A deoxygenation system

Abstract

The present invention relates to a deoxygenation system (DOS) adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising a conduit (CON) comprising an inner lumen (IN_LU), a first end and a second end, the conduit further comprising a gas permeable wall (GAS_P), the inner lumen adapted to contain and transport the liquid from said first end to said second end. The system is adapted for fluidic connection to an associated flow cell (FC) comprising at least one compartment (CO) with a membrane (MEM) and an inlet (IL) and an outlet (OL), the inlet fluidically connected to the second end of the conduit, wherein the gas permeable wall is enveloped in a fluid (FLU). The fluid may be selected from a fluid a)substantially void of oxygen, or b) having an affinity for oxygen higher than that of the liquid, thus enabling the system to move oxygen from the liquid to the fluid to, deoxygenize said liquid.

Description

[0001] A DEOXYGENATION SYSTEM

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a system, device and method for the deoxygenation of a liquid provided to a cell reactor in simulation of biological processes.

[0004] BACKGROUND OF THE INVENTION

[0005] Within the field of in-vitro simulation of in-vivo interaction between e.g. bacteria and animal or human cells, growth medium etc. it is important to simulate as close to real-life (in-vivo) conditions as possible.

[0006] As an example, to simulate in-vivo anaerobic conditions and processes, it is important to provide an anaerobic in-vitro device or system to simulate said in- vivo conditions or processes.

[0007] In the gut, microorganisms are present, which require anaerobic conditions. To better understand such microorganisms and their interaction with the host, an anaerobic environment must be provided.

[0008] Culturing intestinal microorganisms anaerobically on intestinal epithelium is primarily done in gas permeable flow cells placed inside anaerobic containers. The anaerobic container maintains an anaerobic background environment for the flow cell and the anaerobic media flowing over the bacteria / intestinal epithelium culture in the flow cell. This background environment maintains equilibrium anaerobic conditions in the flow cell. The oxygen-requiring intestinal epithelium culture inside this system is maintained by flowing oxygen-rich media through separate tubing from outside of the anaerobic container to the intestinal epithelium inside the anaerobic container.

[0009] This basic principle has drawbacks: 1) the anaerobic background environment must be maintained for the entire setup throughout experiments; 2) physical access to the setup breaches the anaerobic environment; 3) only very low flow rates of the anaerobic media can be used over the bacteria / epithelium culture due to limitations to the amount of accessible anaerobic media. This is due to 1) size constraints inside the anaerobic container where the anaerobic media is placed and kept anaerobic; 3) replacement of the media is infeasible as it breaks the anaerobic barrier of the anaerobic container; 3) anaerobization of the media occurs by passive diffusion of oxygen out of the media while placed in the anaerobic container. This is a very slow process only feasible with small volumes of media, which is incompatible with higher flow rates. Further, known systems create a background anaerobic environment for a gas-permeable flow cell, where epithelial cells are provided with oxygen from a source outside this environment, i.e. wherein the flow cell needs to be contained within an anaerobic environment.

[0010] Additionally, such systems can only use very slow, non-physiological flow rates of the anaerobic media as explained above, thus oxygen seeping from the aerobic compartment into the anaerobic compartment becomes an even bigger problem as the anaerobic media does not flow fast enough to clear or void the oxygen diffusing from the aerobic compartment.

[0011] Overall this compromises the stability of the system and its practical use as the flow cell is not directly accessible for microscopy inspection or for sample harvest without breaking the anaerobic barrier. Moreover, media flow over the culture at rates simulating what is present in the intestine is not feasible due to lack of a direct supply of adequate amounts of anaerobic media.

[0012] Current systems for simulating e.g. flow of a medium over a cell layer, are primarily concerned with e.g. simulating peristaltic flow, due to flow change during peristaltic muscle movement. Even when attempting to simulate anaerobic conditions, usually oxygen seeps in, destroying or skewing the results of the simulation in a matter of hours.

[0013] Hence, a system, device and method of simulating anaerobic conditions in-vitro is proposed. In particular, a more reliable device for continued or prolonged simulation would be advantageous for the study of e.g. anaerobic microorganisms. OBJECT OF THE INVENTION

[0014] It is a further object of the present invention to provide an alternative to the prior art.

[0015] In particular, it may be seen as an object of the present invention to provide a deoxygenation system and method that solves the above mentioned problems of the prior art with prolonged anaerobic in-vivo simulation of cells, cell-layers, bacteria and their environment.

[0016] SUMMARY OF THE INVENTION

[0017] Thus, the above described object and several other objects are intended to be obtained in a first aspect of the invention by providing a deoxygenation system adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising:

[0018] -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, -a flow cell comprising two compartments separated by a membrane or support structure, wherein at least one of the two compartments comprises an inlet and an outlet, the inlet fluid ically connected to the second end of the conduit, wherein the gas permeable wall is enveloped in a fluid, the fluid being : -substantially void of oxygen, or

[0019] -having an affinity for oxygen higher than that of the liquid, the system enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid.

[0020] In an alternative embodiment, the invention is a deoxygenation system adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising:

[0021] -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 -having an affinity for oxygen higher than that of the liquid, the system enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid, and wherein the system is adapted to be connected to an associated flow cell, to provide deoxygenized liquid, such as cell culture medium, to said flow cell.

[0022] In another alternative embodiment, the invention is a deoxygenation system adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising:

[0023] -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:

[0024] -substantially void of oxygen, or

[0025] -having an affinity for oxygen higher than that of the liquid, the system enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid, and wherein the system is adapted to be connected to an associated flow cell, to provide deoxygenized liquid, such as cell culture medium, to said flow cell.

[0026] In yet an alternative embodiment, the invention is a deoxygenation system adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising:

[0027] -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:

[0028] -substantially void of oxygen, or

[0029] -further containing a fluid with an affinity for oxygen higher than that of the liquid, the system enabling oxygen to move from the liquid to the sealed container to deoxygenize said liquid, and wherein the system is adapted to be connected to an associated flow cell, to provide deoxygenized liquid, such as cell culture medium, to said flow cell.

[0030] In an alternative embodiment of the invention, the system may be used for other purposes than simulation of anaerobic in vitro environments, such as anaerobic systems adapted for providing anaerobic media to liquid (flask) cultures of anaerobic microorganisms or for anaerobizing fluid for anaerobic cylinders with agar plate cultures of anaerobic bacteria.

[0031] Other applications outside microbiology may be relevant, such as where one needs to deplete a fluid from oxygen in a fast or compact manner, such as in chemistry, when handling oxygen-sensitive chemicals or materials. Further, the food industry may have processes where the present invention may be advantageous for sustaining an anaerobic environment.

[0032] 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.

[0033] 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.

[0034] It is further to be understood, that the present invention provides a system which is enabled to push an anaerobized environment into a flow cell, whereas known systems prevent oxygen from seeping into the flow cell, i.e. by providing a continuous positive pressure of anaerobized media within the anaerobic compartment of the flow cell.

[0035] 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. 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.

[0036] In the context of the present invention, deoxygenation is to be understood as the removal, subtraction or extraction of oxygen. In particular, the invention is suitable for deoxygenation of fluids, such as liquids, wherein the oxygen content of said fluid is reduced, removed or substantially removed.

[0037] 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.

[0038] 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.

[0039] In the context of the present invention, conduit is to be understood as a tube or other means of transporting a fluid, such as a liquid.

[0040] 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 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. In the context of the present invention, a flow cell is to be understood as a cell reactor suitable for the growth of cells on a membrane or support structure.

[0041] In the context of the present invention, enveloped is to be understood as surrounded by, submerged into or covered by; and other similar synonyms.

[0042] 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 molecules, 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.

[0043] It is further to be understood, that the removal or reduction of oxygen may be a result of diffusion of oxygen, through the gas permeable wall.

