by, submerged into or covered by; and other similar synonyms.

[0075] In the context of the present invention, affinity is to be understood as the degree to which a substance tends to combine with another substance. In this context, it is to be understood as a substance which has a high degree of tendency to attract and retain gas, in particular oxygen. It is to be understood, that oxygen affinity can be influenced by various factors such as pH levels, temperature changes, which the system is adapted to control, based on the fluid which is used for oxygen removal or reduction.

[0076] In a preferred embodiment, the system comprises a gas permeable conduit between the container and the inlet, said gas permeable conduit being arranged at or within the deoxygenation device.

[0077] In another preferred embodiment, the deoxygenation device comprises a fluid with an affinity for oxygen. In yet another preferred embodiment of the invention, the pump is adapted to adjust the flow velocity of nutrient liquid through the first compartment. This embodiment may be particularly advantageous for simulating peristaltic movements to the gas-permeable support and thus the cell layer.

[0078] In a third aspect, the invention relates to a method of simulating anaerobic conditions in vitro, preferably simulating anaerobic intestinal conditions in vitro, the method comprising:

[0079] -providing the cell reactor device according to the first aspect of the invention,

[0080] -seeding the gas-permeable support, on a surface of the gas-permeable support facing the first compartment, with one or more cells, preferably intestinal epithelial cells,

[0081] -providing a flow of nutrient fluid through the first compartment, wherein any oxygen from the nutrient fluid has been extracted prior to flowing through said first compartment, and

[0082] -growing a cell layer on the gas-permeable support, said layer being exposed to an anaerobic environment within the first compartment.

[0083] In a preferred embodiment, the method further comprises:

[0084] -providing at least oxygen to the cell layer from the second compartment through the gas-permeable support.

[0085] In another preferred embodiment the method further comprises:

[0086] -adding a drug or compound to the system and subsequently determining the effect of said drug or compound on the system. This embodiment is particularly advantageous for in vitro research and development of drugs, such as for eliminating side-effects prior to animal or human studies.

[0087] In yet another preferred embodiment, a microorganism, such as a pathogenic microorganism, is added to the system before the drug or compound is applied and the effect of the drug or compound or the microorganism is determined. In an advantageous embodiment, the microorganism is a bacterium, a parasite, a bacteriophage, a fungus, or a virus.

[0088] In some embodiments, the microorganism is selected from the group consisting of: Lactobacillus; Bacterioides; Ruminococcus; Peptococcus; Peptostreptococcus; Bifidobacterium; Escherichia; Achromobacter; Acidaminococcus fermentans;

[0089] Acinetobacter cacoaceticus; Aeromonas; Alcaligenes faecalis; Bacillus;

[0090] Butyriviberio fibrosolvens; Camplyobacter; Campylobacter coli; Clostridium difficile; Clostridium sordelli; Enterobacter cloacae; Enterococcus faecalis;

[0091] Enterococcus faecium; Escherichia coli; Flavobacterium; Mycobacterium;

[0092] Mycoplasma; Plesiomonas shigelloides; Propionibacterium acnes; Pseudomonas aeruginosa; Ruminococcus bromii; Sarcina; Staphylococcus aureus; Streptococcus anginosus; Veillonella; Vibrio; Yersinia enterocolitica; Lactobacillus rhamnosus;

[0093] Lactobacillus rhamnosus GG; Bifidobacterium breve; Bifidobacterium longum;

[0094] Bifidobacterium infantis; Lactobacillus acidophilus; Lactobacillus plantarum;

[0095] Lactobacillus paracasei; Lactobacillus bulgaricus ; and Streptococcus thermophilus.

[0096] In some embodiments, the microorganism is pathogenic.

[0097] In some embodiments, the microorganism is a probiotic.

[0098] In some embodiments, the pathogens are selected from the group consisting of: enterotoxigenic Escherichia coli; Bilophila wadsworthia; Shigella; Yersinia;

[0099] Pleisiomonas; Vibrio; Aeromonas; Campylobacter; Crytosporidia; Coccidosis;

[0100] Salmonella; Helicobacter pylori; Clostridium difficile; Salmonella kedougou;

[0101] Bacteroides; Clostridium; Firmicutes; Shigellia dysenteriae; Salmonella enterica;

[0102] Salmonella typhi; Listeria; Listeria monocytogenes; Vibrio parahaemolyticus; Proteus; Vibrio cholerae; Enterococcus faecalis; Yersinia enterocolitica ; and Campylobacter jejuni ; rotavirus; norwalk-like viruses; adenoviruses; astroviruses; sapporo-like viruses; toroviruses; coronaviruses; picornaviruses; herpes viruses; noroviruses; Candida; Aspergillus; Candida albicans ; single-celled parasites; multi-celled parasites; ameobas; worms; tape worms; protozoans; flukes; roundworms; pinworms; hookworms; Giradia lamblia; cryptosporidium ; and Entamoeba histolytica . Microbial cells can be bacterial cells, including both gram positive and gram negative bacteria. Non-limiting examples of bacterial cells useful in the cell culture system described herein include Lactobacillus; Bacterioides; Ruminococcus; Peptococcus; Peptostreptococcus; Bifidobacterium; Escherichia; Achromobacter; Acidaminococcus fermentans; Acinetobacter cacoaceticus; Aeromonas;

[0103] Alcaligenes faecalis; Bacillus; Butyriviberio fibrosolvens; Camplyobacter; Campylobacter coli, Clostridium difficile; Clostridium sordelli; Enterobacter cloacae; Enterococcus faecalis; Enterococcus faecium; Escherichia coli; Flavobacterium; Mycobacterium; Mycoplasma; Plesiomonas shigelloides; Propionibacterium acnes; Pseudomonas aeruginosa; Ruminococcus bromii; Sarcina; Staphylococcus aureus; Streptococcus anginosus; Veillonella; Vibrio; Yersinia enterocolitica; Lactobacillus rhamnosus, Lactobacillus rhamnosus GG; Bifidobacterium breve; Bifidobacterium longum; Bifidobacterium infantis; Lactobacillus acidophilus; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus bulgaricus; and Streptococcus thermophilus.

[0104] In some embodiments, the microbial cells are pathogenic. In some embodiments, the microbial cells are intestinal pathogens. Non-limiting examples of pathogenic microbial cells include, enterotoxigenic Escherichia coli; Bilophila wadsworthia; Shigella; Yersinia; Pleisiomonas; Vibrio; Aeromonas; Campylobacter;

[0105] Crytosporidia; Coccidosis; Salmonella; Helicobacter pylori; Clostridium difficile; Salmonella kedougou; Bacteroides; Clostridium; Firmicutes; Shigellia dysenteriae; Salmonella enterica; Salmonella typhi; Listeria; Listeria monocytogenes; Vibrio parahaemolyticus; Proteus; Vibrio cholerae; Enterococcus faecalis; Yersinia enterocolitica ; and Campylobacter jejuni . Intestinal pathogens have been well studied and described (see for example. Microbial Pathogenesis and the Intestinal Epithelial Cell— Gail A. Hecht— 2003— ASM press). Intestinal pathogens described in this book are hereby incorporated by reference.

[0106] In some embodiments, the cell culture system comprises pathogens. As used herein, "pathogens" can include viruses, bacteria, fungi, and parasites which are known to cause or be associated with any disorder or disease of the intestine. Microbial pathogens are discussed above herein. Non-limiting examples of viral intestinal pathogens include rotavirus; norwalk-like viruses; adenoviruses; astroviruses; sapporo-like viruses; toroviruses; coronaviruses; picornaviruses; herpes viruses; and noroviruses. Non-limiting examples of fungal intestinal pathogens include Candida, Aspergillus , and Candida albicans . Non-limiting examples, of intestinal parasites include single-celled parasites, multi-celled parasites, ameobas, worms, tape worms, protozoans, flukes (flatworms), roundworms, pinworms, hookworms, Giradia lamblia, cryptosporidium , and Entamoeba histolytica.

[0107] In an embodiment the microorganism is C. difficile and / or B. fragilis. These are both obligate anaerobic.

[0108] In some embodiments, the microorganism is aerobic.

[0109] In some embodiments, the microorganism is anaerobic.

[0110] Preferred drugs / compounds may be selected from one or more of the following: Nutriceutical

[0111] Probiotics, prebiotics

[0112] Pathogens

[0113] Drugs - antibiotics Cancergenous bacteria

[0114] In an embodiment the intestinal epithelial cells are selected from the group consisting of:

[0115] Caco2 cells; HT-29 cells; primary small intestine epithelial cells; primary large intestine epithelial cells; iPS cells; ESC cells; stem cells; paneth cells; crypt cells; and mucus-secreting cells.

