Bioreactor for tissue or cell culture, gas exchange unit and system comprising the bioreactor and the gas exchange unit, and related culture method for culturing a sample comprising tissue or embedded cells

Abstract

The present invention relates to a bioreactor for culturing samples and allowing simultaneous live-cell imaging of the samples, a gas exchange unit for allowing gas exchange of the culture medium during cell or tissue culture, thereby allowing the bioreactor to be kept suitable for long-term storage outside a culture chamber, a system comprising the bioreactor and the gas exchange unit, and a method for culturing samples.

Description

[0001] The present invention relates to a bioreactor for culturing samples and allowing simultaneous live-cell imaging of the samples, a gas exchange unit for allowing gas exchange of the culture medium during cell or tissue culture and allowing the bioreactor to be kept suitable for long-term preservation outside an incubator, a system comprising the bioreactor and the gas exchange unit, and a method for culturing samples. Technical Field

[0002] This invention relates to the field of biotechnology, particularly to the fields of cell culture and tissue culture, and more specifically to the long-term culture of thick and small tissue samples in a specific system, further enabling in vitro live-cell imaging. Background Technology

[0003] Cellular survival depends on a continuous supply of nutrients. Multicellular organisms sustain their individual cells through various means, such as the blood circulation of nutrients and diffusion from the blood into tissues (where cells can take up these nutrients). Cells or tissues separated from their organism can only be maintained with an artificial supply of nutrients. Such an artificial supply of nutrients is particularly challenging for tissues, where cells are densely packed and thus form a strong diffusion barrier.

[0004] In vivo live-cell imaging enables visualization of cellular behavior with high spatiotemporal resolution. One in vivo technique preserves cells in their native environment, minimizing interference from external influences on their behavior. Therefore, this technique has become an important tool in medical science. However, this technique is incompatible with humans simply because it requires the surgical implantation of a transparent imaging window. Furthermore, not only for humans but also for animals, the limited anesthesia time results in short imaging times, low sample throughput, and limited access to the sample. Therefore, the science relies on animal experiments, which have limited comparability and translatability into humans.

[0005] To reduce animal testing and allow such technologies to be applied directly to human cells or tissues, in vitro experiments in bioreactors have received considerable attention in recent years. Bioreactors have been developed that provide nutrients for cells or tissues through surface flow of nutrient-containing liquids and diffusion of nutrients into cells. Furthermore, more advanced techniques targeting the perfusion of tissue samples or cell aggregates are known. However, providing flexible and adaptable bioreactors suitable for both thick and thin tissue samples and enabling live-cell imaging presents numerous challenges.

[0006] WO2014172575A1 discloses a bioreactor system for tissue culture that is compatible with CT or NMR imaging. The tissue is perforated through channels for circulating culture medium, allowing nutrients to diffuse into the tissue. However, such a system is not compatible with standard cell culture equipment, and the internal tissue structure is severely affected by this perforation. Furthermore, the imaging techniques are limited to expensive and highly complex techniques unavailable in standard cell culture laboratories and do not allow imaging of fluorescently labeled cells or structures, which are the most widely used techniques for cell imaging.

[0007] US20220033769A1 and WO2013182574A1 disclose systems including bioreactors for cell and tissue culture suitable for culturing biopsy samples. However, these bioreactors are not suitable for imaging thin and thick tissue samples, especially thin samples, which are either oriented in a way that makes imaging or effective perfusion impossible because the inlet and outlet of the chamber containing the sample are positioned at approximately a 90° angle relative to the imaging plane.

[0008] WO2017025620 discloses a bioreactor for culturing artificial bioprinted tissues, including a movable element for adjusting the volume of a sample chamber in which multiple channels (i.e., inlets and outlets) are opened. However, these channels need to be aligned with blood vessels in natural organs or with a network of artificial channels in artificial tissues. Although the document mentions culturing natural tissue samples, aligning natural blood vessels with channels in this case presents an even greater challenge.

[0009] Therefore, an object of the present invention is to provide an improved system that eliminates or at least reduces the disadvantages of the prior art. In particular, such a system should be suitable for long-term culture of both thin and thick samples, and it should allow for live-cell imaging. This object is fully achieved by the present invention. Summary of the Invention

[0010] In one aspect of the invention, the disadvantages and other disadvantages identified above are overcome by providing a bioreactor for culturing samples comprising tissues or embedded cells, the bioreactor including an inlet configured to introduce fluid into the bioreactor, the inlet comprising:

[0011] The first part includes a sample bed configured to hold the sample within a sample space (where the sample is located) when a sample is inserted.

[0012] The second part, which is movable relative to the sample bed to compress the sample between the second part and the sample bed when the sample has been inserted, and is openable to include a through-hole having a diameter that opens toward the center of the sample space and is configured to direct fluid flow to the center of the sample space, and

[0013] The third part is configured to connect to a fluid supply source.

[0014] According to the present invention, a bioreactor should (as is commonly understood in the art) be understood to include at least one inlet and at least one outlet for connecting the bioreactor to a fluid supply source and a fluid discharge end (e.g., a pipe). Therefore, a bioreactor may include the inlet as defined in claim 1, as well as other inlets. The terms “inlet” and “outlet” must be understood to refer to those devices that can be substantially identically implemented. Thus, an outlet can also be used as an inlet, and vice versa.

[0015] A “section” of a bioreactor can refer to a component of the bioreactor or any defined area of ​​a component that includes at least one section of the bioreactor. A component can be configured to be disassembled and therefore removed from the bioreactor. Any defined area of ​​a component may not be removed from the bioreactor, or may only be removed along with at least one other section or area.

[0016] The sample can be tissue, embedded tissue, or embedded cells. The term "embedded" should be understood as encapsulating a fragment of tissue or cells in a three-dimensional scaffold suitable for providing a certain degree of stability. Preferably, the scaffold is a gel that is poured onto or around a tissue block or cells in a liquid state and then solidifies to form a gel. More preferably, the scaffold is a collagen hydrogel. Collagen hydrogels are advantageously comparable to the natural extracellular matrix.

[0017] Preferably, the scaffold is transparent. This advantageously allows the embedded tissue or embedded cells to be visually observed.

[0018] Preferably, the tissue or embedded tissue is a fragment of liver or ovarian tissue, more preferably a liver biopsy sample or an ovarian biopsy sample. These tissues have been proven suitable for use with the bioreactor according to the invention.

[0019] Throughout this document, the "openability" of the second part should be understood as making the second part "changeable" between an open state and a closed state. This change can be achieved, for example, by moving, deforming, stretching, or by removing and replacing at least a portion of the second part, or by introducing or removing an object into or from the through-hole (the object blocking the through-hole).

[0020] The openability of the second section is advantageous because it allows for the cultivation of thick and thin samples in the bioreactor via perfusion or overflow.

[0021] The term "perfusion" refers to the flow of fluid through the interior of a tissue (which may be embedded) or through the interior of a scaffold in which cells are embedded.

[0022] The second part is further configured to compress the sample. Compressing the sample is advantageous for incubation via perfusion because the fluid is forced to flow completely or almost completely through the sample in this manner. Furthermore, the sample is prevented from being carried away by the fluid flow.

[0023] The through-hole is configured to direct fluid flow to the center of the sample space. It will be clear to those skilled in the art that for this purpose, the third section is in fluid communication with at least the through-hole and the sample bed.

[0024] When the second section is closed, the sample can be cultured by directing fluid flow through the outlet to above or through the sample. The sample in the sample space can be overflowed or perfused. For overflow culture, the second section is preferably closed. For this purpose, the sample is preferably not compressed by the second section. Advantageously, this ensures that the fluid flow overflows the sample.

[0025] When the second section is opened, the sample can be cultured by guiding fluid flow through the through-hole to the top of the sample or through the sample. The sample in the sample space can be overflowed or perfused. For perfusion culture, the second section is preferably opened. For this purpose, the sample is preferably squeezed. Advantageously, this ensures fluid flow perfusion of the sample.

[0026] In a preferred embodiment, the through-hole can be opened by exchanging the closed second portion for the open second portion.

[0027] In another preferred embodiment, the through-hole can be opened by removing a stopper from it. This advantageously allows for easy opening of the through-hole.

[0028] In a preferred embodiment, the through-hole is switchable between the following two states:

[0029] The inverted funnel shape results in a through-hole comprising a narrowest portion and a tapered portion, and also makes the sample space tapered, with the tapered portion forming the apex of the tapered sample space.

[0030] The cylindrical state, wherein the diameter of the through hole in the cylindrical state is greater than the diameter of the through hole at the narrowest part in the inverted funnel-shaped state.

[0031] The conical section and the conical sample space should be understood as being approximately conical in shape, meaning they do not necessarily have to be precisely conical. In particular, the sample space can deviate from a conical shape, as it includes the tip of an approximately conical shape on the top of a component that is approximately cylindrical or of a different shape.

[0032] The inverted funnel shape, resulting in a conical sample space, is advantageous for cultivating samples at least 1 mm thick because the conical shape causes fluid flow to impact a reduced and concentrated central region of the sample, maximizing perfusion into the sample interior. The risk of sample damage is limited due to the sample thickness. Perfusion is particularly enhanced at the center of the sample. For example, the sample can have a thickness of 1 mm, 2 mm, 3 mm, 4 mm, or 5 mm, preferably 2 mm.

[0033] The cylindrical shape is advantageous for cultivating thin samples with a maximum thickness of 1 mm because the fluid flow impacts a large area of ​​the sample uniformly, thus minimizing the risk of sample damage. Due to the limited thickness of the sample, the fluid flow can still fully penetrate the entire interior of the sample.

[0034] In a preferred embodiment, the through-hole is variable by exchanging the open second portion in the inverted funnel shape to the open second portion in the cylindrical shape (and vice versa).