[0044] In a preferred embodiment of the invention, the liquid to be void of oxygen is a cell culture medium. The medium may comprise nutrients, growth factors, pH- buffers, salts, hormones or other relevant ingredients to accommodate the specific environment to simulate.

[0045] In a preferred embodiment of the invention, a section of the conduit, said section comprising at least the gas permeable wall, is coiled. This embodiment is particularly advantageous for increasing the area of the gas permeable wall, to promote the removal or reduction of oxygen from the liquid within the lumen. Further, the coil may enable for a size reduction of the system, to e.g. fit the system onto a table in a laboratory. In some embodiments, the system may fit onto a support plate or within a box, which can be used as a plug-in / turn-key solution.

[0046] In another preferred embodiment of the invention, the conduit comprises a second wall, the second wall being non-permeable to gas and defining a second lumen, the second lumen surrounding at least the gas permeable wall, wherein the fluid is provided within said second lumen.

[0047] In the context of the present invention, the conduit may be construed as a tube with a first lumen situated in the centre of a second lumen, i.e. a two-lumen tube. In other embodiments, the second lumen of the conduit may be of a different geometry than the first lumen, and wherein the first lumen fits into the second lumen, wherein the second lumen is defined by a second wall. In some embodiments, the second wall may be a glass jar or container.

[0048] In yet another preferred embodiment, the invention comprises a container being non-permeable to gas, the container defining a sealable void, wherein at least the gas-permeable wall of the conduit is positioned within said void, the container adapted with a sealed port enabling at least the second end of the conduit to extend outside from within said container, said void adapted to contain the fluid when sealed. This embodiment may be advantageous for improved control of the environment outside the gas-permeable section of the conduit. In some embodiments, the container is adapted for sustaining a low pressure environment for a prolonged period of time.

[0049] It is further to be understood, that the present invention may be suitable for sustaining an anaerobic environment, for simulation of anaerobic conditions, for a prolonged period of time.

[0050] Preferably, the system can sustain an anaerobic environment for more than a day. More preferably, the system can sustain an anaerobic environment for more than a week.

[0051] Even more preferably, the system can sustain an anaerobic environment for more than two weeks, such as a month.

[0052] Most preferably, the system can sustain an anaerobic environment for an indefinite period of time, such as for several months or years.

[0053] In an advantageous embodiment, the invention further comprises a pump and valve device adapted to remove and add fluid to the second lumen of the conduit. In another advantageous embodiment, the invention further comprises a pump and valve device adapted to remove and add fluid to the sealable void of the container when sealed.

[0054] It is to be understood, that the pump may be any type of suitable pressurizing / depressurizing means adapted to interchangeably add or remove a fluid from within a sealed environment.

[0055] In yet another advantageous embodiment of the invention, the fluid comprises a molecule which absorbs oxygen. This embodiment may be particularly suitable for extracting or removing oxygen. In some embodiments, the fluid may be suitable for exclusively removing oxygen, while not removing other gases which are not to be removed.

[0056] In a preferred embodiment of the invention, the fluid is an antioxidant,

[0057] In another preferred embodiment of the invention, the fluid is c-vitamin ascorbate. The inventors have realized, that c-vitamin ascorbate is particularly advantageous for the removal or reduction of oxygen within a liquid, through a gas permeable wall. In a specific embodiment of the invention, the gas permeable wall is a silicone tube which is submerged into a c-vitamin ascorbate bath.

[0058] In some embodiments of the invention, other fluids than c-vitamin ascorbate may be utilized. Below is a list of preferred oxygen scavengers:

[0059] -Ascorbate

[0060] -Erythorbate

[0061] -Sulfites:

[0062] -(Sodium*) sulfite

[0063] -(Sodium*) bisulfite

[0064] -(Sodium*) hydrosulfite

[0065] -(Sodium*) metabisulfite

[0066] (*or corresponding salts with potassium / calcium / ammonium / magnesium)

[0067] Below is a list of other suitable oxygen scavengers: -Sodium thiosulfate

[0068] -Sodium sulfide

[0069] -Gallic acid

[0070] -Pyrogallic acid

[0071] -Catechol

[0072] -Tocopherol

[0073] -Carbohydrazide

[0074] -Hydrazine

[0075] -Hydroquinone

[0076] -Methylethyl ketoxime (MEKO) -Diethylhydroxylamine (DEHA) -(Poly)unsaturated fatty acids (e.g., oleic, linoleic and linolenic acid) -Yeast

[0077] It is to be understood, that in some embodiments according to the invention, any of the above listed oxygen scavengers may be used as the fluid adapted for the removal or quenching of oxygen, for the deoxygenation system according to the present invention.

[0078] In yet another preferred embodiment of the invention, the liquid of the inner lumen is a medium comprising nutrients. This embodiment is particularly advantageous for the continued sustainability of cells or cell layers, as well as any microorganisms, microbiota or bacteria present in the flow cell. Further, the liquid may be used to introduce further substances into the flow cell for study, such as a substance or organism not present and which are to be studied during the simulation. In some cases, a cell layer is grown during the continued use of the system, and wherein, when a targeted growth is achieved, further elements, substances, or environmental parameters are introduced into the flow cell. In a specific embodiment, an epithelial cell layer is grown on the membrane or support structure within the flow cell, by providing nutrients into said flow cell, after which an anaerobic environment is initialized and specific anaerobic bacteria or microbiota / microorganisms are introduced.

[0079] Thus, it is to be understood, that the present invention, according to some embodiments, is enabled to provide an aerobic environment and switch into providing an anaerobic environment, based on the specific simulation. Even further, the present invention is adapted to continuously switch between an aerobic and anaerobic environment in cycles, based on the specific simulation.

[0080] In an advantageous embodiment of the invention, at least the gas permeable wall of the conduit is made from silicone. The inventors have realized, that silicone is a suitable and substantially inert material which is readily available, easy to work with and works well within the present invention.

[0081] In other embodiments, the gas permeable wall may be made from Polyolefins, vinyl and vinylidene polymers, natural and synthetic rubbers, polyesters and polycarbonates, cellulose and cellulose derivatives, fluoropolymers, polyorganosiloxanes including polydimethylsiloxane, polynitriles, polyamides, polyimides, polyurethanes, polyoxides, polysulfones, polyacetylenes, polyacrylics.

[0082] In another advantageous embodiment of the invention, at least the inlet end or outlet end of the conduit is made from stainless steel. It is to be understood, that the inlet and outlet ends of the conduit are to be non-permeable to gas. Hence, other suitable materials may be used, when complying to the following requirements: being inert, bio- and tissue compatible, non or low toxic, non- permeable or substantially non-permeable to gas and preferably but not necessarily: flexible, cost effective, light in weight and easy to fit and adapt.

[0083] In yet another advantageous embodiment of the invention, the liquid within the first lumen is flowing by operation of a pump, impeller, injector / ejector or piston in fluidic connection with said first lumen. This embodiment is particularly advantageous for the continued sustainability of the environment simulated within the flow cell. In some preferred embodiments, the means of generating the flow is adapted to simulate peristaltic flow, i.e. flow generated by peristaltic muscle movement.

[0084] In an embodiment of the invention, the outlet of the flow cell is connected to the first end of the conduit. This embodiment may be advantageous for providing a closed loop, wherein the fluid flowing through the flow cell may be reused. In an advantageous embodiment of the invention, a liquid treatment device is inserted between the outlet of the flow cell and the first end of the conduit, fluidically connecting said outlet with said first end. This embodiment may be suitable to treat the liquid, such as by enriching the liquid with a nutrient, removing nutrients from the liquid or such as for removing undesired particles, molecules or other objects from within the liquid.