[0116] In some embodiments of the cell culture system described herein, at least one layer of intestinal epithelial cells is attached to at least one surface of the membrane. In some embodiments, one or more layers of intestinal epithelial cells are attached to the membrane, e.g. one layer, two layers, three layers, or more layers of intestinal epithelial cells. In some embodiments, intestinal epithelial cells are attached to one side of the membrane. In some embodiments, intestinal epithelial cells are attached to two sides of the membrane. In some embodiments, the intestinal epithelial cells are mammalian cells. In some embodiments, the intestinal epithelial cells are human cells. In some embodiments, the intestinal epithelial cells are primary cells, primary small intestine cells, primary large intestine cells, small intestine cells, large intestine cells, cultured cells, passaged cells, immortalized cells, transgenic cells, genetically modified cells, cancerous cells or cells from an animal with an intestinal cancer, cells from an animal with an intestinal disease or disorder, stem cells, embryonic stem cells (ESCs), induced pluripotent stem cells (IPSCs), paneth cells, crypt cells, mucus-secreting cells, Caco2 cells, or HT-29 cells. In some embodiments, the intestinal epithelial cells in the cell culture system described herein comprise villi structures.

[0117] In an embodiment, intestinal epithelial cells are attached to one side of the membrane and endothelial cells are attached to the other side. This may simulate underlying blood vessels.

[0118] In another embodiment, immune cells are attached to one side of the membrane and endothelial cells are attached to the other side.

[0119] In a fourth aspect, the invention relates to a kit of parts for extracting oxygen from a liquid to be provided to a compartment the cell reactor, said cell reactor adapted to simulate an in vitro environment of a biological process, the kit of parts comprising :

[0120] -a cell reactor according to the first aspect of the invention, -a conduit having a first end and a second end, the conduit defining an inner lumen to contain, and transport said liquid to the compartment of the cell reactor, the conduit further comprising:

[0121] -a gas permeable section,

[0122] -a second section being non-permeable to gas, the second section extending between the gas permeable section of the conduit and the second end of the conduit,

[0123] -a connector at the second end, the connector adapted to fluidica lly connect to the cell reactor,

[0124] -means for extracting oxygen from the liquid within the gas permeable section of the conduit, the means selected from one of: -a fluid adapted to quench oxygen from the liquid by submerging at least the gas permeable section of the conduit in said fluid, or

[0125] -a sealable container with a valve, the container adapted to contain at least the gas permeable section of the conduit and wherein at least oxygen is removed from within said container through the valve, or

[0126] -the conduit comprises a second lumen outside the inner lumen and wherein the second lumen is one of:

[0127] -void of oxygen, or

[0128] -filled with a fluid adapted to quench or catch oxygen.

[0129] In a preferred embodiment of the invention, the kit of parts further comprises a pump adapted to provide flow to the liquid to the cell reactor from the conduit.

[0130] In another preferred embodiment of the invention, the fluid comprises a molecule adapted to absorb oxygen, preferably the fluid is c-vitamin ascorbate.

[0131] In a fifth aspect, the invention relates to the use of any of the other aspect for in vitro simulation of anaerobic conditions in a cell reactor.

[0132] The first, second, third, fourth and fifth aspects of the present invention may each be combined with any of the other aspects. These and other aspects of the invention will be apparent from and elucidated with reference to the embodiments described hereinafter.

[0133] BRIEF DESCRIPTION OF THE FIGURES

[0134] The cell reactor according to the invention will now be described in more detail with regard to the accompanying figures. The figures show one way of implementing the present invention and is not to be construed as being limiting to other possible embodiments falling within the scope of the attached claim set.

[0135] Fig.l shows a sideview of a cell reactor, according to an embodiment of the invention;

[0136] FIG. 2 shows a sideview of a cell reactor, according to another embodiment of the invention; FIG. 3 shows a sideview of a cell reactor, according to yet another embodiment of the invention;

[0137] Fig. 4 shows a schematic overview, in sideview, of a simulation setup, for anaerobic in vitro simulation, according to an embodiment of the invention. Fig. 5 is a flow-chart of a method according to the invention;

[0138] Fig. 6A - Fig. 6C shows the design of a cell reactor and anaerobic system, according to an embodiment of the invention;

[0139] Fig. 7A - Fig. 7E shows Oxygen levels and dependency on flow rate, tube length and chamber conditions;

[0140] Fig. 8A - Fig. 8C shows characterization of the Caco-2 cell layer 13 days post seeding;

[0141] Fig. 9A - Fig. 9C shows transcriptomic characterization of the Caco-2-based DFC model;

[0142] Fig. 10A - Fig. 10E shows colonization with C. difficile and B. fragilis;

[0143] Fig. 11A and Fig. 11B shows bacterial colonization of Caco-2 cells;

[0144] Fig. 12A - Fig. 12C shows images from a four-week study of a Caco-2 cell layer, utilizing a system according to an embodiment of the invention.

[0145] DETAILED DESCRIPTION OF AN EMBODIMENT

[0146] FIG. 1 shows a sideview of a cell reactor CR, according to an embodiment of the invention. The cell reactor CR comprises a housing HOU, with an inlet IL and an outlet OL, fluidically connected by a first compartment CO defined by the housing HOU and the gas-permeable support SUP or support structure. The housing HOU is made from a material which is non-permeable to gas. Within the centre of the housing, HOU, the gas-permeable support SUP or support structure extends along the length of the housing HOU, thus further defining a second compartment CO' within the housing, separated from the first compartment CO, by said gas- permeable support SUP or support structure. The gas-permeable support SUP or support structure is made from a porous polymer with parallel micro channel extending between the first and second compartment CO, CO'. In FIG. 7 a cell layer CL is grown along the bottom of the first compartment CO, on the gas- permeable support SUP or support structure. Thus, the first compartment is sealed from the outside environment by the housing HOU, the gas-permeable support SUP or support structure and the cell layer CL respectively. The second compartment CO' is defined by the housing HOU and the gas-permeable support SUP or support structure respectively and further comprises an inlet IL' and an outlet OL' adapted for connection to a fluidic flow. It is to be understood, that a fluid, such as a liquid or gas comprising oxygen may be provided in the second compartment CO'. When the cell reactor CR is used with the deoxygenation device DOD, shown in FIG. 4, the inlet is connected to a liquid connection, wherein the liquid is void of oxygen. Thus, any oxygen to reach the cell layer CL, must pass through the gas-permeable support SUP or support structure from the second compartment CO', thereby providing a first compartment CO within the cell reactor CR void of oxygen, i.e. an anaerobic environment for anaerobic in vitro simulation of biological processes, such as in vitro intestinal simulation. In some embodiments, the housing may be made from a transparent material, such as polycarbonate, thus enabling for microscopy through the upper surface US of the housing HOU. In some embodiments, the cell reactor CR may comprise a light source adapted at a bottom portion of the housing HOU, thus providing light from below, for improved microscopy of the cell layer CL or a liquid within the first compartment CO. In some embodiments, the gas-permeable support SUP or support structure may be an integral part of the housing, such as manufactured from a single element or sandwiched between two half-shells of the housing HOU.

[0147] FIG. 2 shows a sideview of a cell reactor CR, according to another embodiment of the invention. The cell reactor CR comprises a housing HOU, with an inlet IL and an outlet OL, fluidica lly connected by a first compartment CO defined by the housing HOU and the gas-permeable support SUP or support structure. The housing HOU is made from a material which is non-permeable to gas. Within the centre of the housing HOU, the gas-permeable support SUP or support structure extends along the length of the housing HOU, thus further defining a second compartment CO' within the housing, separated from the first compartment CO, by said gas-permeable support SUP or support structure. The gas-permeable support SUP or support structure is made from a silicone or silicone hydrogel composition. In FIG. 2 a cell layer CL is grown along the bottom of the first compartment CO, on the gas-permeable support SUP or support structure. Thus, the first compartment is sealed from the outside environment by the housing HOU, the gas-permeable support SUP or support structure and the cell layer CL respectively. The second compartment CO' is defined by the housing HOU and the gas-permeable support SUP or support structure respectively and further comprises an opening OP to an outside environment. The opening OP may be plugged (not shown), thus sealing the second compartment from the outside environment. It is to be understood, that a fluid, such as a liquid or gas comprising oxygen may be provided in the second compartment CO'. When the cell reactor CR is used with the deoxygenation device DOD, shown in FIG. 4, the inlet is connected to a liquid connection, wherein the liquid is void of oxygen. Thus any oxygen to reach the cell layer CL, must pass through the gas-permeable support SUP or support structure from the second compartment CO', thereby providing a first compartment CO within the cell reactor CR void of oxygen, i.e. an anaerobic environment for anaerobic in vitro simulation of biological processes, such as in vitro intestinal simulation. In some embodiments, the housing may be made from a transparent material, such as polycarbonate, thus enabling for microscopy through the upper surface US of the housing HOU. In some embodiments, the cell reactor CR may comprise a light source adapted at a bottom portion of the housing HOU, thus providing light from below, for improved microscopy of the cell layer CL or a liquid within the first compartment CO.