[0035] In a preferred embodiment, the funnel-shaped through-hole can be dumbbell-shaped.

[0036] In another preferred embodiment, at least one opening is formed between the second portion and the sample bed, the opening being arranged circumferentially around the sample space.

[0037] The phrase "at least one opening arranged circumferentially around the sample space" should be understood as such that at least one opening opens at least 60% (more preferably 75%, even more preferably 90%) of the perimeter of the sample space.

[0038] It is advantageous to have at least one opening arranged circumferentially around the sample space, because such an opening allows fluid flow to exit the sample space uniformly in multiple or all directions.

[0039] When a single opening is provided, fluid flow can enter the sample space from one direction and exit in another direction via the same opening. When more than one opening is provided, fluid flow can enter the sample space from one direction and exit in another direction via the same opening or another opening.

[0040] When the second part is opened, fluid flow can enter the sample space through the through-hole and exit uniformly in multiple or all directions through at least one opening arranged circumferentially around the sample space.

[0041] When the second part is closed, fluid flow can enter the sample space from the first direction through at least one opening and exit the sample space in the second direction. Preferably, the first direction and the second direction are parallel to each other, and the fluid flow enters on the first side of the sample space and exits on the second opposite side of the sample space.

[0042] In a preferred embodiment, at least one opening is a single annular opening. The annular opening advantageously allows fluid flow to exit the sample space in all directions.

[0043] In another preferred embodiment, at least one opening is two separate sickle-shaped openings.

[0044] The sickle-shaped opening advantageously allows fluid to flow out of the sample space in almost all directions. Furthermore, the sickle-shaped opening is particularly advantageous when the second section is closed and fluid is introduced via the outlet, as the flow of fluid from the side of the sample is blocked.

[0045] In another preferred embodiment, when the second section is open, the channel is straight and has a central axis. The central axis is preferably at an angle α of 90° to at least one opening that circumferentially surrounds the sample space.

[0046] In another preferred embodiment, the first portion includes a first hollow cylinder, including a first internal thread on the inner surface of the first hollow cylinder, and wherein the second portion includes a first external thread that can be screwed into the first internal thread, wherein the second portion is movable by screwing it into or out of the first internal thread.

[0047] This advantageously allows the second part to remain movable by screwing, while also allowing it to remain openable by exchanging it. Further advantageously, the degree of sample compression can be easily controlled by screwing the second part into the first hollow cylinder. When the second part is removed from the first hollow cylinder, this further advantageously allows easy access to the sample bed for cleaning or sample insertion.

[0048] In this embodiment, the second part and the third part are preferably a single component.

[0049] In another preferred embodiment, the first hollow cylinder includes a second external thread located on the outer surface of the first hollow cylinder, and the third portion includes a second internal thread that can be screwed onto the second external thread.

[0050] This advantageously allows for convenient fluid communication between the third section and the through-hole. Furthermore, when the second and third sections are removed from the first hollow cylinder, this further advantageously allows for easy access to the sample bed for cleaning or sample insertion.

[0051] In another preferred embodiment, the sample bed is at least partially transparent, allowing the sample to be visually observed.

[0052] Visual observation can be performed, for example, by observing the sample with the naked eye or via a magnifying optical system (e.g., a microscope). Those skilled in the art will understand that the sample can be observed in this or other embodiments, with or without a partially transparent sample bed, using imaging techniques (e.g., NMR or CT).

[0053] The transparency of the sample bed advantageously allows for visual observation of the samples, particularly for imaging. More advantageously, it allows for observation of the samples during culture, thus enabling in vitro live-cell imaging using fluorescent cell markers. Live-cell imaging using fluorescent cell markers is advantageous because it allows for high spatial and temporal resolution, enabling visualization and tracking of individual cells and subcellular compartments, and it is a widely established technique that is relatively easy to implement in laboratories equipped with standard equipment. Equipment used for NMR or CT is not considered standard laboratory equipment. Furthermore, fluorescent cell markers allow for the simultaneous use of multiple cell, compartment, or protein-specific markers to specifically visualize and identify a wide variety of different sites within tissues or cells.

[0054] In this embodiment, the compression of the sample between the second part and the sample bed is further advantageous for allowing imaging of the sample, because in this way the sample does not move in the fluid flow and can remain stationary on the object plane of the potential imaging unit for observing the sample from outside the bioreactor.

[0055] In this embodiment, the invention advantageously allows for visual observation of both thin and thick samples. In embodiments where at least one opening circumferentially surrounds the sample space, particularly when the at least one opening is a single annular opening or two separate sickle-shaped openings, the field of view of the imaging unit is not obstructed, hindered, or otherwise impaired by means of means (e.g., outlets or pipes) for draining fluid flowing through the at least one opening. Prior art bioreactors have difficulty allowing live-cell imaging, particularly of thin samples, because the inlet and outlet of the sample space are positioned at approximately a 90° angle relative to the imaging plane.

[0056] The sample bed, which is at least partially transparent, can be made of glass or plastic, and is preferably flat glass, more preferably a microscope coverslip. This advantageously allows for easy microscopic observation of living samples.

[0057] In another preferred embodiment, the bioreactor includes a mesh located between the sample bed and the second portion, wherein the sample (when inserted) is enclosed between the mesh and the sample bed.

[0058] The mesh advantageously allows the sample to be secured to the sample bed, minimizing accidental slippage of the sample, sample movement (especially uncompressed samples) due to fluid flow, or sideflow of the sample during the compression process. Simultaneously, by implementing this securing device in the form of a mesh, easy infusion or diffusion of fluid through the sample is ensured.

[0059] In another preferred embodiment, the bioreactor is manufactured via 3D printing. This allows for personalized manufacturing (potentially tailored to the specific needs of the user), enabling the bioreactor to be adapted to the specific requirements of the sample to be cultured.

[0060] In another preferred embodiment, the material used to prepare the bioreactor is biocompatible, preferably a biocompatible medical resin. The loss of components or substances from the material due to fluid flow should be minimized, as these components or substances will come into contact with the sample. Such selection of bioreactor materials advantageously avoids negative impacts of the bioreactor material on the sample to be cultured.

[0061] In another preferred embodiment, the materials used to prepare the bioreactor are autoclaved and sterilizable. This advantageously allows the bioreactor to be sterilized and reused.

[0062] In another preferred embodiment, the bioreactor may include plugs for insertion into the openings of the bioreactor for autoclaving. Such plugs advantageously ensure the sterility of the bioreactor's interior after autoclaving and before use. Preferably, plugs are placed at all openings representing connections to the interior of the bioreactor during the autoclaving process.

[0063] In another preferred embodiment, the second part can be configured to be screwed on with a screwdriver, which advantageously allows for easy assembly and disassembly of the bioreactor. Furthermore, a stand for the screwdriver can be provided, which advantageously allows for the safe and convenient storage of the screwdriver without placing it on a workbench (which could compromise its sterility).

[0064] In another aspect, the present invention relates to a gas exchange unit for allowing gas exchange in cell culture or tissue culture medium during cell culture or tissue culture, the gas exchange unit comprising:

[0065] The subject, which includes:

[0066] Tubular inner lumen;

[0067] The first connector is configured to connect to the container and allow gas to pass freely;

[0068] The second connector is configured to be at least indirectly connected to the gas supply source and to allow gas to pass freely; and

[0069] Gas filtration membrane,

[0070] The gas filter membrane is impermeable to microorganisms but permeable to gases, and spans a tubular cavity between the first connector and the second connector, allowing gas passing through the tubular cavity from the second connector to the first connector to pass through the gas filter membrane.

[0071] According to the invention, a container connected by a first connector contains fluid to be circulated through the bioreactor. Such fluid is preferably a liquid, more preferably a cell culture medium or tissue culture medium, both of which are water-based liquids supplemented with at least nutrients for the corresponding tissue or cells.

[0072] Throughout this specification, the term "fluid-system" refers to the volume of the internal volume of a container extending through at least one outlet, a sample space, and a first connecting member. This volume preferably extends further through pipes and pumps interconnecting the bioreactor with the gas exchange unit and through an inlet. Fluid to be circulated through the bioreactor is also circulated through the fluid-system and will be referred to as the circulating fluid.

[0073] Throughout this specification, the term "gas volume" refers to the volume that is discharged from the fluid system and extends adjacent to the side of the gas filter membrane that is not facing the fluid system.

[0074] The “tubular” interior should be understood as the internal volume of the body, within which gas can enter and exit from the outside of the body at at least two different locations.

[0075] "Connector" should be understood as a structure that includes means for releasably connecting two objects. Examples of connectors are threaded, snap-fit, plug-in, or pneumatic connections.

[0076] The releasability of the first connector advantageously allows for easy replacement of the container connected to it. Furthermore, it provides easy access to the fluid to be circulated, enabling convenient replacement, replenishment, filling, and removal.

[0077] The releasability of the second connector advantageously allows the gas exchange unit to be kept inside or outside an incubator cabinet and connected to a gas supply source.

[0078] The gas exchange unit advantageously allows for the regulated exchange of gas between the fluid to be circulated and the gas volume. In particular, the CO2 and O2 levels within the fluid system must be regulated, for example, to maintain the required pH level and to allow cellular respiration, respectively.

[0079] Furthermore, the gas exchange unit allows such gas exchange to occur both inside and outside the incubator cabinet. Typically, cell culture flasks, tissue culture flasks, or bioreactors are maintained within an incubator cabinet in an atmosphere of, for example, 5% v / v CO2, which is standard cell or tissue culture equipment and provides such an atmosphere. Outside this atmosphere (i.e., outside the incubator cabinet), CO2 evaporates from the cell or tissue culture medium, causing an increase in pH. The gas exchange unit according to the invention advantageously allows connection to a gas supply source, enabling it to remain outside the incubator cabinet for extended periods while preventing undesirable changes in the pH of the culture medium.