[0085] In an embodiment of the invention, the liquid treatment device is a mechanical filter or a UV filter.

[0086] In a second aspect, the invention relates to a method for in vitro simulation of anaerobic conditions in a flow cell, preferably in vitro simulation of anaerobic intestinal conditions in a flow cell, the method comprising:

[0087] -providing a conduit to transport a liquid within a lumen, the conduit comprising a gas permeable section,

[0088] -providing a flow cell comprising two compartments separated by a membrane or support structure, wherein at least one of the two compartments comprises an inlet and an outlet,

[0089] -fluidically connecting the conduit to the inlet of the at least one compartment,

[0090] -removing oxygen at or near the gas permeable section of the conduit, -flowing the liquid through the conduit, and

[0091] -providing the liquid substantially void of oxygen to the inlet of the at least one compartment of the flow cell.

[0092] In a preferred embodiment, the method further comprises:

[0093] -seeding the membrane or support structure with a plurality of cells or a cell layer.

[0094] In a preferred embodiment, the method comprises the steps of starting the systems according to the first aspect, by introducing the cell layer in an aerobic environment, and after culturing into a cell laye after which, the anaerobic environment is introduced. When the cell layer is matured or ready for anaerobic (such as gut-bacteria co-culturing) simulation, a bacteria culture is then introduced into the anaerobic simulation system. This embodiment is particularly advantageous for simulating e.g. the effect of bacteria on e.g. drugs or probiotics within an anaerobic environment and for studying the transfer of e.g. drugs through an intestinal cell layer. It is to be understood, that the cell layer is to be grown on the membrane or support structure, thus effectively sealing the at least two compartments of the flow cell.

[0095] This and other embodiments according to the invention may further be advantageous for sustaining tissue, such as intestinal tissue explanted from a patient, or in vitro cultured tissue intended for later transplantation, by, instead of a cell layer, positioning or culturing the tissue on the support.

[0096] In another preferred embodiment, the method further comprises:

[0097] -seeding the membrane or support structure with one or more epithelial cells.

[0098] In yet another preferred embodiment, the method further comprises: -removing the oxygen at or near the gas permeable section of the conduit by surrounding the gas permeable section of the conduit with a fluid, said fluid being adapted to extract oxygen from the surrounding environment.

[0099] In yet another preferred embodiment, the method further comprises: -providing a second fluid to the one of the two compartments which is not provided with the liquid substantially void of oxygen.

[0100] In an advantageous embodiment, the method further comprises: -providing a sealable container, the container comprising a port adapted to fluidically connect the conduit to the flow cell, and a valve adapted to connect an inside of the container to an outside environment of the container,

[0101] -positioning at least the gas permeable section of the conduit within the container,

[0102] -seal the container, -provide a fluid to within the container through the valve of the container, said fluid being adapted to extract oxygen from the surrounding environment.

[0103] In another advantageous embodiment, the method further comprises:

[0104] -removing gas from within the container, through the valve, prior to providing the fluid, and optionally:

[0105] -repeating the steps of removing and providing gas or fluids from within the container.

[0106] In a preferred embodiment, the method further comprises:

[0107] -providing a second conduit enveloping at least the gas permeable section of the conduit, the second conduit defining a lumen wherein at least said gas permeable section is positioned, and

[0108] -providing a fluid within the lumen of the second conduit between the gas permeable section of the conduit and the second conduit, the fluid adapted to extract oxygen from the surrounding environment.

[0109] In another preferred embodiment the method further comprises:

[0110] -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.

[0111] 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.

[0112] In an advantageous embodiment, the microorganism is a bacterium, a parasite, a bacteriophage, a fungus, or a virus.

[0113] In some embodiments, the microorganism is selected from the group consisting of: Lactobacillus; Bacterioides; Ruminococcus; Peptococcus; Peptostreptococcus; Bifidobacterium; Escherichia; Achromobacter; Acidaminococcus fermentans;

[0114] Acinetobacter cacoaceticus; Aeromonas; Alcaligenes faecalis; Bacillus; Butyriviberio fibrosolvens; Camplyobacter; Campylobacter coli; Clostridium difficile; Clostridium sordelli; Enterobacter cloacae; Enterococcus faecalis;

[0115] Enterococcus faecium; Escherichia coli; Flavobacterium; Mycobacterium;

[0116] Mycoplasma; Plesiomonas shigelloides; Propionibacterium acnes; Pseudomonas aeruginosa; Ruminococcus bromii; Sarcina; Staphylococcus aureus; Streptococcus anginosus; Veillonella; Vibrio; Yersinia enterocolitica; Lactobacillus rhamnosus;

[0117] Lactobacillus rhamnosus GG; Bifidobacterium breve; Bifidobacterium longum;

[0118] Bifidobacterium infantis; Lactobacillus acidophilus; Lactobacillus plantarum;

[0119] Lactobacillus paracasei; Lactobacillus bulgaricus ; and Streptococcus thermophilus.

[0120] In some embodiments, the microorganism is pathogenic.

[0121] In some embodiments, the microorganism is a probiotic.

[0122] In some embodiments, the pathogens are selected from the group consisting of: enterotoxigenic Escherichia coli; Bilophila wadsworthia; Shigella; Yersinia;

[0123] Pleisiomonas; Vibrio; Aeromonas; Campylobacter; Crytosporidia; Coccidosis;

[0124] Salmonella; Helicobacter pylori; Clostridium difficile; Salmonella kedougou;

[0125] Bacteroides; Clostridium; Firmicutes; Shigellia dysenteriae; Salmonella enterica;

[0126] Salmonella typhi; Listeria; Listeria monocytogenes; Vibrio parahaemolyticus;

[0127] 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 .

[0128] 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;

[0129] Peptococcus; Peptostreptococcus; Bifidobacterium; Escherichia; Achromobacter;

[0130] Acidaminococcus fermentans; Acinetobacter cacoaceticus; Aeromonas;

[0131] 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.

[0132] 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;

[0133] 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.

[0134] 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. In an embodiment the microorganism is C. difficile and / or B. fragilis. These are both obligate anaerobic.

[0135] In some embodiments, the microorganism is aerobic.

[0136] In some embodiments, the microorganism is anaerobic.

[0137] Preferred drugs / compounds may be selected from one or more of the following: Nutriceutical

[0138] Probiotics, prebiotics

[0139] Pathogens

[0140] Drugs - antibiotics Cancerogenous bacteria

[0141] In an embodiment the intestinal epithelial cells are selected from the group consisting of:

[0142] Caco2 cells; HT-29 cells; T84 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.

[0143] 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.

[0144] 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.

[0145] In another embodiment, immune cells are attached to one side of the membrane and endothelial cells are attached to the other side.

[0146] The embodiments of the method stated above, have the particular advantage of providing long-term anaerobic conditions for continued simulation and study of cell layers developing, while being subjected to anaerobic conditions. Further, this allows for users to study anaerobic organisms, and their development. Even further, this allows for users to study the solubility, toxicity or absorption of different molecules, such as drugs or probiotics within such an anaerobic environment.

[0147] In a third aspect, the invention relates to a kit of parts for extracting oxygen from a liquid to be provided to a compartment of a flow cell, said flow cell adapted to simulate an in vitro environment of a biological process, the kit of parts comprising:

[0148] -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 flow cell, the conduit further comprising:

[0149] -a gas permeable section,

[0150] -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,

[0151] -a connector at the second end, the connector adapted to fluidica lly connect to a flow cell,

[0152] -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

[0153] -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

[0154] -the conduit comprises a second lumen outside the inner lumen and wherein the second lumen is one of:

[0155] -void of oxygen, or

[0156] -filled with a fluid adapted to quench or catch oxygen.

[0157] In the context of the present invention, quench or quenching is to be understood as catching, absorbing or by other means removing. Thus, the fluid is adapted to catch, absorb or by other means remove oxygen.