[0148] FIG. 3 shows a sideview of a cell reactor CR, according to yet another embodiment of the invention. The cell reactor CR comprises a housing HOU, with an inlet IL and an outlet OL, fluidically connected by a compartment CO defined within the housing HOU. The housing HOU is made from a material which is non- permeable to gas. At the bottom of the cell reactor CR, a gas-permeable support SUP or support structure is positioned, abutting the housing HO and compartment CO. The gas-permeable support SUP or support structure is made from a silicone or silicone hydrogel composition. In FIG. 3 a cell layer CL is grown along the bottom of the compartment CO, on the gas-permeable support SUP or support structure. Thus, the compartment is sealed from the outside environment by the housing HOU, the gas-permeable support SUP or support structure and the cell layer CL respectively. When the cell reactor CR is used with the deoxygenation device DOD, shown in FIG. 4, the inlet is connected to a liquid connection, wherein the liquid is void of oxygen. Thus any oxygen to reach the cell layer CL, must pass through the gas-permeable support SUP or support structure, thereby providing a compartment CO within the cell reactor CR void of oxygen, i.e. an anaerobic environment for anaerobic in vitro simulation of biological processes, such as in vitro intestinal simulation. In some embodiments, the housing may be made from a transparent material, such as polycarbonate, thus enabling for microscopy through the upper surface US of the housing HOU.

[0149] Fig.4 shows a schematic overview, in sideview, of a simulation setup SIM_SET, for anaerobic in vitro simulation, according to an embodiment of the invention. In FIG. 4, at centre, the cell reactor CR is positioned. The cell reactor CR comprises a first and second compartment (not visible) encased within a sealed housing. The cell reactor CR has an inlet IL and an outlet OL, connected to the first compartment (not visible) and an inlet IL' and an outlet OL' connected to the second compartment (not visible). The cell reactor CR compartments is clearly shown and explained in various embodiments, in FIGS. 1, 2 and 3. The outlets OL, OL' are fluidically connected to a flow generator FG, operated by e.g. a pump, impeller piston or similar. In other embodiments, the flow generator FG, may be positioned between medium MED, MED' and the inlets IL, IL'. In FIG. 4, flow directions of medium MED, MED' are shown with arrows. A medium MED, is fluidically connected to the cell reactor CR via a conduit CON. The conduit CON goes through a deoxygenation device DOD, into the first compartment of the cell reactor CR. Thus the medium MED is transported through the deoxygenation device DOD, through the inlet IL, into the first compartment of the cell reactor CR, out of the cell reactor CR through the outlet OL, through the flow generator FG and into a container for discarded medium D_MED. The same applies for the second compartment of the cell reactor CR, being fed with a medium MED' being transported from the container marked MED', through the inlet IL' to the second compartment of the cell reactor CR, through the outlet OL', through the flow generator FG and back into the container for medium MED'. In other embodiments, the medium MED' may be discarded.

[0150] Thus, from FIG. 4 is can be seen, that a medium MED is being anaerobized (oxygen is removed from the medium MED), prior to entering the cell reactor CR.

[0151] Fig. 5 is a flow-chart of a method according to the invention, for simulating anaerobic conditions in vitro, preferably simulating anaerobic intestinal conditions in vitro, the method comprising the following steps SI to S4:

[0152] Sl-providing the cell reactor device according to the first aspect, 52-seeding the gas-permeable support, on a surface of the gas-permeable support facing the first compartment, with one or more cells, preferably intestinal epithelial cells,

[0153] 53-providing a flow of nutrient fluid through the first compartment, wherein any oxygen from the nutrient fluid has been extracted prior to flowing through said first compartment, and

[0154] 54-growing a cell layer on the gas-permeable support, said layer being exposed to an anaerobic environment within the first compartment.

[0155] Figs. 6A - C shows the design of a cell reactor and anaerobic system, according to an embodiment of the invention. Fig. 6A shows 3D images of the dual flow chamber. Fig. 6B shows real life image of the dual chamber during an aerobic experiment. Fig. 6C shows a schematic presentation of the system.

[0156] Figs. 7A - E shows oxygen levels and dependency on flow rate, tube length and chamber conditions. Fig. 7A shows a schematic presentation of the single and dual flow chambers showing the connection sites of the oxygen sensor (inlet and exit). Fig. 7B shows inlet oxygen percentage based on different lengths of the silicone coil and at various flow rates. Fig. 7C shows oxygen percentage at inlet and exit side in a single flow chamber (p-slide I luer 0.4mm, Ibidi) at different flow speeds. Fig. 7D shows oxygen percentage in effluent from dual flow chamber with Caco-2 cells cultured under anaerobic conditions for 6 days with a flow of 320pl / min. The data is based on an hourly average from three individual flow chambers. Mean ±range. E) Reabsorption of oxygen into the apical flow chamber when flow is stopped, dependent on the duration of the pause.

[0157] Figs. 8A - C shows characterization of the Caco-2 cell layer 13 days post seeding. Fig. 8A shows representative confocal Z-stack images of Caco-2 cells grown under static (cell culture inserts) or flow conditions (dual flow chamber). The anaerobization unit was connected to the apical inlet on day 7 post seeding to create an oxygen gradient throughout the cell layer (-02). Cells were stained for Mucin 2 (MUC2), the tight junction protein Occludin (OCLN) and F-actin. Fig. 8B shows height of the Caco-2 cell layer grown under different conditions. The height was determined by confocal microscopy. Fig. 8C shows staining of neutral (Shift's reagent) and acidic (Alcian blue) mucins. Figs. 9A - C shows transcriptomic characterization of the Caco-2-based DFC model. Caco-2 cells were cultured for 13 days in the DFC model, both aerobically (DFC+O2) and anaerobically (DFC-O2), as well as in cell culture inserts under aerobic conditions (Static). RNA sequencing was then performed to analyze gene expression profiles. Fig. 9A shows differentially expressed genes (DEGs) between Caco-2 cells cultured in the aerobic DFC and the static cell culture inserts, as well as between cells cultured in the anaerobic and aerobic DFC models. Fig. 9B shows pathway enrichment analysis of Reactome gene sets for up- and downregulated genes in the aerobic DFC model compared to the cell culture inserts, and in the anaerobic DFC model compared to the aerobic DFC model. Here, top 5 enriched pathways are shown, unless fewer pathways were significantly enriched. Fig. 9C shows a clustered heatmap showing scaled marker gene expression profiles of six major intestinal cell types: goblet cells, paneth cells, enteroendocrine cells (EEC), intestinal stem cells (ISC), absorptive enterocytes (AE), and absorptive coloncytes (AC). This comparison includes data from Caco-2 models of this study, a Caco-2- based 3D gut model from Cheng et al. (2023) and single-cell and bulk RNA sequencing data of intestinal cell types (goblet, paneth, EEC, distal enterocytes, and proximal enterocytes) and tissues (colon and small intestine (SI)) derived from the Human Protein Atlas.

[0158] Figs. 10A - E shows colonization with C. difficile and B. fragilis. Caco-2 cells were infected with C. difficile and B. fragilis 7 days post seeding in the dual flow chamber (DFC). On the third day post infection, cultures were treated with 6 pg / ml vancomycin (VAN). Fig. 10A shows CFU / mL in effluent from the apical channel of the DFC. Fig. 10B shows CFU / mL harvested from the cell layer 5 days post colonization. Error bars represent the mean ± standard deviation. Statistical comparisons were made using an unpaired Wilcoxon test. Fig. 10C shows oxygen saturation (%) measured in the exit site before and five days post colonization. Black lines indicate mean values. Statistical comparisons were made using a paired t-test. Fig. 10D shows 4x magnification confocal images of LIVE / DEAD stained Caco-2 cell layers 5 days post colonization. Live cells are stained green, while dead cells appear red. Combined z-stack images. Fig. 10E shows gram stain of effluent from apical channel of the DFC 5 days post colonization. The gram- positive C. difficile is stained blue (not visible) while the gram-negative B. fragilis is stained red (not visible).

[0159] Figs 11A and B shows bacterial colonization of Caco-2 cells. Caco-2 cells were inoculated with C. difficile and B. fragilis 7 days post seeding in the dual flow chamber (DFC). On the third day post inoculation, cultures were treated with 6 pg / ml vancomycin (VAN). Fig. 11A shows 60x magnification confocal images of Live / dead stained bacteria and Caco-2 cell layers 5 days post inoculation. Green (not visible): live cells, Red (not visible): dead cells, Blue: DAPI (Nuclei). Fig. 11B shows electron microscopy of bacteria and Caco-2 cell layers 5 days post inoculation. Bacteria are present both intra- and extracellular. Dark arrows indicate areas with colonizing bacteria. Light arrows indicate microvilli on the surface of a Caco-2 cells. Black arrows indicate what appears as lysed bacteria.

[0160] Figs. 12A - C shows a images from a three-week study of Caco-2 cell layer.

[0161] Fig. 12A shows Caco-2 cell layer observed by brightfield microscopy. Between day 19 and 28 post seeding, there was an accumulation of areas with only one layer of cells. Therefore, the experiment was terminated on day 28 post seeding.

[0162] Fig. 12B shows a LIVE / DEAD stain, wherein the stain showed that the vast majority of cells were viable after 21 days of anaerobic culture.