[0080] The gas filter membrane is gas-permeable but microbial-impermeable, and spans a tubular lumen between the first and second connectors, allowing gas passing through the tubular lumen from the second connector to the first connector to pass through the gas filter membrane. Therefore, advantageously, when the container (i.e., the fluid system) is connected to the first connector, any gas entering the container is aseptically filtered. This advantageously keeps the fluid system and the sample sterile, while allowing gas exchange between the fluid system and the gas volume. It will be apparent to those skilled in the art that, for this purpose, the gas filter membrane is fixed to, for example, the body along all its edges in a microbiologically sealed manner.

[0081] In a preferred embodiment, such fixation is achieved by attaching all edges of the gas filter membrane to the body, for example by adhesive bonding, plastic welding, or clamping (most preferably by clamping). Clamping advantageously allows for replacement of the gas filter membrane when needed.

[0082] In another preferred embodiment, the gas exchange unit includes an inlet pipe configured to introduce fluid to be circulated into a container connected by a first connector, and an outlet pipe configured to discharge the fluid from the container. This advantageously allows the gas exchange unit to be connected to a fluid system.

[0083] In a preferred embodiment, the inlet pipe extends through the plane of the gas filter membrane and the second connector, and the outlet pipe extends from the bottom of the container connected by the first connector through the plane of the gas filter membrane and the second connector. Advantageously, when introduced, the fluid drips or falls downwards from the inlet pipe toward the bottom of the container. Unbound by theory, this is presumably enhanced to improve gas exchange between the fluid and gas volumes. The outlet pipe advantageously extends from the bottom of the container, preventing any gas above the fluid contained in the container from escaping from the container, for example, into a bioreactor. This advantageously allows the fluid to be circulated to be pumped or drawn out of the container and through the fluid system. Further advantageously, in this embodiment, the gas exchange unit also functions as a bubble trap, which allows for steady-state fluid flow.

[0084] In a preferred embodiment, the gas exchange unit includes: a counter bridge comprising a first fluid channel and a second fluid channel, each fluid channel extending through or through the plane of the gas filter membrane and through a second connector; and an uptake-tube extending from the second fluid channel to the bottom of a container connected to the first connector, wherein an inlet tube is connected to the first fluid channel and extends within the second connector, and an outlet tube is connected to the second fluid channel and extends within the second connector. In this embodiment, the inlet and outlet tubes do not extend through the plane of the gas filter membrane, which advantageously facilitates the connection and disconnection of the inlet and outlet tubes from the gas exchange unit, as they do not need to be blocked by the gas filter membrane and fixed together with it. Furthermore, advantageously, the inlet and outlet tubes do not need to be permanently fixed to the gas filter membrane, which facilitates replacement and / or cleaning.

[0085] In a preferred embodiment, the inlet pipe and the outlet pipe each extend through the second connector by extending through a pipe hole in the second connector or through a groove at the edge of the second connector.

[0086] In a preferred embodiment, the inlet pipe and outlet pipe, or the first fluid channel and the second fluid channel, each extend through the plane of the gas filter membrane by extending through an opening in the second connector or a groove at the edge of the gas filter membrane.

[0087] In a further preferred embodiment, the first connector includes threads, preferably a third internal thread, such that the first connector is configured to be screwed onto a threaded container.

[0088] The first connector includes threads that advantageously allow the gas exchange unit to be connected to a container that is otherwise closed by a threaded cap, a widely adopted option for closing containers.

[0089] Preferably, the container connected to the first connector is a standard laboratory-grade plastic container, such as a centrifuge container. Such containers are typically closed with a threaded cap. Advantageously, this makes the gas exchange unit compatible with standard laboratory equipment and provides flexibility in use. Furthermore, the container can be easily and flexibly replaced, replenished, filled, and removed.

[0090] In another preferred embodiment, the second connector is configured to be indirectly connected to the gas supply source.

[0091] The "intermediary" connection of the second connector should be understood as connecting the second connector to a component including the third connector, which is directly connected to the gas supply source. The "direct" connection should be understood as connecting the connector itself to the gas supply source.

[0092] In another preferred embodiment, the gas exchange unit includes a cover configured to connect to a second connector, the cover including a third connector configured to connect directly to a gas supply source. Therefore, in this embodiment, the second connector is configured to connect indirectly to the gas supply source via the cover.

[0093] Such a cover is particularly advantageous when the second or third connector does not allow free gas passage or restricts gas passage (e.g., due to a small diameter for free gas passage), and when the corresponding connector is not connected to a gas supply source. Advantageously, when removed from the second connector, the cover ensures free gas passage between the gas volume and the gas exchange unit.

[0094] In a preferred embodiment, the third connector preferably includes a pneumatic connector.

[0095] The third connection includes a pneumatic connector that advantageously allows the gas exchange unit to be easily connected to a conduit, such as a gas supply source, even if the conduit is under pressure. The gas supply source is preferably a gas supply source for the environmental chamber at the top of the microscope stage, which enhances compatibility with the microscope stage-top incubator.

[0096] In another preferred embodiment, the second connector is configured to be directly connected to the gas supply source.

[0097] The second connector, which connects directly to the gas supply source, advantageously reduces the number of components included in the gas exchange unit. This facilitates operation.

[0098] In a preferred embodiment, the second connector includes threads, preferably a third external thread, such that the second connector is configured to be connected indirectly or directly to a gas supply source via a threaded connection.

[0099] The second connector includes threads that advantageously allow for a quick, easy, and stable connection between the gas exchange unit and the gas supply source.

[0100] In a preferred embodiment, the body and / or cover have a grip-friendly profile on their outer surfaces. This advantageously facilitates operation, particularly the connection of the connectors.

[0101] In a further preferred embodiment, at least the body of the gas exchange unit and, under specified conditions, the lid, are manufactured by 3D printing. 3D printing allows for personalized manufacturing, potentially carried out by the respective user of the bioreactor, enabling the gas exchange unit to be adapted to the specific needs of the fluid system (in which culture medium is contained).

[0102] In a further preferred embodiment, the material used to prepare at least the body of the gas exchange unit and, under specified conditions, the cover, is biocompatible, preferably a biocompatible medical resin. This minimizes the components or substances that the fluid flow washes away from the material, as these components or substances will come into contact with the sample. Such selection of bioreactor materials advantageously avoids the negative impact of the bioreactor material on the sample to be cultured.

[0103] In another preferred embodiment, the material used to prepare at least the main body of the gas exchange unit and, under specified conditions, the cover, is autoclaved. This advantageously allows the gas exchange unit to be sterilized and reused.

[0104] The present invention also relates to a system for culturing samples comprising tissues or embedded cells, the system comprising:

[0105] At least one bioreactor,

[0106] At least one gas exchange unit, and

[0107] Piping including the pump.

[0108] At least one bioreactor may be a bioreactor according to the present invention, and / or at least one gas exchange unit may be a gas exchange unit according to the present invention.

[0109] The conduit can be made of rubber, preferably silicone. The conduit may comprise multiple tubes (or pipes) with walls having a wall thickness and an inner diameter, the walls defining an internal volume of the tube configured to allow fluid passage. The inner diameter can be up to 10 mm, preferably ranging from 1 mm to 6 mm, more preferably from 2 mm to 4 mm.

[0110] Preferably, the pump is a peristaltic pump. Peristaltic pumps advantageously provide uniform and continuous fluid circulation without the need for valves. They further allow the fluid system to be kept closed, i.e., completely isolated from the gas volume, the surrounding environment, or pump components, thereby keeping at least the fluid and sample sterile.

[0111] In a preferred embodiment, at least a portion of the bioreactor and piping is configured to be inserted together into an incubator atop a partially transparent microscope stage, and the gas exchange unit is configured to be inserted into a heated water bath.

[0112] At least a portion of the bioreactor, gas exchange unit, and piping are configured to be inserted together into the incubator cabinet.

[0113] Such a system configuration is advantageous because it allows for the operation of the system both inside and outside the incubator cabinet, and its maintenance for extended periods. Keeping the system inside the cabinet advantageously allows for storage, for example, overnight, or when no observation of the samples is required. Keeping the system outside the cabinet advantageously allows for monitoring the system's functionality, manipulating fluids and / or samples, and / or visual observation of the samples.

[0114] Holding the bioreactor inside the incubator atop the microscope stage advantageously allows it to be heated, for example, to 37°C, while it is being held outside the incubator cabinet.

[0115] In a preferred embodiment, the system further includes a support configured to support a container connected to the gas exchange unit via a first connector. This advantageously allows the container to be placed upright on a flat surface (e.g., a workbench or the bottom of a heatable water bath). More preferably, the support is configured to be placed in a heatable water bath together with the corresponding container.

[0116] In another preferred embodiment, the system comprises two bioreactors. This advantageously allows for the simultaneous cultivation of two identical or different samples under the same or different conditions.

[0117] In this embodiment, the sample bed of each bioreactor is preferably at least partially transparent. This advantageously allows for simultaneous visual observation and / or imaging of two samples. For example, one sample can be used as a control while the other is cultured under conditions where its effect on the sample should be examined.

[0118] In a preferred embodiment, the system further includes an imaging unit configured to image the sample.

[0119] The imaging unit may include a device for optically magnifying the sample, such as a microscope. This advantageously allows for observation of the sample not only at the macroscopic level but also at the microscopic level. For example, the microstructure of the sample, or the behavior or properties of cells, can be observed. The device for optical magnification may be, for example, a fluorescence microscope, a total internal reflection microscope, a confocal microscope, a two-photon microscope, a multiphoton microscope, or a light sheet microscope. Preferably, the imaging unit includes a multiphoton microscope, which advantageously allows imaging of the sample at the cellular and / or subcellular level at depths up to 1 mm or more.