[0158] In a preferred embodiment, the kit of parts further comprises a flow cell. The flow cell may be in accordance with the fifth aspect of the invention.

[0159] In another preferred embodiment, the kit of parts further comprises a pump adapted to provide flow to the liquid to the flow cell from the conduit.

[0160] In yet another preferred embodiment, the fluid of the kit of parts comprises a molecule adapted to absorb oxygen, preferably the fluid is c-vitamin ascorbate.

[0161] In a fourth aspect, the invention relates to an apparatus for extracting oxygen from a liquid, the apparatus comprising:

[0162] -a conduit having at least a first end and a second end, the conduit comprising an inner wall and an outer wall, the inner wall defining an inner lumen and the outer wall defining an outer lumen, the inner wall positioned within the outer lumen, wherein the inner wall is permeable to gas and the outer wall is not permeable to gas, wherein the inner lumen is adapted to convey the liquid from which oxygen is to be extracted and the outer lumen is adapted to:

[0163] -contain an environment void of oxygen, or

[0164] -a fluid adapted to quench or catch oxygen. In a fifth aspect, the invention relates to a system, method or kit of parts according to any of the first, second and third aspect respectively, further comprising a specific flow cell. The flow cell will be described, according to the following embodiments:

[0165] In a preferred embodiment, the flow cell is adapted to simulate anaerobic conditions in vitro, preferably simulation of anaerobic intestinal conditions in vitro, the flow cell comprising :

[0166] -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:

[0167] -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 or hydrophilic- treated or coated silicone to enhance cell adhesion, and wherein the housing is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment.

[0168] This aspect 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.

[0169] In particular, this aspect 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.

[0170] Further, this aspect 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. It is an aim of the fifth aspect, to provide an improved flow cell 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.

[0171] In the context of the present invention, track etched is to be understood as pores of a nano scale size.

[0172] In a preferred embodiment of the fifth aspect, 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.

[0173] I another preferred embodiment, the track etched pores are created by a chemical etching process.

[0174] In some embodiments, the ion treatment and chemical etching may be combined, to provide the track etched pores.

[0175] 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).

[0176] 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.

[0177] The porous membrane may be selected from a suitable polymer, such as polydimethylsiloxane, tris-(trimethyl-silyl-propyl-methacrylate) or similar material. 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.

[0178] 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.

[0179] The first, second, third, fourth and fifth aspect 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.

[0180] BRIEF DESCRIPTION OF THE FIGURES

[0181] The deoxygenation system 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.

[0182] Fig.l shows a schematic overview, in sideview, of a simulation setup, for anaerobic in vitro simulation, according to an embodiment of the invention; FIG.2 shows a schematic sideview of the deoxygenation system and the flow cell according to an embodiment of the invention;

[0183] FIG. 3 shows a schematic sideview of a deoxygenation system and flow cell according to another embodiment of the invention;

[0184] FIG. 4 shows a schematic sideview of a deoxygenation system and flow cell according to yet another embodiment of the invention;

[0185] FIG. 5 shows a sideview of a flow cell, according to an embodiment of the invention;

[0186] FIG. 6 shows a sideview of a flow cell, according to another embodiment of the invention;

[0187] FIG. 7 shows a sideview of a flow cell, according to yet another embodiment of the invention;

[0188] Fig. 8 is a flow-chart of a method according to the invention; Fig. 9A - Fig. 9C shows the design of a cell reactor and anaerobic system, according to an embodiment of the invention;

[0189] Fig. 10A - Fig. 10E shows Oxygen levels and dependency on flow rate, tube length and chamber conditions;

[0190] Fig. 11A - Fig. 11C shows characterization of the Caco-2 cell layer 13 days post seeding;

[0191] Fig. 12A - Fig. 12C shows transcriptomic characterization of the Caco-2 cell layer under anaerobic condition with gut-mimicking flow;

[0192] Fig. 13A - Fig. 13E shows colonization with C. difficile and B. fragilis;

[0193] Fig. 14A and Fig. 14B shows bacterial colonization of Caco-2 cells;

[0194] Fig. 15A - Fig. 15C shows images from a four-week study of a Caco-2 cell layer, utilizing a system according to an embodiment of the invention.

[0195] DETAILED DESCRIPTION OF AN EMBODIMENT

[0196] Fig.l 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. 1, at centre, the flow cell FC is positioned. The flow cell FC comprises a first and second compartment (not visible) encased within a sealed housing. The flow cell FC 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 flow cell FC compartments will be clearly shown and explained in various embodiments, in FIGS. 6 to 7. 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. 1, flow directions of medium MED, MED' are shown with arrows. A medium MED, is fluidically connected to the flow cell FC via a conduit CON. The conduit CON goes through a deoxygenation system DOS, into the first compartment of the flow cell FC. Thus the medium MED is transported through the deoxygenation system DOS, through the inlet IL, into the first compartment of the flow cell FC, out of the flow cell FC 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 flow cell FC, being fed with a medium MED' being transported from the container marked MED', through the inlet IL' to the second compartment of the flow cell FC, 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.

[0197] Thus, from FIG. 1 is can be seen, that a medium MED is being anaerobized (oxygen is removed from the medium MED), prior to entering the flow cell FC. The specific of the deoxygenation system will be discussed in FIGS. 2 to 4.

[0198] FIG.2 shows a schematic sideview of the deoxygenation system DOS and the flow cell FC according to an embodiment of the invention. In Fig. 2 the deoxygenation system comprises a vessel VES containing a fluid FLU. A conduit CON adapted with a lumen to transport a liquid (not visible) is partially immersed in the fluid FLU. A flow direction of the liquid within the conduit CON, is shown with arrows on FIG.2. The conduit comprises a gas-permeable section GAS_P, which is coiled to increase a surface area of the gas permeable section GAS_P, to enable for a higher diffusion rate of oxygen from the liquid within the gas permeable section GAS_P, to the fluid FLU. When the liquid flows into the gas permeable portion GAS_P of the conduit CON, which is immersed in the fluid FLU, at least oxygen is removed or quenched from the liquid. After the liquid exits the gas permeable section GAS_P of the conduit CON, the liquid enters a non-gas permeable section N_GAS_P of the conduit CON. Thus, the deoxygenation system prevents oxygen from a surrounding environment to enter the fluid, after the oxygen has been removed and prior to the fluid entering the inlet IL of the flow cell FC. The flow cell FC further comprises an outlet OL, to enable the liquid to exit the flow cell FC, thus enabling a flow of deoxygenized liquid within the flow cell FC. It should be noted, that the flow cell FC, may have further inlets and outlets, to a second compartment within said flow cell, such as shown in FIG 3.

[0199] FIG. 3 shows a schematic sideview of a deoxygenation system DOS and flow cell FC according to another embodiment of the invention. In FIG. 3, the deoxygenation system DOS comprises a sealed container S_CON adapted with a valve device VD and a sealed port SP. The deoxygenation system DOS further comprises a conduit CON partly positioned within and partly outside the sealed container S_CON. A portion of the conduit CON, within the sealed container S_CON is permeable to gas GAS_P, thus enabling for a liquid (not visible) flowing within a lumen of the conduit CON, to be quenched of oxygen, when a fluid FLU inside the sealed container S_CON is provided, wherein the fluid FLU is adapted to remove oxygen from a surrounding environment, such as by diffusion of oxygen from the liquid, through the gas permeable wall GAS_P of the conduit, to the fluid FLU. Within the sealed container S_CON, the gas permeable wall GAS_P is coiled, to increase the surface area between the fluid FLU and the liquid within. Between the sealed container S_CON and the inlet IL of the flow cell FC, the conduit CON is non-permeable to gas N_GAS_P, to prevent oxygen to enter the liquid, prior to the liquid entering the flow cell FC. The flow cell is illustrated with two inlets IL, IL' and two outlets OL, OL'. It is to be understood, that within the flow cell, which is encased in a housing which is non-permeable to gas, a first and second compartment is adapted, separated by a support structure or membrane, and wherein respective inlets IL, IL' and outlets OL, OL' enables a flow in each of said compartments. In some embodiments, the sealed container S_CON comprises a second sealed port, to provide the conduit CON a sealed passage into said sealed container S_CON.