[0163] Fig. 12C shows macroscopic observation of the cells also revealing a confluent layer of Caco-2 cells.

[0164] In short, the invention relates to a cell reactor CR device adapted to simulate anaerobic conditions in vitro, preferably simulation of anaerobic intestinal conditions in vitro. The cell reactor CR comprises a housing HOU, the housing HOU defining a vessel and wherein said vessel comprises a first and second compartment CO, CO' divided by a gas-permeable support SUP. The first compartment CO comprises an inlet IL and an outlet OL to provide a flow of a deoxygenized liquid MED within said first compartment, wherein the gas- permeable support SUP is selected from one of a) a porous membrane made from a semi-rigid polymer with track etched pores, such as polyethylene or polycarbonate; or b) a hydrophilic support comprising silicone and hydrogel, and wherein the housing HOU is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment, such as polycarbonate.

[0165] Although the present invention has been described in connection with the specified embodiments, it should not be construed as being in any way limited to the presented examples. The scope of the present invention is set out by the accompanying claim set. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Also, the mentioning of references such as "a" or "an" etc. should not be construed as excluding a plurality. The use of reference signs in the claims with respect to elements indicated in the figures shall also not be construed as limiting the scope of the invention. Furthermore, individual features mentioned in different claims, may possibly be advantageously combined, and the mentioning of these features in different claims does not exclude that a combination of features is not possible and advantageous.

[0166] The invention will now be described in further details in the following non-limiting examples, wherein the cell reactor according to the invention may be referenced as a flow chamber or dual flow chamber:

[0167] Example 1 - Materials and methods Fabrication of the dual flow chamber The dual flow chamber (DFC) was made by sticking two sticky-slide I Luer (Ibid i® ) on both sides of a 12 pm thick tissue culture treated transparent polyester (PET) membrane with a pore size of 0.45 pm and a porosity of 0.6% (it4ip, 2000M12 / 640N453). To prevent leakage from the flow chamber, an even amount of pressure was applied to the sticky-slides using screw clamps and plastic blocks with predrilled holes for the inlet and exit for 10-15 seconds, see Fig. 11A. Both sides of the DFC were filled with 70% ethanol and left for 20 minutes to sterilize followed by curing of the glue at 37°C overnight.

[0168] Anaerobization unit (AU)

[0169] To ensure a continuous supply of anoxic media, an ascorbate solution was made by adding 2 grams of sodium L-ascorbate to 100 ml_ of a 0.1 M NaOH solution. Inside the solution, a 150 cm long silicone tube (Helixmark®; mat.no: 456350287, inner diameter (ID): 0.99 mm, wall thickness (WT): 0.31 mm) was placed. The small size of the silicone tube was chosen to ensure complete deoxygenation of the media. To prevent reabsorption of oxygen into the media, the anoxic media was connected to the DFC and oxygen sensor using stainless steel pipes. Before connecting to the DFC, culture media was flushed through the anaerobization unit for at least one hour for the system to stabilize.

[0170] Cell culture

[0171] Caco-2 HTB-37™ cells (ATCC) were cultured in Dulbecco's Modified Eagles Media (DMEM; Gibco) supplemented with 20% heat-inactivated fetal bovine serum (HI FBS) and 1% penicillin-streptomycin (PS) (Stock: 10,000 units / mL Penicillin and 10,000 pg / mL Streptomycin). Cells were split when 60-90% confluent (twice- thrice weekly) and used for experiments in passage 32-54. All cultures were placed in a standard CO2 incubator.

[0172] Prior to cell seeding in the DFC and cell culture insert (Falcon; 353090), the apical side of the membrane was coated with Collagen Coating Solution (SAFC®; 125- 50) by filling the apical channel with the solution, incubating at 37°C for 30 minutes and washing thrice with phosphate buffered saline (PBS). Caco-2 cells were seeded in the apical channel at a density of l-2xl05cells / cm2and left to adhere for four hours before starting the flow at 15 pL / min (SS: 0.014 dynes / cm2). One day post seeding (DPS) the flow was set to 60 pL / min (SS: 0.057 dynes / cm2) and left at this flow rate until six DPS. On the 6thday the flow rate was set to 320 pL / min (SS: 0.3 dynes / cm2) with pulsating flow (one min flow followed by a nine-minute break) to imitate peristaltic motions. At 7 DPS, the medium was serum-reduced to 2% HI FBS and flow set to 320 pL / min (SS: 0.3 dynes / cm2) with continuous flow to maintain stable anaerobic conditions in the apical channel. For the static cultures, cell medium was changed every third day and changed to serum-reduced medium on day 7 post seeding.

[0173] Oxygen measurements

[0174] Oxygen measurements were performed using an O2 MicroOptode installed in a PEEK flow cell connected to a single channel O2 UniAmp (UniSense, Denmark). Measurements were made every 10-60 seconds by flowing media through the flow cell at a flow rate between 30-1440 pL / min. Measurements were logged using SensorTrace Suite Logger software (UniSense, Denmark). The sensor was calibrated using the manufacturers guidelines. For oxygen measurement with the DFC, the sensor was connected to the exit-site using stainless steel pipes to prevent diffusion of oxygen into the media.

[0175] The oxygen percentage at the inlet of the DFC depends on the length of the silicone coil and flow rate, thus these can be adapted to fit the desired oxygen concentration. For <0.5% oxygen at the inlet, the media must be in the silicone coil for at least two minutes. In Fig. 7A the relationship between minutes the coil is submerged in the ascorbate solution and oxygen percentage is illustrated. The length of the coil can then be adapted to fit the desired flow rate, by using the following formula :

[0176] Immunohistochemical staining

[0177] Before staining, cells were washed with Hank's balanced salt solution (HBSS; Gibco) thrice and fixated with a 10% neutral buffered formalin solution (Sigma- Aldrich) for 15 minutes. Formalin was removed and cells washed thrice with PBS. Cells were permeabilized with 0,1% Triton-X in PBS for 15 minutes, washed thrice with PBS and blocked with 5% bovine serum albumin (Sigma-Aldrich) in PBS for 60 minutes. Cells were stained at room temperature with a mixture of 2 pg / mL of Occludin Monoclonal Antibody, Alexa Fluor 488 (Invitrogen; OC-3F10) and 2 pg / mL MUC2 Antibody, Alexa Fluor 405 (Novus Biologicals; 944 / 152) in 1% BSA in PBS for 3 hours. Cells were washed with PBS and F-actin stained with 100 nM Acti-stain™ 555 Phalloidin (Cytoskeleton) for 30 minutes before washing thrice with PBS. The membranes were removed from the sticky slides using scalpels and assembled in silicone frames on microscopy slides, mounted with Fluorescence Mounting Medium (Dako) and sealed with a coverslip.

[0178] Mucus staining

[0179] The Alcian Blue / PAS staining kit (Artisan; AR16992-2) were used to visualize mucins in formalin fixated Caco-2 cells. For staining of neutral mucins, cells were first incubated with periodic acid for 2 minutes, followed by washing thrice with PBS. Cells were then incubated with Shift's reagent for 10 minutes and washed with PBS thrice to remove excess staining solution. For staining of acidic mucins, cells were incubated with Alcian Blue for 15 minutes, followed by washing one time with distilled water and trice with 3% acetic acid. Membranes were stored in PBS until imaging to prevent drying.

[0180] LIVE / DEAD stain

[0181] Viability of cells were visualized using LIVE / DEAD™ Viability / Cytotoxicity Kit (Invitrogen; L3224) by adding 2 pL Calcein-AM and 2 pL Ethidium homodimer-1 to 2 mL of HBSS before transferring it to the apical channel of the DFC. Cells were stained for 30 minutes before a 15-minute fixation step in formalin. The cells were washed thrice with HBSS before each step. The membranes were removed from the sticky slides using scalpels and assembled in silicone frames on microscopy slides, mounted with SlowFade™ Diamond Antifade Mountant with DAPI (Invitrogen; S36968) and sealed with a coverslip.

[0182] RNA isolation

[0183] To isolate RNA from Caco-2 cells grown in DFCs, culture medium was aspirated, and ice-cold lysis buffer (4 M guanidinium thiocyanate (GITC), 0.02 mM Tris-HCI (pH 7.5), 10 mM NaAcetate pH 4.5, 25 mM EDTA, 0.1 % Triton X-100, 2 mM DTT) was added. The cells were sheared from the DFC by rapidly moving a syringe, fitted into the Luer port of the apical channel outlet, up and down. The lysate was then transferred to RNase-free 1.5 mL tubes and snap-frozen in liquid nitrogen. RNA extraction was subsequently performed using a phenol-chloroform method. Briefly, 300 pL of lysate was mixed with 150 pL of solution 2 (10 mM Na-acetate pH 4.5, and 2% SDS), 700 pL of acidic phenol (pH 4.5) and 300 pL of chloroform. Tubes were inverted and heated at 80°C for 3-4 minutes, then cooled on ice. After centrifugation at 10,000 x g for 5 min, the aqueous phase was transferred to 96 % ethanol with Na-acetate (37.5 mM) and precipitated overnight (ON). RNA was pelleted by centrifugation (20,000 x g for 45 minutes), washed in ice-cold ethanol, resuspended in RNase-free H2O, and stored at -20°C or -80°C.