[0120] In addition, the imaging unit may include means for imaging the sample, i.e., means for acquiring images or videos of the sample. The means for imaging the sample can be used with any imaging technique, such as CT, NMR, or live-cell imaging using a microscope.

[0121] Preferably, the imaging unit includes means for performing live-cell imaging of the sample. Live-cell imaging advantageously allows for imaging and observation of living samples, and thus allows for spatiotemporal resolution of the behavior of cells or tissues under specific conditions, such as bioreactors. In particular, when tissue samples are cultured in a bioreactor, live-cell imaging allows for the observation and imaging of the behavior or characteristics of cells in the tissue within their native environment.

[0122] Most preferably, live-cell imaging can be performed during sample culture. This advantageously allows culture to continue during and after imaging. Therefore, samples can be repeatedly imaged at different durations of culture and / or continuously (e.g., under different conditions). Advantageously, this avoids the need to interrupt or pause culture and / or remove samples from the bioreactor for imaging.

[0123] The present invention also relates to a method for culturing samples comprising tissues or embedded cells with a thickness exceeding 1 mm, the method comprising the following steps:

[0124] The sample is fixed at the base of the conical sample space;

[0125] By guiding the fluid flow into the sample through the tip of the conical sample space and allowing the fluid flow to exit the sample through the edge of the conical sample space base, the fluid flow is concentrated at the center of the sample; and

[0126] Maintain the sample for the required period of time.

[0127] As the inventors recognize, reliable perfusion of samples with a thickness of at least 1 mm requires enhanced perfusion, especially at the center of the sample (where the culture medium is less accessible compared to, for example, the edges of the sample).

[0128] It is advantageous to fix the sample at the base of the conical sample space and concentrate the fluid flow through the tip of the conical space, as this allows for perfusion of the sample, and especially its center.

[0129] Perfusion is maintained by allowing fluid to exit the sample via the edge of the conical sample space base. Since the fluid flows away from the sample at the edge of the base, it must perfuse most (or even all) of the sample.

[0130] In another embodiment, the method includes using a bioreactor.

[0131] In another embodiment, the method includes using a bioreactor according to the invention or a system according to the invention.

[0132] The use of the bioreactor or system according to the invention advantageously allows for the cultivation of samples with a thickness of at least 1 mm, as well as thinner samples. For example, the sample thickness can be 1 mm, 2 mm, 3 mm, 4 mm or 5 mm, preferably 2 mm.

[0133] In another preferred embodiment, when the system includes an imaging unit, the method further includes an image imaging step. The same advantages applicable to systems including an imaging unit also apply to methods including an imaging step.

[0134] It should be understood that, without departing from the scope of the invention, the features mentioned above and those to be explained below can be used not only in the specific combinations given, but also in other combinations or individually. In particular, the features, characteristics, embodiments, and advantages mentioned with respect to bioreactors are similarly applicable to systems including such bioreactors.

[0135] The invention will now be described and explained in further detail with reference to the following non-limiting embodiments and accompanying drawings. Attached Figure Description

[0136] In the attached diagram:

[0137] Figure 1A A schematic diagram of a bioreactor (including a closed second section) for culturing samples comprising tissues or embedded cells is shown.

[0138] Figure 1B A schematic diagram of a bioreactor (including an open second section) for culturing samples comprising tissues or embedded cells is shown.

[0139] Figure 1C A schematic diagram of the second part of the bioreactor in an inverted funnel shape is shown.

[0140] Figure 2 A bioreactor for culturing samples including tissues or embedded cells is shown;

[0141] Figure 3 shows the second part of a bioreactor used to culture samples including tissues or embedded cells.

[0142] Figure 4 A bioreactor (in cross-section) is shown for culturing samples including tissues or embedded cells.

[0143] Figure 5A A gas exchange unit is shown for allowing gas exchange in cell culture or tissue culture medium during cell culture or tissue culture.

[0144] Figure 5B The second compression ring of the opposed bridge of the gas exchange unit is shown (top view).

[0145] Figure 6 A gas exchange unit (in cross-section) is shown for allowing gas exchange in cell culture or tissue culture medium during cell culture or tissue culture.

[0146] Figure 7A A schematic diagram of a system for culturing samples including tissues or embedded cells is shown, the system including a bioreactor (which includes an open second section).

[0147] Figure 7B A schematic diagram of a system for culturing samples including tissues or embedded cells is shown, the system including a bioreactor (which includes a closed second part).

[0148] Figure 8 The simulation shows the fluid flow through the inverted funnel-shaped through-holes in the second part of the bioreactor.

[0149] Figure 9A The simulation of fluid flow through the sample space of the bioreactor is shown when the through-hole is in a cylindrical or inverted funnel shape (top view of the sample space).

[0150] Figure 9B A visualization of the DRAQ5 concentration, as a measure of fluid flow through the sample space of the bioreactor when the orifice is in a cylindrical or inverted funnel shape, is shown (as a top view over the sample space).

[0151] Figure 10A A graph is shown that illustrates the total number of lysed caspase-3 positive cells in liver biopsy samples after two days of culture (normalized to the total tissue area of ​​the top, middle, and bottom tissue sections).

[0152] Figure 10B The spatial distribution of lysed caspase-3 positive cells in liver biopsy samples after two days of culture is shown (assigned to 40×40 bins for top, middle and bottom tissue sections).

[0153] Figure 11 The time projection plot is shown from a 30-minute video obtained during the culture of an ovarian biopsy sample 4 days after culture. Detailed Implementation

[0154] 1. The bioreactor of the present invention

[0155] Figure 1A and Figure 1B The diagram shows a bioreactor 10 for culturing sample 12, the bioreactor 10 including an inlet configured to introduce fluid into the bioreactor 10, the inlet including a first portion 16, a second portion 18 and a third portion 20.

[0156] The first part 16 includes a sample bed 22 configured to hold the sample 12 in a sample space 24 (where the sample 12 is positioned).

[0157] The sample bed 22 can be a microscope coverslip made of glass with a thickness of 170 µm, and the transparent area for visual observation of the sample can have a diameter of 10 mm.

[0158] The second part 18 is movable relative to the sample bed 22 to press the sample 12 between the second part 18 and the sample bed 22. The second part 18 is openable.

[0159] Part 3, 20, is configured to connect to a fluid supply source.

[0160] Figure 1A Bioreactor 10 is shown, with the second section 18 closed.

[0161] Figure 1B A bioreactor 10 is shown, wherein a second portion 18 is open, thereby including a through-hole 26 having a diameter x, the through-hole 26 being open toward the center of a sample space 24 and configured to direct fluid flow toward the center of the sample space 24.

[0162] In a preferred embodiment, the through-hole 26 has an inverted funnel shape, such that the through-hole 26 includes a narrowest portion 29 and a tapered portion 30, and the sample space 24 is tapered, wherein the tapered portion 30 forms the tip 32 of the tapered sample space 24. The narrowest portion 29 refers to the portion that includes the smallest diameter x compared to any other portion of the through-hole 26 in the inverted funnel shape.

[0163] In a preferred embodiment, the through hole 26 has a cylindrical shape, wherein the diameter x of the through hole 26 in the cylindrical shape is greater than the diameter x of the through hole 26 at the narrowest part 29 in the inverted funnel shape.

[0164] The bioreactor 10 also includes at least one opening 36 formed between the second portion 18 and the sample bed 22. The at least one opening 36 is arranged circumferentially around the sample space 24 and allows fluid to flow out of or into the sample space 24.

[0165] Figure 2 A bioreactor 100 is shown. The bioreactor 100 includes an inlet comprising a first section 16, a second section 18, and a third section 20. Features identical to those shown in Figure 1 are referred to by the same reference numerals.

[0166] The first part 16 of the bioreactor 100 includes a base 38 and a first hollow cylinder 40.

[0167] The base 38 may be a semi-circular plate with a radius of 57 mm and includes a hexagonal chamber 42 and two outlets 44. The chamber 42 may have a bottom 46 and six sides 48a, 48b, 48c, 48d, 48e, and 48f. The bottom 46 of the chamber 42 may include a sample bed 22. The sample bed 22 may be a square region located between the sides 48a, 48b, 48c, and 48d. The bottom 46 may also include two regions 47 located between the sides 48a, 48b, and 48e and 48c, 48d, and 48f, respectively. The regions 47 form an obtuse angle (angle β) relative to the sample bed 22 so that the chamber 42 tapers towards the outlets 44. The sides 48e and 48f may lead to the outlets 44. Each outlet 44 may each include a hollow channel 50 and a port 52, the hollow channel 50 being configured to guide fluid flow from the chamber 42 to the port 52 and vice versa. Port 52 is configured to connect to a conduit (not shown), for example, by means of at least partially entering the end of the conduit. Any outer edge of the base 38 may be rounded.

[0168] In other preferred embodiments, the plate forming the base 38 can have any shape. For example, the plate can be circular, square, polygonal, elliptical, or irregular in shape.

[0169] In other preferred embodiments, multiple bioreactors can be connected together. For example, two semi-circular plates forming a base 38 can be connected or integrated to form a circular multi-channel base 39. Such a multi-channel base 39 may include more than two bases 38.

[0170] In an embodiment of the bioreactor 100, the first hollow cylinder 40 may include a first open end 54, a second open end 56, a first internal thread 58 located on the inner surface of the first hollow cylinder 40, and a second external thread 60 located on the outer surface of the first hollow cylinder 40. The first internal thread 58 may be an M10×1.5 thread. The second external thread 60 may be an M16×2 thread. The first hollow cylinder 40 can access the chamber 42 through the second open end 56.