[0200] In some embodiments, a vessel containing the liquid is positioned within the sealed container, and wherein the fluid is in direct contact with said liquid. In some embodiments, the sealed container is adapted with a negative pressure, such as by a vacuum device, to remove oxygen from within the conduit CON, at the gas permeable wall section GAS_P.

[0201] FIG. 4 shows a schematic sideview of a deoxygenation system DOS and flow cell FC according to yet another embodiment of the invention. In FIG. 4, the deoxygenation system DOS comprises a conduit CON shown in sideview, and in cross-sectional view CS_CON. The conduit CS_CON comprises an inner wall IN_WA, said inner wall IN_WA defining an inner lumen; IN_LU surrounded by an outer lumen OU_LU, defined by an outer wall OU_WA. The inner lumen IN_LU is fluidically connected to a liquid inlet LIQ_IN, adapted to provide a liquid, such a cell culture medium (not visible) and further fluidically connected to an inlet IL of a flow cell FC. The flow cell further comprises a housing and an outlet OL. The outer lumen OU_LU is fluidically connected to a fluidic inlet FLU_IN and a fluidic outlet FLU_OU, thus enabling a fluid to flow around the inner lumen IN_LU. A portion of the inner lumen IN_LU is comprised of a gas permeable wall section, thus enabling oxygen from within a liquid of the inner lumen to pass through said gas-permeable wall section, into a fluid within the outer lumen OU_LU. It should be noted, that the flow cell FC, may have further inlets and outlets, to a second compartment within said flow cell, such as shown in FIG 3.

[0202] FIG. 5 shows a sideview of a flow cell FC, according to an embodiment of the invention. The flow cell 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, or substantially non-permeable to gas. At the bottom of the flow cell FC, a membrane MEM or support structure is positioned, abutting the housing HO and compartment CO. The membrane MEM or support structure is made from a silicone or silicone hydrogel composition. In FIG. 5 a cell layer CL is grown along the bottom of the compartment CO, on the membrane MEM or support structure. Thus, the compartment is sealed from the outside environment by the housing HOU, the membrane MEM or support structure and the cell layer CL respectively. When the flow cell FC is used with the deoxygenation system DOS, shown in FIGS. 2 to 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 membrane MEM or support structure, thereby providing a compartment CO within the flow cell FC 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.

[0203] It is to be understood, at least 98% of the oxygen, to reach the cell layer in the first compartment, will be supplied through the gas-permeable support. Thus, in preferred embodiments, as low as between 2 to 0 % oxygen will be provided from other parts of the system, such as from the conduit or the liquid.

[0204] FIG. 6 shows a sideview of a flow cell FC, according to another embodiment of the invention. The flow cell 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 membrane MEM 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 membrane MEM 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 membrane MEM or support structure. The membrane MEM or support structure is made from a silicone or silicone hydrogel composition. In FIG. 6 a cell layer CL is grown along the bottom of the first compartment CO, on the membrane MEM or support structure. Thus, the first compartment is sealed from the outside environment by the housing HOU, the membrane MEM or support structure and the cell layer CL respectively. The second compartment CO' is defined by the housing HOU and the membrane MEM 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 flow cell FC is used with the deoxygenation system DOS, shown in FIGS. 2 to 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 membrane MEM or support structure from the second compartment CO', thereby providing a first compartment CO within the flow cell FC 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 flow cell 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.

[0205] FIG. 7 shows a sideview of a flow cell FC, according to yet another embodiment of the invention. The flow cell 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 membrane MEM 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 membrane MEM 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 membrane MEM or support structure. The membrane MEM 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 membrane MEM or support structure. Thus, the first compartment is sealed from the outside environment by the housing HOU, the membrane MEM or support structure and the cell layer CL respectively. The second compartment CO' is defined by the housing HOU and the membrane MEM 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 flow cell FC is used with the deoxygenation system DOS, shown in FIGS. 2 to 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 membrane MEM or support structure from the second compartment CO', thereby providing a first compartment CO within the flow cell FC 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 flow cell 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 membrane MEM 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.

[0206] Fig. 8 is a flow-chart of a method according to the invention, for in vitro simulation of anaerobic conditions in a flow cell, preferably in vitro simulation of anaerobic intestinal conditions in a flow cell, the method comprising the following steps SI to S6:

[0207] 51-providing a conduit to transport a liquid within a lumen, the conduit comprising a gas permeable section,

[0208] 52-providing a flow cell comprising two compartments separated by a membrane or support structure, wherein at least one of the two compartments comprises an inlet and an outlet, 53-fluid ica lly connecting the conduit to the inlet of the at least one compartment,

[0209] 54-removing oxygen at or near the gas permeable section of the conduit,

[0210] 55-flowing the liquid through the conduit, and

[0211] 56-providing the liquid substantially void of oxygen to the inlet of the at least one compartment of the flow cell.

[0212] In an embodiment of the method, a further step is to provide cells on the membrane, prior to S3.

[0213] It is further to be understood, that the cell layer may be grown or matured in an aerobic environment, after which, the anaerobic environment is introduced. When the anaerobic environment is flowing within the compartment sustaining the cell layer, bacteria or other substances, such as drugs, may be introduced.

[0214] Figs. 9A - C shows the design of a cell reactor and anaerobic system, according to an embodiment of the invention. Fig. 9A shows 3D images of the dual flow chamber. Fig. 9B shows real life image of the dual chamber during an aerobic experiment. Fig. 9C shows a schematic presentation of the system.

[0215] Figs. 10A - E shows Oxygen levels and dependency on flow rate, tube length and chamber conditions. Fig. 10A shows a schematic presentation of the single and dual flow chambers showing the connection sites of the oxygen sensor (inlet and exit). Fig. 10B shows inlet oxygen percentage based on different lengths of the silicone coil and at various flow rates. Fig. 10C 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. 10D 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.

[0216] Figs. 11A - C shows characterization of the Caco-2 cell layer 13 days post seeding. Fig. 11A 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. 11B shows height of the Caco-2 cell layer grown under different conditions. The height was determined by confocal microscopy. Fig. 11C shows staining of neutral (Shift's reagent) and acidic (Alcian blue) mucins.

[0217] Figs. 12A - C shows transcriptomic characterization of the Caco-2 cell layer under anaerobic conditions with gut-mimicking flow. Caco-2 cells were cultured for 13 days in the dual flow chamber (DFC) model, both aerobically (DFC+02) and anaerobically (DFC-02), as well as in cell culture inserts under aerobic conditions (Static). RNA sequencing was then performed to analyze gene expression profiles. Fig. 12A 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. 12B 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. 12C 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; DOI: 10.34133 / research.0058) 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.

[0218] Figs. 13A - 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. 13A shows CFU / mL in effluent from the apical channel of the DFC. Fig. 13B 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. 13C 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. 13D 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. 13E shows gram stain of effluent from apical channel of the DFC 5 days post colonization. The grampositive C. difficile is stained blue (not visible) while the gram-negative B. fragilis is stained red (not visible).

[0219] Figs 14A 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. 14A 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. 14B 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.

[0220] Figs. 15A - C shows a images from a three-week study of Caco-2 cell layer.

[0221] Fig. 15A 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.