[0184] RNA-sequencing

[0185] RNA samples from Caco-2 cultures grown in DFCs were depleted of ribosomal RNA using the NEBNext rRNA Depletion Kit (Human / Mouse / Rat). RNA sequencing library preparation was performed with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) and paired-end sequencing was conducted on a NovaSeq 6000 System (Illumina). Raw paired-end reads were aligned to the human genome assembly GRCh38 / hg38 using STAR (version 2.7.11a). Primary alignments were filtered, sorted, and indexed using samtools (version 1.19). For feature counting, only protein-coding genes were considered. Read counts within exons were determined using featurecounts from the subread package (version 2.0.6), with multimapping reads included and counted fractionally. Differential gene expression analysis was conducted with edgeR (version 4.2.1), identifying genes with | log2(FC) | > 2 and FDR < 0.05 as significantly differentially expressed. Pathway enrichment on significant DEGs was conducted using the webtool MetaScape with Reactome gene sets18. For marker gene expression analysis, lineage marker genes from Burclaff et al.54were used. Normalized gene expression data (normalized transcript per million, nTPM) from the "RNA single cell type data" and "RNA consensus tissue gene data" datasets from The Human Protein Atlas were used for comparison55-56. Clustered heatmaps were generated with the R package pheatmap (version 1.0.12), using Ward's method ("ward.D2") for hierarchical clustering.

[0186] Colonization with Clostridioides difficile and Bacteroides fragilis

[0187] Caco-2 cells were matured aerobically for seven days in the DFC, as previously described. On the day of inoculation, the media was replaced with DMEM containing 2% heat-inactivated FBS (HI), and the anaerobization unit was connected to the inlet of the apical channel. Antibiotic-free media was flushed through both channels of the DFC for one hour before infection. Bacterial suspensions of C. difficile (ATCC 700057) and B. fragilis (ATCC 25285), grown anaerobically overnight on 5% blood agar plates, were adjusted to an OD600 of 0.2 in deoxygenated Hanks' Balanced Salt Solution (HBSS, Gibco). A mixture of B. fragilis and C. difficile was made from the solutions (OD ratio 1:9) and introduced through the apical channel at a flow rate of 320 pl / min until the suspension reached the top of the outlet reservoir. The CFU / mL in the inoculum was 5*106for C .difficile and l*108for B. fragilis. The flow was then stopped for 10 minutes to allow for initial bacterial attachment, after which the flow rate was resumed at 320 pl / min. Daily effluent samples were plated on selective agar plates (CHROMID® C. difficile agar plates and BHIS plates with 6 pg / mL vancomycin) to measure colony-forming units (CFU). On day 3, the medium in one of the DFCs was changed to DMEM with 2% HI FBS and 6 pg / mL vancomycin. On day 5, loose biofilm was aspirated, and the cell layers of the DFCs were harvested using Trypsin-EDTA (0.25%) with 0.1% Triton X-100, then plated for CFU measurements. Gram staining was performed on loose biofilm harvested from the apical channels. For microscopy (LIVE / DEAD or electron microscopy), the infected cell layers were not harvested but instead prepared according to the appropriate protocol.

[0188] Microscopy

[0189] Live cultures of Caco-2 cells were imaged using an Olympus CKX53 inverted microscope connected to an Olympus SC50 camera and using CellSens software. PAS-stained cultures were imaged using a Laica DM4 B microscope using LAS X software. Gram-stained cultures were imaged using a Laica DM3000 LED microscope connected to a Flexacam Cl and using LAS X software.

[0190] Immunohistochemical stained and LIVE / DEAD stained cultures were imaged using a Nikon AX or Olympus FV1000 confocal single- or multiphoton laser scanning microscope.

[0191] Electron microscopy

[0192] The Caco-2 cells were matured in flow chambers for seven days followed by inoculation with C. difficile and B. fragilis and treatment with vancomycin as described previously. The cell cultures were fixed using an 2% glutaraldehyde solution in 0.04 M phosphate buffer. The membranes with adhering cell cultures were removed from the flow chambers, washed with 0.1 M phosphate buffer, and stained with 1% osmium for 90 min. Following staining the membranes were washed in phosphate buffer and water, dehydrated in ethanol series and acetone, and infiltrated with TAAB 812 Embedding Resin (T030, TAAB).

[0193] Ultrathin (60 nm) sections were cut on a Leica Ultracut UCT microtome. The sections were collected on formvar support film copper grids (FF2010-CU-50, Electron Microscopy Sciences). The grids were stained with 3% uranyl acetate for 15 min. at 60°C and 3% lead citrate (Leica Ultrostain 2) for 6 min. at room temperature. The cells were photographed using JEM-1400 Plus electron microscope, equipped with Quemsa TEM CCD camera and Radius imaging software. Statistics

[0194] Statistical analyses were performed using R (version 4.3.2). Normality of the differences in paired oxygen concentration measurements was assessed using the Shapiro-Wilk test. Based on this, a paired t-test was conducted using the compare_means function from the ggpubr package (method = "t.test"). For the comparison of CFUs between treated and untreated DFCs, a non-parametric test (Wilcoxon rank-sum test) (method = "wilcox.test") was applied.

[0195] Example 2 - Establishment of the hypoxic intestinal lumen environment Aim of study

[0196] To establish a physiologically relevant model of the human intestine and to create a system that mimics the shear stress conditions and low oxygen environment present in the intestine, while also facilitating the growth of an intestinal cell layer.

[0197] Materials and methods

[0198] See also Example 1 for details.

[0199] We chose the dual flow channel principle for the model but contrary to gold standard PDMS soft lithography chips, we opted for hard-plastic systems that are considerably cheaper and can be used in ambient air or CO2 incubator, without the need for enclosure in an anaerobic container or additional mounting of glass coverslips.

[0200] A challenge with known systems is to ensure a fast and unlimited supply of anaerobic media to the upper compartment for a stable anaerobic environment. We aimed for a liquid shear stress (SS) in the upper channel of minimum 0.1 dynes / cm2and stable oxygen levels below 1% saturation both of which approaches the conditions in the intestine. Rather than relying on time-consuming passive diffusion of oxygen from the media into a surrounding anaerobic environment, that requires the whole system to be enclosed in an anaerobic container, we designed a device for fast online anaerobization of media before it enters the upper channel. This device exploits the fast diffusion of oxygen through silicone rubber and contains submerged ultra-thin silicone tube coiled within a container filled with a strong aqueous antioxidant solution. Passing liquid media through this system results in a complete depletion of oxygen in the cell media within a few minutes. The dual flow chamber (DFC) was made from two commercially available flow chambers from Ibidi® (sticky slides) mounted back- to-back with a thin, porous polyester membrane in between, see Fig. 6. This created apical and basolateral flow channels in the DFC where the outer walls provide an effective oxygen barrier whereas oxygen readily diffuses over the culture membrane from the lower channel to the intestinal cell culture. To create a hypoxic environment in the apical channel, anaerobic media from the anaerobization unit (AU) passes directly to the inlet of the apical channel through stainless steel tubing to prevent reabsorption of oxygen into the cell media. Oxygen is supplied to the cells via the basolateral channel using silicone tubing passing through atmospheric air with 5% CO2 in a standard cell incubator. To test that the system met the desired oxygen concentrations below 1% saturation, an optical oxygen sensor was used (O2 MicroOptode, Unisense Denmark). The sensor was connected to the exit site of the AU to test the efficacy of the anaerobization process, and to the exit site of the apical channel to monitor any reabsorption of oxygen into the flow chamber during passage, see Fig. 7A.

[0201] Results and conclusion

[0202] We measured highly efficient oxygen depletion of the media passing through the AU which allowed the high flow rates necessary for simulating a physiological liquid shear in the chip. The oxygen depletion efficacy depended on several factors such as wall thickness of the silicone coil, lumen diameter of the silicone coil, length of the silicone coil, and flow rate of the cell media, see Fig. 7.

[0203] Example 3 - Optimization of tubing size Aim of study

[0204] To optimize the tubings for optimal deoxygenation.

[0205] Materials and methods

[0206] Silicone tubing, spooled into coil and inserted into anaerobic container, container evacuated by applying negative pressure followed by filling with nitrogen gas. i. ID 1.00mm, WT 1.00mm ii. ID 0.64mm, WT 0.28mm iii. ID 0.76mm, WT 0.45mm iv. ID 0.99mm, WT 0.31mm

[0207] Lengths, starting from 7 meters were tested. Results

[0208] Targeting an oxygen percentage of <0.4% at inlet of cell reactor / flow cell, tube i needs to be 7 metres whereas tubing iv is as effective at 3 metres, thus internal diameter of 0.99 mm and wall thickness of 0.31 mm was used.