[0171] Port 52 is configured to connect to a pipe (not shown) with an inner diameter ranging from 2 to 4 mm.

[0172] The third part 20 of the bioreactor 100 may include a planar circular body 62, a second hollow cylinder 64, a second internal thread 66, a hollow channel 68, and a port 70.

[0173] The planar circular body 62 may include a hollow channel 68 configured to guide fluid flow from port 70 to the second hollow cylinder 64.

[0174] The second hollow cylinder 64 may include a first end 72, a second end 74 and a second internal thread 66 adjacent to the second end 74, and extends vertically from the planar circular body 62, which thereby closes the first end 72 of the second hollow cylinder 64.

[0175] The second internal thread 66 can be an M16×2 thread and can be screwed onto the second external thread 60 of the first hollow cylinder 40.

[0176] Port 70 is configured to connect to a fluid supply source (not shown), preferably to a pipe for the fluid supply source with an inner diameter ranging from 2 to 4 mm, for example, by at least partially introducing it into the end of the pipe.

[0177] The second portion 18 of the bioreactor 100 may include a first external thread 76 on its outer surface. The first external thread 76 may be an M10×1.5 thread and may be screwed into a first internal thread 58, wherein the second portion 18 is movable by screwing it into or out of the first internal thread 58.

[0178] Figure 3 illustrates three preferred embodiments of the second portion 18. In all three embodiments, the second portion may be generally cylindrical and includes an inlet end 78, an outlet end 80, and a cylindrical housing 82. The inlet end 78 is configured to allow fluid flow into the through-hole 26 when the second portion 18 is open. The outlet end 80 is configured to allow fluid flow out of the through-hole 26 when the second portion is closed. At the outlet end 80, the second portion 18 may taper. A first external thread 76 may cover approximately half of the cylindrical housing 82 and is located immediately adjacent to the inlet end 78.

[0179] The second portion 18 can be configured to be screwed on with a screwdriver (not shown). In a preferred embodiment, the second portion 18 includes two recesses 79 on opposite sides of the inlet end 78. Figure 2 As shown in the image, these grooves are configured to provide a grip for the screwdriver.

[0180] In other embodiments, the first external thread 76 may cover 20% to 100% of the cylindrical housing 82.

[0181] Figure 3A A preferred embodiment of the second portion 18 is shown. In this embodiment, the second portion 18 is closed such that it does not include the through hole 26.

[0182] Figure 3B A preferred embodiment of the second portion 18 is shown. In this embodiment, the second portion 18 is open, thereby including a through-hole 26. The through-hole 26 is in an inverted funnel shape, in which the diameter x of the through-hole 26 can be 6 mm at the inflow end 78, 5 mm at the outflow end 80, and 4 mm at the narrowest part 29 of the through-hole 26.

[0183] Figure 3C A preferred embodiment of the second portion 18 is shown. In this embodiment, the second portion 18 is open, thereby including a through hole 26. The through hole 26 is cylindrical, and in this state, the diameter of the through hole 26 can be 6 mm.

[0184] In an embodiment of bioreactor 100, the second portion can be opened by replacing the second portion 18 with a second portion 18 of a different specific embodiment, and the through hole 26 can be changed in the manner described above.

[0185] In other preferred embodiments, the second portion 18 may include a through-hole 26 that is blocked when the second portion 18 is closed. In such an embodiment, closing can be achieved by inserting a plug or cap into the through-hole 26. Opening can be achieved by removing such a plug or cap.

[0186] In other preferred embodiments, the second portion 18 can be opened by moving, deforming or stretching at least a portion of the second portion 18, and the through hole 26 can be changed in the manner described above.

[0187] The bioreactor 100 may further include a sealing ring 84 formed of silicone, which may have an inner diameter of 9 mm and an outer diameter of 13 mm. The sealing ring 84 may be located between the first portion 16 and the third portion 20, more specifically adjacent to the first open end 54 of the first hollow cylinder 40 and the planar circular body 60, and when the second internal thread 66 is screwed onto the second external thread 60, the sealing ring 84 serves as a fluid tightness seal between the first portion 16 and the third portion 20.

[0188] In other embodiments, the sealing ring 84 may be attached to the first portion or the third portion.

[0189] The bioreactor 100 may also include a mesh 85, which may be circular and have a diameter of 8 mm and a mesh size of 100 µm. The mesh 85 is located between the sample bed 22 and the second part 18. The sample 12 is enclosed between the mesh 85 and the sample bed 22 when inserted.

[0190] Figure 4 A bioreactor 100 (in cross-section) for culturing a sample 12, including tissues or embedded cells, is shown in an assembled state. The second part 18 can be opened to include a through-hole 26, which can be in an inverted funnel shape, and the sample 12 can be placed on a sample bed 22 in the chamber 42 of the first part 16.

[0191] The second part 18 can be screwed into the first internal thread 58 of the first hollow cylinder 40 using the first external thread 76, so that the sample 12 can be squeezed between the first part 16 and the second part 18, and so that at least one opening 36 can be formed as two sickle-shaped openings 86.

[0192] In other embodiments, opening 36 may be, for example, an annular opening.

[0193] The third part 20 can be screwed onto the second external thread 60 of the first hollow cylinder 40 using the second internal thread 66, and the sealing ring 84 can seal the bioreactor.

[0194] A fluid (not shown), preferably a liquid, more preferably a tissue culture medium or cell culture medium, can be circulated through the bioreactor 100 in a fluid flow (indicated by arrow 171; for clarity, not every arrow 171 is labeled with reference numeral 171). The fluid can be introduced via an inlet. More specifically, the fluid can first be introduced into a hollow channel 68 via port 70, which guides the fluid flow from port 70 to a second hollow cylinder 64.

[0195] Next, the fluid flow can pass through the sealing ring 84 and enter the through hole 26 of the second part 18 via the inlet end 78, pass through the through hole 26, and exit the through hole 26 via the outlet end 80.

[0196] Upon exiting through-hole 26, fluid at least partially enters and passes through sample 12, thereby perfusing sample 12 (i.e., entering, passing through, and thus perfusing sample space 24). Furthermore, fluid can exit sample 12 at opening 36, passing through said opening 36 and chamber 42. Fluid flow can be introduced into outlet 44 via sides 48e and 48f of chamber 42. More specifically, fluid flow can be introduced into hollow channel 50 and guided from chamber 42 to port 52. Finally, fluid flow can exit bioreactor 100 via said port 52.

[0197] The through-hole 26 may have a central axis 88. The central axis 88 may be at an angle α of 90° relative to at least one opening 36 that circumferentially surrounds the sample space 24.

[0198] In other embodiments, the angle α can be 60° to 90°.

[0199] In other preferred embodiments, the means for assembling the first part 16 with the second part 18 and / or the means for assembling the first part 16 with the third part 20 (specifically implemented as a threaded connection in the embodiment of bioreactor 100) can be any other means that allows the second part 18 to be held movable relative to the sample bed. Examples are snap-fit ​​connections, sliding connections with locking screws, or magnetic connections.

[0200] In other preferred embodiments, at least the second part 18 and the third part 20 may be a single component.

[0201] 2. The gas exchange unit of the present invention

[0202] Figure 5 illustrates a gas exchange unit 200 for allowing gas exchange in cell culture or tissue culture media during cell culture or tissue culture. The gas exchange unit 200 includes a body 90 and a gas filter membrane 92, and may include a first compression ring 94, a C-shaped retaining ring 96, a cap 98, an inlet tube (not shown), an outlet tube (not shown), and an intake tube (not shown).

[0203] The main body 90 includes a tubular inner cavity, a first connector 108 and a second connector 110, and may include an inner surface 104, an outer surface 106, a first connector opening 112, a second connector opening 114, a counter bridge 116, a first pipe hole 120 and a second pipe hole (not shown).

[0204] The body 90 can be formed in a generally cylindrical shape and includes a plurality of protrusions and grooves on its outer surface 106. These protrusions and grooves advantageously form a grip-friendly profile to facilitate operation of the gas exchange unit.

[0205] The first connector 108 may include a third internal thread 109 on the inner surface 104 adjacent to the opening 112 of the first connector, which allows gas to pass freely through the first connector 108. The first connector 108 may be configured to connect to a standard laboratory 50 mL centrifuge tube (not shown). However, it will be apparent to those skilled in the art that the container may have any volume, preferably at least 15 mL.

[0206] The second connector 110 may include a third external thread 111 on the outer surface 106 adjacent to the second connector opening 114, which allows gas to pass freely through the second connector 110. The third external thread 111 may be an M32×2 thread. The second connector 110 may be configured to be connected at least indirectly to a gas supply source (not shown).

[0207] In other preferred embodiments, the first connector 108 and / or the second connector 110 may include any means for releasably connecting the two objects. Examples are threaded, snap-fit, plug-in, or pneumatic connections.

[0208] The opposed bridge 116 may include a second compression ring 124, a first fluid channel 126 and a second fluid channel 128, and may be located between the first connector 108 and the second connector 110.

[0209] The first fluid passage 126 and the second fluid passage 128 may each be a channel or pipe including first ports 130a, 132a and second ports 130b, 132b for connection to a conduit. The first ports 130a, 132a may extend from the second compression ring 124 into the first connector 108 and may be configured to connect to a conduit having a diameter in the range of 4 to 6 mm. The second ports 130b, 132b may extend into the second connector 110 and may be configured to connect to a conduit having a diameter in the range of 2 to 4 mm. Ports 130a, 132a, 130b, and 132b may all be connected to the conduit by at least partially introducing the respective port into the end of the conduit.