[0222] Fig. 15B shows a LIVE / DEAD stain, wherein the stain showed that the vast majority of cells were viable after 21 days of anaerobic culture.

[0223] Fig. 15C shows macroscopic observation of the cells also revealing a confluent layer of Caco-2 cells.

[0224] The invention will now be described in further details in the following non-limiting examples, wherein the flow cell according to the invention may be referred to as a flow chamber or dual flow chamber:

[0225] Example 1 - Materials and methods

[0226] 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. 14A. 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.

[0227] Anaerobization unit (AU)

[0228] 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.

[0229] Cell culture

[0230] 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.

[0231] 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.

[0232] Oxygen measurements

[0233] 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.

[0234] 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. 10A 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 :

[0235] Immunohistochemical staining

[0236] 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.

[0237] Mucus staining

[0238] 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.

[0239] LIVE / DEAD stain

[0240] 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.

[0241] RNA isolation

[0242] 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.

[0243] RNA-sequencing

[0244] 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 sets. For marker gene expression analysis, lineage marker genes from Burclaff et al.

[0245] (DOI: were 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 comparison. Clustered heatmaps were generated with the R package pheatmap (version 1.0.12), using Ward's method ("ward.D2") for hierarchical clustering.

[0246] Colonization with Clostridioides difficile and Bacteroides fragilis

[0247] 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.

[0248] Microscopy 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. Immunohistochemical stained and LIVE / DEAD stained cultures were imaged using a Nikon AX or Olympus FV1000 confocal single- or multiphoton laser scanning microscope.

[0249] Electron microscopy

[0250] 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).

[0251] 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.

[0252] Statistics

[0253] 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.

[0254] Example 2 - Establishment of the hypoxic intestinal lumen environment Aim of study

[0255] 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.

[0256] Materials and methods

[0257] See also Example 1 for details.

[0258] 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. 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. 9. 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. 10A.

[0259] Results and conclusion

[0260] 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. 10. Example 3 - Optimization of tubing size

[0261] Aim of study

[0262] To optimize the tubings for optimal deoxygenation.

[0263] Materials and methods

[0264] 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

[0265] Lengths, starting from 7 meters were tested.

[0266] Results

[0267] 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 is used.

[0268] Conclusion

[0269] 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.

[0270] Example 4 - Optimization of flow rate and coil length

[0271] Aim of study

[0272] To optimize the flow rate and coil length for optimal deoxygenation.

[0273] To test if the anaerobic container with nitrogen gas could be replaced with an antioxidant solution.

[0274] Materials and methods

[0275] 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. Results

[0276] 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.

[0277] Conclusion

[0278] 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. 10B.

[0279] Example 5 - Real-time oxygen levels in flow cell during experiments

[0280] Aim of study

[0281] To test the performance of the flow chambers to maintain anaerobic conditionsduring experiments with and without cells.

[0282] Materials and methods

[0283] 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.

[0284] Results

[0285] 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. 10C. 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. 10D. 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. 10E. Oxygen levels decreased to <1% within a few minutes after resuming the flow.

[0286] Conclusion

[0287] 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.

[0288] Example 6 - Caco-2 cells matured in the DFC demonstrates in vivo characteristics of the intestine

[0289] Aim of study

[0290] 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

[0291] See example 1.

[0292] Results

[0293] 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. 11. 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. 11B. By comparison, the average height of the cells cultured statically for 13 days was approximately 20 pm, see Fig. 11B. 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. 11A. 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. 11C. 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. 11C. 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).

[0294] Conclusion

[0295] 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.

[0296] Example 7 - Transcriptomic profiling

[0297] Aim of study

[0298] 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.

[0299] Materials and methods

[0300] See also example 1

[0301] 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.

[0302] Results

[0303] 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 epithelium.

[0304] 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. 12A. 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. 12B. 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 model. The clustered heatmap, see Fig. 12C, reveals that all the Caco-2 models most closely resemble (proximal) enterocytes of the small intestine, as reported in the literature. 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. (DOI: 10.34133 / research.0058), demonstrating that microfluidic-based Caco-2 models share similar molecular characteristics.

[0305] Conclusion

[0306] Transcriptom ic 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.

[0307] Example 8 - Co-culture of obligate anaerobes and Caco-2 cells in the DFC Aim of study

[0308] 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.

[0309] Materials and methods

[0310] See also example 1.

[0311] 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.

[0312] Results

[0313] 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. 13A. Following bacterial colonization, the viability of the intestinal epithelium was examined by confocal laser scanning microscopy (CLSM).

[0314] Conclusion 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. 13D.

[0315] Example 9 - Vancomycin treatment of the C. difficile colonized epithelium Aim of study

[0316] 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.

[0317] Materials and methods

[0318] See also example 1

[0319] Treatment with vancomycin (6 ug / ml) was initiated three days post infection (DPI).

[0320] Results

[0321] After two days of treatment, the CFU count of C. difficile in the effluent had reduced almost 1000-fold, see Fig. 13A, but some bacteria were still present in the biofilm clumps harvested from the chamber, see Fig. 13E. Interestingly, a 10- fold reduction in the amount of B. fragilis was observed in the effluent, compared to the untreated control, see Fig. 13A. 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. 13B.

[0322] The amount of C. difficile associated with the cell layer was reduced approximately 25-fold following vancomycin treatment, compared to the control, see Fig. 13B. 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. 13E. 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. 13C, indicating that the integrity of the cell layer is not compromised by the colonizing bacteria. Conclusion

[0323] 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.

[0324] Example 10 - Invasion of the intestinal epithelium

[0325] Aim of study

[0326] 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.

[0327] Materials and methods

[0328] See example 1

[0329] Results

[0330] In the untreated control, colonies of bacteria were found in the crypts, see Fig. 14A, indicated by arrows, and both extra- and intracellular bacteria were identified by EM, see Fig. 13F, 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. 14A and 11B. The extracellular colonies contained what appeared to be lysed bacteria, see Fig. 14B, 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. 14B, indicated by light arrows).

[0331] Conclusion

[0332] 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.

[0333] Example 11 - Longevity of model according to the present invention

[0334] Purpose:

[0335] 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. Methods:

[0336] 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.

[0337] Results:

[0338] 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. 15A. 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. 15B. Macroscopic observation of the cells also revealed a confluent layer of Caco-2 cells, see Fig. 15C.

[0339] Example 12 - a comparison of membranes

[0340] 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.

[0341] In short, the present invention relates to a deoxygenation system DOS adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising a conduit CON comprising an inner lumen IN_LU, a first end and a second end, the conduit further comprising a gas permeable wall GAS_P, the inner lumen adapted to contain and transport the liquid from said first end to said second end. The system is adapted for fluidic connection to an associated flow cell FC comprising at least one compartment CO with a membrane MEM and an inlet IL and an outlet OL, the inlet fluidica lly connected to the second end of the conduit, wherein the gas permeable wall is enveloped in a fluid FLU. The fluid may be selected from a fluid a)substantially void of oxygen, or b) having an affinity for oxygen higher than that of the liquid, thus enabling the system to move oxygen from the liquid to the fluid to, deoxygenize said liquid.

[0342] 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.

[0343] The following is a list of itemized embodiments, according to the invention :

[0344] 1.1. A deoxygenation system DOS adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising:

[0345] -a conduit CON comprising an inner lumen IN_LU, a first end and a second end, the conduit further comprising a gas permeable wall GAS_P, the inner lumen adapted to contain and transport the liquid from said first end to said second end,

[0346] -a flow cell FC comprising two compartments CO, CO' separated by a membrane MEM, wherein at least one of the two compartments comprises an inlet IL and an outlet OL, the inlet fluidically connected to the second end of the conduit, wherein the gas permeable wall is enveloped in a fluid FLU, the fluid being: -substantially void of oxygen, or -having an affinity for oxygen higher than that of the liquid, the system enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid.