[0209] Conclusion

[0210] After testing various silicone tubing sizes, a lumen diameter of 0.99 mm and a wall thickness of 0.31 mm was selected for the AU.

[0211] Example 4 - Optimization of flow rate and coil length

[0212] Aim of study

[0213] To optimize the flow rate and coil length for optimal deoxygenation.

[0214] To test if the anaerobic container with nitrogen gas could be replaced with an antioxidant solution.

[0215] Materials and methods

[0216] Tube iv was tested, but wherein the container void of oxygen was replaced with a strong aqueous antioxidant solution consisting of 0.1 M vitamin C (ascorbate) in 0.1 M NaOH. Deoxygenation was tested at flow rates 60-800 pL / min, with coillengths 50-300 cm.

[0217] Results

[0218] Using tubing of type iv and coil-lengths of >150 cm resulted in oxygen levels below 1 % at all tested flow rates. Coil-length of 100 cm resulted in oxygen levels below 1 % at flow rates 60-640 pL / min, whereas coil-length of 50 cm only achieved oxygen levels below 1 % at low flow rates < 180 pL / min.

[0219] Conclusion

[0220] A strong antioxidant solution is an efficient replacement to the anaerobic container. For sufficient deoxygenation (<0.5 %) at flow rates between 120-640 pl / min (corresponding to physiological liquid shear stress of 0.1-0.6 dyne / cm2in the apical channel), a coil length of at least 150 cm is required, see Fig. 7B. Example 5 - Real-time oxygen levels in flow cell during experiments Aim of study

[0221] To test the performance of the flow chambers to maintain anaerobic conditions during experiments with and without cells.

[0222] Materials and methods

[0223] Tubing of type iv with a length of 150cm placed in the antioxidant vitamin C solution was used to generate oxygen depleted media. A single-channel flow chamber from Ibidi® (matching the DFC's apical channel dimensions) was used to test the diffusion of oxygen through the walls of the flow chambers without the disturbance of oxygen-consuming cells. The oxygen concentration in the presence of a confluent layer of Caco-2 cells were tested using the DFC.

[0224] Results

[0225] To test the reabsorption of oxygen during the media's passage through the apical channel, a single-channel flow chamber from Ibidi® (matching the DFC's apical channel dimensions) was used, showing minimal oxygen variation and the lowest O2 levels at a flow rate of 320 pl / min (shear stress of 0.3 dyne / cm2), see Fig. 7C. Oxygen levels in the DFC with a confluent Caco-2 cell layer (matured aerobically for 7 days) were monitored, confirming persistent anaerobic conditions during a six-day experiment, with oxygen saturation consistently ranging from 0.1% to 1%, see Fig. 7D. During flow interruptions, oxygen concentration measurements revealed that flow could be paused for up to 60 seconds before oxygen levels at the exit site exceeded 1%, and up to 5 minutes before surpassing 3%, see Fig. 7E. Oxygen levels decreased to < 1% within a few minutes after resuming the flow.

[0226] Conclusion

[0227] In conclusion, the DFC system effectively maintains anaerobic conditions (< 1% oxygen) for at least 6 days. Flow pauses of up to 5 minutes cause only a temporary, small increase in oxygen levels, which quickly return to baseline upon resumption of flow, allowing for a variety of experimental interventions to be carried out without disturbing the anaerobic environment. Example 6 - Caco-2 cells matured in the DFC demonstrates in vivo characteristics of the intestine.

[0228] Aim of study

[0229] After confirming that the model met the requirements to SS and oxygen levels, the characteristics of the cell layer cultured in the DFC under both aerobic and anaerobic conditions were investigated and compared to static culture in cell culture inserts. Previous studies have demonstrated that Caco-2 cells cultivated under flow conditions matures into 3D cell layers, that includes the formation of crypt- and villus-like structures, mucus production, and enhanced barrier function. Materials and methods See example 1.

[0230] Results

[0231] Caco-2 cells cultured for 13 days in the DFC, formed a complex layer with cryptand villus-like structures, reaching approximately 135 pm in height, see Fig. 8. Under anaerobic conditions in the apical channel, a greater variation in the height of the cell layer ranging between 50-115 pm was observed, see Fig. 8B. By comparison, the average height of the cells cultured statically for 13 days was approximately 20 pm, see Fig. 8B. Under both aerobic and anaerobic flow, the mature Caco-2 layer stained positive for both the tight junction protein occludin and the cytoskeleton component F-actin, indicating establishment of an intestinal barrier, see Fig. 8A. To assess mucus production by the Caco-2 cells, they were stained for both neutral and acidic mucins, as well as immunostained for MUC2. Both aerobic and anaerobic flow conditions revealed a higher signal of neutral mucins compared to the static control, see Fig. 8C. Due to the thickness of the cell layer, it was difficult to make firm conclusions on the presence of acidic mucins, but some blue stains did appear under all three culture conditions, see Fig. 8C. The presence of MUC2 by immunostaining was detected in the Caco-2 cells cultured under aerobic and anaerobic conditions in the DFC. MUC2 appeared mostly in vesicles within some of the cells in the top of the cultured epithelium. The signal was low, indicating that only small levels of MUC2 was produced by the cells. The presence of MUC2 was subsequently examined by ELISA and found to be below the lower detection limit (0.78ng / mL, data not shown).

[0232] Conclusion Caco-2 cells cultured in the DFC with anaerobic conditions in the apical chamber differentiate to form 3D structures with tight cell junctions and mucin production.

[0233] Example 7 - Transcriptomic profiling

[0234] Aim of study

[0235] To comprehensively investigate the transcriptional profile of the intestinal epithelium under flow culture conditions, transcriptomic analyses on Caco-2 cells cultured for 13 days under both aerobic and anaerobic conditions in the DFC model, and statically under aerobic conditions in cell culture inserts were conducted.

[0236] Materials and methods

[0237] See also example 1

[0238] Differentially expressed genes (DEGs) were identified and analyzed by pathway enrichment analysis using Metascape. Additionally, the expression of marker genes for intestinal cell types was compared to publicly available single-cell and bulk RNA-sequencing datasets from human intestinal cells and tissues, with the results visualized in a clustered heatmap.

[0239] Results

[0240] Among the 817 upregulated genes in the aerobic DFC model relative to the static model, several were associated with the 'HDACs deacetylate histones' Reactome pathway, including multiple histone genes, see Figs 9A and 9B. Upregulation of histone genes, a marker of entry into the S phase of the cell cycle, may indicate an increase in the number of actively dividing cells in the aerobic flow chamber models, aligning with the observed increase in 3D growth compared to the static model. Additionally, pathways related to small molecule transport (R-HSA-382551 and R-HSA-425407) and lipid metabolism (R-HSA-556833) were enriched, pointing to enhanced absorptive function of the enterocytes. Conversely, among the 1,182 downregulated genes, pathways related to cilia development and function (R-HSA-5617833 and R-HSA-5620912) were significantly enriched. Cilia, membrane-bound sensory organelles that detect signals such as shear stress, are typically present in quiescent cells and are generally absent in rapidly renewing tissues, including the intestinal epithelium20. By contrast, only 174 genes were differentially expressed between the anaerobic and aerobic DFC models. The minor differences in transcriptional activity when removing oxygen above the cultured epithelium suggests that the system adequately provides oxygen to the Caco-2 cells in the anaerobic model for sustaining a normal, vital state of growth, see Fig. 9A. The upregulation of several metallothionein-encoding genes (R-HSA-5661231), which are involved in metal homeostasis and protection against oxidative stress, alongside the downregulation of genes related to the electron transport chain (R-HSA-611105), suggests a hypoxic response, as expected. Additionally, several pathways related to mitosis (R-HSA-3214815, R-HSA-2467813, R-HSA-156711, and R-HSA-174178) were also enriched among the downregulated genes, indicating a decrease in cell proliferation compared to the aerobic DFC model, see Fig. 9B.

[0241] Although derived from the colon, Caco-2 monolayers exert many of the same properties as absorptive enterocytes of the small intestine. To determine whether DFC conditions promote maturation toward a more differentiated intestinal epithelium, their transcriptomic profiles were compared to various intestinal cell types and tissues. To determine the cell type resemblance, we analyzed marker gene expression for six major intestinal cell types: goblet cells, Paneth cells, enteroendocrine cells (EEC), intestinal stem cells (ISC), absorptive enterocytes (AE), and absorptive colonocytes (AC). We compared our data with single-cell RNA-sequencing and bulk RNA-sequencing datasets, as well as a recently described Caco-2-based anaerobic 3D gut model22. The clustered heatmap, see Fig. 9C, reveals that all the Caco-2 models most closely resemble (proximal) enterocytes of the small intestine, as reported in the literature21-23. Notably, the anaerobic DFC model exhibited higher expression of AE-specific markers APOA4, ALPI, MTTP, RBP2, MAF, and LCT) compared to the static model, indicating a stronger AE profile. Additionally, our anaerobic DFC model closely resembles the Caco-2-based anaerobic 3D gut model described by Cheng et al., demonstrating that microfluidic-based Caco-2 models share similar molecular characteristics.