[0210] In other embodiments, the diameters of ports 130a, 132a, 130b, and 132b can range from 2 mm to 10 mm, depending on the specific conduit used.

[0211] Figure 5B A top view of the second compression ring 124 is shown. The second compression ring 124 may include a first ring 134, a second ring 136, and a third ring 138, a first support 140, a second support 142, a third support 144, and a fourth support 146. These rings and supports may be in a single plane, such that the shape of the second compression ring may be an overall disc shape. The second compression ring 124 may also include a second gas passage 147, which includes a first aperture 147a, a second aperture 147b, a third aperture 147c, and a fourth aperture 147d, said apertures allowing gas to pass freely.

[0212] like Figure 5A As shown, the first ring 134 can be circumferentially attached to the first fluid channel 126. The second ring 136 can be circumferentially attached to the second fluid channel 128. The third ring 138 can be attached to the inner surface 104 along the periphery of the body 90.

[0213] The first support 140 can extend from the third ring 138 at two radially opposite points at an angle of 180°. The second support 142 can extend from the third ring 138 to the first ring 134 at a 90° angle relative to the first support 140. The third support 144 can extend from the third ring 138 to the second ring 136 at a point radially opposite to the second support 142 at an angle of 180°, and can also be at a 90° angle relative to the first support 140. The fourth support 146 can extend from the first ring 134 to the second ring 136, intersecting the first support 140 at a right angle. A second gas passage 147 is formed between the supports and the rings.

[0214] The gas filter membrane 92 is impermeable to microorganisms but permeable to gases. It can be circular, have a first opening 150 and a second opening 152, and can be made of PTFE. The gas filter membrane 92 can be positioned adjacent to the second compression ring 124 such that the second port 130b of the first fluid channel 126 extends through the first opening 150, the second port 132b of the second fluid channel 128 extends through the second opening 152, the inner diameter of the first ring 134 is aligned with the periphery of the first opening 150, the inner diameter of the second ring 136 matches the periphery of the second opening 152, and the outer diameter of the third ring 138 matches the periphery of the gas filter membrane 92. The gas filter membrane 92 can be positioned between the opposing bridge 116 and the first compression ring 94.

[0215] The first compression ring 94 may include a fourth ring 154, a fifth ring 156, and a sixth ring 158 aligned with rings 134, 136, and 138 of the opposing bridge 116, and a fifth support 160, a sixth support 162, a seventh support 164, and an eighth support 166 aligned with supports 140, 142, 144, and 146 of the opposing bridge 116, respectively. The rings and supports may lie in a single plane, allowing the second compression ring to be generally disc-shaped. The first compression ring 94 may also include a first gas passage 167, which includes a fifth hole 167a, a sixth hole 167b, a seventh hole 167c, and an eighth hole 167d, allowing gas to pass freely. The holes of the first gas passage 167 are formed between the supports and the rings of the first compression ring 94. The first compression ring 94 can be positioned close to the gas filter membrane 92, such that the second port 130b of the first fluid channel 126 extends through the fourth ring 154, the second port 132b of the second fluid channel 128 extends through the fifth ring 156, the inner diameter of the fourth ring 154 matches the periphery of the first opening 150 of the gas filter membrane, the inner diameter of the fifth ring 156 matches the periphery of the second opening 152 of the gas filter membrane 92, and the outer diameter of the sixth ring 158 matches the periphery of the gas filter membrane 92.

[0216] The C-shaped retaining ring 96 can conform to DIN 472 with a diameter of 26 mm and can be positioned adjacent to the first compression ring 94 to secure the gas filter membrane 92 between the opposing bridge 116 and the first compression ring 94.

[0217] When the C-shaped retaining ring 96 is inserted, the alignment of the rings 134, 136, 138 and the supports 140, 142, 144, 146 of the opposing bridge 116 with the corresponding rings 154, 156, 158 and the supports 160, 162, 164, 166 of the first compression ring 94, as well as the corresponding periphery and openings 150, 152 of the gas filter membrane 92, advantageously allows for the microbial sealing and stable fixation of the gas filter membrane 92 within the body 90.

[0218] In other preferred embodiments, the struts and / or rings of the opposing bridge 116 and the first compression ring 94 may be positioned in an alternative manner, provided that at least these rings remain aligned with the corresponding peripheries and openings 150, 152 of the gas filter membrane 92 and that the gas filter membrane 92 remains microbiologically sealed and stably fixed. For example, the first ring 134 and the second ring 136 may be tangentially positioned relative to the third ring 138, and the fourth ring 154 and the fifth ring 156 may be tangentially positioned relative to the sixth ring 158. In such an embodiment, the gas filter membrane 92 does not include the first opening 150 and the second opening 152, but instead includes a first groove and a second groove (not shown), through which a first fluid channel and a second fluid channel extend. In such an embodiment, the struts may be omitted.

[0219] The first pipe hole 120 and the second pipe hole can each be a circular hole on the second connector 110, with a diameter of 5 mm, and can be bridged by a pipe (not shown). The inlet pipe is configured to introduce the fluid to be circulated into the container, and the outlet pipe is configured to discharge the fluid from the container. The first pipe hole 120 can be bridged by an inlet pipe connected to a second port 130b of the first flow channel 126. The second pipe hole can be bridged by an outlet pipe connected to a second port 132b of the second flow channel 128. The first port 132a of the second flow channel 128 can be connected to an intake pipe (not shown) that extends to the bottom of the container.

[0220] In other preferred embodiments, the pipe hole 120 and / or the second pipe hole can have any shape (e.g., elliptical or polygonal) and the diameter can be in the range of 2 mm to 10 mm, depending on the specific pipe used.

[0221] The cover 98 may include a third connector 168, a circular plate 170, and a cylindrical wall 172.

[0222] Figure 6 A gas exchange unit 200 (in cross-section) for allowing gas exchange in cell culture medium or tissue culture medium is shown in an assembled state.

[0223] The circular plate 170 may have a first side 174, a second side 176, and a through hole 178 including a fourth internal thread. A third connector 168 (which may be a pneumatic connector for a 4 mm pipe) can be screwed into the fourth internal thread at the first side 174. The fourth internal thread may be an M5 thread. At the periphery of the circular plate 170, a cylindrical wall 172 may extend at the second side 176. This cylindrical wall 172 may include a fifth internal thread 179 configured to connect to the second connector 110. The fifth internal thread 179 may be an M32×2 thread.

[0224] The cylindrical wall 172 can be formed in a generally cylindrical shape and can include a plurality of protrusions and grooves on its outer surface. These protrusions and grooves advantageously form a grip-friendly profile, thereby facilitating operation of the gas exchange unit.

[0225] In other preferred embodiments, the cylindrical wall 172 may have other shapes and / or other types of grip-friendly profiles, such as a cube. In such embodiments, the circular plate may also have other shapes, such as a square shape.

[0226] In other preferred embodiments, the third connector 168 can be any device for connecting two objects. Examples of connectors are threaded, snap-fit, and plug-in connections.

[0227] In other preferred embodiments, where the second connector 110 has no external threads, the cover 98 may not include the fifth internal thread 179 configured to connect to the second connector 110, but instead include an alternative structure corresponding to the type of the second connector 110.

[0228] In other preferred embodiments, the second connector 110 may be configured to connect directly to a gas supply source. In this case, the opening 114 of the second connector preferably has a large diameter, thereby allowing sufficient gas to pass through when the second connector 110 is not connected to a gas supply source (i.e., when the gas exchange unit 200 is placed inside the incubator cabinet). More preferably, in this case, the second connector 110 is a threaded connection, a snap-fit ​​connection, or a plug-in connection.

[0229] A gas filter membrane 92 can be placed on the opposing bridge 116 such that a first fluid channel 126 extends through a first opening 150 of the gas filter membrane 92, and a second fluid channel 128 extends through a second opening 152 of the gas filter membrane 92. The periphery of the gas filter membrane 92 and the periphery of the openings 150 and 152 of the gas filter membrane respectively contact the opposing bridge 116 and the rings 134, 136, 138, 154, 156, and 158 of the first compression ring 94, which can be secured between the above structures by pressure applied by a C-shaped retaining ring 96. A first connector 108 can be screwed onto a threaded 50 mL centrifuge container filled with liquid to be circulated through the fluid system. A second connector 110 can be screwed into a cap 98 and thus indirectly connected to a gas supply source via the cap 98. A third connector can be directly connected to a gas supply source.

[0230] In use, gas enters the gas exchange unit 200 via the third connector 168 and then the second connector 110. The gas then passes through the second gas channel 167. Afterward, the gas passes through the gas filter membrane 92 and then through the first gas channel 147 to enter a 50 mL centrifuge container. Gas flow is indicated by arrows 169. For clarity, not every arrow 169 is labeled with reference numeral 169. Within the container, the gas can come into contact with the fluid to be circulated, allowing gas exchange with the fluid.

[0231] Furthermore, when connected to a pipeline and in use, the fluid to be circulated can be introduced into the container from the inlet pipe via the first fluid channel 126, and can be drawn out of the container via the intake pipe and the second fluid channel 128 into the outlet pipe. Fluid flow is indicated by arrows 171. For clarity, not every solid arrow 171 is labeled with reference numeral 171.

[0232] 3. The system of the present invention

[0233] Figure 7A A schematic diagram of a system 300 for culturing a sample 12 comprising tissue or embedded cells is shown. The system 300 includes a bioreactor 100, a gas exchange unit 200, and a conduit 180 including a pump 182.

[0234] In use, the liquid to be circulated through the fluid system (usually cell culture medium or tissue culture medium) is circulated by pump 182. The liquid can flow from gas exchange unit 200 into bioreactor 100 via pipe 180, pass through bioreactor 100, leave bioreactor 100 and enter pipe 180, pass through pipe 180, and then re-enter gas exchange unit 200.