[0347] 1.2. The system according to Item 1 wherein a section of the conduit, said section comprising at least the gas permeable wall, is coiled.

[0348] 1.3. The system according to Item 1 or 2, the conduit comprising a second wall OU_WA, the second wall being non-permeable to gas and defining a second lumen OU_LU, the second lumen surrounding at least the gas permeable wall, wherein the fluid is provided within said second lumen.

[0349] 1.4. The system according to Item 1 or 2 further comprising a container S_CON being non-permeable to gas, the container defining a sealable void, wherein at least the gas-permeable wall of the conduit is positioned within said void, the container adapted with a sealed port SP enabling at least the second end of the conduit to extend outside from within said container, said void adapted to contain the fluid when sealed.

[0350] 1.5. The system according to Item 1, the system further comprising a pump and valve device VD adapted to remove and add fluid to:

[0351] -the second lumen according to Item 3, or

[0352] -the sealable void of the container when sealed, according to Item 4.

[0353] 1.6. The system according to any of the preceding Items, wherein the fluid comprises a molecule which absorbs oxygen.

[0354] 1.7. The system according to any of the preceding Items wherein the fluid is an antioxidant, preferably c-vitamin ascorbate.

[0355] 1.8. The system according to any of the following Items wherein the liquid of the inner lumen is a media comprising nutrients. 1.9. The system according to any of the preceding Items, wherein at least the gas permeable wall of the conduit is made from silicone, in particular from a biocompatible silicone.

[0356] 1.10. The system according to any of the preceding Items, wherein at least the inlet end or outlet end of the conduit is made from stainless steel.

[0357] 1.11. The system according to any of the preceding Items, wherein the liquid within the first lumen is flowing by operation of a flow generator FG, such as a pump, impeller, injector or piston, in fluidic connection with said first lumen.

[0358] 1.12. The system according to any of the preceding Items, wherein the outlet of the flow cell is connected to the first end of the conduit.

[0359] 1.13. The system according to Item 12, wherein a liquid treatment device is inserted between the outlet of the flow cell and the first end of the conduit, fluidically connecting said outlet with said first end.

[0360] 1.14. The system according to Item 13, wherein the liquid treatment device is a mechanical filter or a UV filter.

[0361] 1.15. A method for in vitro simulation of anaerobic conditions in a flow cell, preferably in vitro simulation of anaerobic intestinal conditions in a flow cell, the method comprising:

[0362] -providing a conduit to transport a liquid within a lumen, the conduit comprising a gas permeable section,

[0363] -providing a flow cell comprising two compartments separated by a membrane, wherein at least one of the two compartments comprises an inlet and an outlet,

[0364] -fluidically connecting the conduit to the inlet of the at least one compartment,

[0365] -removing oxygen at or near the gas permeable section of the conduit, -flowing the liquid through the conduit, and

[0366] -providing the liquid substantially void of oxygen to the inlet of the at least one compartment of the flow cell. 1.16. The method according to Item 15, the method further comprising:

[0367] -seeding the membrane with a plurality of cells or a cell layer.

[0368] 1.17. The method according to Item 15 or 16, the method further comprising:

[0369] -seeding the membrane with one or more epithelial cells.

[0370] 1.18. The method according to any of Items 15 to 17, the method further comprising:

[0371] -removing the oxygen at or near the gas permeable section of the conduit by surrounding the gas permeable section of the conduit with a fluid, said fluid being adapted to extract oxygen from the surrounding environment.

[0372] 1.19. The method according to any of Items 15 to 18 further comprising:

[0373] -providing a second fluid to the one of the two compartments which is not provided with the liquid substantially void of oxygen.

[0374] 1.20. The method according to Item 15 further comprising:

[0375] -providing a sealable container, the container comprising a port adapted to fluidically connect the conduit to the flow cell, and a valve adapted to connect an inside of the container to an outside environment of the container,

[0376] -positioning at least the gas permeable section of the conduit within the container,

[0377] -seal the container,

[0378] -provide a fluid to within the container through the valve of the container, said fluid being adapted to extract oxygen from the surrounding environment.

[0379] 1.21. The method according to Item 20 further comprising:

[0380] -removing gas from within the container, through the valve, prior to providing the fluid, and optionally:

[0381] -repeating the steps of removing and providing gas or fluids from within the container. 1.22. The method according to Item 15 further comprising:

[0382] -providing a second conduit enveloping at least the gas permeable section of the conduit, the second conduit defining a lumen wherein at least said gas permeable section is positioned, and

[0383] -providing a fluid within the lumen of the second conduit between the gas permeable section of the conduit and the second conduit, the fluid adapted to extract oxygen from the surrounding environment.

[0384] 1.23. A kit of parts for extracting oxygen from a liquid to be provided to a compartment of an associated flow cell, said associated flow cell adapted to simulate an in vitro environment of a biological process, the kit of parts comprising:

[0385] -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 flow cell, the conduit further comprising:

[0386] -a gas permeable section,

[0387] -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,

[0388] -a connector at the second end, the connector adapted to fluidically connect to a flow cell,

[0389] -means for extracting oxygen from the liquid within the gas permeable section of the conduit, the means selected from one of:

[0390] -a fluid adapted to quench oxygen from the liquid by submerging at least the gas permeable section of the conduit in said fluid, or

[0391] -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

[0392] -the conduit comprises a second lumen outside the inner lumen and wherein the second lumen is one of:

[0393] -void of oxygen, or

[0394] -filled with a fluid adapted to quench or catch oxygen. 1.24. The kit of parts according to Item 23, further comprising a pump adapted to provide flow to the liquid to the flow cell from the conduit.

[0395] 1.25. The kit of parts according to any of Items 23 or 24, wherein the fluid comprises a molecule adapted to absorb oxygen, preferably the fluid is c-vitamin ascorbate.

[0396] 1.26. An apparatus for extracting oxygen from a liquid, the apparatus comprising:

[0397] -a conduit having at least a first end and a second end, the conduit comprising an inner wall and an outer wall, the inner wall defining an inner lumen and the outer wall defining an outer lumen, the inner wall positioned within the outer lumen, wherein the inner wall is permeable to gas and the outer wall is not permeable to gas, wherein the inner lumen is adapted to convey the liquid from which oxygen is to be extracted and the outer lumen is adapted to: -contain an environment void of oxygen, or -a fluid adapted to quench or catch oxygen.

[0398] 1.27. The system according to any of Items 1 to 15, the flow cell further comprising:

[0399] -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:

[0400] -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.

[0401] 1.28. The flow cell according to Item 27, wherein the pores of the porous membrane are substantially perpendicular to a surface plane of said porous membrane. 1.29. The flow cell according to Item 27 or 28 wherein the porous membrane is substantially transparent.

[0402] 1.30. The flow cell according to any of Items 27 to 29, wherein the porous membrane has a thickness of between 10 and 50 pm.

[0403] 1.31. The flow cell according to Item 27, wherein the hydrophilic support has a thickness of between 0.5 and 3 mm.

[0404] 1.32. The flow cell according to Item 27 or 31, wherein the hydrophilic support is a chemical blend or a co-polymer or an interpenetrating polymer network.

[0405] 1.33. The flow cell according to any of Items 27 or 31 to 32, wherein the hydrophilic support comprises between 10 and 40% hydrogel.

[0406] 1.34. The flow cell according to any of Items 27 to 33, 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.

[0407] 1.35. The flow cell according to any Items 27 to 34, 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.

[0408] 1.36. The flow cell according to any of Items 27 to 34, 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.

[0409] 1.37. The flow cell according to any of Items 27 to 34, wherein the second compartment comprises an inlet and an outlet adapted to provide a flow of a fluid through said second compartment. 1.38. The flow cell according to any of Items 27 to 37, wherein at least a portion of the housing is transparent.