[0242] Conclusion

[0243] Transcriptomic profiling of DFC-cultured Caco-2 cell layers reveals characteristics that resemble enterocytes of the small intestine, with increased expression of several enterocyte marker genes compared to statically cultured Caco-2 cells, indicating a more mature and differentiated cell phenotype. Example 8 - Co-culture of obligate anaerobes and Caco-2 cells in the DFC Aim of study

[0244] To test if the model supported the growth of obligate anaerobes, it was used to simulate colonization with the obligate anaerobic bacterial species C. difficile. The obligate anaerobe B. fragilis, a prominent member of the normal microbiota, was included to represent the commensal population.

[0245] Materials and methods

[0246] See also example 1.

[0247] The DFCs were inoculated with a mix of C. difficile and B. fragilis suspensions at a 600 nm optical density (OD600) ratio of 9: 1, a proportion found to be optimal for co-colonization (own observations). Bacterial colonization was monitored both macro- and microscopically and by culturing the effluent from the apical channel daily.

[0248] Results

[0249] Three days post inoculation, areas resembling biofilm was visible by the naked eye, and the bacterial counts in the effluent had reached >107CFU / ml for B. fragilis and >105CFU / ml for C. difficile, demonstrating that the system provides favorable growth conditions for these obligate anaerobic microorganisms, see Fig. 10A. Following bacterial colonization, the viability of the intestinal epithelium was examined by confocal laser scanning microscopy (CLSM).

[0250] Conclusion

[0251] This analysis showed that the anaerobic DFC model supported the growth of obligate anaerobic species, including C. difficile (non-toxigenic) and B. fragilis, and that Caco-2 cell layers remained viable, see Fig. 10D.

[0252] Example 9 - Vancomycin treatment of the C. difficile colonized epithelium Aim of study

[0253] To assess the DFC model's applicability for testing antimicrobial treatment regimens, a vancomycin treatment experiment was conducted. Vancomycin, a common antibiotic for C. difficile infections, is effective against C. difficile, while B. fragilis tolerates the drug. Materials and methods

[0254] See also example 1

[0255] Treatment with vancomycin (6 ug / ml) was initiated three days post infection (DPI).

[0256] Results

[0257] After two days of treatment, the CFU count of C. difficile in the effluent had reduced almost 1000-fold, see Fig. 10A, but some bacteria were still present in the biofilm clumps harvested from the chamber, see Fig. 10E. Interestingly, a 10- fold reduction in the amount of B. fragilis was observed in the effluent, compared to the untreated control, see Fig. 10A. However, when harvesting the cell layer to enumerate the sessile population, the same amount of B. fragilis was present in the vancomycin treated DFC, compared to the untreated control, see Fig. 10B.

[0258] The amount of C. difficile associated with the cell layer was reduced approximately 25-fold following vancomycin treatment, compared to the control, see Fig. 10B. Despite this, high numbers of C. difficile was still present in the vancomycin- treated DFC (>10,000 CFU / cm2). This may be attributed to C. difficile being shielded in dense B. fragilis biofilm aggregates, as observed in Gram stains of supernatant samples collected from the apical channel of the vancomycin-treated DFC, see Fig. 10E. The oxygen concentration was measured before and after the five-day experiment, showing a mean decrease in oxygen saturation from 0.6 to 0.2 %, see Fig. 10C, indicating that the integrity of the cell layer is not compromised by the colonizing bacteria.

[0259] Conclusion

[0260] In conclusion, the DFC model effectively simulates C. difficile infection and response to vancomycin, highlighting the persistence of C. difficile in biofilms despite treatment and the model's suitability for antimicrobial studies.

[0261] Example 10 - Invasion of the intestinal epithelium

[0262] Aim of study

[0263] To investigate the colonization patterns and potential invasion of the intestinal epithelial cells by the bacteria, CLSM and electron microscopy (EM) was performed on the colonized epithelium. Materials and methods

[0264] See example 1

[0265] Results

[0266] In the untreated control, colonies of bacteria were found in the crypts, see Fig.

[0267] IIA, indicated by arrows, and both extra- and intracellular bacteria were identified by EM, see Fig. 10F, indicated by arrows. In the vancomycin treated bacterial co-culture, only a few small colonies were observed in the crypts, but numerous intracellular bacteria were observed by EM, see Fig. 11A and 11B. The extracellular colonies contained what appeared to be lysed bacteria, see Fig. 11B, indicated by dark arrows. Additionally, EM also revealed a consistent layer of microvilli on the lumen of the Caco-2 cell layer at 5 days post infection, see Fig.

[0268] IIB, indicated by light arrows).

[0269] Conclusion

[0270] The obligate anaerobic bacteria were found to effectively colonize the intestinal epithelium, with bacteria found both extra- and intracellular. The bacteria might evade vancomycin treatment by hiding intracellular.

[0271] Example 11 - Longevity of model according to the present invention

[0272] Purpose:

[0273] To observe how long Caco-2 cells can be cultured in the double flow chamber while maintaining anaerobic conditions on the apical side of the cells and aerobic conditions in the basolateral side.

[0274] Methods:

[0275] Caco-2 cells were seeded at a density of 3xl06cells / mL and grown aerobically for seven days before changing to anaerobic conditions in the apical chamber. The following flow was applied: 15 pl / min on day one, 60 pl / min on day 2-5, 320 pl / min (1 min flow and 4 min. pause) for day 6-7. After connecting the anaerobization unit on day 7, the flow was set to 320 pl / min continuously. After 21 days of culture with anaerobic conditions, cells were stained with LIVE / DEAD® Viability / Cytotoxicity Kit for Mammalian Cells (Invitrogen) and imaged using brightfield (BF) and fluorescence microscopy. A 3x0.5cm piece of the membrane with adhering cells was scanned using CellSens Dimension and a motorized stage. Results:

[0276] Caco-2 cell layers remained confluent for three weeks, while maintaining anaerobic conditions <0.5% oxygen. Observed by brightfield microscopy, there was an accumulation of areas with only one layer of cells between day 19 and 28 post seeding, see Fig. 12A. Therefore, the experiment was terminated on day 28 post seeding. The LIVE / DEAD stain showed that the vast majority of cells were viable after 21 days of anaerobic culture, see Fig. 12B. Macroscopic observation of the cells also revealed a confluent layer of Caco-2 cells, see Fig. 12C.

[0277] Example 12 - a comparison of membranes

[0278] Below table 1 can be seen, comparing different porous polycarbonate and polyester membranes. As can be seen, the membrane in column two has the most advantages. The following is a list of itemized embodiments, according to the invention:

[0279] 1.1. A cell reactor device adapted to simulate anaerobic conditions in vitro, preferably simulation of anaerobic intestinal conditions in vitro, the device comprising:

[0280] -a housing, the housing defining a vessel, the vessel comprising a first and second compartment divided by a gas-permeable support, the first compartment comprising an inlet and an outlet to provide a flow of a deoxygenized liquid within said first compartment, wherein the gas- permeable support is selected from one of:

[0281] -a porous membrane made from a semi-rigid polymer with track etched pores, such as polyethylene or polycarbonate; or

[0282] -a hydrophilic support comprising silicone and hydrogel, and wherein the housing is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment.

[0283] 1.2. The device according to Item 1, wherein the pores of the porous membrane are substantially perpendicular to a surface plane of said porous membrane.

[0284] 1.3. The device according to Item 1 or 2 wherein the porous membrane is substantially transparent.

[0285] 1.4. The device according to any of Items 1 to 3, wherein the porous membrane has a thickness of between 10 and 50 pm.

[0286] 1.5. The device according to Item 1, wherein the hydrophilic support has a thickness of between 0.5 and 3 mm.

[0287] 1.6. The device according to Item 1 or 5, wherein the hydrophilic support is a chemical blend or a co-polymer or an interpenetrating polymer network.

[0288] 1.7. The device according to any of Items 1 or 5 to 6, wherein the hydrophilic support comprises between 10 and 40% hydrogel. 1.8. The device according to any of the preceding Items, wherein the inlet and outlet comprises connectors, said connectors adapted to provide a fluidic connection with associated conduits, said connectors being non-permeable or substantially non-permeable to at least oxygen from a surrounding atmospheric environment.

[0289] 1.9. The device according to any of the preceding Items, wherein the second compartment is enclosed and sealed within walls of the housing and the gas- permeable support respectively, preferably wherein oxygen is provided within the second compartment.

[0290] 1.10. The device according to any of Items 1 to 8, wherein the second compartment has an opening to an outside environment and / or atmospheric air, the gas-permeable support providing a gas-permeable sealing between the first compartment and said opening.

[0291] 1.11. The device according to any of Items 1 to 8, wherein the second compartment comprises an inlet and an outlet adapted to provide a flow of a fluid through said second compartment.

[0292] 1.12. The device according to any of the preceding Items, wherein at least a portion of the housing is transparent.