[0235] Pump 182 may be a peristaltic pump. In other embodiments, other types of pumps may be used, preferably pumps that do not come into direct contact with the fluid to be circulated, i.e., pumps that allow for closed, sterile fluid systems.

[0236] When the second section 18 of the bioreactor 100 is opened, liquid can enter the bioreactor 100 through the inlet (including the first section 16, the second section 18, and the third section 20) and exit through the outlet 44, such as... Figure 7A As shown.

[0237] Figure 7B A system 400 is shown, which differs from system 300 in that the second part 18 is closed. In such an embodiment, liquid can enter the bioreactor 100 via one outlet 44 and exit via another outlet 44.

[0238] In other embodiments, system 300 may include a bioreactor or a gas exchange unit that is not based on the invention.

[0239] In other preferred embodiments, system 300 or 400 may further include an imaging unit (not shown). In such an embodiment, the sample bed 22 is at least partially transparent, allowing visual observation of the sample 12. It will be apparent to those skilled in the art that the imaging unit is positioned close to the transparent sample bed 22.

[0240] In other preferred embodiments, system 300 or 400 may include more than one bioreactor 100, and / or more than one gas exchange unit 200, and / or more than one pipe 180 or pump 182. System 300 or 400 may also include more than one imaging unit. Preferably, system 300 or 400 includes two bioreactors 100 and two gas exchange units 200.

[0241] 4. Manufacturing

[0242] The bioreactor 100 and gas exchange unit 200 can be manufactured by 3D printing using a Rapid Shape D30 II SLA printer. More specifically, the first part 16, the second part 18, and the third part 20 of the bioreactor 100, as well as the body 90, compression ring 94, and cover 98 (excluding pneumatic connectors) of the gas exchange unit 200, can be 3D printed. Development and design can be carried out in 3D computer-aided design (3D CAD) software. The raw material used for printing can be a biocompatible medical resin.

[0243] The bioreactor 100, made of biocompatible medical resin, has been proven to be leak-free and suitable for repeated high-temperature and high-pressure sterilization.

[0244] Furthermore, the devices configured for use in the assembly and / or autoclaving of the bioreactor 100 and the gas exchange unit 200 can be 3D printed. These advantageously allow for the aseptic assembly of the bioreactor 100 and the gas exchange unit 200.

[0245] In a preferred embodiment, these devices may be a screwdriver for screwing the second part 18 into the first part 16, a stand for the screwdriver, and / or a plug for sealing any port (e.g., port 52 and / or 70) or pipe before autoclaving.

[0246] 5. Simulation of fluid flow

[0247] Figure 8 The simulation shows the fluid flow through a bioreactor 100 comprising a second section 18, which is opened to include a through-hole 26 in an inverted funnel shape. Black indicates high-speed fluid flow. Arrows indicate the direction of fluid flow. The input parameters used for the simulation are listed in Table 1.

[0248] Table 1: Input parameters for fluid flow simulation

[0249]

[0250] In this embodiment, the fluid flow can be introduced into the bioreactor 100 at an angle (approximately 90°) relative to the central axis 88 of the through-hole 26. As the fluid flow enters the second hollow cylinder 64 (leaving the hollow channel 68), it is slowed due to the increased internal volume of the second hollow cylinder 64 compared to the hollow channel 68. The fluid flow can then pass through the through-hole 26, which allows for laminar flow, collimation, and acceleration, directing the fluid flow towards the center of the sample 12 and guiding it into the sample 12 via the tip of the conical sample space 24. The fluid flow is allowed to exit the sample space 24 uniformly in all directions through at least one opening 36 circumferentially surrounding it, i.e., through the edge of the base of the conical sample space 24, as indicated by the fluid flow vector 183. Thus, the fluid flow is concentrated at the center of the sample 12.

[0251] Figure 9A The simulation of fluid flow through sample space 24 is shown when through-hole 26 is in a cylindrical state (top view, 184) or an inverted funnel-shaped state (bottom view, 186) (top view on sample 12). Black indicates higher filling. Sample 12 has an edge 188 and a center 190.

[0252] When the through-hole 26 is in a cylindrical state (see above), the fluid flow is highest at the edge 188 of the sample 12 (near at least one opening 36), and therefore the infusion is most intense. At the center 190 of the sample 12, the fluid flow is low.

[0253] When the through-hole 26 is in an inverted funnel shape (see figure below), the fluid flow is generally more uniform, and is particularly enhanced at the center 190 of sample 12. At the edge 188 of sample 12, the fluid flow is reduced compared to when the through-hole 26 is in a cylindrical shape, but is still not low.

[0254] 6. Cell Culture

[0255] Cell culture was performed on bioreactor 100. Sample 12 consisted of cells expressing nuclear green fluorescent protein (GFP), embedded in a collagen hydrogel with a diameter of 6 mm and a thickness of 2 mm. Sample 12 was inserted into sample bed 22, and bioreactor 100 was assembled. System 300 was then assembled using bioreactor 100, gas exchange unit 200, and tubing 180 including a peristaltic pump. Sample 12 was cultured in system 300 for 5 minutes in cell culture medium supplemented with the nuclear dye DRAQ5 (1,5-bis{[2-(di-methylamino)-ethyl]amino}-4,8-dihydroxyanthracene-9,10-dione) at a perfusion rate of 1.5 mL / min. Part 2 18 included through-holes 26 in either an inverted funnel or cylindrical configuration. As controls, the same sample 12 was cultured in cell culture dishes for 5 minutes (negative control) or 12 hours (positive control) using cell culture medium supplemented with DRAQ5. All samples were cleaned, fixed, and optically transparentized, and then 3D imaged using an Ultramicroscope II light sheet system (covering the entire sample within the field of view). Signals from GFP and DRAQ5 were detected in separate channels. To avoid chromatic aberration and other optical artifacts, the fluorescence intensity of positive cell nuclei was measured for each channel (at the central voxel of the maximum intensity identified using the Difference of Gaussian (DoG) algorithm and processed using a 2D Gaussian filter convolution). To eliminate light absorption and scattering effects, the perfusion-dependent signal from DRAQ5 was normalized to the perfusion-independent GFP signal, and the resulting image stack was summed along the z-axis.

[0256] In other embodiments, the thickness of the sample may be, for example, 1 mm, 2 mm, 3 mm, 4 mm or 5 mm, preferably 2 mm.

[0257] Figure 9B A visualization (top view on sample 12) is shown, serving as a measure of the concentration of DRAQ5 as a measure of samples 12 cultured in system 300 (top left circle 192 and bottom left circle 194). Additionally, a negative control (top right circle 196) and a positive control (bottom right circle 198) are shown for comparison. Black indicates a higher concentration of DRAQ5. In the negative control, perfusion was generally very low at the edge 188 and center 190, while in the positive control, the sample space was uniformly and highly stained with DRAQ5 at both the edge region 188 and the center region 190. The experiment confirmed the presence of DRAQ5 from... Figure 9AThe simulated data shown. While the through-hole 26 in the cylindrical filling state (upper left circle) maintains the injection (especially at the edge 188), the through-hole 26 in the inverted funnel shape (lower left circle) maintains a more uniform injection and a wider range of injection (especially at the center 190), and reaches a sufficient degree at the edge 188.

[0258] 7. Tissue culture

[0259] Tissue culture was performed on system 300. Sample 12 was a live mouse liver biopsy sample with a diameter of 6 mm and a thickness of 2 mm. Sample 12 was inserted into sample bed 22 and bioreactor 100 was assembled. Sample 12 was secured by mesh 85 and by compression of a second part 18. The second part 18 was opened to include a through-hole 26. The through-hole 26 had an inverted funnel shape. Thus, sample 12 was secured at the base of a conical sample space 24. Subsequently, system 300 was assembled by bioreactor 100, gas exchange unit 200, and tubing 180 including pump 182 (a peristaltic pump). The fluid flow was concentrated at the center of sample 12 by guiding the fluid flow through the tip of the conical sample space 24 into sample 12 and allowing the fluid flow to exit through the edge of the base of the conical sample space 24. Figure 8 As shown), sample 12 was cultured in System 300 for 2 days using tissue culture medium at a perfusion rate of 1.5 mL / min. As a control, the same sample 12 was cultured in cell culture dishes for 2 days using tissue culture medium (static control), or fixed immediately at the start of culture (d0 culture). All samples 12 were fixed and embedded in paraffin. Serial sections were prepared.

[0260] In other embodiments, the thickness of the sample may be, for example, 1 mm, 2 mm, 3 mm, 4 mm or 5 mm, preferably 2 mm.

[0261] From all samples 12, one slice each from the top (facing the outflow end of section 18), middle, and bottom (facing the coverslip) of biopsy samples at the same height was selected. These slices were immunofluorescence stained for lysis caspase-3 (a marker of apoptosis (cell death)). The slices were imaged using epifluorescence microscopy with a mosaic scan covering the entire tissue area. For all slices, lysis caspase-3 positive cells were segmented and separated using the same threshold. The total number of lysis caspase-3 positive cells was normalized to the total area of ​​the slice (…). Figure 10A Alternatively, local summarization can be performed within a 40×40 pixel merged array to visualize apoptosis hotspots. Figure 10BThe more cleavable caspase-3 positive cells present, the lower the sample viability.