Claims

CLAIMS1. A deoxygenation system (DOS) adapted to deoxygenize a liquid flowing to an in vitro simulation environment, the system comprising:-a conduit (CON) comprising an inner lumen (IN_LU), a first end and a second end, the conduit further comprising a gas permeable wall (GAS_P), the inner lumen adapted to contain and transport the liquid from said first end to said second end,-a flow cell (FC) comprising two compartments (CO, CO') separated by a membrane (MEM), wherein at least one of the two compartments comprises an inlet (IL) and an outlet (OL), the inlet fluidically connected to the second end of the conduit, wherein the gas permeable wall is enveloped in a fluid (FLU), the fluid being : -substantially void of oxygen, or-having an affinity for oxygen higher than that of the liquid, the system enabling oxygen to move from the liquid to the fluid to deoxygenize said liquid.

2. The system according to claim 1 wherein a section of the conduit, said section comprising at least the gas permeable wall, is coiled.

3. The system according to claim 1 or 2, the conduit comprising a second wall (OU_WA), the second wall being non-permeable to gas and defining a second lumen (OU_LU), the second lumen surrounding at least the gas permeable wall, wherein the fluid is provided within said second lumen.

4. The system according to claim 1 or 2 further comprising a container (S_CON) being non-permeable to gas, the container defining a sealable void, wherein at least the gas-permeable wall of the conduit is positioned within said void, the container adapted with a sealed port (SP) enabling at least the second end of the conduit to extend outside from within said container, said void adapted to contain the fluid when sealed.

5. The system according to claim 1, the system further comprising a pump and valve device (VD) adapted to remove and add fluid to:-the second lumen according to claim 3, or-the sealable void of the container when sealed, according to claim 4.

6. The system according to any of the preceding claims, wherein the fluid comprises a molecule which absorbs oxygen.

7. The system according to any of the preceding claims wherein the fluid is an antioxidant, preferably c-vitamin ascorbate.

8. The system according to any of the following claims wherein the liquid of the inner lumen is a media comprising nutrients.

9. The system according to any of the preceding claims, wherein at least the gas permeable wall of the conduit is made from silicone.

10. The system according to any of claims 1 to 8, wherein at least the gas permeable wall of the conduit is made from an inert material.

11. The system according to any of the preceding claims, wherein at least the inlet end or outlet end of the conduit is made from stainless steel.

12. The system according to any of the preceding claims, wherein the liquid within the first lumen is flowing by operation of a flow generator (FG), such as a pump, impeller, injector or piston, in fluidic connection with said first lumen.

13. The system according to any of the preceding claims, wherein the outlet of the flow cell is connected to the first end of the conduit.

14. The system according to claim 13, wherein a liquid treatment device is inserted between the outlet of the flow cell and the first end of the conduit, fluidically connecting said outlet with said first end.

15. The system according to claim 14, wherein the liquid treatment device is a mechanical filter or a UV filter.

16. A method for in vitro simulation of anaerobic conditions in a flow cell, preferably in vitro simulation of anaerobic intestinal conditions in a flow cell, the method comprising:-providing a conduit to transport a liquid within a lumen, the conduit comprising a gas permeable section,-providing a flow cell comprising two compartments separated by a membrane, wherein at least one of the two compartments comprises an inlet and an outlet,-fluid ically connecting the conduit to the inlet of the at least one compartment,-removing oxygen at or near the gas permeable section of the conduit, -flowing the liquid through the conduit, and-providing the liquid substantially void of oxygen to the inlet of the at least one compartment of the flow cell.

17. The method according to claim 16, further comprising:-seeding the membrane with a plurality of cells or a cell layer.

18. The method according to claim 16 or 17 further comprising:-seeding the membrane with one or more epithelial cells.

19. The method according to any of claim 16 to 18 further comprising:-removing the oxygen at or near the gas permeable section of the conduit by surrounding the gas permeable section of the conduit with a fluid, said fluid being adapted to extract oxygen from the surrounding environment.

20. The method according to any of claim 16 to 19 further comprising:-providing a second fluid to the one of the two compartments which is not provided with the liquid substantially void of oxygen.

21. The method according to any of claims 16 to 20 further comprising:-providing a sealable container, the container comprising a port adapted to fluidically connect the conduit to the flow cell, and a valve adapted to connect an inside of the container to an outside environment of the container,-positioning at least the gas permeable section of the conduit within the container,-seal the container,-provide a fluid to within the container through the valve of the container, said fluid being adapted to extract oxygen from the surrounding environment.

22. The method according to claim 21 further comprising:-removing gas from within the container, through the valve, prior to providing the fluid, and optionally:-repeating the steps of removing and providing gas or fluids from within the container.

23. The method according to claim 16 further comprising:-providing a second conduit enveloping at least the gas permeable section of the conduit, the second conduit defining a lumen wherein at least said gas permeable section is positioned, and-providing a fluid within the lumen of the second conduit between the gas permeable section of the conduit and the second conduit, the fluid adapted to extract oxygen from the surrounding environment.

24. The method according to any of claims 16 to 23 further comprising:-adding a drug or compound to the system and subsequently determining the effect of said drug or compound on the system.

25. The method according to claim 24, wherein 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.

26. The method according to claim 25 wherein the microorganism is a bacterium, a parasite, a bacteriophage, a fungus or a virus.

27. A kit of parts for extracting oxygen from a liquid to be provided to a compartment of a flow cell, said flow cell adapted to simulate an in vitro environment of a biological process, the kit of parts comprising:-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 flow cell, 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 a flow cell,-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.

28. The kit of parts according to claim 27, further comprising a pump adapted to provide flow to the liquid to the flow cell from the conduit.

29. The kit of parts according to any of claims 27 or 28, wherein the fluid comprises a molecule adapted to absorb oxygen, preferably the fluid is c- vitamin ascorbate.

30. An apparatus for extracting oxygen from a liquid, the apparatus comprising :-a conduit having at least a first end and a second end, the conduit comprising an inner wall and an outer wall, the inner wall defining an innerlumen and the outer wall defining an outer lumen, the inner wall positioned within the outer lumen, wherein the inner wall is permeable to gas and the outer wall is not permeable to gas, wherein the inner lumen is adapted to convey the liquid from which oxygen is to be extracted and the outer lumen is adapted to:-contain an environment void of oxygen, or-a fluid adapted to quench or catch oxygen.

31. The system according to any of claims 1 to 15, the flow cell further comprising:-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:-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, or hydrophilic-treated or coated silicone to enhance cell adhesion, and wherein the housing is made from a material being non-permeable to at least oxygen, from a surrounding atmospheric environment.

32. The system according to claim 31, wherein the pores of the porous membrane are substantially perpendicular to a surface plane of said porous membrane.

33. The system according to claim 31 or 32 wherein the porous membrane is substantially transparent.

34. The system according to any of claims 31 to 33, wherein the porous membrane has a thickness of between 10 and 50 pm.

35. The system according to claim 31, wherein the hydrophilic support has a thickness of between 0.5 and 3 mm.

36. The system according to claim 31 or 35, wherein the hydrophilic support is a chemical blend or a co-polymer or an interpenetrating polymer network.

37. The system according to any of claims 31 or 35 to 36, wherein the hydrophilic support comprises between 10 and 40% hydrogel.

38. The system according to any of claims 31 to 37, 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.

39. The system according to any of claims 31 to 38, 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.

40. The system according to any of claims 31 to 38, 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.

41. The system according to any of claims 31 to 38, wherein the second compartment comprises an inlet and an outlet adapted to provide a flow of a fluid through said second compartment.

42. The system according to any of claims 31 to 41, wherein at least a portion of the housing is transparent.

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