[0293] 1.13. A system adapted to simulate anaerobic conditions in vitro, preferably anaerobic intestinal conditions, the system comprising:

[0294] -the cell reactor device according to any of Items 1 to 12,

[0295] -a container comprising a nutrient liquid,

[0296] -a deoxygenation device adapted to remove or reduce any oxygen from the nutrient liquid,

[0297] -a pump, the pump fluid ically connected to at least the container and the first compartment, the pump adapted to create a flow of the nutrient liquid through the first compartment. 1.14. The system according to Item 13 further comprising a gas permeable conduit between the container and the inlet, said gas permeable conduit being arranged at or within the deoxygenation device.

[0298] 1.15. The system according to Item 13 or 14, wherein the deoxygenation device comprises a fluid with an affinity for oxygen.

[0299] 1.16. The system according to any of Items 13 to 15 wherein the pump is adapted to adjust the flow velocity of nutrient liquid through the first compartment.

[0300] 1.17. A method of simulating anaerobic conditions in vitro, preferably simulating anaerobic intestinal conditions in vitro, the method comprising:

[0301] -providing the cell reactor device according to any of Items 1 to 12, -seeding the gas-permeable support, on a surface of the gas-permeable support facing the first compartment, with one or more cells, preferably intestinal epithelial cells,

[0302] -providing a flow of nutrient fluid through the first compartment, wherein any oxygen from the nutrient fluid has been extracted prior to flowing through said first compartment, and

[0303] -growing a cell layer on the gas-permeable support, said layer being exposed to an anaerobic environment within the first compartment.

[0304] 1.18. The method according to Item 17, the method further comprising:

[0305] -providing at least oxygen to the cell layer from the second compartment through the gas-permeable support.

[0306] 1.19. A kit of parts for extracting oxygen from a liquid to be provided to a compartment of a cell reactor, said cell reactor adapted to simulate an in vitro environment of a biological process, the kit of parts comprising :

[0307] -a cell reactor according to any of Items 1 to 12,

[0308] -a conduit having a first end and a second end, the conduit defining an inner lumen to contain, and transport said liquid to the compartment of the cell reactor, the conduit further comprising :

[0309] -a gas permeable section, -a second section being non-permeable to gas, the second section extending between the gas permeable section of the conduit and the second end of the conduit,

[0310] -a connector at the second end, the connector adapted to fluidica lly connect to the cell reactor,

[0311] -means for extracting oxygen from the liquid within the gas permeable section of the conduit, the means selected from one of:

[0312] -a fluid adapted to quench oxygen from the liquid by submerging at least the gas permeable section of the conduit in said fluid, or

[0313] -a sealable container with a valve, the container adapted to contain at least the gas permeable section of the conduit and wherein at least oxygen is removed from within said container through the valve, or

[0314] -the conduit comprises a second lumen outside the inner lumen and wherein the second lumen is one of:

[0315] -void of oxygen, or

[0316] -filled with a fluid adapted to quench or catch oxygen.

[0317] 1.20. The kit of parts according to Item 19, further comprising a pump adapted to provide flow to the liquid to the cell reactor from the conduit.

[0318] 1.21. The kit of parts according to any of Items 19 or 20, wherein the fluid comprises a molecule adapted to absorb oxygen, preferably the fluid is c-vitamin ascorbate.

Claims

CLAIMS1. A cell reactor (CR) device adapted to simulate anaerobic conditions in vitro, preferably simulation of anaerobic intestinal conditions in vitro, the device comprising:-a housing (HOU), the housing defining a vessel, the vessel comprising a first and second compartment (CO, CO') divided by a gas-permeable support (SUP), the first compartment (CO) comprising an inlet (IL) and an outlet (OL) to provide a flow of a deoxygenized liquid (MED) within said first compartment, wherein the gas-permeable support is selected from one of:-a porous membrane made from a semi-rigid polymer with track etched pores, such as polyethylene or polycarbonate; or-a hydrophilic support comprising silicone and hydrogel, and wherein the housing is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment.

2. The device according to claim 1, wherein the pores of the porous membrane are substantially perpendicular to a surface plane of said porous membrane.

3. The device according to claim 1 or 2 wherein the porous membrane is substantially transparent.

4. The device according to claim 1, 2 or 3, wherein the porous membrane has a thickness of between 10 and 50 pm.

5. The device according to claim 1, wherein the hydrophilic support has a thickness of between 0.5 and 3 mm.

6. The device according to claim 1 or 5, wherein the hydrophilic support is a chemical blend or a co-polymer or an interpenetrating polymer network.

7. The device according to any of claims 1 or 5 to 6, wherein the hydrophilic support comprises between 10 and 40% hydrogel.

8. The device according to any of the preceding claims, wherein the inlet and outlet comprises connectors, said connectors adapted to provide a fluidic connection with associated conduits, said connectors being non-permeable or substantially non-permeable to at least oxygen from a surrounding atmospheric environment.

9. The device according to any of the preceding claims, wherein the second compartment (CO') is enclosed and sealed within walls of the housing and the gas-permeable support respectively, preferably wherein oxygen is provided within the second compartment.

10. The device according to any of claims 1 to 8, wherein the second compartment has an opening to an outside environment and / or atmospheric air, the gas- permeable support providing a gas-permeable sealing between the first compartment and said opening.

11. The device according to any of claims 1 to 8, wherein the second compartment comprises an inlet and an outlet adapted to provide a flow of a fluid (MED') through said second compartment.

12. The device according to any of the preceding claims, wherein at least a portion of the housing is transparent.

13. The device according to any of claims 1 to 4 or 8 to 12, wherein the porous membrane is made from polyester.

14. A system adapted to simulate anaerobic conditions in vitro, preferably anaerobic intestinal conditions, the system comprising:-the cell reactor (CR) device according to any of claims 1 to 13, -a container comprising a nutrient liquid (MED),-a deoxygenation device (DOD) adapted to remove or reduce any oxygen from the nutrient liquid,-a pump (FG), the pump fluidically connected to at least the container and the first compartment (CO), the pump adapted to create a flow of the nutrient liquid through the first compartment.

15. The system according to claim 14 further comprising a gas permeable conduit between the container and the inlet, said gas permeable conduit being arranged at or within the deoxygenation device.

16. The system according to claim 14 or 15, wherein the deoxygenation device comprises a fluid with an affinity for oxygen.

17. The system according to any of claims 14 to 16 wherein the pump is adapted to adjust the flow velocity of nutrient liquid through the first compartment.

18. A method of simulating anaerobic conditions in vitro, preferably simulating anaerobic intestinal conditions in vitro, the method comprising:-providing the cell reactor (CR) device according to any of claims 1 to 13, -seeding the gas-permeable support (SUP), on a surface of the gas- permeable support facing the first compartment (CO), with one or more cells, preferably intestinal epithelial cells,-providing a flow of nutrient fluid (MED) through the first compartment, wherein any oxygen from the nutrient fluid has been extracted prior to flowing through said first compartment, and-growing a cell layer (CL) on the gas-permeable support, said layer being exposed to an anaerobic environment within the first compartment.

19. The method according to claim 18, the method further comprising:-providing at least oxygen to the cell layer from the second compartment through the gas-permeable support.

20. The method according to any of claims 18 or 19 further comprising:-adding a drug or compound to the first compartment and subsequently determining the effect of said drug or compound on an environment of said first compartment.

21. The method according to claim 20, wherein a microorganism, such as a pathogenic microorganism, is added to the first compartment before the drugor compound is applied and the effect of the drug or compound or the microorganism is determined.

22. The method according to claim 21 wherein the microorganism is a bacterium a parasite, a bacteriophage, a fungus or a virus.

23. A kit of parts for extracting oxygen from a liquid to be provided to a compartment of a cell reactor, said cell reactor adapted to simulate an in vitro environment of a biological process, the kit of parts comprising :-a cell reactor according to any of claims 1 to 13,-a conduit having a first end and a second end, the conduit defining an inner lumen to contain, and transport said liquid to the compartment of the cell reactor, the conduit further comprising:-a gas permeable section,-a second section being non-permeable to gas, the second section extending between the gas permeable section of the conduit and the second end of the conduit,-a connector at the second end, the connector adapted to fluidica lly connect to the cell reactor,-means for extracting oxygen from the liquid within the gas permeable section of the conduit, the means selected from one of:-a fluid adapted to quench oxygen from the liquid by submerging at least the gas permeable section of the conduit in said fluid, or-a sealable container with a valve, the container adapted to contain at least the gas permeable section of the conduit and wherein at least oxygen is removed from within said container through the valve, or-the conduit comprises a second lumen outside the inner lumen and wherein the second lumen is one of:-void of oxygen, or-filled with a fluid adapted to quench or catch oxygen.

24. The kit of parts according to claim 23, further comprising a pump adapted to provide flow to the liquid to the cell reactor from the conduit.

25. The kit of parts according to claim 23 or 24, wherein the fluid comprises a molecule adapted to absorb oxygen, preferably the fluid is c-vitamin ascorbate.

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