[0262] Figure 10A The viability of the samples in the d0 control (dashed line) was approximately the same across all tissue sections. In the top tissue sections (group 202 on the x-axis), bioreactor 100 (solid line) showed a slight increase in viability relative to the d0 control. Static culture (dotted line) resulted in a slight decrease in viability compared to the d0 control. In the middle tissue sections (group 204 on the x-axis), static culture significantly reduced viability, while culture in bioreactor 100 resulted in only a slight decrease in viability. In the bottom tissue sections (group 206 on the x-axis), the viability of samples in static culture was the same as or mildly reduced compared to samples cultured in bioreactor 100.

[0263] Figure 10B The viability of the d0 control (column 208) samples was approximately the same across all tissue sections. In the top tissue sections (row 214), bioreactor 100 (column 212) showed enhanced viability compared to the d0 control and the static control (column 210). In the middle tissue sections (row 216), static culture severely reduced viability. Particularly at center 190, spots of lysed caspase-3 positive (i.e., dead) cells were observed. Culture in bioreactor 100 resulted in a reduction and more uniform distribution of these spots. Particularly at center 190, only a very small number of lysase-3 positive cells were detected. In the bottom tissue sections (row 218), the viability of samples cultured statically was the same as or only slightly reduced compared to samples cultured in bioreactor 100.

[0264] Therefore, particularly in the middle slice, i.e. at the center 190 of sample 12, the viability of sample 12 can be enhanced by using the bioreactor according to the invention.

[0265] 8. Live-cell imaging

[0266] Figure 11 The time projection of a 30-minute video taken during the 4-day culture of an ovarian biopsy sample in System 300 is shown.

[0267] A live biopsy sample from a mouse ovary containing fluorescently labeled metastatic breast cancer cells (MMPV-PyMT-ML1B1B1) was inserted into sample bed 22, and bioreactor 100 was assembled. Sample 12 was secured by mesh 85 and by compression of a second section 18. The second section 18 was opened to include a through-hole 26. The through-hole 26 was in an inverted funnel shape. Thus, sample 12 was secured at the base of the conical sample space 24. The second connector 110 of the gas exchange unit 200 had no connector cover and no gas supply source. The gas exchange unit 200 was connected to a 50 mL centrifuge container containing tissue culture medium via a first connector 108. System 300 (except for pump 182 and part of tubing 180) was placed in an incubator cabinet, and the pump was activated. The fluid flow was concentrated at the center of sample 12 by guiding the fluid flow through the tip of the conical sample space 24 into sample 12 and allowing the fluid flow to exit through the edge of the base of the conical sample space 24. Figure 8 As shown), sample 12 was cultured in system 300 at a perfusion rate of 1.5 mL / min for four days. Afterward, bioreactor 100 was transferred to a microscope stage top incubator (PECON) located atop an inverted multiphoton microscope, and gas exchange unit 200, along with its supporting frame, was transferred to a heated water bath assembled from water-filled beakers placed within a bottle heater sleeve (PECON). It should be understood that the piping and pump are correspondingly arranged. A cap is connected to a second connector 110. A CO2 supply source is connected to gas exchange unit 200 via a third connector 168 (a pneumatic connector). The culture continues.

[0268] Imaging was performed externally to the incubator chamber using an inverted multiphoton microscope. Connective tissue (collagen) was detected using second harmonic generation (SHG) signals. Breast cancer metastases were detected using fluorescence signals from mCherry. Figure 11 The brighter the detected signal from mCherry, the later it appears in the 30-minute video. Figure 11 In the image, circle 220 highlights a group of cells that underwent strong contraction during the 30-minute video. Circle 222 highlights cell movement. Circle 224 highlights stationary cells.

[0269] Therefore, sample 12 can be visually observed during tissue culture or cell culture in system 300 (i.e., in bioreactor 100). Furthermore, in vitro live cell imaging can be performed during tissue culture or cell culture. Additionally, sample 12 can be imaged to generate images and / or videos.

[0270] 9. Conclusion

[0271] The inventors provide a highly flexible system 300 or 400 including a bioreactor 100 and a gas exchange unit 200, which allows for cell or tissue culture of thick and thin samples 12 in both overflow and perfusion modes, and also allows for in vitro live cell imaging.

Claims

1. A bioreactor (100) for culturing a sample (12) comprising tissue or embedded cells, the bioreactor (100) including an inlet configured to introduce fluid into the bioreactor (100), the inlet comprising: - A first part (16), the first part (16) includes a sample bed (22) configured to hold the sample (12) in the sample space (24) where the sample (12) is located when the sample (12) is inserted; - A second portion (18), which is movable relative to the sample bed (22) such that when the sample (12) has been inserted, it presses the sample (12) between the second portion (18) and the sample bed (22), and the second portion (18) is openable, thereby including a through hole (26) having a diameter x, the through hole (26) opening toward the center of the sample space (24) and being configured to guide fluid flow to the center of the sample space (24), and - The third part (20) is configured to be connected to a fluid supply source.

2. The bioreactor (100) as claimed in claim 1, wherein the through-hole (26) is adjustable between an inverted funnel shape and a cylindrical shape (34). In the inverted funnel-shaped configuration, the through-hole (26) includes a narrowest portion (29) and a tapered portion (30), and the sample space (24) is tapered, wherein the tapered portion (30) forms the tip (32) of the tapered sample space (24), and In the cylindrical state (34), the diameter x of the through hole (26) in the cylindrical state (34) is wider than the diameter x of the through hole (26) at the narrowest part (29) in the inverted funnel state.

3. The bioreactor (100) as claimed in any of the preceding claims, wherein at least one opening (36) is formed between the second portion (18) and the sample bed (22), the opening being arranged circumferentially around the sample space (24).

4. The bioreactor (100) of claim 3, wherein the at least one opening (36) is two separate sickle-shaped openings (86).

5. The bioreactor (100) as claimed in any of the preceding claims, wherein the first portion (16) comprises a first hollow cylinder (40), the first hollow cylinder (40) comprising a first internal thread (58) located on the inner surface of the first hollow cylinder (40), and The second part (18) includes a first external thread (76) that can be screwed into the first internal thread (58), wherein the second part (18) is movable by screwing into or out of the first internal thread (58).

6. The bioreactor (100) of claim 5, wherein the first hollow cylinder (40) includes a second external thread (60) located on the outer surface of the first hollow cylinder (40), and the third portion (20) includes a second internal thread (66) capable of being screwed onto the second external thread (60).

7. The bioreactor (100) as claimed in any of the preceding claims, wherein the sample bed (22) is at least partially transparent, such that the sample (12) can be visually observed.

8. The bioreactor (100) according to any of the preceding claims, the bioreactor (100) comprising a mesh (85) wherein the mesh (85) is located between the sample bed (22) and the second portion (18), and wherein the sample (12) is enclosed between the mesh (85) and the sample bed (22) when inserted.

9. A gas exchange unit (200) for allowing gas exchange in cell culture or tissue culture medium during cell culture or tissue culture, said gas exchange unit (200) comprising: - Body (90), said body (90) comprising: tubular inner lumen, The first connector (108) is configured to connect to the container and allow gas to pass freely. The second connector (110) is configured to be at least indirectly connected to a gas supply source and to allow free passage of gas; and - Gas filter membrane (92). The gas filter membrane is impermeable to microorganisms but permeable to gases, and spans a tubular cavity between the first connector (108) and the second connector (110), such that gas passing through the tubular cavity from the second connector (110) to the first connector (108) passes through the gas filter membrane (92).

10. The gas exchange unit (200) of claim 9, wherein the gas exchange unit (200) comprises: An inlet pipe is configured to introduce the fluid to be circulated into a container connected to the first connector (108); An outlet pipe is configured to discharge the fluid from the container; and preferably Opposite bridge (116), said opposite bridge (116) comprising: A first fluid channel (126) and a second fluid channel (128), each fluid channel extending through the plane of the gas filter membrane, and An intake tube extends from the second fluid channel (128) to the bottom of a container to which the first connector (108) is connected. The inlet pipe is connected to the first fluid channel (126) and extends inside the second connector (110), and the outlet pipe is connected to the second fluid channel (128) and extends inside the second connector (110), wherein the inlet pipe and the outlet pipe preferably extend via a pipe hole (120) in the second connector (110), and wherein the first fluid channel (126) and the second fluid channel (128) preferably extend via openings (150, 152) in the gas filter membrane.

11. The gas exchange unit (200) of any one of claims 9 to 10, the gas exchange unit (200) comprising a cover (98); the cover (98) comprising a third connector (168) for direct connection to a gas supply source, wherein the first connector (108), the second connector (110) and the third connector (168) each preferably comprise threads, such that the first connector (108) is configured to be screwed onto a threaded container, and the second connector (110) is configured to be connected to the gas supply source via a threaded connection with the cover (98), wherein the third connector (168) is preferably a pneumatic connector.

12. A system (300; 400) for culturing a sample (12) comprising tissue or embedded cells, said system (300; 400) comprising: - At least one bioreactor (100) as claimed in any one of claims 1 to 8. - At least one gas exchange unit (200) as claimed in any one of claims 9 to 11; and - Piping (180) including pump (182).

13. The system of claim 12, wherein the system (300; 400) further comprises an imaging unit configured to image the sample (12).

14. A method for culturing a sample (12) with a thickness greater than 1 mm and comprising tissue or embedded cells, the method comprising the following steps: - Fix the sample (12) at the base of the conical sample space (24); - By guiding the fluid flow through the tip of the conical sample space (24) into the sample (12) and allowing the fluid flow to exit the sample (12) through the edge of the base of the conical sample space (24), the fluid flow is concentrated at the center of the sample (12); and - The time period required to incubate the sample (12).

15. The method of claim 14, wherein the method further comprises using a bioreactor (100) as claimed in any one of claims 1 to 8 or a system (300; 400) as claimed in any one of claims 12 to 13; and preferably, when the system (300; 400) includes the imaging unit, the method further comprises the step of imaging the sample (12).

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